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2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels
Abstract
The action of 2-aminoethoxydiphenyl borate (2-APB) on Ca(2+) signalling in HeLa cells and cardiac myocytes was investigated. Consistent with other studies, we found that superfusion of cells with 2-APB rapidly inhibited inositol 1,4,5-trisphosphate (InsP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOC). In addition to abrogating hormone-evoked Ca(2+) responses, 2-APB could antagonise Ca(2+) signals evoked by a membrane permeant InsP(3) ester. 2-APB also slowed the recovery of intracellular Ca(2+) signals consistent with an effect on Ca(2+) ATPases. The inhibitory action of 2-APB on InsP(3) receptors (InsP(3)Rs), SOC channels and Ca(2+) pumps persisted for several minutes after washout of the compound. Application of 2-APB to unstimulated cells had no effect on subsequent Ca(2+) responses suggesting that it has a use-dependent action. Mitochondria in cells treated with 2-APB showed a rapid and slowly reversible swelling. 2-APB did not cause the mitochondria to depolarise, but it reduced the extent of mitochondrial calcium uptake. Although 2-APB has been demonstrated not to affect voltage-operated Ca(2+) channels or ryanodine receptors, we found that it gave a concentration-dependent long-lasting inhibition of Ca(2+) signalling in electrically-stimulated cardiac myocytes, where InsP(3)Rs and SOC channels do not play a significant role. Our data suggest that 2-APB has multiple cellular targets, a use-dependent action, is difficult to reverse and may affect Ca(2+) signalling in cell types where InsP(3) and SOC are not active.
... Therefore, we performed semi-quantitative immunoblotting to assess protein levels of STIM1 in RyR2 KO , IRBIT KO , and control INS-1 cells, and found no difference between the three cell lines ( Fig 4F). 2-APB is reported to have multiple targets [41] and to induce mitochondrial swelling [42], so we performed the SOCE assays under conditions that minimized exposure of the cells to 2-APB. In this modified assay, 2-APB was co-applied to cells with thapsigargin, and extracellular Ca 2+ was added back to cells 5 minutes after thapsigargin + 2-APB application. ...
... Our finding that deletion of RyR2 reduces the magnitude of SOCE in INS-1 cells is consistent with Gustafsson et al. [59], who found that RyR activation stimulates Ca 2+ influx via 2-APB-sensitive TRP channels in INS-1E cells. Experiments utilizing 2-APB must be interpreted cautiously, since it can inhibit IP 3 receptors and Ca 2+ -ATPases, in addition to SOCE channels, and can induce mitochondrial swelling [42]. However, in this study, the effects of RyR2 deletion on the magnitude of the SOCE and PLC activity were evident in the absence of 2-APB, and it was used in both control and RyR2 KO cells to inhibit SOCE and PLC activity in well-established assays. ...
The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 μM carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates β-cell electrical activity by controlling Cav current density and SK channel activation.
... In the present study, both STICs and STDs in murine CCSMC were inhibited by CPA, consistent with a role for Ca 2+ release from the SR in the generation of this activity (Craven et al., 2004). STICs and STDs were also abolished by tetracaine and 2APB, antagonists of ryanodine receptors (Rae et al., 1991) and IP 3 receptors (Peppiatt et al., 2003), respectively. Therefore, it appears that the spontaneous activation of TMEM16A channels in murine CCSMC relies on a combination of Ca 2+ release from both RyRs and IP 3 Rs. ...
... Although Na V 1.5 channels are present in mouse CCSM, most would be inactivated at the resting membrane potential (−50 mV to -60 V), providing little contribution to myogenic activity at these potentials, under normal circumstances (Eggermont, 2004). The IP 3 R inhibitor, 2APB has been shown to inhibit store operated Ca 2+ (SOC) entry (Peinelt et al., 2006;Peppiatt et al., 2003). Unfortunately, almost every IP 3 R antagonist has limitations and their selectivity has been questioned. ...
Penile detumescence is maintained by tonic contraction of corpus cavernosum smooth muscle cells (CCSMC), but the underlying mechanisms have not been fully elucidated. The purpose of this study was to characterize the mechanisms underlying activation of TMEM16A Ca2+ -activated Cl- channels in freshly isolated murine CCSMC. Male C57BL/6 mice aged 10-18 weeks were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg.kg-1 ). Whole-cell patch clamp, pharmacological, and immunocytochemical experiments were performed on isolated CCSM. Tension measurements were performed in whole tissue. TMEM16A expression in murine corpus cavernosum was confirmed using immunocytochemistry. Isolated CCSMC developed spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarizations (STDs) in current clamp mode of the whole cell, perforated patch clamp technique. STICs reversed close to the predicted Cl- equilibrium potential and both STICs and STDs were blocked by the TMEM16A channel blockers, Ani9 and CaCC(inh)-A01. These events were also blocked by GSK7975A (ORAI inhibitor), cyclopiazonic acid (CPA, sarcoplasmic reticulum [SR] Ca2+- ATPase blocker), tetracaine (RyR blocker), and 2APB (IP3 R blocker), suggesting that they were dependent on Ca2+ release from intracellular Ca2+ stores. Nifedipine (L-type Ca2+ channel blocker) did not affect STICs, but reduced the duration of STDs. Phenylephrine induced transient depolarizations and transient inward currents which were blocked by Ani9. Similarly, phenylephrine induced phasic contractions of intact corpus cavernosum muscle strips and these events were also inhibited by Ani9. This study suggests that contraction of CCSM is regulated by activation of TMEM16A channels and therefore inhibition of these channels could lead to penile erection.
... As it has been shown that 2-APB interferes with intracellular Ca 2+ dynamics by the antagonization of several channels and receptors [31], and because Ca 2+ overload due to glutamate-induced neuronal toxicity may lead to mitochondrial dysfunction and oxidative stress, especially after reperfusion [32], we wondered whether the neuroprotective effect of this molecule could be associated with a possible reduction in oxidative stress, since prolonged ischemia without restoration of cerebral blood flow (CBF) can also produce an accumulation of reactive oxygen species [33]. To this end, we analyzed the in vivo content of superoxide anion (O 2 •−) in 2-APB-treated stroke mice. ...
... A limitation of our study is attributable to the lack of specificity of 2-APB. It has been described that 2-APB is a membrane-permeable inhibitor of IP3 receptors [31,53], a recognized antagonist of Ca 2+ entry [54,55] and an activator of transient receptor potential family channels [56]. The involvement of 2-APB in Ca 2+ signaling has been related to its thromboprotective properties [26], and neuroprotective roles after ischemia-reperfusion [24,25]. ...
Brain stroke is a highly prevalent pathology and a main cause of disability among older adults. If not promptly treated with recanalization therapies, primary and secondary mechanisms of injury contribute to an increase in the lesion, enhancing neurological deficits. Targeting excitotoxicity and oxidative stress are very promising approaches, but only a few compounds have reached the clinic with relatively good positive outcomes. The exploration of novel targets might overcome the lack of clinical translation of previous efficient preclinical neuroprotective treatments. In this study, we examined the neuroprotective properties of 2-aminoethoxydiphenyl borate (2-APB), a molecule that interferes with intracellular calcium dynamics by the antagonization of several channels and receptors. In a permanent model of cerebral ischemia, we showed that 2-APB reduces the extent of the damage and preserves the functionality of the cortical territory, as evaluated by somatosensory evoked potentials (SSEPs). While in this permanent ischemia model, the neuroprotective effect exerted by the antioxidant scavenger cholesteronitrone F2 was associated with a reduction in reactive oxygen species (ROS) and better neuronal survival in the penumbra, 2-APB did not modify the inflammatory response or decrease the content of ROS and was mostly associated with a shortening of peri-infarct depolarizations, which translated into better cerebral blood perfusion in the penumbra. Our study highlights the potential of 2-APB to target spreading depolarization events and their associated inverse hemodynamic changes, which mainly contribute to extension of the area of lesion in cerebrovascular pathologies.
... 2-Aminoethoxydiphenyl borate (2-APB) rapidly inhibits IP3Rs (Maruyama et al., 1997). 2-APB may also affect Ca 2+ refilling to the ER during store operated Ca 2+ entry (SOCE) (Peppiatt et al., 2003), possibly because activation of IP3Rs is important for SOCE (Ma et al., 2000). 2-APB during Ca 2+ refilling to the lysosome significantly blocked Ca 2+ refilling ( Fig. 4.4A,B). ...
... 2-APB during Ca 2+ refilling to the lysosome significantly blocked Ca 2+ refilling ( Fig. 4.4A,B). 2-APB can be difficult to washout (Peppiatt et al., 2003), which might be the cause of the diminished response to the third application of ML-SA1 that we observed (Fig. 4.4A). ...
Lysosomes are acidic intracellular vesicles containing hydrolases that degrade intracellular and extracellular debris delivered through endocytic trafficking and autophagy. Lysosome function requires the establishment of luminal ionic homeostasis for ions including H+ and Ca2+, which are 1,000-5,000 times more concentrated in the lysosome lumen than in the cytosol. Lysosomal H+ homeostasis is required to activate hydrolases and Ca2+ efflux through lysosomal ion channels serves as signals required for precise delivery of hydrolases and cargo and the timely removal of catabolites. Impaired lysosomal Ca2+ homeostasis results in lysosomal dysfunction, lysosomal storage diseases (LSDs), and has been implicated more broadly in neurodegenerative phenotypes. The molecular mechanisms by which lysosomes acquire and refill Ca2+ are unknown. We developed a physiological assay to monitor lysosomal Ca2+ store refilling using specific activators of lysosomal Ca2+ channel TRPML1 to repeatedly induce lysosomal Ca2+ release. In contrast to the prevailing view that lysosomal acidification drives Ca2+ into the lysosome, inhibiting the V-ATPase H+ pump did not prevent Ca2+ refilling. Instead, pharmacological and genetic depletion or chelation of endoplasmic reticulum (ER) Ca2+ prevented lysosomal Ca2+ stores from refilling. More specifically, antagonists of ER IP3 receptors rapidly and completely blocked Ca2+ refilling to lysosomes. Reducing ER Ca2+ or blocking IP3 receptors resulted in a dramatic lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca2+ to the lysosome. These findings may clarify seemingly overlapping ER and lysosome Ca2+ stores in some studies and shed light on why ER Ca2+ homeostasis is often involved in LSDs and neurodegenerative diseases.
... Another molecule that enhanced the human HSPC content is 2-Aminoethoxydiphenyl borate (2-APB) identi ed as an antogonist of inositol 1,4,5-trisphosphate (InsP3R). This molecule inhibits InsP3-induced Ca+2 release from mouse cerebellar membranes in a concentration-dependent manner and also, found as a blocker of storeoperated Ca+2 entry (SOCE) independently of InsP3 (35,36). Moreover, it promotes the gradual release of Ca2+ stores through inhibiting sarco-endoplasmic reticulum Ca2+ ATPases (SERCAs) pumps. ...
... 2-APB is an antogonist of inositol 1,4,5-trisphosphate (InsP3R) This molecule inhibits InsP3-induced Ca+2 release from mouse cerebellar membranes (35). HSCs highly express calcium sensing receptors (CaRs) which is a G protein-coupled receptor to provide sensitivity against to calcium abundance in response to niche(37). ...
Hematopoietic stem cells (HSC), known for their ability to multipotent and self-renew, are often used in HSC transplantation for the treatment of hematological diseases and malignancies. Umbilical cord blood (UCB) and mobilized peripheral blood (mPB), which are alternatives to bone marrow (BM) for HSC transplantation, have reduced HSC. To address this restriction, the ex vivo expansion of HSCs is a highly promising therapeutic approach rather than the use of a double-cord blood unit that induces delays in hematopoietic recovery. We have previously shown that knockout of HSC quiescence genes could increase the HSC pool in vivo. Thus, we thought that targeting HSC quiescence regulators using small molecules could be used for ex vivo expansion of both mPB and UCB-HSC. The goal was to identify novel hematopoietic small molecules (HSMs) and their combinations, and to enhance performance of human HSC expansion medium. We identified and analyzed 35 possible HSMs targeting HSC quiescence factors. We assessed their impact on human HSPC activity, including expansion, quiescence, multilineage capacity, cycling capability and metabolism. We have also investigated their cytotoxic and genotoxic effects on human HSPCs. On the other hand, a transplantation study was performed on immunocompromised mice for the evaluation of the repopulation and engraftment capacities of the expanded cells. We observed that BML-260 and TAME molecules robustly increased both the mPB and UCB-HSPC content and activated the re-entry of HSCs into the cell cycle. Colony Forming Unit (CFU) assay confirmed their improved multilineage capacity. BML-260 proved safer for the viability of expanded cells based on cytotoxicity and genotoxicity assays. In addition, gene expression analysis showed that BML-260 and TAME molecules contributed to HSC expansion by modulating cell cycle kinetics, including p27, Skp2 and Cdh1. In conjunction with these in vitro results, we have observed that BML-260-expanded HSCs had a strong hematopoietic reconstitution capacity. After the determination of the most effective molecule as BML-260, a comparative study of chemically defined media, including various supplements, was analyzed in addition to the BML-260 molecule. These results from in vitro and xenotransplantation experiments have shown that BML-260 molecules can be used therapeutically for human HSC expansion and regulation of HSC activity. In addition, the medium composition found may be a novel platform for human HSC expansion to used in clinical trials.
... With the extension of culture time, the colonies increased, expanded, fused with each other, and covered the bottom of the culture flask by the second day. The passaged cells adhered quickly, distributed 14 uniformly in fusiform, and proliferated rapidly (Fig. 5). Cells from the third passage were selected for this experiment. ...
... It was also found that compared with the natural recovery group, the related indexes of micro-CT were improved after intervention with 2-APB and dantrolene. We 19 consistently found that after intervention with 2-APB and dantrolene, the concentration of Ca 2+ in BMSCs was significantly decreased and ALP activity was increased, which is consistent with the results of Claire et al. [14] . The possible reasons for these results are as follows. ...
Objective To observe the effects of arsenic and the protective effects of 2-aminoethoxydiphenyl borate (2-APB) and dantrolene on the trabeculae of Sprague Dawley (SD) rats.
Methods Thirty-six SD rats were randomly divided into a control group and an arsenic poisoning group. After 12 weeks of arsenic exposure, six rats in the arsenic poisoning group were randomly selected for sacrifice. The remaining rats were randomly assigned to four groups (n=6 per group): natural recovery after arsenic exposure, dantrolene intervention after arsenic exposure, 2-APB intervention after arsenic exposure, and 2-APB + dantrolene intervention after arsenic exposure. After 21 days of treatment by oral gavage every other day, the bilateral femurs and tibias of the rats were scanned using micro-computed tomography (micro-CT). Bone marrow stromal cells (BMSCs) were isolated and cultured, and Ca²⁺ concentration and alkaline phosphatase (ALP) activity of BMSCs was assessed.
Results Compared with the control group, bone mineralization density (BMD), bone volume (BV)-to-total volume (TV) ratio (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and degree of anisotropy (DA) decreased, while trabecular separation (Tb.Sp), trabecular pattern factor (TBpf), and structural model index (SMI) increased in the arsenic poisoning group (P<0.05); additionally, Ca²⁺ concentration increased and ALP activity decreased significantly in the arsenic poisoning group (P<0.05). Compared with the natural recovery group, after arsenic exposure, the indices of micro-CT recovered to some extent, the Ca²⁺ concentration of BMSCs decreased significantly, and the ALP activity of BMSCs increased significantly after intervention with 2-APB and dantrolene (P<0.05).
Conclusion Arsenic can lead to significant changes in the structure of trabeculae and osteoporosis in rats, and these changes can be improved by intervention with 2-APB and dantrolene.
... In vitro testing methods provide reliable tools to quantify the effects of metals on human cells. Metal ions can interact with cell surface receptors, calcium (Ca 2? ) influx channels and intracellular Ca 2? transport systems (Peppiatt et al. 2003;Limke et al. 2003). This interaction causes stress, which leads to increase in intracellular calcium levels. ...
... This elevation in fluorescence ratio with time suggests maximum binding of fura-2 with intracellular Ca 2? . Metal ions can interact with cell surface receptors, Ca 2? influx channels and intracellular Ca 2? transport systems and mimic the intracellular Ca 2? signaling by increasing IP 3 concentrations and releasing Ca 2? from the endoplasmic reticulum (Peppiatt et al. 2003). However, later phase intense increase in fura-2 fluorescence intensity in response to metal ions indicates the participation of more than one intracellular Ca 2? source (Limke et al. 2003). ...
Chlorella is a green alga consumed as dietary food supplement in pulverized form. In addition to its high nutritional value, it is reported as an excellent detoxifying agent. The pulverized Chlorella is partially soluble in water and insoluble portion has been reported for removal of mercury, cadmium and radioactive strontium from body. Chlorella contains a variety of metal-binding functional groups such as carboxyl, amino, phosphoryl, hydroxyl and carbonyl groups, which has high affinity towards various metal ions. The present study was envisaged to evaluate the chelating effect of water soluble fraction of Chlorella powder (AqCH) on metal ions. Fura-2 fluorescence ratio (F340/F380) was measured by fluorescence spectrometer (FS) after the exposure of chloride salt of metals viz., strontium, cobalt, barium, cesium, thallium and mercury to lymphocytes. Pretreatment of AqCH (0.1–20 mg mL−1) was given to evaluate the attenuating effect on fura-2 fluorescence ratio induced by metal ions. The intracellular levels of these metal ions were analyzed by atomic absorption spectrophotometer (AAS) and fluorescence microscopy (FM). Pretreatment with AqCH significantly attenuated the metal induced fluorescence ratio in dose-dependent manner. The results of AAS and FM were found in coherence with fura-2 fluorescence ratio which emphasized that AqCH significantly prevented the metal ions internalization. The present study suggests AqCH chelates with these metal ions and prevents its interaction with cells thereby reducing the intracellular mobilization of Ca2+.
... 2-APB is an IP 3 R receptor antagonist, which can act on IP 3 R of the ER and inhibit Ca 2+ release [30]. The study found that 2-APB could block the increase of Ca 2+ concentration in Plasmodium berndii sporozoites [31] and inhibit the release of Ca 2+ from HeLa cells and cardiomyocytes [32]. The present study found that TROP2 knockdown upregulated the expression of calcium release channel protein IP 3 R, and increased the Ca 2+ concentration of TNBC cells, and a large amount of enlarged ER was observed in the cytoplasm of TNBC cells. ...
Objective: To explore the molecular mechanisms of tumor-associated calcium signal transduction factor 2 (TROP2) affecting the occurrence and development of triple-negative breast cancer (TNBC). Methods: The TCGA database, immunohistochemical staining, and qRT-PCR were used to analyze the expression of TROP2 in TNBC tissues and cells. The protein expressions of TROP2 and inositol 1,4,5-trisphosphate receptor (IP3R) after TROP2 knockdown were detected by western blot (WB). Cell proliferation was detected by CCK8 and colony formation assay, Annexin V-APC/PI flow cytometry was used to detect apoptosis, and intracellular calcium ion (Ca²⁺) was detected by flow cytometry with Fura 2-AM fluorescent probe. Finally, the morphological changes of the endoplasmic reticulum (ER) were observed by transmission electron microscopy, and the expression of ER stress (ERS)-related proteins was detected by WB and immunofluorescence staining. Results: TROP2 was up-regulated in TNBC tumor tissues and cells. Silencing TROP2 decreased the proliferation rate and clone formation number, and increased the apoptosis rate and the Ca²⁺ level in TNBC cells. These phenomena were reversed after the addition of 2-APB. In addition, after TROP2 knockdown, the expressions of IP3R and ERS-related proteins were up-regulated, the ER was cystic dilated, and ERS was activated. And the addition of 2-APB significantly inhibited the activation of ERS induced by TROP2 knockdown. Conclusion: TROP2 regulated the proliferation and apoptosis of TNBC cells through a Ca²⁺-dependent ERS signaling pathway.
... Although few studies have reported the involvement of SOCE in NMDAR-mediated calcium response (26,46,60,61), the role of IP3Rs in the context of NMDAR stimulation is largely unexplored. In our study, we used 2-APB, which is reported to block IP3Rs and SOCE (62,63,64) to investigate the role of both these pathways in NMDAR-mediated translation response. In the presence of 2-APB, NMDAR stimulation resulted in hyperphosphorylation of eEF2 at 5 min which recovered by 20 min. ...
Calcium signaling is integral for neuronal activity and synaptic plasticity. We demonstrate that the calcium response generated by different sources modulates neuronal activity–mediated protein synthesis, another process essential for synaptic plasticity. Stimulation of NMDARs generates a protein synthesis response involving three phases—increased translation inhibition, followed by a decrease in translation inhibition, and increased translation activation. We show that these phases are linked to NMDAR-mediated calcium response. Calcium influx through NMDARs elicits increased translation inhibition, which is necessary for the successive phases. Calcium through L-VGCCs acts as a switch from translation inhibition to the activation phase. NMDAR-mediated translation activation requires the contribution of L-VGCCs, RyRs, and SOCE. Furthermore, we show that IP3-mediated calcium release and SOCE are essential for mGluR-mediated translation up-regulation. Finally, we signify the relevance of our findings in the context of Alzheimer’s disease. Using neurons derived from human fAD iPSCs and transgenic AD mice, we demonstrate the dysregulation of NMDAR-mediated calcium and translation response. Our study highlights the complex interplay between calcium signaling and protein synthesis, and its implications in neurodegeneration.
... Studies have pointed out that inositol is involved in the synthesis of phosphatidyl inositol, and phosphatidyl inositol could generate inositol triphosphate through phosphorylation, which could regulate the concentration of calcium ions and control the release of acetylcholine. Acetylcholine could make intestinal contraction frequent through neural pathways, accelerate Aquaculture Nutrition gastrointestinal emptying, shorten the satiation time, and increase the food intake of fish, thus increasing the WGR [29]. Inositol is also a cyclohexane derivative and has a strong affinity with lipids, which could promote lipid metabolism, and improve the utilization of feed lipid, thus improving feed efficiency and accelerating growth. ...
The present study explored the effects of inositol on growth performance, body composition, antioxidant performance, and lipid metabolism of largemouth bass (Micropterus salmoides). Six isonitrogenous and isolipidic diets containing 0 mg/kg (G1, control), 125 mg/kg (G2), 250 mg/kg (G3), 375 mg/kg (G4), 500 mg/kg (G5), and 625 mg/kg (G6) inositol were prepared and fed to cultured fish (initial weight: 110 ± 1 g) for 8 weeks in recirculating the aquaculture systems. The results indicated that compared with G1 group, the weight gain rate (WGR), specific growth rate (SGR), and feed efficiency rate (FER) in the G3 group were significantly higher. The crude lipid content of the whole fish and the liver of cultured fish was significantly reduced with increasing dietary inositol inclusion. However, no significant effects on moisture, crude protein, and ash contents of fish were observed among the different groups. Dietary inositol supplementation significantly increased muscular crude protein. However, muscular total lipid contents were decreased when the inclusion level was higher than 250 mg/kg (G3–G6 groups). As dietary inositol supplemental level increased, serum triglyceride (TG), and cholesterol (TC) contents showed an increasing trend and reached the maximum value in the G3 group. Additionally, serum low-density lipoprotein cholesterol (LDL-C) in G2, G3, G4, and G5 groups was significantly upregulated by increasing inositol. While, there was no significant change in serum high-density lipoprotein cholesterol (HDL-C) among the treatments. Inositol inclusion also significantly reduced the serum alkaline phosphatase (AKP), glutamic–pyruvic transaminase (ALT), and glutamic–oxaloacetic transaminase (AST) activities as well as serum malondialdehyde (MDA) content but significantly increased serum catalase (CAT), superoxide dismutase (SOD) activities, and total antioxidant capacity (T-AOC). Compared with the control group, the activities of hepatic total lipase (TL) and lipoprotein lipase (LPL) were significantly elevated in the G3, G4, and G5 groups. Above all, dietary inositol supplementation could improve growth performance and antioxidant capacity, and reduce the liver fat content of largemouth bass, and the optimal supplementation level of inositol in feed is estimated to be 250.31–267.27 mg/kg.
... These disruptors impair ER-mitochondrion calcium signaling and mitochondrial function by targeting VDAC and GRP78/BiP but do not release the ER from the attached mitochondrion. [50][51][52][53] In line with our results described above, CM have twice the oxidative capacity of PDM ( Figure 7H). Administration of the MAM disruptor 2-APB or clotrimazole decreased FA oxidation in CM, but oxidation in PDM was not affected ( Figure 7H), indicating CM are more dependent on MAM interactions for FA oxidation than PDM. ...
Organelle interactions play a significant role in compartmentalizing metabolism and signaling. Lipid droplets (LDs) interact with numerous organelles, including mitochondria, which is largely assumed to facilitate lipid transfer and catabolism. However, quantitative proteomics of hepatic peridroplet mitochondria (PDM) and cytosolic mitochondria (CM) reveals that CM are enriched in proteins comprising various oxidative metabolism pathways, whereas PDM are enriched in proteins involved in lipid anabolism. Isotope tracing and super-resolution imaging confirms that fatty acids (FAs) are selectively trafficked to and oxidized in CM during fasting. In contrast, PDM facilitate FA esterification and LD expansion in nutrient-replete medium. Additionally, mitochondrion-associated membranes (MAM) around PDM and CM differ in their proteomes and ability to support distinct lipid metabolic pathways. We conclude that CM and CM-MAM support lipid catabolic pathways, whereas PDM and PDM-MAM allow hepatocytes to efficiently store excess lipids in LDs to prevent lipotoxicity.
... 2-aminoethoxy diphenylborinate (2APB), a known inhibitor of inositol 1,4,5-trisphosphate (IP3)-mediated Ca 2+ release (Peppiatt et al., 2003), can also inhibit the NLRP3 inflammasome (Baldwin et al., 2017). Nijami et al revealed that EC144, a specific inhibitor of heat shock protein 90 (HSP 90) can also target the assembly of NLRP inflammasome and prevents IL-1β secretion with an IC 50 value of 99 nM in macrophages (Nizami et al., 2021). ...
Alzheimer's disease (AD), a progressive neurodegenerative disorder, has emerged as the most common form of dementia in the elderly. Several pathological hallmarks have been identified, including neuroinflammation. A comprehensive insight into the underlying mechanisms that can fuel the development of novel therapeutic approaches is necessary due to the alarmingly rapid increase in the frequency of incidence. Recently, NLRP3 inflammasome was identified as a critical mediator of neuroinflammation. Activation of nucleotide‐binding domain (NOD)‐like receptor protein 3 (NLRP3) inflammasome by amyloid, neurofibrillary tangles, impaired autophagy and endoplasmic reticulum stress, triggers the release of pro‐inflammatory cytokines such as IL‐1β and IL‐18. Subsequently, these cytokines can promote neurodegeneration and cognitive impairment. It is well established that genetic or pharmacological ablation of NLRP3 alleviates AD related pathological features in in vitro and in vivo models. Therefore, several synthetic and natural compounds have been identified that exhibit the potential to inhibit NLRP3 inflammasome and alleviate AD associated pathology. The current review article will highlight the various mechanisms by which activation of NLRP3 inflammation occurs during Alzheimer's disease, and how it influences neuroinflammation, neurodegeneration and cognitive impairment. Moreover, we will summarize the different small molecules that possess the potential to inhibit NLRP3 and can pave the path for developing novel therapeutic interventions for AD.
... Il a été caractérisé initialement comme un inhibiteur des IP 3 R (Maruyama et al. 1997, Prakriya et Lewis 2001, puis par la suite comme modulant l'activité de divers membres de la famille TRP (Voets et al. 2001, Trebak et al. 2002, Hu et al. 2004, Li et al. 2006, et de la pompe SERCA (Missiaen et al. 2001, Peppiatt et al. 2003. ...
Durant les processus métastatiques mammaires, les cellules acquièrent des capacités migratoires, notamment par un remaniement du cytosquelette, mais également un remodelage des capacités adhésives hautement dépendantes de la concentration calcique intracellulaire. Depuis deux décennies, plusieurs travaux ont montré l'implication des canaux calciques dans la modulation des capacités métastatiques mammaires. Récemment, plusieurs études ont permis d'établir un rôle du canal calcique Orai3 dans plusieurs processus tumoraux tels que la résistance à la chimiothérapie et la prolifération ainsi que l'apoptose des cellules cancéreuses exprimant les récepteurs aux œstrogènes. Cependant, le rôle du canal Orai3 dans l'agressivité tumorale mammaire tel que le processus métastatique demeure méconnu.Dans un premier temps, nous avons caractérisé l'entrée de calcium, par le canal Orai3, dans les lignées cancéreuses de type basal (MDA-MB-231 et MDA-MB-231 BrM2). En effet, nous avons montré, pour la première fois, que le canal Orai3 est activé au niveau basal, indépendamment des stocks calciques réticulaires, et régule la concentration calcique intracellulaire. D'autre part, nous avons démontré que cette signature calcique régule la migration des cellules cancéreuses mammaires. De plus, nous avons établi que cette modulation des capacités migratoires dépend des facultés adhésives cellulaires. Durant ce processus, la calpaïne (protéase hautement dépendante du calcium) voit son activité modifiée selon l'expression d'Orai3. Lorsque le canal Orai3 est moléculairement invalidé, l'activité de la calpaïne chute entrainant ainsi une adhésivité des cellules importante corrélée à une migration cellulaire réduite.D'autre part, nous avons mis en évidence une corrélation inverse entre l'expression d'Orai3 et la morphologie cellulaire des lignées cancéreuses utilisées. En effet, la diminution d'expression d'Orai3 est associée à une morphologie cellulaire arrondie. De façon intéressante, nous avons découvert que cette régulation de la morphologie cellulaire dépendante d'Orai3 passe par un remaniement du cytosquelette d'actine de façon indépendante du calcium suggérant un effet impliquant Orai3 en tant que protéine (et non pas son activité canalaire). Ce résultat suggère l'interaction d'Orai3 avec des protéines de signalisation impliquées dans le remodelage du cytosquelette d'actine. En effet, l'inhibition moléculaire d’Orai3 est associée à une diminution de l'expression de Focal Adhesion Kinase (FAK) sans pour autant moduler son activité.En conclusion, nos résultats révèlent le rôle pivot d'Orai3 dans les processus migratoires des cellules cancéreuses mammaires basales, via une modulation de l'adhésion cellulaire dépendante du calcium et du remodelage du cytosquelette d’actine indépendamment du calcium
... Like the aforementioned inhibitors, off target effects of 2-APB on store-operated Ca 2+ entry, mitochondria and SERCA pumps have however been reported. Careful titration of 2-APB in cardiomyocytes, showed that when applied at a low concentration of ∼2 µM, selective inhibition of InsP 3 Rs is achieved with no effects on Ca 2+ transients or SR store loading detected [30,33,144]. Caffeine also inhibits InsP 3 Rs but owing to its potent activation of RyRs has limited use in cardiomyocytes [138]. As a complement to experiments involving InsP 3 R inhibition, InsP 3 R may also be activated pharmacologically. ...
The contraction of cardiac muscle underlying the pumping action of the heart is mediated by the process of excitation-contraction coupling (ECC). While triggered by Ca ²⁺ entry across the sarcolemma during the action potential, it is the release of Ca ²⁺ from the sarcoplasmic reticulum (SR) intracellular Ca ²⁺ store via ryanodine receptors (RyRs) that plays the major role in induction of contraction. Ca ²⁺ also acts as a key intracellular messenger regulating transcription underlying hypertrophic growth. Although Ca ²⁺ release via RyRs is by far the greatest contributor to the generation of Ca ²⁺ transients in the cardiomyocyte, Ca ²⁺ is also released from the SR via inositol 1,4,5-trisphosphate (InsP 3 ) receptors (InsP 3 Rs). This InsP 3 -induced Ca ²⁺ release modifies Ca ²⁺ transients during ECC, participates in directing Ca ²⁺ to the mitochondria, and stimulates the transcription of genes underlying hypertrophic growth. Central to these specific actions of InsP 3 Rs is their localization to responsible signalling microdomains, the dyad, the SR-mitochondrial interface and the nucleus. In this review, the various roles of InsP 3 R in cardiac (patho)physiology and the mechanisms by which InsP 3 signalling selectively influences the different cardiomyocyte cell processes in which it is involved will be presented.
This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.
... Second, the degree of 2-APB-dependent inhibition could depend on the histamine concentration employed to characterize the Ca 2+ response in WI-38 lung fibroblasts. For instance, early work carried out in HeLa cells showed that 100 µM 2-APB was able to completely inhibit the ATP-evoked Ca 2+ response at all the tested concentrations, while histamine-evoked Ca 2+ signals were only slightly reduced at high agonist doses (i.e., 100 µM) (Peppiatt et al., 2003). It is likely that a higher concentration of 2-APB is required to block InsP 3 R recruited by histamine in WI-38 lung fibroblasts. ...
Histamine is an inflammatory mediator that can be released from mast cells to induce airway remodeling and cause persistent airflow limitation in asthma. In addition to stimulating airway smooth muscle cell constriction and hyperplasia, histamine promotes pulmonary remodeling by inducing fibroblast proliferation, contraction, and migration. It has long been known that histamine receptor 1 (H1R) mediates the effects of histamine on human pulmonary fibroblasts through an increase in intracellular Ca ²⁺ concentration ([Ca ²⁺ ] i ), but the underlying signaling mechanisms are still unknown. Herein, we exploited single-cell Ca ²⁺ imaging to assess the signal transduction pathways whereby histamine generates intracellular Ca ²⁺ signals in the human fetal lung fibroblast cell line, WI-38. WI-38 fibroblasts were loaded with the Ca ²⁺ -sensitive fluorophore, FURA-2/AM, and challenged with histamine in the absence and presence of specific pharmacological inhibitors to dissect the Ca ²⁺ release/entry pathways responsible for the onset of the Ca ²⁺ response. Histamine elicited complex intracellular Ca ²⁺ signatures in WI-38 fibroblasts throughout a concentration range spanning between 1 µM and 1 mM. In accord, the Ca ²⁺ response to histamine adopted four main temporal patterns, which were, respectively, termed peak, peak-oscillations, peak-plateau-oscillations, and peak-plateau. Histamine-evoked intracellular Ca ²⁺ signals were abolished by pyrilamine, which selectively blocks H1R, and significantly reduced by ranitidine, which selectively inhibits H2R. Conversely, the pharmacological blockade of H3R and H4R did not affect the complex increase in [Ca ²⁺ ] i evoked by histamine in WI-38 fibroblasts. In agreement with these findings, histamine-induced intracellular Ca ²⁺ signals were initiated by intracellular Ca ²⁺ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate (InsP 3 ) receptors (InsP 3 R) and sustained by store-operated Ca ²⁺ channels (SOCs). Conversely, L-type voltage-operated Ca ²⁺ channels did not support histamine-induced extracellular Ca ²⁺ entry. A preliminary transcriptomic analysis confirmed that WI-38 human lung fibroblasts express all the three InsP 3 R isoforms as well as STIM2 and Orai3, which represent the molecular components of SOCs. The pharmacological blockade of InsP 3 and SOC, therefore, could represent an alternative strategy to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients.
... 2-aminoethyldiphenyl borate (2APB), a boron containing compound, is known to inhibit IP 3 -mediated calcium release, store-operated calcium entry, and disrupt other calciumdependent pathways in HeLa cells and cardiac myocytes, which may affect the ion flux balance required to activate NLRP3 (Peppiatt et al., 2003). Further studies demonstrated that 2APB can inhibit nigericin-and ATP-induced NLRP3 inflammasome signaling cascade independently of its effect on calcium flux (Katsnelson et al., 2015). ...
The NLRP3 inflammasome is a multiprotein complex that plays a pivotal role in regulating the innate immune system and inflammatory signaling. Upon activation by PAMPs and DAMPs, NLRP3 oligomerizes and activates caspase-1 which initiates the processing and release of pro-inflammatory cytokines IL-1β and IL-18. NLRP3 is the most extensively studied inflammasome to date due to its array of activators and aberrant activation in several inflammatory diseases. Studies using small molecules and biologics targeting the NLRP3 inflammasome pathway have shown positive outcomes in treating various disease pathologies by blocking chronic inflammation. In this review, we discuss the recent advances in understanding the NLRP3 mechanism, its role in disease pathology, and provide a broad review of therapeutics discovered to target the NLRP3 pathway and their challenges.
... Next, we tested whether IP3R is the channel by which CALRdel52expressing cells mediate efflux of ER Ca 2+ to sustain ER Ca 2+ depletion. To do so, we utilized the allos teric IP3R inhibitor, 2aminoethoxydiphenyl borate (2APB), which inhibits IP3R Ca 2+ release (41). U2OS cells transiently cotransfected with CALR variants and the CEPIA1er Ca 2+ sensor were treated with vehicle or 2ABP for 90 seconds and imaged as previously described in Figs. 2 and 3. We found that CALRdel52expressing cells treated with 2APB dem onstrated significantly increased ER Ca 2+ levels compared with CALRdel52 cells treated with vehicle, as measured by fluorescence of the ER Ca 2+ sensor ( Fig. 5E; Supplementary Fig. S5A). ...
Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca2+ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain unexplored. Here, we show that the loss of Ca2+ binding residues in the type I mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type CALR-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression in vivo.
Significance:
Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.
... To determine whether GC-induced activation of NMII and Ca 2+ flux contributed to GCinduced actin reorganization in polarized epithelial cells, we utilized an MLC kinase (MLCK) inhibitor, ML-7 [71], and a Ca 2+ flux inhibitor, 2APB [72], which have been shown to effectively inhibit GC-induced activation of NMII and Ca 2+ flux [14]. We evaluated their effects on the Factin reduction at adherent sites of Pil+Opa+ GC in polarized epithelial cells, using 3D-CFM and the FIR of F-actin at GC adherent sites relative to the no GC surface area (Fig 4E and 4F). ...
Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.
... 39 It should be noted, however, that several studies have reported that 2-APB have effects on Ca 2+ mobilization not related to inhibition of IP 3 -induced Ca 2+ release, such as blockade of store-operated Ca 2+ entry pathways. [40][41][42] A role for the store operated Ca 2+ channel, Orai1, in mammary gland MEC contraction and lactation has been elegantly demonstrated by Davis et al. 43 Future studies should address the role of Ca 2+ channels in lacrimal gland MEC contraction and tearing. ...
Purpose:
We reported that oxytocin (OXT), added to freshly prepared lacrimal gland lobules, induced myoepithelial cell (MEC) contraction. In other systems, OXT activates phospholipase C (PLC) generating Inositol 1,4,5-trisphosphate (IP3) which increases intracellular calcium concentration ([Ca2+]i) causing contraction. The aim of the current study was to investigate the role of this pathway in OXT-induced contraction of MEC.
Methods:
Tear volume was measured using the cotton thread method. Lacrimal gland MEC were isolated and propagated from α-smooth muscle actin (SMA)-green fluorescent protein (GFP) mice, in which MEC express GFP making them easily identifiable. RNA and protein samples were prepared for RT-PCR and Western blotting for G protein expression. Changes in [Ca2+]i were measured in Fura-2 loaded MEC using a ratio imaging system. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software.
Results:
OXT applied either topically to surgically exposed lacrimal glands or delivered subcutaneously resulted in increased tear volume. OXT stimulated lacrimal gland MEC contraction in a dose-dependent manner, with a maximum response at 10-7 M. MEC express the PLC coupling G proteins, Gαq and Gα11, and their activation by OXT resulted in a concentration-dependent increase in [Ca2+]i with a maximum response at 10-6 M. Furthermore, the activation of the IP3 receptor to increase [Ca2+]i is crucial for OXT-induced MEC contraction since blocking the IP3 receptor with 2-APB completely abrogated this response.
Conclusions:
We conclude that OXT uses the PLC/Ca2+ pathway to stimulate MEC contraction and increase lacrimal gland secretion.
... To this end, IICR was either stimulated by ET-1 application or suppressed by co-application of the InsP 3 R inhibitor 2-aminoethoxydiphenyl borate (2-APB) according to the protocol indicated (Fig. 4B). 2-APB was used at a relatively low concentration of 2 µM previously shown to inhibit InsP 3 Rs without effects on pacing-induced Ca 2+ transients in cardiomyocytes (Peppiatt et al., 2003). Consistent with the hypothesis that dyadic Ca 2+ dynamics were influenced by ET-1stimulated IICR, 2-APB abrogated the increased maximal Ca 2+ release flux integrated across all dyadic sites during the Ca 2+ transient elicited by its application (Fig. 4C,Di). ...
Excitation–contraction coupling (ECC) relies on temporally synchronized sarcoplasmic reticulum (SR) Ca2+ release via ryanodine receptors (RyRs) at dyadic membrane compartments. Neurohormones, such as endothelin-1 (ET-1), that act via Gαq-associated G protein-coupled receptors (GPCRs) modulate Ca2+ dynamics during ECC and induce SR Ca2+ release events involving Ca2+ release via inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs). How the relatively modest Ca2+ release via InsP3Rs elicits this action is not resolved. Here, we investigated whether the actions of InsP3Rs on Ca2+ handling during ECC were mediated by a direct influence on dyadic Ca2+ levels and whether this mechanism contributes to the effects of ET-1. Using a dyad-targeted genetically encoded Ca2+ reporter, we found that InsP3R activation augmented dyadic Ca2+ fluxes during Ca2+ transients and increased Ca2+ sparks. RyRs were required for these effects. These data provide the first direct demonstration of GPCR and InsP3 effects on dyadic Ca2+, and support the notion that Ca2+ release via InsP3Rs influences Ca2+ transients during ECC by facilitating the activation and recruitment of proximal RyRs. We propose that this mechanism contributes to neurohormonal modulation of cardiac function.
This article has an associated First Person interview with the first author of the paper.
... Hence, we treated the cells with blockers of the two ER channels (Fig. 5). The IP3R blocker 2-aminoethoxydiphenyl borate (2-APB, [42,43]) did slightly reduce the inhibition of GlyT2 by carbachol (Fig. 4a), but this was only significant at high concentrations of the compound (100 µM), suggesting no or minimal involvement of the IP3R pathway in GlyT2 inhibition. In fact, concentrations of 2-APB around 100 µM have been shown to inhibit store operated calcium channels (SOC, see below). ...
The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission and plays a key role in regulating nociceptive signal progression. The cholinergic system acting through muscarinic acetylcholine receptors (mAChRs) also mediates important regulations of nociceptive transmission being the M2 subtype the most abundantly expressed in the spinal cord. Here we studied the effect of M2 mAChRs stimulation on GlyT2 function co-expressed in a heterologous system with negligible levels of muscarinic receptor activity. We found GlyT2 is down-regulated by carbachol in a calcium-dependent manner. Different components involved in cell calcium homeostasis were analysed to establish a role in the mechanism of GlyT2 inhibition. GlyT2 down-regulation by carbachol was increased by thapsigargin and reduced by internal store depletion, although calcium release from endoplasmic reticulum or mitochondria had a minor role on GlyT2 inhibition. Our results are consistent with a GlyT2 sensitivity to intracellular calcium mobilized by M2 mAChRs in the subcortical area of the plasma membrane. A crucial role of the plasma membrane sodium calcium exchanger NCX is proposed.
... Besides the inhibition of ORAI1, 2-APB at 50 µM concentration showed weak inhibitory effect of ORAI2, and significantly potentiated the activity of ORAI3 channels in a store-independent manner (Lis et al., 2007;Peinelt et al., 2008). 2-APB was also shown to inhibit SERCA pumps (Bilmen et al., 2002;Peppiatt et al., 2003) and to modulate the activity of members of the TRP channel superfamily, including members of TRPM subfamily, TRPM6, TRPM7 and TRPM8 (Hu et al., 2004), TRPV subfamily, TRPV1, TRPV2, and TRPV3 (Hu et al., 2004) and members of TRPC subfamily, TRPC3 (Trebak et al., 2002) and TRPC6 (Hu et al., 2004). Its multiple targets and limited specificity promoted the need to develop analogues of 2-APB with improved selectivity and potency. ...
In the adult, vascular smooth muscle cells (VSMC) are normally physiologically quiescent, arranged circumferentially in one or more layers within blood vessel walls. Remodelling of native VSMC to a proliferative state for vascular development, adaptation or repair is driven by platelet-derived growth factor (PDGF). A key effector downstream of PDGF receptors is store-operated calcium entry (SOCE) mediated through the plasma membrane calcium ion channel, ORAI1, which is activated by the endoplasmic reticulum (ER) calcium store sensor, stromal interaction molecule-1 (STIM1). This SOCE was shown to play fundamental roles in the pathological remodelling of VSMC. Exciting transgenic lineage-tracing studies have revealed that the contribution of the phenotypically-modulated VSMC in atherosclerotic plaque formation is more significant than previously appreciated, and growing evidence supports the relevance of ORAI1 signalling in this pathologic remodelling. ORAI1 has also emerged as an attractive potential therapeutic target as it is accessible to extracellular compound inhibition. This is further supported by the progression of several ORAI1 inhibitors into clinical trials. Here we discuss the current knowledge of ORAI1-mediated signalling in pathologic vascular remodelling, particularly in the settings of atherosclerotic cardiovascular diseases (CVDs) and neointimal hyperplasia, and the recent developments in our understanding of the mechanisms by which ORAI1 coordinates VSMC phenotypic remodelling, through the activation of key transcription factor, nuclear factor of activated T-cell (NFAT). In addition, we discuss advances in therapeutic strategies aimed at the ORAI1 target.
... Given that TRPM4 channels are robustly expressed in cardiomyocytes and underlie human cardiac phenotypes ranging from conduction abnormalities to cardiac hypertrophy [33,46], it would be important to further understand the mechanism of action for BTP-2 in cardiac electrophysiology [112]. Another pharmacologic agent that blocks SOCE with high potency in cardiomyocytes is 2-aminoethyldiphenyl borate (2-ABP) [74,108]. A curious aspect of 2-ABP is the ability to activate Orai3 channels, independent of STIM1, or store depletion. ...
Store-operated Ca2+ entry (SOCE) is an ancient and ubiquitous Ca2+ signaling pathway that is present in virtually every cell type. Over the last two decades, many studies have implicated this non-voltage dependent Ca2+ entry pathway in cardiac physiology. The relevance of the SOCE pathway in cardiomyocytes is often questioned given the well-established role for excitation contraction coupling. In this review, we consider the evidence that STIM1 and SOCE contribute to Ca2+ dynamics in cardiomyocytes. We discuss the relevance of this pathway to cardiac growth in response to developmental and pathologic cues. We also address whether STIM1 contributes to Ca2+ store refilling that likely impacts cardiac pacemaking and arrhythmogenesis in cardiomyocytes.
... To a certain extent, these drugs, such as thapsigargin, provide only methodological implications for the study of these channels [182]. The small molecule 2-aminoethyldiphenyl borinate (2-APB) enhances SOCE in the 1-10 µM range and blocks SOCE in the 25-100 µM range [183] and has many intracellular targets, including the InsP 3 R and ORAI channels [184,185]. Although the mechanism of 2-APB as a SOCE activator has been reported, its efficacy in AD is unclear. ...
Alzheimer’s disease (AD) is the most common neurodegenerative disorder. Although the pathological hallmarks of AD have been identified, the derived therapies cannot effectively slow down or stop disease progression; hence, it is likely that other pathogenic mechanisms are involved in AD pathogenesis. Intracellular calcium (Ca2+) dyshomeostasis has been consistently observed in AD patients and numerous AD models and may emerge prior to the development of amyloid plaques and neurofibrillary tangles. Thus, intracellular Ca2+ disruptions are believed to play an important role in AD development and could serve as promising therapeutic intervention targets.
One of the disrupted intracellular Ca2+ signaling pathways manifested in AD is attenuated storeoperated Ca2+ entry (SOCE). SOCE is an extracellular Ca2+ entry mechanism mainly triggered by intracellular Ca2+ store depletion. Maintaining normal SOCE function not only provides a means for the cell to replenish ER Ca2+ stores but also serves as a cellular signal that maintains normal neuronal functions, including excitability, neurogenesis, neurotransmission, synaptic plasticity, and gene expression. However, normal SOCE function is diminished in AD, resulting in disrupted neuronal spine stability and synaptic plasticity and the promotion of amyloidogenesis. Mounting evidence suggests that rectifying diminished SOCE in neurons may intervene with the progression of AD. In this review, the mechanisms of SOCE disruption and the associated pathogenic impacts on AD will be discussed. We will also highlight the potential therapeutic targets or approaches that may help ameliorate SOCE deficits for AD treatment.
... Next, to build into this finding, we performed functional studies in the presence of 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of the IP3r. Noteworthy, it is known that 2-APB also inhibits receptor-and storeoperated Ca 2+ entry 23,24 , and therefore, this inhibitor can be used to study phasic and tonic vascular responses to α -1 adrenergic stimulation 12 . In this set of experiments, aiming at blocking the IP3r, 2-APB was added at the moment we replaced the Krebs' solution with 0[Ca 2+ ] Krebs' solution. ...
Heat-shock protein 70 (HSP70) is a ubiquitously expressed molecular chaperone with various biological functions. Recently, we demonstrated that HSP70 is key for adequate vascular reactivity. However, the specific mechanisms targeted by HSP70 to assist in this process remain elusive. Since there is a wealth of evidence connecting HSP70 to calcium ( $$\hbox {Ca}^{2+}$$ Ca 2 + ), a master regulator of contraction, we designed this study to investigate whether blockade of HSP70 disrupts vascular contraction via impairment of $${\text{Ca}}^{2+}$$ Ca 2 + handling mechanisms. We performed functional studies in aortas isolated from male Sprague Dawley rats in the presence or absence of exogenous $$\hbox {Ca}^{2+}$$ Ca 2 + , and we determined the effects of VER155008, an inhibitor of HSP70, on $$\hbox {Ca}^{2+}$$ Ca 2 + handling as well as key mechanisms that regulate vascular contraction. Changes in the intracellular concentration of $$\hbox {Ca}^{2+}$$ Ca 2 + were measured with a biochemical assay kit. We report that blockade of HSP70 leads to $$\hbox {Ca}^{2+}$$ Ca 2 + mishandling in aorta stimulated with phenylephrine, decreasing both phasic and tonic contractions. Importantly, in $$\hbox {Ca}^{2+}$$ Ca 2 + free Krebs’ solution, inhibition of HSP70 only reduced the $$\hbox {E}_{\mathrm{max}}$$ E max of the phasic contraction if the protein was blocked before IP3r-mediated $$\hbox {Ca}^{2+}$$ Ca 2 + release, suggesting that HSP70 has a positive effect towards this receptor. Corroborating this statement, VER155008 did not potentiate an IP3r inhibitor’s outcomes, even with partial blockade. In another set of experiments, the inhibition of HSP70 attenuated the amplitude of the tonic contraction independently of the moment VER155008 was added to the chamber (i.e., whether it was before or after IP3r-mediated phasic contraction). More compelling, following re-addition of $$\hbox {Ca}^{2+}$$ Ca 2 + , VER155008 amplified the inhibitory effects of a voltage-dependent $$\hbox {Ca}^{2+}$$ Ca 2 + channel blocker, but not of a voltage-independent $$\hbox {Ca}^{2+}$$ Ca 2 + channel inhibitor, indicating that HSP70 has a positive impact on the latter. Lastly, the mechanism by which HSP70 modulates vascular contraction does not involve the $$\hbox {Ca}^{2+}$$ Ca 2 + sensitizer protein, Rho-kinase, nor the SERCA pump, as blockade of these proteins in the presence of VER155008 almost abolished contraction. In summary, our findings shed light on the processes targeted by HSP70 during vascular contraction and open research avenues for potential new mechanisms in vascular diseases.
... Thus, it regulates population-level behavior and cell communication [25]. On the other hand, as in some other kingdoms, calcium is a key regulator in fungi; in this sense, 2-aminoethyl-diphenylborinic acid (2-APB), and calcium channel blockers [26,27], particularly transient potential receptor channels [3,[28][29][30][31][32][33][34]), are attractive BCCs, as they decreases zoospore cleavage by intracellular calcium modulation by almost 40 % that inhibits sporogenesis and zoosporulation [35,36], probably by a mechanism involving blocking channels, as has been described for limiting growth in Choletrotichum graminicola [37]. Additionally, it is known that fungi have a calcium biosensor for chitosan production, and chitosan seems to be a regulator of the production and activity of fungal proteins [38,39]. ...
BACKGROUND:The number of known boron-containing compounds (BCCs) is
increasing due to their identification in nature and innovative synthesis procedures.
Their effects on the fungal kingdom are interesting, and some of their mechanisms of
action have recently been elucidated. METHODS:In this review, scientific reports
from relevant chemistry and biomedical databases were collected and analyzed.
RESULTS: It is notable that several BCC actions in fungi induce social and economic
benefits for humans. In fact, boric acid was traditionally used for multiple purposes, but
some novel synthetic BCCs are effective antifungal agents, particularly in their action
against pathogen species, and some were recently approved for use in humans.
Moreover, most reports testing BCCs in fungal species suggest a limiting effect of
these compounds on some vital reactions. CONCLUSIONS:New BCCs have been
synthesized and tested for innovative technological and biomedical emerging
applications, and new interest is developing for discovering new strategic compounds
that can act as environmental or wood protectors, as well as antimycotic agents that let us improve food acquisition and control some human infections. Free access before march 4, 2021: https://authors.elsevier.com/a/1cPC3,LXbk55zh
... Importantly, 2-APB also has a similar biphasic effect on the IP3R [12,13] but also inhibits the ER Ca 2+ pumps (SERCA [14,15]) and can directly open Orai3 channels, allowing an Orai3-dependent SOCE [16][17][18]. These data all underscore that one limit to the use of 2-APB and its analogs is their lack of specificity, implying that any new interesting molecules derived from them must be tested for its effects on other potential targets like IP3R and SERCA. ...
The store-operated calcium entry, better known as SOCE, forms the main Ca2+ influx pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation and the immune response. During the past decades, several inhibitors were developed, but they lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate (2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups. Among them, our compound P11 showed the best inhibitory capacity with a Ki around 75 nM. Furthermore, below 1 µM, P11 was devoid of any inhibitory activity on the two other main cellular targets of 2-APB, the IP3 receptors, and the SERCA pumps. Interestingly, Jurkat T cells secrete interleukin-2 under phytohemagglutinin stimulation but undergo cell death and stop IL-2 synthesis when stimulated in the presence of increasing P11 concentrations. Thus, P11 could represent the first member of a new and potent family of immunosuppressors.
... Therefore, calcium retention in the ER relieves mitochondrial calcium overload in PS1 mutants which avoids abnormal MPTP opening. This effect can be achieved via mitochondria-ER interaction, given calcium exchange between these two organelles as previously suggested by Toglia et al. in their model 46 and the known effect of 2-APB in mitochondrial calcium 47 . Mitochondrial function seems to be also altered in AD affecting lipid synthesis, respiratory chain, and calcium homeostasis 21 . ...
Presenilin 1 (PS1) mutations are the most common cause of familial Alzheimer’s disease (FAD). PS1 also plays a role in cellular processes such as calcium homeostasis and autophagy. We hypothesized that mutant presenilins increase cellular vulnerability to stress. We stably expressed human PS1, mutant PS1E280A and mutant PS1Δ9 in mouse neuroblastoma N2a cells. We examined early signs of stress in different conditions: endoplasmic reticulum (ER) stress, calcium overload, oxidative stress, and Aβ 1–42 oligomers toxicity. Additionally, we induced autophagy via serum starvation. PS1 mutations did not have an effect in ER stress but PS1E280A mutation affected autophagy. PS1 overexpression influenced calcium homeostasis and generated mitochondrial calcium overload modifying mitochondrial function. However, the opening of the mitochondrial permeability transition pore (MPTP) was affected in PS1 mutants, being accelerated in PS1E280A and inhibited in PS1Δ9 cells. Altered autophagy in PS1E280A cells was neither modified by inhibition of γ-secretase, nor by ER calcium retention. MPTP opening was directly regulated by γ-secretase inhibitors independent on organelle calcium modulation, suggesting a novel direct role for PS1 and γ-secretase in mitochondrial stress. We identified intrinsic cellular vulnerability to stress in PS1 mutants associated simultaneously with both, autophagic and mitochondrial function, independent of Aβ pathology.
... 2-Aminoethoxydiphenyl borate (2-APB), which inhibits Ca 2+ release by blocking IP3 receptors in the endoplasmic reticulum (ER), has been extensively used to reduce Ca 2+ release [9]. 2-APB exerts an effect of altering the IP3-induced Ca 2+ release and can pass through the ER membrane. ...
Objective:
We conducted this study to explore the possible protective effect of 2-aminoethoxydiphenyl borate (2-APB) on experimentally induced optic nerve injury in an acute ischemia-reperfusion (AIR) model.
Materials and methods:
A total of 30 Wistar albino rats were randomly divided into sham, AIR, and AIR+treatment (AIR10) groups. In the sham group, AIR model was not created. In the AIR group, AIR model was created without the administration of drug. In the AIR10 group, 2-APB was administered 10 min before reperfusion.
Results:
Tissue samples were subjected to histological, immunohistochemical, and electron microscopic procedures. Histopathological examination revealed intense hypertrophic cells, more glial cells, capillary dilatation, and intense demyelination areas in the AIR group compared to those in the sham and AIR10 groups. Immunohistochemical staining demonstrated an increase in Orai1 and STIM1 immunoreactivity in the AIR group but less intense staining in the AIR10 group. Electron microscopy revealed injury in optic nerve axons in the AIR group, whereas this type of injury occurred to a lesser extent in the AIR10 group.
Conclusion:
In rats, store-operated Ca2+ entry in the cell had an essential role in optic nerve ischemia-reperfusion injury, and 2-ABP may have a protective effect on optic nerve injury caused due to AIR.
... Previously (Sugita et al. 2016), we found that bath application of 2-aminoethoxydiphenyl borate (2-APB), an antagonist of IP 3 Rs and/or store-operated calcium channels (Maruyama et al. 1997;Iwasaki et al. 2001;Bootman et al. 2002;Peppiatt et al. 2003), during priming HFS significantly decreased the magnitude of depotentiation in hippocampal CA1 neurons, and suggested that IP 3 Rs remain activated after HFS and that the modulation of IP 3 R activity induced during and/or after the subsequent LFS dephosphorylates postsynaptic proteins, leading to a decrease in LTP amplitude. We also found that the induction of depotentiation at CA1 synapses was inhibited by the application of FK506, a calcineurin inhibitor (Liu et al. 1991), to CA1 neurons for 20 min from the end of LFS, and suggested that the dephosphorylation of postsynaptic proteins as a result of calcineurin activation, which occurs during LFS, is maintained by test synaptic stimulation after LFS (Sugita et al. 2016). ...
In CA1 neurons of guinea pig hippocampal slices, long-term potentiation (LTP) was induced in field excitatory postsynaptic potentials (EPSPs) or population spikes (PSs) by the delivery of high-frequency stimulation (HFS, 100 pulses at 100 Hz) to CA1 synapses, and was reversed by the delivery of a train of low-frequency stimulation (LFS, 1000 pulses at 2 Hz) at 30 min after HFS (depotentiation), and this effect was inhibited when test synaptic stimulation was halted for a 19-min period after HFS or for a 20-min period after LFS or applied over the same time period in the presence of an antagonist of N-methyl-D-aspartate receptors (NMDARs), group I metabotropic glutamate receptors (mGluRs), or inositol 1, 4, 5-trisphosphate receptors (IP3Rs). Depotentiation was also blocked by the application of a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor or a calcineurin inhibitor applied in the presence of test synaptic input for a 10-min period after HFS or for a 20-min period after LFS. These results suggest that, in postsynaptic neurons, the coactivation of NMDARs and group I mGluRs due to sustained synaptic activity following LTP induction results in the activation of IP3Rs and CaMKII, which leads to the activation of calcineurin after LFS and depotentiation of CA1 synaptic responses.
... unc-77(e625) animals exposed to 2-APB at both 10 and 40 μM concentrations looked indistinguishable from wild-type animals ( Although we obtained 2-APB for its K + channel activator properties, this drug has a variety of additional pharmacological actions as can be seen in Fig. 3. It inhibits inositol trisphosphate receptors (IP3R), transient receptor potential channels, store-operated calcium channels, and gap junctions made of connexin26 or connexin32 (Bootman et al., 2002;Peppiatt et al., 2003;Bai et al., 2006). Because 2-APB was the only K + channel activator to restore the performance of unc-77(e625) animals to wildtype levels, we reasoned that 2-APB might act instead on calcium channels or other types of channel to rescue locomotion. ...
The Na leak-current channel (NALCN) regulates the resting membrane potential in excitable cells, thus determining the likelihood of depolarization in response to incoming signals. Gain-of-function (gf) mutations in this channel are associated with severe dystonic movement disorders in man. Currently, there are no known pharmacological antagonists or selective modulators of this important channel. A gain-of-function mutation in NALCN of C. elegans [known as unc-77(e625)] causes uncoordinated, hyperactive locomotion. We hypothesized that this hyperactive phenotype can be rescued with pharmacological modulators. Here, we summarize the results of targeted drug screening aimed at identification of drugs that corrected locomotion deficits in unc-77(e625) animals. To assay hyperactive locomotion, animals were acutely removed from food and characteristic foraging movements were quantified. Drug screening revealed that 2-aminoethoxydiphenyl borate (2-ABP), nifedipine, nimodipine, flunarizine and ethoxzolamide significantly decreased abnormal movements in unc-77(e625) animals. 2-APB also corrected egg release and coiling deficits in this strain. In addition, serotonin and dopamine both reduced hyperactive locomotion, consistent with regulatory interactions between these systems and the NALCN. 2-APB induced movement phenotypes in wild-type animals that faithfully mimicked those observed in NALCN knockout strains, which suggested that this drug may directly block the channel. Moreover, 2-APB and flunarizine showed significant structural similarities suggestive of overlap in their mode of action. Together, these studies have revealed new insights into regulation of NALCN function and led to the discovery of a potential pharmacological antagonist of the NALCN.
... To a certain extent, these drugs, such as thapsigargin, provide only methodological implications for the study of these channels [182]. The small molecule 2-aminoethyldiphenyl borinate (2-APB) enhances SOCE in the 1-10 µM range and blocks SOCE in the 25-100 µM range [183] and has many intracellular targets, including the InsP 3 R and ORAI channels [184,185]. Although the mechanism of 2-APB as a SOCE activator has been reported, its efficacy in AD is unclear. ...
Mitochondria absorb calcium ²⁺ at the expense of the electrochemical gradient generated during respiration. The influx of Ca ²⁺ into the mitochondrial matrix helps to maintain metabolic function and results in increased cytosolic Ca ²⁺ during intracellular Ca ²⁺ signaling. Mitochondrial Ca ²⁺ homeostasis is tightly regulated by proteins located in the inner and outer mitochondrial membranes and by cross-talk with endoplasmic reticulum Ca ²⁺ signals. Increasing evidence indicates that mitochondrial Ca¹2+ overload is a pathological phenotype associated with Alzheimer’s disease (AD). As intracellular Ca¹2+ dysregulation can be observed before the appearance of typical pathological hallmarks of AD, it is believed that mitochondrial Ca ²⁺ overload may also play an important role in AD etiology. Exaggerated mitochondrial Ca ²⁺ uptake can easily compromise neuronal functions and exacerbate AD progression by impairing mitochondrial respiration, increasing reactive oxygen species formation and inducing apoptosis. Additionally, mitochondrial Ca ²⁺ overload can damage mitochondrial recycling via mitophagy. This review will discuss the molecular players involved in mitochondrial Ca ²⁺ dysregulation and the pharmacotherapies that target this dysregulation. As most of the current AD therapeutics is based on amyloidopathy, tauopathy, and the cholinergic hypothesis, they achieve only symptomatic relief. Thus, determining how to reestablish mitochondrial Ca ²⁺ homeostasis may aid in the development of novel AD therapeutic interventions.
... Addition of a low concentration of 2-Aminoethyl diphenylborinate (2-APB) (2.5 µmol/L), which is low enough to inhibit IP3-dependent effects in cardiomyocytes without altering cellular Ca 2+ transient amplitude or SERCA function (Maruyama et al., 1997;Bootman et al., 2002;Peppiatt et al., 2003;Ju et al., 2011), had no significant effect on right atrial beating All rights reserved. No reuse allowed without permission. ...
Inositol trisphosphate (IP3) is a major Ca2+-mobilising second messenger and atrial IP3 receptor (IP3R) expression is greatly increased in atrial fibrillation (AF). Cardiac atrial and sino-atrial node myocytes also express Ca2+-stimulated adenylyl cyclases (AC1 and AC8); however the physiological pathways underlying AC1 and AC8 activation are not known. We investigated whether IP3 signalling in cardiac atria and sino-atrial node utilises ACs. Immunocytochemistry in isolated guinea pig atrial myocytes showed type 2 IP3Rs co-localised with AC8, while AC1 was located in close vicinity. UV photorelease of IP3 significantly enhanced Ca2+ transient amplitudes following stimulation of atrial myocytes (31 ± 6 % increase 60 s post photorelease, n=16), an effect abolished by 3 μmol/L MDL-12,330 (MDL), to inhibit ACs, or 1 μmol/L H89, to inhibit PKA. The maximum rate change observed in spontaneously-beating murine right atrial preparations exposed to phenylephrine (14.7 ± 0.5 %, n=10) was significantly reduced by 2.5 μmol/L 2-APB (to 4.7 ± 0.2 %, n=7, P<0.05 compared to control) and abolished by a low dose of MDL (1 μmol/L, n=5). These observations are consistent with a functional interaction between IP3 and cAMP signalling involving Ca2+ stimulation of ACs in cardiac atria and sino-atrial node. Structural evidence supports AC8 as the most likely effector. This signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly AF.
... 2-APB is a broad spectrum inhibitor that, although effective in blocking IP3Rs in native endothelial cells, may inhibit Ca 2+ entry pathways (e.g. Peppiatt et al., 2003;Trebak et al., 2002;Voets et al., 2001;Wilson et al., 2016a). However, in the present experimental conditions, 2-APB did not reduce the slow global Ca 2+ rise evoked by TRPV4 activation suggesting that it is not inhibiting influx in the present experiments. ...
Background and Purpose
The TRPV4 ion channels are Ca²⁺ permeable, non‐selective cation channels that mediate large, but highly localized, Ca²⁺ signals in the endothelium. The mechanisms that permit highly localized Ca²⁺ changes to evoke cell‐wide activity are incompletely understood. Here, we tested the hypothesis that TRPV4‐mediated Ca²⁺ influx activates Ca²⁺ release from internal Ca²⁺ stores to generate widespread effects.
Experimental Approach
Ca²⁺ signals in large numbers (~100) of endothelial cells in intact arteries were imaged and analysed separately.
Key Results
Responses to the TRPV4 channel agonist GSK1016790A were heterogeneous across the endothelium. In activated cells, Ca²⁺ responses comprised localized Ca²⁺ changes leading to slow, persistent, global increases in Ca²⁺ followed by large propagating Ca²⁺ waves that moved within and between cells. To examine the mechanisms underlying each component, we developed methods to separate slow persistent Ca²⁺ rise from the propagating Ca²⁺ waves in each cell. TRPV4‐mediated Ca²⁺ entry was required for the slow persistent global rise and propagating Ca²⁺ signals. The propagating waves were inhibited by depleting internal Ca²⁺ stores, inhibiting PLC or blocking IP3 receptors. Ca²⁺ release from stores was tightly controlled by TRPV4‐mediated Ca²⁺ influx and ceased when influx was terminated. Furthermore, Ca²⁺ release from internal stores was essential for TRPV4‐mediated control of vascular tone.
Conclusions and Implications
Ca²⁺ influx via TRPV4 channels is amplified by Ca²⁺‐induced Ca²⁺ release acting at IP3 receptors to generate propagating Ca²⁺ waves and provide a large‐scale endothelial communication system. TRPV4‐mediated control of vascular tone requires Ca²⁺ release from the internal store.
... 2APB also shifted the current balance during the AP in the outward direction, a change compatible with an increased contribution of I NCX during IP 3 R activation (Signore et al., 2013). Direct I CaL blockade by 2APB is unlikely because of the slow kinetics of 2APB effect (Figure 6) and of failure 2APB alone to affect Ca 2+ dynamics in ventricular myocytes (Peppiatt et al., 2003). The present results suggest that IP 3 R activation, possibly through an increment of cytosolic Ca 2+ , may indeed facilitate Ca 2+ waves under the conditions generated by APD prolongation and β-adrenergic stimulation. ...
Introduction: Increases in action potential duration (APD), genetic or acquired, and arrhythmias are often associated; nonetheless, the relationship between the two phenomena is inconstant, suggesting coexisting factors. β-adrenergic activation increases sarcoplasmic reticulum (SR) Ca²⁺-content; angiotensin II (ATII) may increase cytosolic Ca²⁺ and ROS production, all actions stimulating RyRs opening. Here we test how APD interacts with β-adrenergic and AT-receptor stimulation in facilitating spontaneous Ca²⁺ release events (SCR).
Methods: Under “action potential (AP) clamp”, guinea-pig cardiomyocytes (CMs) were driven with long (200 ms), normal (150 ms), and short (100 ms) AP waveforms at a CL of 500 ms; in a subset of CMs, all the 3 waveforms could be tested within the same cell. SCR were detected as inward current transients (ITI) following repolarization; ITI incidence and repetition within the same cycle were measured under increasing isoprenaline concentration ([ISO]) alone, or plus 100 nM ATII (30 min incubation+superfusion).
Results: ITI incidence and repetition increased with [ISO]; at longer APs the [ISO]-response curve was shifted upward and ITI coupling interval was reduced. ATII increased ITI incidence more at low [ISO] and under normal (as compared to long) APs. Efficacy of AP shortening in suppressing ITI decreased in ATII-treated myocytes and at higher [ISO].
Conclusions: AP prolongation sensitized the SR to the destabilizing actions of ISO and ATII. Summation of ISO, ATII and AP duration effects had a “saturating” effect on SCR incidence, thus suggesting convergence on a common factor (RyRs stability) “reset” by the occurrence of spontaneous Ca²⁺ release events.
This work examined the effect of 2-aminoethoxydiphenyl borate (2-APB) on the functioning of isolated mouse skeletal muscle mitochondria and modeled its putative interaction with mitochondrial proteins. We have shown that 2-APB is able to dose-dependently suppress mitochondrial respiration in state 3 and 3UDNP driven by substrates of complex I and II. This effect of 2-APB was accompanied by a slight dose-dependent decrease in mitochondrial membrane potential and appears to be due to inhibition of complex I and complex III of the electron transport chain (ETC) with IC50 values of 200 and 120 μM, respectively. The results of molecular docking identified putative 2-APB interaction sites in these ETC complexes. 2-APB was shown to dose-dependently inhibit both mitochondrial Ca2+ uptake and Ca2+ efflux, which seems to be caused by a decrease in the membrane potential of the organelles. We have found that 2-APB has no significant effect on mitochondrial calcium retention capacity. On the other hand, 2-APB exhibited antioxidant effect by reducing mitochondrial hydrogen peroxide production but without affecting superoxide generation. It is concluded that the effect of 2-APB on mitochondrial targets should be taken into account when interpreting the results of cell and in vivo experiments.
Boron-containing compounds (BCC) have emerged as important pharmacophores. To date, five BCC drugs (including boronic acids and boroles) have been approved by the FDA for the treatment of cancer, infections, and atopic dermatitis, while some natural BCC are included in dietary supplements. Boron’s Lewis acidity facilitates a mechanism of action via formation of reversible covalent bonds within the active site of target proteins. Boron has also been employed in the development of fluorophores, such as BODIPY for imaging, and in carboranes that are potential neutron capture therapy agents as well as novel agents in diagnostics and therapy. The utility of natural and synthetic BCC has become multifaceted, and the breadth of their applications continues to expand. This review covers the many uses and targets of boron in medicinal chemistry.
Aims:
CRISPR/Cas9 gene-edits of cardiac ryanodine receptor (RyR2) in human induced pluripotent stem cells derived-cardiomyocytes (hiPSC-CMs) provides a novel platform for introducing mutations in RyR2 Ca2+ binding residues and examining the resulting EC-coupling remodeling consequences.
Methods and results:
Ca2+-signaling phenotypes of mutations in RyR2 Ca2+ binding site residues associated with cardiac arrhythmia (RyR2-Q3925E) or not proven to cause cardiac pathology (RyR2-E3848A) were determined using ICa- and caffeine-triggered Ca2+ releases in voltage-clamped and TIRF-imaged wild type (WT) and mutant cardiomyocytes infected with SR-targeted ER-GCaMP6 probe. 1) ICa- and caffeine-triggered Fura-2 or ER-GCaMP6 signals were suppressed, even when ICa was significantly enhanced in Q3925E and E3848A mutant cardiomyocyte; 2) Spontaneous beating (Fura-2 Ca2+-transients) persisted in mutant cells without the SR-release signals; 3) While 5-20mM caffeine failed to trigger Ca2+-release in voltage-clamped mutant-cells, only ∼20% to ∼70% of intact myocytes responded respectively to caffeine; 4) 20mM caffeine-transients, however, activated slowly, were delayed, and variably suppressed by 2-APB, FCCP, or ruthenium red.
Conclusion:
Mutating RyR2 Ca2+ binding residues, irrespective of their reported pathogenesis, suppressed both ICa- and caffeine-triggered Ca2+ releases, suggesting interaction between Ca2+ and caffeine binding-sites. Enhanced transmembrane calcium influx and remodeling of EC-coupling pathways may underlie the persistence of spontaneous beating in CICR-suppressed mutant myocytes.
Pressure-dependent chronotropy of murine lymphatic collecting vessels relies on the activation of the Ca²⁺-activated chloride channel encoded by Anoctamin 1 (Ano1) in lymphatic muscle cells. Genetic ablation or pharmacological inhibition of ANO1 results in a significant reduction in basal contraction frequency and essentially complete loss of pressure-dependent frequency modulation by decreasing the rate of the diastolic depolarization phase of the ionic pacemaker in lymphatic muscle cells (LMCs). Oscillating Ca²⁺ release from sarcoendoplasmic reticulum Ca²⁺ channels has been hypothesized to drive ANO1 activity during diastole, but the source of Ca²⁺ for ANO1 activation in smooth muscle remains unclear. Here, we investigated the role of the inositol triphosphate receptor 1 (Itpr1; Ip3r1) in this process using pressure myography, Ca²⁺ imaging, and membrane potential recordings in LMCs of ex vivo pressurized inguinal-axillary lymphatic vessels from control or Myh11CreERT2;Ip3r1fl/fl (Ip3r1ismKO) mice. Ip3r1ismKO vessels had significant reductions in contraction frequency and tone but an increased contraction amplitude. Membrane potential recordings from LMCs of Ip3r1ismKO vessels revealed a depressed diastolic depolarization rate and an elongation of the plateau phase of the action potential (AP). Ca²⁺ imaging of LMCs using the genetically encoded Ca²⁺ sensor GCaMP6f demonstrated an elongation of the Ca²⁺ flash associated with an AP-driven contraction. Critically, diastolic subcellular Ca²⁺ transients were absent in LMCs of Ip3r1ismKO mice, demonstrating the necessity of IP3R1 activity in controlling ANO1-mediated diastolic depolarization. These findings indicate a critical role for IP3R1 in lymphatic vessel pressure-dependent chronotropy and contractile regulation.
The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGFRa+ cells. Together these cells form the SIP syncytium. ICC and PDGFRa+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRa+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transientsoccur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarization (STDs). Neurotransmission increases or decreases Ca2+transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract, suggest the term myogenic should be replaced by the term, SIPgenic, as this review discusses.
Tuberous Sclerosis Complex (TSC) tumors are presently incurable despite a cytostatic response to mTOR pathway inhibition since recurrence of disease occurs after discontinuation of treatment. Here, we explored the hypothesis that inhibiting tyrosine kinase activity in mesenchymal lineage specific platelet derived growth factor receptor beta (PDGFRβ) signaling in TSC tumors is cytocidal and attenuates tumorigenesis at significantly higher levels than treatment with an mTOR inhibitor. Rapamycin- versus tyrosine kinase inhibitor (TKI)-induced renal angiomyolipoma (AML) and pulmonary lymphangioleiomyomatosis (LAM) tumor cells were comparatively analyzed using cell survival assays, and RNA sequencing and bioinformatics to distinguish tumoricidal mechanisms adopted by each drug type. The efficacy of imatinib therapy was validated against spontaneously developing renal cystadenomas in Tuberous Sclerosis Tsc2+/- mouse models (C57BL/6J mice; N=6; 400mg/kg/day; oral gavage) compared to Tsc2+/- mice treated with phosphate buffered saline (PBS) (C57BL/6J mice; N=6). Our study revealed that TKIs imatinib and nilotinib were cytocidal to both pulmonary LAM and renal AML cell cultures through the downregulation of the glycoprotein GPVI pathway and resultant disruption in mitochondrial permeability, increased cytosolic cytochrome C, and caspase 3 activation. Importantly, renal tumor growth was significantly attenuated in imatinib-treated Tsc2+/- mice compared to PBS treatment. The preclinical studies reported here provide evidence documenting the effectiveness of TKIs in limiting LAM and AML cell growth and viability with important clinical potential. Furthermore, these drugs elicit their effects by targeting a PDGF pathway-dependent apoptotic mechanism supporting the investigation of these drugs as a novel class of TSC therapeutics.
Malfunctions in airway smooth muscle Ca2+-signalling leads to airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Ca2+-release from intracellular stores is important in mediating agonist-induced contractions, but the role of influx via l-type Ca2+ channels is controversial. We re-examined roles of the sarcoplasmic reticulum Ca2+ store, refilling of this store via store-operated Ca2+ entry (SOCE) and l-type Ca2+ channel pathways on carbachol (CCh, 0.1-10 µM)-induced contractions of mouse bronchial rings and intracellular Ca2+ signals of mouse bronchial myocytes. In tension experiments, the ryanodine receptor (RyR) blocker dantrolene (100 µM) reduced CCh-responses at all concentrations, with greater effects on sustained rather than initial components of contraction. 2-Aminoethoxydiphenyl borate (2-APB, 100 μM), in the presence of dantrolene, abolished CCh-responses, suggesting the sarcoplasmic reticulum Ca2+ store is essential for contraction. The SOCE blocker GSK-7975A (10 µM) reduced CCh-contractions, with greater effects at higher (e.g. 3 and 10 µM) CCh concentrations. Nifedipine (1 µM), abolished remaining contractions in GSK-7975A (10 µM). A similar pattern was observed on intracellular Ca2+-responses to 0.3 µM CCh, where GSK-7975A (10 µM) substantially reduced Ca2+ transients induced by CCh, and nifedipine (1 µM) abolished remaining responses. When nifedipine (1 µM) was applied alone it had less effect, reducing tension responses at all CCh concentrations by 25% - 50%, with greater effects at lower (e.g. 0.1 and 0.3 µM) CCh concentrations. When nifedipine (1 µM) was examined on the intracellular Ca2+-response to 0.3 µM CCh, it only modestly reduced Ca2+ signals, while GSK-7975A (10 µM) abolished remaining responses. In conclusion, Ca2+-influx from both SOCE and l-type Ca2+ channels contribute to excitatory cholinergic responses in mouse bronchi. The contribution of l-type Ca2+ channels was especially pronounced at lower doses of CCh, or when SOCE was blocked. This suggests l-type Ca2+ channels might be a potential target for bronchoconstriction under certain circumstances.
Hematopoietic stem cells (HSCs), which are multipotent and have the ability to self‐renew, are frequently used in the treatment of hematological diseases and cancer. Small molecules that target HSC quiescence regulators could be used for ex vivo expansion of both mobilized peripheral blood (mPB) and umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPC). We identified and investigated 35 small molecules that target HSC quiescence factors. We looked at how they affected HSC activity, such as expansion, quiescence, multilineage capacity, cycling ability, metabolism, cytotoxicity, and genotoxicity. A transplantation study was carried out on immunocompromised mice to assess the expanded cells' repopulation and engraftment abilities. 4‐[(5Z)‐5‐benzylidene‐4‐oxo‐2‐sulfanylidene‐1,3‐thiazolidin‐3‐yl]benzoic acid (BML)‐260 and tosyl‐l‐arginine methyl ester (TAME) significantly increased both mPB and UCB‐HSPC content and activated HSC re‐entry into the cell cycle. The improved multilineage capacity was confirmed by the colony forming unit (CFU) assay. Furthermore, gene expression analysis revealed that BML‐260 and TAME molecules aided HSC expansion by modulating cell cycle kinetics, such as p27, SKP2, and CDH1. In addition to these in vitro findings, we discovered that BML‐260‐expanded HSCs had a high hematopoietic reconstitution capacity with increased immune cell content after xenotransplantation into immunocompromised mice. In addition to the BML‐260 molecule, a comparison study of serum‐containing and serum‐free chemically defined media, including various supplements, was performed. These in vitro and xenotransplantation results show that BML‐260 molecules can be used for human HSC expansion and regulation of function. Furthermore, the medium composition discovered may be a novel platform for human HSPC expansion that could be used in clinical trials.
Electrical slow waves, generated by interstitial cells of Cajal (ICC), cause spontaneous contractions of human stomach. Software was developed to measure muscle tone and eleven different parameters defining these contractions in human stomach, displaying data as radar plots. A pilot study assessed the effects of potential modulators, selected from among compounds known to influence ICC activity; n=4-7 each concentration tested/compound. Human distal stomach (corpus-antrum) muscle strips were suspended in tissue baths for measuring myogenic (non-neuronal) contractions in the presence of tetrodotoxin (10⁻⁶M). Initial characterization: Contractions (amplitude 4±0.4mN, frequency 3±0.1 min⁻¹, n=49) were unchanged by ꭃ-conotoxin GVIA (10⁻⁷M) or indomethacin (10⁻⁶M) but abolished by nifedipine (10⁻⁴M). Carbachol (10⁻⁷M) increased contraction rate and amplitude; 10⁻⁶-10⁻⁵M increased tone and caused large, irregular contractions. [Ca²⁺]i modulators: Ryanodine (10⁻⁵-10⁻⁴M) increased muscle tone accompanied by inhibition of myogenic contractions. Xestospongin-C (10⁻⁶M; IP3 channel inhibitor) had no effects. SERCA pump inhibitors, 2-APB and cycloplazonic acid (10⁻⁵-10⁻⁴M) increased tone and myogenic contraction amplitude before abolishing contractions; thapsigargin was weakly active. CaCC blockers: MONNA and CaCCinh-A01 had little-or-no effects on tone but reduced myogenic contractions; MONNA (10⁻⁴M) was more effective, reducing amplitude (77.8±15.2%) and frequency. CaV3.1/3.2/3.3 channel block: Mibefradil reduced tone and myogenic contraction amplitude (pIC50 4.8±0.9). Inward-rectifying K⁺-channel inhibitor: E-4031 (10⁻⁴M) increased contraction duration (17.4±5.8%). Conclusions: (1) Measurement of multiple parameters of myogenic contractions identified subtle differences between compounds, (2) only E-4031 and CaCC blockers influenced myogenic contractions, not muscle tone, (3) studies are needed with compounds with known and/or improved selectivity/potency for human targets affecting ICC functions.
In hippocampal CA1 neurons of wild-type mice, a short tetanus (15 or 20 pulses at 100 Hz) or a standard tetanus (100 pulses at 100 Hz) to a naive input pathway induces long-term potentiation (LTP) of the responses. Low-frequency stimulation (LFS; 1000 pulses at 1 Hz) 60 min after the standard tetanus reverses LTP (depotentiation [DP]), while LFS applied 60 min prior to the standard tetanus suppresses LTP induction (LTP suppression). We investigated LTP, DP, and LTP suppression of both field excitatory postsynaptic potentials and population spikes in CA1 neurons of mice lacking the inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R)-binding protein released with IP 3 (IRBIT). The mean magnitudes of LTP induced by short and standard tetanus were not different in mutant and wild-type mice. In contrast, DP and LTP suppression were attenuated in mutant mice, whereby the mean magnitude of responses after LFS or tetanus were significantly greater than in wild-type mice. These results suggest that, in hippocampal CA1 neurons, IRBIT is involved in DP and LTP suppression, but is not essential for LTP. The attenuation of DP and LTP suppression in mice lacking IRBIT indicates that this protein, released during or after priming stimulations, determines the direction of LTP expression after the delivery of subsequent stimulations.
Mitochondria-associated ER membranes (MAMs) are formed by close and specific components in the contact sites between the endoplasmic reticulum (ER) and mitochondria, which participate in several cell functions, including lipid metabolism, autophagy, and Ca²⁺ signaling. Particularly, the presence of α-synuclein (α-syn) in MAMs was previously demonstrated, indicating a physical interaction among some proteins in this region and a potential involvement in cell dysfunctions. MAMs alterations are associated with neurodegenerative diseases such as Parkinson's disease (PD) and contribute to the pathogenesis features. Here, we investigated the effects of α-syn on MAMs and Ca²⁺ transfer from the ER to mitochondria in WT- and A30P α-syn-overexpressing SH-SY5Y or HEK293 cells. We observed that α-syn potentiates the mitochondrial membrane potential (Δψm) loss induced by rotenone, increases mitophagy and mitochondrial Ca²⁺ overload. Additionally, in α-syn-overexpressing cells, we found a reduction in ER–mitochondria contact sites through the impairment of the GRP75–IP3R interaction, however, with no alteration in VDAC1–GRP75 interaction. Consequently, after Ca²⁺ release from the ER, α-syn-overexpressing cells demonstrated a reduction in Ca²⁺ buffering by mitochondria, suggesting a deregulation in MAM activity. Taken together, our data highlight the importance of the α-syn/MAMs/Ca²⁺ axis that potentially affects cell functions in PD.
Hydrogen sulfide (H2S) has a variety of physiological functions. H2S reportedly increases intracellular Ca²⁺ concentration ([Ca²⁺]i) in astrocytes. However, the precise mechanism and functional role of this increase are not known. Here, we examined the effects of H2S on [Ca²⁺]i in astrocytes from the rat spinal cord and whether H2S affects ATP-induced Ca²⁺ signaling, which is known to be involved in synaptic function. Na2S (150 μM), an H2S donor, produced a nontoxic increase in [Ca²⁺]i. The [Ca²⁺]i increase by Na2S was inhibited by Ca²⁺ depletion in the endoplasmic reticulum (ER) but not by removal of extracellular Ca²⁺, indicating that H2S releases Ca²⁺ from the ER. On the other hand, Na2S inhibited ATP-induced [Ca²⁺]i increase when Na2S clearly increased [Ca²⁺]i in the astrocytes, which was not suppressed by a reducing agent. In addition, Na2S had no effect on intracellular cyclic AMP (cAMP) level. These results indicate that oxidative post-translational modification of proteins and cAMP are not involved in the inhibitory effect of H2S on ATP-induced Ca²⁺ signaling. We conclude that H2S indirectly inhibits ATP-induced Ca²⁺ signaling by decreasing Ca²⁺ content in the ER in astrocytes. In this way, H2S may influence intercellular communication between astrocytes and neurons, thereby contributing to neuronal signaling in the nervous system.
Inositol trisphosphate (IP 3 ) is a Ca ²⁺ - mobilizing second messenger shown to modulate atrial muscle contraction, and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP 3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP 3 -mediated Ca ²⁺ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca ²⁺ -stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sino-atrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified co-localization of type 2 IP 3 Rs with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP 3 by UV light significantly enhanced the amplitude of the Ca ²⁺ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6 % increase 60 s post photorelease, n=16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP 3 . In mouse spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP 3 mediated Ca ²⁺ release, increased the maximum beating rate by 14.7 ± 0.5 %, n=10. This effect was substantially reduced by 2.5 µmol/L 2-APB and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP 3 and cAMP signaling involving Ca ²⁺ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP 3 receptor pathways and Ca ²⁺ -stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias.
Background
Inflammation is the fast coordinating response of body’s immune system to irritants caused by pathogens, external injuries and chemical or radiation effects. The NLRP3 (nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome is a critical component of the innate immune system. Malfunction of NLRP3 inflammasome will contribute to various pathogenesis of complex diseases, such as uncontrolled infection, autoimmune diseases, neurodegenerative diseases, and metabolic disorders. This review aims to describe recent progress on discovery of NLRP3 inflammasome inhibitors and their therapeutic potential.
Methods
Based on the mechanism of NLRP3 activation, several types of NLRP3 inhibitors were described and summarized according to their origins, structures, bioactivity and mechanism of action. Structure-activity-relationship (SAR) was also listed for different scaffolds, as well as effective pharmacophore.
Results
Over one-hundred papers were included in the review. The development of NLRP3 inhibitors was described from the earliest glyburide in 2001 to the latest progress in 2019. Several series of inhibitors were categorized, such as JC-series based on glyburide and BC-series based on 2APB. Many other small molecules as NLRP3 inhibitors were also listed. SAR, application in related therapeutic models and five different action mechanisms were described.
Conclusion
The findings of this review confirmed the importance of developing NLRP3 inflammasome inhibitors. Various NLRP3 inhibitors have been discovered as effective therapeutic treatments to multiples diseases such as type II diabetes, experimental autoimmune encephalomyelitis, stress-related mood disorders, etc. The development of a full range of NLRP3 inflammasome inhibitors is still at its foundational phase. We are looking forward to identification of inhibitory agents which provide the most potent therapeutic strategies and efficiently treat NLRP3 inflammasome-related inflammatory diseases.
NLRP3 is a cytosolic receptor member of the nucleotide-binding and oligomerization domain NOD-like receptor (NLR) family that surveys the intracellular environment for the presence of infection, pathogens, and metabolic alarms. While the surveillance activity of NLRP3 is required to protect the host from several pathogens, uncontrolled activity can be detrimental to the host. Pharmacological and genetic strategies limiting NLRP3 inflammasome activation have been shown to be beneficial in a wide range of experimental models, from common pathologies such as arthritis, cardiovascular disease, and metabolic syndromes, to rare genetic disorders such as cryopyrin-associated-periodic-syndrome (CAPS). Thus, compounds that prevent NLRP3 inflammasome activation are of common interest with relevant therapeutic potential. The focus of this review is recent developments in NLRP3 inflammasome inhibitors.
Since its introduction to Ca2+ signaling in 1997, 2-aminoethoxydiphenyl borate (2-APB) has been used in many studies to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2-APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2-APB has thrown up some interesting results, particularly with respect to store-operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2-APB can be useful in probing certain aspects of Ca2+ signaling.—Bootman, M. D., Collins, T. J., Mackenzie, L., Roderick, H. L., Berridge, M. J., Peppiatt, C. M. 2-Aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3-induced Ca2+ release.
The thiol reagent, thimerosal, has been shown to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in several cell types, and to cause Ca2+ spikes in unfertilized hamster eggs. Using single cell video-imaging we have shown that thimerosal evokes repetitive Ca2+ spikes in intact Fura-2-loaded HeLa cells that were similar in shape to those stimulated by histamine. Both thimerosal- and histamine-stimulated Ca2+ spikes occurred in the absence of extracellular (Ca2+ o), suggesting that they result from mobilization of Ca2+ from intracellular stores. Whereas histamine stimulated formation of inositol phosphates, thimerosal, at concentrations that caused sustained Ca2+ spiking, inhibited basal and histamine-stimulated formation of inositol phosphates. Thimerosal-evoked Ca2+ spikes are therefore not due to the stimulated production of inositol 1,4,5-trisphosphate (InsP3). The effects of thimerosal on Ca2+ spiking were probably due to alkylation of thiol groups on intracellular proteins because the spiking was reversed by the thiol-reducing compound dithiothreitol, and the latency between addition of thimerosal and a rise in [Ca2+]i was greatly shortened in cells where the intracellular reduced glutathione concentration had been decreased by preincubation with DL-buthionine (S,R)-sulfoximine. In permeabilized cells, thimerosal caused a concentration-dependent inhibition of Ca2+ accumulation, which was entirely due to inhibition of Ca2+ uptake into stores because thimerosal did not affect unidirectional 45Ca2+ efflux from stores preloaded with 45Ca2+. Thimerosal also caused a concentration-dependent sensitization of InsP3-induced Ca2+ mobilization: half-maximal mobilization of Ca2+ stores occurred with 161 +/- 20 nM InsP3 in control cells and with 62 +/- 5 nM InsP3 after treatment with 10 microM thimerosal. We conclude that thimerosal can mimic the effects of histamine on intracellular Ca2+ spiking without stimulating the formation of InsP3 and, in light of our results with permeabilized cells, suggest that thimerosal stimulates spiking by sensitizing cells to basal InsP3 levels.
Low caffeine concentrations were unable to completely release the caffeine- and ryanodine-sensitive intracellular Ca2+ pool in intact adrenal chromaffin cells. This 'quantal' Ca2+ release is the same as that previously observed with inositol Ins(1,4,5)P3-induced Ca2+ release. The molecular mechanism underlying quantal Ca2+ release from the ryanodine receptor was investigated using fura-2 imaging of single chromaffin cells. Our data indicate that the intracellular caffeine-sensitive Ca2+ pool is composed of functionally discrete stores, that possess heterogeneous sensitivities to caffeine. These stores are mobilized by caffeine in a concentration-dependent fashion, and, when stimulated, individual stores release their Ca2+ in an 'all-or-none' manner. Such quantal Ca2+ release may be responsible for graded Ca2+ responses in single cells.
The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.
The mechanism for coupling between Ca2+ stores and store-operated channels (SOCs) is an important but unresolved question. SOC-mediated Ca2+ entry is complex and may reflect more than one type of channel and coupling mechanism. To assess such possible divergence
the function and coupling of SOCs was compared with two other distinct yet related Ca2+ entry mechanisms. SOC coupling in DDT1MF-2 smooth muscle cells was prevented by the permeant inositol 1,4,5-trisphosphate (InsP3) receptor blockers, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C. In contrast, Ca2+ entry induced by S-nitrosylation and potentiated by store depletion (Ma, H-T., Favre, C. J., Patterson, R. L., Stone, M. R., and Gill, D. L.
(1999)J. Biol. Chem. 274, 35318–35324) was unaffected by 2-APB, suggesting that this entry mechanism is independent of InsP3 receptors. The cycloalkyl lactamimide, MDL-12,330A (MDL), prevented SOC activation (IC50 10 μm) and similarly completely blockedS-nitrosylation-mediated Ca2+ entry. Ca2+ entry mediated by the TRP3 channel stably expressed in HEK293 cells was activated by phospholipase C-coupled receptors but
independent of Ca2+ store depletion (Ma, H.-T., Patterson, R. L., van Rossum, D. B., Birnbaumer, L., Mikoshiba, K., and Gill, D. L. (2000) Science 287, 1647–1651). Receptor-induced TRP3 activation was 2-APB-sensitive and fully blocked by MDL. Direct stimulation of TRP3
channels by the permeant diacylglycerol derivative, 1-oleoyl-2-acetyl-sn-glycerol, was not blocked by 2-APB, but was again prevented by MDL. The results indicate that although the activation and
coupling processes for each of the three entry mechanisms are distinct, sensitivity to MDL is a feature shared by all three
mechanisms, suggesting there may be a common structural feature in the channels themselves or an associated regulatory component.
The compound 2-aminoethyl diphenylborate (2-APB), an inhibitor of Ins(1,4,5)P(3) receptor action in some cell types, has been used to assess the role of Ins(1,4,5)P(3) receptors in the activation of store-operated Ca2+ channels (SOCs) [Ma, Patterson, van Rossum, Birnbaumer, Mikoshiba and Gill (2000) Science 287, 1647-1651]. In freshly-isolated rat hepatocytes, 2-APB inhibited thapsigargin- and vasopressin-stimulated Ca2+ inflow (measured using fura-2) with no detectable effect on the release of Ca2+ from intracellular stores. The concentration of 2-APB which gave half-maximal inhibition of Ca2+ inflow was approx. 10 microM. 2-APB also inhibited Ca2+ inflow initiated by a low concentration of adenophostin A but had no effect on maitotoxin-stimulated Ca2+ inflow through non-selective cation channels. The onset of the inhibitory effect of 2-APB on thapsigargin-stimulated Ca2+ inflow was rapid. When 2-APB was added to rat hepatocytes in the presence of extracellular Ca2+ after a vasopressin-induced plateau in the cytoplasmic free Ca2+ concentration ([Ca2+](cyt)) had been established, the kinetics of the decrease in [Ca2+](cyt) were identical with those induced by the addition of 50 microM Gd(3+) (gadolinium). 2-APB did not inhibit the release of Ca2+ from intracellular stores induced by the addition of Ins(1,4,5)P(3) to permeabilized hepatocytes. In the H4-IIE rat hepatoma cell line, 2-APB inhibited thapsigargin-stimulated Ca2+ inflow (measured using fura-2) and, in whole-cell patch-clamp experiments, the Ins(1,4,5)P(3)-induced inward current carried by Ca2+. It was concluded that, in liver cells, 2-APB inhibited SOCs through a mechanism which involved the binding of 2-APB to either the channel protein or an associated regulatory protein. 2-APB appeared to be a novel inhibitor of SOCs in liver cells with a mechanism of action which, in this cell type, is unlikely to involve an interaction of 2-APB with Ins(1,4,5)P(3) receptors. The need for caution in the use of 2-APB as a probe for the involvement of Ins(1,4,5)P(3) receptors in the activation of SOCs in other cell types is briefly discussed.
The mechanism for coupling between Ca2+ stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP3R) was examined using the permeant InsP3R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP3R-mediated store release and carbachol-induced Sr2+ entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP3Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP3R-independent since InsP3Rs are dispensable for the light response. We used triple InsP3R knockout DT40 chicken B-cells to further assess the role of InsP3Rs in SOC activation. 45Ca2+ flux analysis revealed that although DT40 wild-type cells retained normal InsP3Rs mediating 2-APB-sensitive Ca2+ release, the DT40InsP3R-k/o cells were devoid of functional InsP3Rs. Using intact cells, all parameters of Ca 2+ store function and SOC activation were identical in DT40wt and DT40InsP3R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC50) were identical. The results indicate that (a) the action of 2-APB on Ca2+ entry is not mediated by the InsP3R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian storeoperated channels.
In nonexcitable cells, the predominant mechanism for regulated entry of Ca(2+) is capacitative calcium entry, whereby depletion of intracellular Ca(2+) stores signals the activation of plasma membrane calcium channels. A number of other regulated Ca(2+) entry pathways occur in specific cell types, however, and it is not know to what degree the different pathways interact when present in the same cell. In this study, we have examined the interaction between capacitative calcium entry and arachidonic acid-activated calcium entry, which co-exist in HEK293 cells. These two pathways exhibit mutual antagonism. That is, capacitative calcium entry is potently inhibited by arachidonic acid, and arachidonic acid-activated entry is inhibited by the pre-activation of capacitative calcium entry with thapsigargin. In the latter case, the inhibition does not seem to result from a direct action of thapsigargin, inhibition of endoplasmic reticulum Ca(2+) pumps, depletion of Ca(2+) stores, or entry of Ca(2+) through capacitative calcium entry channels. Rather, it seems that a discrete step in the pathway signaling capacitative calcium entry interacts with and inhibits the arachidonic acid pathway. The findings reveal a novel process of mutual antagonism between two distinct calcium entry pathways. This mutual antagonism may provide an important protective mechanism for the cell, guarding against toxic Ca(2+) overload.
We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP(3)) in capacitative calcium entry and calcium release-activated calcium current (I(crac)) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with blocked calcium entry and I(crac) activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I(crac). The membrane-permeant IP(3) receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I(crac). However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP(3) receptor because the drug was equally effective in wild-type DT40 B-cells and in DT40 B-cells whose genes for all three IP(3) receptors had been disrupted. Intracellular application of another potent IP(3) receptor antagonist, heparin, failed to inhibit activation of I(crac). Finally, the inhibition of I(crac) activation by or wortmannin was not reversed or prevented by direct intracellular application of IP(3). These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP(3) and IP(3) receptor in this mechanism, at least in these particular cell types.
Drosophila phototransduction is an important model system for studies of inositol lipid signaling. Light excitation in Drosophila photoreceptors depends on phospholipase C, because null mutants of this enzyme do not respond to light. Surprisingly, genetic elimination of the apparently single inositol trisphosphate receptor (InsP(3)R) of Drosophila has no effect on phototransduction. This led to the proposal that Drosophila photoreceptors do not use the InsP(3) branch of phospholipase C (PLC)-mediated signaling for phototransduction, unlike most other inositol lipid-signaling systems. To examine this hypothesis we applied the membrane-permeant InsP(3)R antagonist 2-aminoethoxydiphenyl borate (2-APB), which has proved to be an important probe for assessing InsP(3)R involvement in various signaling systems. We first examined the effects of 2-APB on Xenopus oocytes. We found that 2-APB is efficient at reversibly blocking the robust InsP(3)-mediated Ca(2+) release and store-operated Ca(2+) entry in Xenopus oocytes at a stage operating after production of InsP(3) but before the opening of the surface membrane Cl(-) channels by Ca(2+). We next demonstrated that 2-APB is effective at reversibly blocking the response to light of Drosophila photoreceptors in a light-dependent manner at a concentration range similar to that effective in Xenopus oocytes and other cells. We show furthermore that 2-APB does not directly block the light-sensitive channels, indicating that it operates upstream in the activation of these channels. The results indicate an important link in the coupling mechanism of vertebrate store-operated channels and Drosophila TRP channels, which involves the InsP(3) branch of the inositol lipid-signaling pathway.
The universality of calcium as an intracellular messenger depends on its
enormous versatility. Cells have a calcium signalling toolkit with many components
that can be mixed and matched to create a wide range of spatial and temporal
signals. This versatility is exploited to control processes as diverse as
fertilization, proliferation, development, learning and memory, contraction
and secretion, and must be accomplished within the context of calcium being
highly toxic. Exceeding its normal spatial and temporal boundaries can result
in cell death through both necrosis and apoptosis.
Arteriolar myogenic tone shows a marked dependency on extracellular Ca2+. The contribution played by mechanisms such as intracellular Ca2+ release and capacitative entry, however, are less certain. The present studies aimed to demonstrate functional evidence for involvement of such mechanisms in myogenic tone and reactivity.
Single cremaster arterioles were denuded of endothelium, pressurized under no-flow conditions and loaded with fura 2-AM for measurement of changes in intracellular Ca2+ [Ca2+]i. The cell permeable, putative, IP3 receptor antagonist 2APB (2 aminoethoxydiphenyl borate) was used to determine the possible role of IP3 receptor-mediated mechanisms in arteriolar myogenic tone and reactivity.
Arterioles dilated in response to increasing concentrations of 2APB (1 – 300 μM) without a concomitant change in global [Ca2+]i. Also 2APB (50 μM) completely inhibited the myogenic constriction in response to a step change in luminal pressure (50 – 120 mmHg) with no apparent effect on pressure-mediated increases in [Ca2+]i. 2APB markedly attenuated the constrictor response and [Ca2+]i increase stimulated by phenylephrine but not KCl.
Capacitative Ca2+ influx in arterioles was demonstrated either by re-addition of extracellular [Ca2+] following pre-treatment with 1 or 10 μM nifedipine in Ca2+ free buffer or exposure of vessels to thapsigargin (1 μM) to induce store depletion. In both cases 2APB inhibited the increase in [Ca2+]i. Capacitative Ca2+ entry showed an inverse relationship with intraluminal pressure over the range 10 – 120 mmHg.
Consistent with an effect on a Ca2+ entry pathway, 2APB had no effect on intracellular (caffeine releasable) Ca2+ stores while decreasing the rate of Mn2+ quench of fura 2 fluorescence.
The results provide functional evidence for capacitative Ca2+ entry in intact arteriolar smooth muscle. The effectiveness of 2APB in inhibiting both non-voltage gated Ca2+ entry and responsiveness to an acute pressure step is consistent with the involvement of an axis involving IP3-mediated and or capacitative Ca2+ entry mechanisms in myogenic reactivity. Given the lack of effect of 2APB on pressure-induced changes in global [Ca2+]i it is suggested that such mechanisms participate on a localized level to couple the myogenic stimulus to contraction.
British Journal of Pharmacology (2001) 134, 247–256; doi:10.1038/sj.bjp.0704270
Elementary Ca(2+) signals, such as "Ca(2+) puffs", which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca(2+) signalling. We characterized Ca(2+) puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca(2+) puffs in these cell types suggests that they are a generic elementary Ca(2+) signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca(2+) signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca(2+) puffs. The amplitude and duration of Ca(2+) puffs were substantially reduced. In addition, the number of Ca(2+) puff sites active during the onset of a Ca(2+) wave declined. The consequence of the changes in Ca(2+) puff properties was that cells displayed a lower propensity to trigger regenerative Ca(2+) waves. Therefore, Ca(2+) puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses.
Since its introduction to Ca2+ signaling in 1997, 2-aminoethoxydiphenyl borate (2-APB) has been used in many studies to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2-APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2-APB has thrown up some interesting results, particularly with respect to store-operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2-APB can be useful in probing certain aspects of Ca2+ signaling.
Ca2+ is a universal second messenger used to regulate a wide range of cellular processes such as fertilization, proliferation, contraction, secretion, learning and memory. Cells derive signal Ca2+ from both internal and external sources. The Ca2+ flowing through these channels constitute the elementary events of Ca2+ signalling. Ca2+ can act within milliseconds in highly localized regions or it can act much more slowly as a global wave that spreads the signal throughout the cell, Various pumps and exchangers are responsible for returning the elevated levels of Ca2+ back to the resting state. The mitochondrion also plays a critical role in that it helps the recovery process by taking Ca2+ up from the cytoplasm. Alterations in the ebb and flow of Ca2+ through the mitochondria can lead to cell death. A good example of the complexity of Ca2+ signalling is its role in regulating cell proliferation, such as the activation of lymphocytes. The Ca2+ signal needs to be present for over two hours and this prolonged period of signalling depends upon the entry of external Ca2+ through a process of capacitative Ca2+ entry. The Ca2+ signal stimulates gene transcription and thus initiates the cell cycle processes that culminate in cell division.
The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca²⁺) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca²⁺ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.
Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca2+ channels and store-operated Ca2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2-APB onthe SERCA Ca2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 µm, respectively, measured at pH 7.2). The Ca2+-ATPase is also more potently inhibited at lower pH (IC50 = 70 µm for SERCA1A at pH 6). 2-APB decreases the affinity for Ca2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca2+-ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca2+ to their binding sites.
• The effects of the IP3-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca2+ release-activated Ca2+ current ( ICRAC ) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined. • 2-APB elicited both stimulatory and inhibitory effects on Ca2+ influx through CRAC channels. At concentrations of 1–5 m, 2-APB enhanced Ca2+ entry in intact cells and increased ICRAC amplitude by up to fivefold. At levels ≥ 10 m, 2-APB caused a transient enhancement of ICRAC followed by inhibition. • 2-APB altered the kinetics of fast Ca2+-dependent inactivation of ICRAC . At concentrations of 1–5 m, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (≥ 10 m) reduced or completely blocked inactivation. • 2-APB inhibited Ca2+ efflux from mitochondria. • 2-APB inhibited ICRAC more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action. • Neither ICRAC activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 g ml−1 heparin. • ICRAC is present in wild-type as well as mutant DT40 B cells lacking all three IP3 receptor isoforms. 2-APB also potentiates and inhibits ICRAC in both cell types, indicating that 2-APB exerts its effects independently of IP3 receptors. • Our results show that CRAC channel activation does not require physical interaction with IP3 receptors as proposed in the conformational coupling model. Potentiation of ICRAC by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.
Two classes of intracellular Ca(2+)-release channels, the ryanodine receptor and the inositol (1,4,5)-trisphosphate (IP3) receptor, are essential for spatio-temporal Ca2+ signalling in cells. Heparin and caffeine have been widely used to study these channels. It was originally thought that caffeine acts solely as an agonist for the ryanodine receptor and heparin acts solely as an inhibitor for the IP3 receptor. However, recent experiments indicate that these compounds have multiple effects, and are discussed in this review by Barbara Ehrlich and colleagues. In the same concentration range, caffeine activates the ryanodine receptor and inhibits the IP3 receptor, and heparin inhibits the IP3 receptor and activates the ryanodine receptor. More specific pharmacological tools that are suitable for studies of ryanodine and IP3 receptors are now beginning to emerge.
Xestospongins (Xe's) A, C, D, araguspongine B, and demethylxestospongin B, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species, are shown to be potent blockers of IP3-mediated Ca2+ release from endoplasmic reticulum vesicles of rabbit cerebellum. XeC blocks IP3-induced Ca2+ release (IC50 = 358 nM) without interacting with the IP3-binding site, suggesting a mechanism that is independent of the IP3 effector site. Analysis of Pheochromocytoma cells and primary astrocytes loaded with Ca2+-sensitive dye reveals that XeC selectively blocks bradykinin- and carbamylcholine-induced Ca2+ efflux from endoplasmic reticulum stores. Xe's represent a new class of potent, membrane permeable IP3 receptor blockers exhibiting a high selectivity over ryanodine receptors. Xe's are a valuable tool for investigating the structure and function of IP3 receptors and Ca2+ signaling in neuronal and nonneuronal cells.
The effects of a novel membrane-penetrable modulator, 2APB (2-aminoethoxy diphenyl borate), on Ins(1,4,5)P3-induced Ca2+ release were examined. 2APB inhibited Ins(1,4,5)P3-induced Ca2+ release from rat cerebellar microsomal preparations without affecting [3H]Ins(1,4,5)P3 binding to its receptor. The IC50 value (concentration producing 50% inhibition) of 2APB for inhibition of Ins(1,4,5)P3 (100 nM) induced Ca2+ release was 42 microM. Further increase in the concentration of 2APB (more than 90 microM) caused a gradual release of Ca2+ from cerebellar microsomal preparations. Addition of 2APB to the extracellular environment inhibited the cytosolic Ca2+ ([Ca2+]c) rise in intact cells such as human platelets and neutrophils stimulated by thromboxane-mimetic STA2 or thrombin, and leukotriene B4 (LTB4) or formyl-methionine-leucine-phenylalanine (FMLP), respectively. 2APB inhibited the contraction of thoracic aorta isolated from rabbits induced by angiotensin II (AII), STA2, and norepinephrine in a non-competitive manner, but showed no effect on the contraction of potassium-depolarized muscle. 2APB had no effect on the Ca2+ release from the ryanodine-sensitive Ca2+ store prepared from rat leg skeletal muscle and heart. Although the specificity of 2APB with respect to the intracellular signaling system was not fully established, 2APB is the first candidate for a membrane-penetrable modulator of Ins(1,4,5)P3 receptor, and it should be a useful tool to investigate the physiological role of the Ins(1,4,5)P3 receptor in various cells.
This communication reports the results of investigations on the effect of low molecular weight heparin (LMWH) on intraneuronal calcium release, and considers its possible relevance to the treatment of ischemic stroke. It previously was shown that intraneuronal injection of conventional heparin (MW 12,000) in vitro prevents glutamate-induced calcium release from intracellular stores through its blocking action on IP3 (inositol-1,4,5-triphosphate) receptors, and thus interferes with events occurring in the ischemic cascade. In the experiments reported herein, a LMWH of MW 4500 was shown to have these same effects when injected into a Purkinje cell in an in vitro cerebellar slice preparation, and also when administered externally (bath application). By contrast, conventional heparin works only when injected into the cell; bath application has no effect. The results are interpreted to mean that the larger conventional heparin molecule cannot pass through the cell membrane, while the smaller LMWH molecule does indeed enter the cell. In a clinical trial, LMWH begun within 48 hours of ischemic stroke onset in humans improved outcome at 6 months; conventional heparin given in a similar trial was without benefit. That one anticoagulant was beneficial while another failed suggests the possibility that the difference was independent of effect on the clotting system. The experimental data herein reported support the view that LMWH may benefit stroke victims by an action directly cytoprotective against the consequences of neuronal ischemia.
The complex changes in intracellular Ca2+ concentration that follow cell stimulation reflect the concerted activities of Ca2+ channels in the plasma membrane and in the membranes of intracellular stores, and the opposing actions of the mechanisms that extrude Ca2+ from the cytosol. Disentangling the roles of each of these processes is hampered by the lack of adequately selective pharmacological tools. In this review, Colin Taylor and Lisa Broad summarize the more serious problems associated with some of the commonly used drugs, and describe specific situations in which the multiple effects of drugs on Ca2(+)-signalling pathways have confused analysis of these pathways.
The receptors for the second messenger inositol 1,4,5-trisphosphate (IP3) constitute a family of Ca2+ channels responsible for the mobilization of intracellular Ca2+ stores. Three different gene products (types I-III) have been isolated, encoding polypeptides which assemble as large tetrameric structures. Recent molecular studies have advanced our knowledge about the structure, regulation and function of IP3 receptors. For example, several Ca(2+)-binding sites and a Ca(2+)-calmodulin-binding domain have been mapped within the type I IP3 receptor, and studies on purified cerebellar IP3 receptors propose a second Ca(2+)-independent calmodulin-binding domain. In addition, minimal requirements for the binding of immunophilins and the formation of tetramers have been identified. Overexpression of IP3 receptors has provided further clues to the regulation of individual IP3 receptor isoforms present within cells, and the role that they play in the generation of IP3-dependent Ca2+ signals. Inhibition of IP3 receptor function and expression, and analysis of mutant IP3 receptors, suggests that IP3 receptors are involved in such diverse cellular processes as proliferation and apoptosis and are thus, necessary for normal development. Our understanding of the complex spatial and temporal nature of cytosolic Ca2+ increases and the role that these Ca2+ signals play in cell function depend upon our knowledge of the structure and the regulation of IP3 receptors. This review focuses on the molecular properties of these ubiquitous intracellular Ca2+ channels.
These studies were performed to evaluate the effect of 2-aminoethoxydiphenyl borate (2-APB), a novel membrane-permeable inositol 1,4,5-trisphosphate-receptor inhibitor on agonist-induced, spontaneous, and KCl-stimulated in vitro myometrial contractions. 2-APB significantly inhibited spontaneous myometrial contractions as well as phasic contractions induced by various uterotonic agonists. Confiriming its effects on intracellular calcium release, 2-APB inhibited phasic contractions in the absence of extracellular calcium. 2-APB had little effect on the tonic response to KCl stimulation, implicating its insignificant effect on voltage-gated calcium channels. The inhibitory effect of 2-APB on phasic contractions was completely reversed by washout. In summary, 2-APB effectively penetrated uterine tissue and significantly inhibited myometrial events previously shown to be mediated through activation of the PI-signaling pathway.
Elementary Ca2+ signals, such as 'Ca2+ puffs', that arise from the activation of clusters of inositol 1 ,4,5,-trisphosphate (InsP3) receptors are the building blocks for local and global Ca2+ signalling. We previously found that one, or a few, Ca2+ puff sites within agonist-stimulated cells act as 'pacemakers' to initiate global Ca2+ waves. The factors that distinguish these pacemaker Ca2+ puff sites from the other Ca2+ release sites that simply participate in Ca2+ wave propagation are unknown.
The spatiotemporal properties of Ca2+ puffs were investigated using confocal microscopy of fluo3-loaded HeLa cells. The same pacemaker Ca2+ puff sites were activated during stimulation of cells with different agonists. The majority of agonist-stimulated pacemaker Ca2+ puffs originated in a perinuclear location. The positions of such Ca2+ puff sites were stable for up to 2 hours, and were not affected by disruption of the actin cytoskeleton. A similar perinuclear distribution of Ca2+ puff sites was also observed when InsP3 receptors were directly stimulated with thimerosal or membrane-permeant InsP3 esters. Immunostaining indicated that the perinuclear position of pacemaker Ca2+ puffs was not due to the localised expression of InsP3 receptors.
The pacemaker Ca2+ puff sites that initiate Ca2+ responses are temporally and spatially stable within cells. These Ca2+ release sites are distinguished from their neighbours by an intrinsically higher InsP3 sensitivity.
2-Aminoethoxydiphenyl borate (2APB) is a membrane-permeable blocker of the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release in bi-directional Ca2+ -flux conditions. We have now studied the effects of 2APB on the 45Ca2+ uptake into, and on the basal and IP(3)-stimulated unidirectional 45Ca2+ efflux from the non-mitochondrial Ca2+ stores in permeabilized A7r5 smooth-muscle cells. 2APB inhibited the IP3 -induced Ca2+ release, with a half maximal inhibition at 36 microM 2APB, without affecting [3H]IP3 binding to the receptor. This inhibition did not depend on the IP3, ATP or free Ca2+ concentration. The Ca2+ pumps of the non-mitochondrial Ca2+ stores were half-maximally inhibited at 91microM 2APB. Higher concentrations of 2APB increased the non-specific leak of Ca2+ from the stores. We conclude that 2APB can not be considered as a selective blocker of the IP3 -induced Ca2+ release. Our results can explain the various effects of 2APB observed in intact cells.
Many inflammatory mediators activate neutrophils (PMN) partly by increasing cytosolic calcium concentration ([Ca2+]i). Modulation of PMN [Ca2+]i might therefore be useful in regulating inflammation after shock or sepsis. The hemodynamic effects of traditional Ca2+ channel blockade, however, could endanger unstable patients. Store-operated calcium influx (SOCI) is known now to contribute to Ca2+ flux in "nonexcitable" cells. Therefore, we studied the role of SOCI in human PMN responses to the proinflammatory ligand PAF. PMN [Ca2+]i was studied by spectrofluorometry with and without external calcium. We studied the effects o
One popular model for the activation of store-operated Ca2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP3) receptors on the stores to bind to, and thus activate, store-operated Ca2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I(CRAC), the most widely distributed and best characterised store-operated Ca2+ current, in a model system, the RBL-1 rat basophilic cell line. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I(CRAC). Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I(CRAC) was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores. Pre-treatment with the membrane-permeable InsP3 receptor antagonist 2-APB blocked the activation of I(CRAC). However, intracellular dialysis with 2-APB failed to prevent I(CRAC) from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I(CRAC) had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La3+. Studies with the more conventional InsP3 receptor antagonist heparin revealed that occupation of the intracellular InsP3-sensitive receptors was not necessary for the activation or maintenance of I(CRAC). Similarly, the InsP3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I(CRAC). Exposure to Li+, which reduces InsP3 levels by interfering with inositol monophosphatase, also failed to alter I(CRAC). Caffeine and Li+ did not affect the size of the intracellular Ca2+ signal that arose when external Ca2+ was re-admitted to cells which had been pre-exposed to thapsigargin/ionomycin in Ca2+-free external solution. Our findings demonstrate that the cytoskeleton does not seem to regulate calcium influx and that functional InsP3 receptors are not required for activation of I(CRAC). If the secretion-like coupling model indeed accounts for the activation of I(CRAC) in RBL-1 cells, then it needs to be revised significantly. Possible modifications to the model are discussed.
We have investigated Ca(2+) release and receptor- and store-operated Ca(2+) influxes in Chinese hamster ovary-K1 (CHO) cells, SH-SY5Y human neuroblastoma cells and RBL-1 rat basophilic leukemia cells using Fura-2 and patch-clamp measurements. Ca(2+) release and subsequent Ni(2+)-sensitive, store-operated influx were induced by thapsigargin and stimulation of G protein-coupled receptors. The alleged noncompetitive IP3 receptor inhibitor,2-aminoethoxydiphenyl borate (2-APB) rapidly blocked a major part of the secondary influx response in CHO cells in a reversible manner. It also reduced Mn(2+) influx in response to thapsigargin. Inhibition of Ca(2+) release was also seen but this was less complete, slower in onset, less reversible, and required higher concentration of 2-APB. In RBL-1 cells, I(CRAC) activity was rapidly blocked by extracellular 2-APB whereas intracellular 2-APB was less effective. Store-operated Ca(2+) influxes were only partially blocked by 2-APB. In SH-SY5Y cells, Ca(2+) influxes were insensitive to 2-APB. Ca(2+) release in RBL-1 cells was partially sensitive but in SH-SY5Y cells the release was totally resistant to 2-APB. The results suggest, that 2-APB (1) may inhibit distinct subtypes of IP3 receptors with different sensitivity, and (2) that independently of this, it also inhibits some store-operated Ca(2+) channels via a direct, extracellular action.
The subcellular mechanisms regulating stimulus-contraction coupling in detrusor remain to be determined. We used Ca2+-free solutions, Ca2+ channel blockers, cyclopiazonic acid (CPA), and RhoA kinase (ROK) inhibitors to test the hypothesis that Ca2+ influx and Ca2+ sensitization play primary roles.
In rabbit detrusor, peak bethanechol (BE)-induced force was inhibited 90% by incubation for 3 min in a Ca2+-free solution. By comparison, a 20 min incubation of rabbit femoral artery in a Ca2+-free solution reduced receptor-induced force by only 5%.
In detrusor, inhibition of sarcoplasmic reticular (SR) Ca2+ release by 2APB, or depletion of SR Ca2+ by CPA, inhibited BE-induced force by only 27%. The CPA-insensitive force was abolished by LaCl3. By comparison, 2APB inhibited receptor-induced force in rabbit femoral artery by 71%.
In the presence of the non-selective cation channel (NSCC) inhibitor, LOE-908, BE did not produce an increase in [Ca2+]i but did produce weak increases in myosin phosphorylation and force.
Inhibitors of ROK-induced Ca2+ sensitization, HA-1077 and Y-27632, inhibited BE-induced force by ∼50%, and in combination with LOE-908, nearly abolished force.
These data suggest that two principal muscarinic receptor-stimulated detrusor contractile mechanisms include NSCC activation, that elevates [Ca2+]i and ROK activation, that sensitizes cross bridges to Ca2+.
British Journal of Pharmacology (2001) 134, 78–87; doi:10.1038/sj.bjp.0704241
The structures, and mechanisms of activation, of plasma membrane intracellular-messenger-activated, non-selective cation channels in animal cells are not well understood. The PC12 adrenal chromaffin cell line is a well-characterized example of a nerve cell. In PC12 cells, 1-oleolyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, initiated the inflow of Ca(2+), Mn(2+) and Sr(2+). Acetylcholine and thapsigargin initiated the inflow of Ca(2+) and Mn(2+), but not of Sr(2+). The activation of bivalent cation inflow by OAG: (i) was mimicked by another membrane-permeant diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, but not by the membrane-impermeant analogue 1-stearoyl-2-arachidonyl-sn-glycerol; (ii) was not blocked by staurosporin or chelerythrine, inhibitors of protein kinase C; (iii) was enhanced by RHC80267 and R50922, inhibitors of diacylglycerol lipase and diacylglycerol kinase respectively; and (iv) was inhibited by extracellular Ca(2+). When OAG was added after acetylcholine, the effect of OAG on Ca(2+) inflow was over-and-above that induced by acetylcholine. 2-Aminoethyl diphenylborate (2-APB) inhibited Ca(2+) inflow initiated by either acetylcholine or thapsigargin, but not that initiated by OAG. Flufenamic acid inhibited OAG-initiated, but not acetylcholine-initiated, Ca(2+) and Mn(2+) inflow. OAG-initiated Ca(2+) inflow was less sensitive to inhibition by SK&F96365 than acetylcholine-initiated Ca(2+) inflow. In polyadenylated RNA prepared from PC12 cells, mRNA encoding TRP (transient receptor potential) proteins 1-6 was detected by reverse transcriptase (RT)-PCR, and in lysates of PC12 cells the endogenous TRP-6 protein was detected by Western blot analysis. It is concluded that PC12 cells express a diacylglycerol-activated, non-selective cation channel. Expression of this channel function correlates with expression of the TRP-3 and TRP-6 proteins, which have been shown previously to be activated by diacylglycerol when expressed heterologously in animal cells [Hofmann, Obukhov, Schaefer, Harteneck, Gudermann, and Schultz (1999) Nature (London) 397, 259-263].
This study examined the transduction pathways activated by epinephrine in the pacemaker region of the toad heart. Recordings of membrane potential, force, and intracellular Ca(2+) concentration ([Ca(2+)](i)) were made from arrested toad sinus venosus. Sympathetic nerve stimulation activated non-alpha-, non-beta-adrenoceptors to evoke a membrane depolarization and a transient increase in [Ca(2+)](i). In contrast, the beta-adrenoceptor agonist isoprenaline (10 microM) caused membrane hyperpolarization and decreased [Ca(2+)](i). The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM) mimicked the isoprenaline-evoked membrane hyperpolarization. Epinephrine (10-50 microM) caused an initial membrane depolarization and an increase in [Ca(2+)](i) followed by membrane hyperpolarization and decreased [Ca(2+)](i). The membrane depolarizations evoked by sympathetic nerve stimulation or epinephrine were abolished either by the phospholipase C inhibitor U-73122 (20 microM) or by the blocker of D-myo-inositol 1,4,5,-trisphosphate-induced Ca(2+) release, 2-aminoethoxydiphenyl borate (2-APB, 60 microM). Neither U-73122 nor 2-APB had an affect on the membrane hyperpolarization evoked by beta-adrenoceptor activation. These results suggest that in the toad sinus venosus, two distinct transduction pathways can be activated by epinephrine to cause an increase in heart rate.
It has been reported that store-mediated Ca2+ entry (SMCE) in human platelets is likely to be mediated by a secretion-like coupling mechanism. Recently, 2-aminoethoxydiphenylborate (2-APB) has been used in the investigation of SMCE. Here, the mechanism of action of 2-APB is investigated in human platelets. In a Ca2+-free medium (EGTA added), addition of 0.1 U/ml thrombin caused an elevation in [Ca2+]i. Preincubation with 100 microM 2-APB for 170s abolished the release of internal Ca2+. In platelets whose internal Ca2+ stores had been depleted by treatment with 200 nM thapsigargin, addition of extracellular Ca2+ caused an elevation in [Ca2+]i indicative of SMCE. Preincubation with 2-APB decreased SMCE by 95.5+/-1.1%. After activation of SMCE, addition of 2-APB rapidly decreased [Ca2+]i to basal levels; in contrast, the coupling between Trp1 and IP3RII, which has been shown to play an important role in SMCE in platelets, remained intact at the same time points. The rate of decrease of [Ca2+]i and the absence of measurable latency in the effect of 2-APB were comparable to the effects of La3+ (a cation channel blocker). These data suggest that 2-APB may act as a blocker of Ca2+ permeable plasma membrane channels. These data provide further information regarding the mechanism and site of action of 2-APB and highlight the necessity of careful interpretation of work performed using this molecule.
Capacitative calcium entry (CCE), the mechanism that replenishes intracellular calcium stores after depletion, is essential to intracellular calcium signaling. CCE is mediated by the channels in the plasma membrane generally referred to as "store operated channels (SOCs)". However, the molecular identity of the SOCs has never been determined, and the mechanism of the activation of SOCs remains to be elucidated. Recent studies have demonstrated that 2-aminoethoxydiphenyl borate (2-APB), which has been found to be an antagonist of inositol 1,4,5-trisphosphate receptors (IP3Rs), inhibits CCE, suggesting that IP3Rs channel activity is essential to the generation of CCE. However, CCE has also been reported to occur normally in IP3R-deficient cells. In order to resolve this discrepancy, we investigated the effect of 2-APB on CCE in IP3Rs-deficient cells. In response to store depletion with thapsigargin or N,N,N',N'-tetrakis (2-pyridylmethyl) ethylene diamine (TPEN), CCE was generated in IP3Rs-deficient cells the same as in wild-type cells, however, 2-APB abolished CCE in IP3Rs-deficient cells, despite the fact that this cell line does not possess functional IP3Rs. We also examined the effect of 2-APB on several types of TRP Ca2+ channels, which exhibit properties similar to those of SOCs. 2-APB had a different inhibitory effect on spontaneous and thapsigargin-induced Ba2+ influx in cells that transiently expressed individual TRP subtypes. These results suggest that the channel activity of IP3Rs is not essential to the generation of CCE in this cell line and that 2-APB inhibits CCE independently of the function of IP3Rs.
Various cardio-active stimuli, including endothelin-1 (ET-1), exhibit potent arrhythmogenicity, but the underlying cellular mechanisms of their actions are largely unclear. We used isolated rat atrial myocytes and related changes in their subcellular Ca(2+) signalling to the ability of various stimuli to induce diastolic, premature extra Ca(2+) transients (ECTs). For this, we recorded global and spatially resolved Ca(2+) signals in indo-1- and fluo-4-loaded atrial myocytes during electrical pacing. ET-1 exhibited a higher arrhythmogenicity (arrhythmogenic index; ratio of number of ECTs over fold-increase in Ca(2+) response, 8.60; n = 8 cells) when compared with concentrations of cardiac glycosides (arrhythmogenic index, 4.10; n = 8 cells) or the beta-adrenergic agonist isoproterenol (arrhythmogenic index, 0.11; n = 6 cells) that gave similar increases in the global Ca(2+) responses. Seventy-five percent of the ET-1-induced arrhythmogenic Ca(2+) transients were accompanied by premature action potentials, while for digoxin this proportion was 25 %. The beta-adrenergic agonist failed to elicit a significant number of ECTs. Direct activation of inositol 1,4,5-trisphosphate (InsP(3)) receptors with a membrane-permeable InsP(3) ester (InsP(3) BM) mimicked the effect of ET-1 (arrhythmogenic index, 14.70; n = 6 cells). Inhibition of InsP(3) receptors using 2 microM 2-aminoethoxydiphenyl borate, which did not display any effects on Ca(2+) signalling under control conditions, specifically suppressed the arrhythmogenic action of ET-1 and InsP(3) BM. Immunocytochemistry indicated a co-localisation of peripheral, junctional ryanodine receptors with InsP(3)Rs. Thus, the pronounced arrhythmogenic potency of ET-1 is due to the spatially specific recruitment of Ca(2+) sparks by subsarcolemmal InsP(3)Rs. Summation of such sparks efficiently generates delayed after depolarisations that trigger premature action potentials. We conclude that the particular spatial profile of cellular Ca(2+) signals is a major, previously unrecognised, determinant for arrhythmogenic potency and that the InsP(3) signalling cassette might therefore be a promising new target for understanding and managing atrial arrhythmia.
The action of Xestospongin C (XeC) on calcium concentration in the cytosol ([Ca2+]i) and within the lumen of endoplasmic reticulum (ER) ([Ca2+]L) was studied using cultured dorsal root ganglia (DRG) neurones. Application of 2.5 microM of XeC triggered a slow [Ca2+]i transient as measured by Fura-2 video-imaging. The kinetics and amplitude of XeC-induced [Ca2+]i response was similar to that triggered by 1 microM thapsigargin (TG). The [Ca2+]L was monitored in cells loaded with low-affinity Ca2+ indicator Mag-Fura-2. The cytosolic portion of Mag-Fura-2 was removed by permeabilisation of the plasmalemma with saponin. Application of XeC to these permeabilised neurones resulted in a slow depletion of the ER Ca2+ store. XeC, however, failed to inhibit inositol 1,4,5-trisphosphate (InsP3)-induced [Ca2+]L responses. We conclude that XeC is a potent inhibitor of sarco(endo)plasmic reticulum calcium ATPase, and it cannot be regarded as a specific inhibitor of InsP3 receptors in cultured DRG neurones.
2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca2+ channels and store-operated Ca2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 micro m, respectively, measured at pH 7.2). The Ca2+-ATPase is also more potently inhibited at lower pH (IC50 = 70 micro m for SERCA1A at pH 6). 2-APB decreases the affinity for Ca2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca2+-ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca2+ to their binding sites.
Recent studies have expanded the number of channel types and messengers that lead to Ca(2+) signals within cells. Furthermore, we are beginning to understand the complex interplay between different sources of Ca(2+).
2-Aminoethoxydiphenylborate (2-APB) inhibits the extent of inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) release from cerebellar microsomes with a potency that is dependent upon the InsP(3) concentration used. At high InsP(3) concentrations (10 microM), the concentration of 2-APB required to cause half-maximal InsP(3)-induced Ca(2+) release (IC(50)) was greater than 1 mM, while at 0.25 microM InsP(3) this reduced to 220 microM. The fact that the inhibition of the extent of InsP(3)-induced Ca(2+) release (IICR) by 2-APB was not restored to control levels by high concentrations of InsP(3), in addition to the fact 2-APB did not substantially inhibit [3H]InsP(3) binding to its receptor, indicates that the inhibition is not competitive in nature. Since the cooperativity of IICR as a function of InsP(3) was reduced in the presence of 2-APB (Hill coefficient changing from 1.9 in the absence of 2-APB to 1.4 in the presence of 1 mM 2-APB), this suggests that it is acting as an allosteric inhibitor. 2-APB also reduces the rate constants for IICR. In cerebellar microsomes this release process is biphasic in nature, with a fast and slow phase. 2-APB appears particularly to affect the fast-phase component. Although 2-APB does not inhibit the ryanodine receptor, it does inhibit the Ca(2+) ATPase activity as well store-operated Ca(2+) entry channels, which may limit its use as a specific membrane permeant InsP(3) receptor inhibitor.
2-Aminoethoxydiphenyl borate (2-APB) is a putative, membrane-permeable inhibitor of inositol 1,4,5-trisphosphate (InsP(3)) receptors, but it is the case that little is known about its action at the InsP(3) receptor level. Thus, we examined the effects of 2-APB on InsP(3) receptor-mediated effects in a range of cell types expressing different complements of InsP(3) receptor types. In experiments with permeabilized cells we found that 2-APB could inhibit InsP(3)-induced release of stored Ca(2+), but also that it released Ca(2+), and that the prevalence of these two effects varied between different cell types and did not correlate with the expression of a particular receptor type. These effects of 2-APB reflected an interaction distal to the ligand binding site of InsP(3) receptors, since InsP(3) binding was unaffected by 2-APB. In intact cells, we found only inhibitory effects of 2-APB on Ca(2+) mobilization, and that variation between cell types in the characteristics of this inhibition appeared to be due to differential entry of 2-APB. 2-APB also inhibited InsP(3) receptor ubiquitination and proteasomal degradation, which again was cell type dependent. In total, these data reveal a remarkable degree of variation between cell types in the effects of 2-APB, showing that its usefulness as a specific and universal inhibitor of InsP(3) receptors is limited. However, the ability of 2-APB to inhibit InsP(3) receptor ubiquitination and degradation indicates that 2-APB may block InsP(3)-induced conformational changes in the receptor, resulting in perturbation of multiple regulatory events.
A common mechanism underlies vertebrate calcium signalling and Drosophila phototransduction The inositol trisphosphate receptor antagonist 2-aminoethoxydiphenylborate (2-APB) blocks Ca 2+ entry channels in human platelets: cautions for its use in studying Ca 2+ influx
- I Chorna
- T Ornan
- Joel
- H C Almagor
- S Ben-Ami
- B Frechter
- Z Gillo
- D L Selinger
- B Gill
- Minke
I. Chorna-Ornan, T. Joel-Almagor, H.C. Ben-Ami, S. Frechter, B. Gillo, Z. Selinger, D.L. Gill, B. Minke, A common mechanism underlies vertebrate calcium signalling and Drosophila phototransduction, J. Neurosci. 21 (2001) 2622–2629. [25] J.M. Diver, S.O. Sage, J.A. Rosado, The inositol trisphosphate receptor antagonist 2-aminoethoxydiphenylborate (2-APB) blocks Ca 2+ entry channels in human platelets: cautions for its use in studying Ca 2+ influx, Cell Calcium 30 (2001) 323–329.
Role of the phospholipase C-inositol 1,4,5-trisphosphate pathway in calcium release-activated calcium current and capacitative calcium entry
- L M Broad
- F.-J Braun
- J.-P Lievremont
- G J St
- T Bird
- J W Kurosaki
- Putney
L.M. Broad, F.-J. Braun, J.-P. Lievremont, G.St.J. Bird, T. Kurosaki,
J.W. Putney, Role of the phospholipase C-inositol 1,4,5-trisphosphate
pathway in calcium release-activated calcium current and capacitative
calcium entry, J. Biol. Chem. 276 (2001) 15945–15952.
Evidence that 2-aminoethoxydiphenyl borate is a novel inhibitor of store-operated Ca2+ channels in liver cells, and acts through a mechanism which does not involve inositol trisphosphate receptors
- Gregory
Role of the phospholipase C-inositol 1,4,5-trisphosphate pathway in calcium release-activated calcium current and capacitative calcium entry
- Broad
A diacylglycerol-activated Ca2+ channel in PC12 cells (an adrenal chromaffin cell line) correlates with expression of the TRP-6 (transient receptor potential) protein
- Tesfai