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Ion Channels in Vascular Endothelium
Abstract
The functional impact of ion channels in vascular endothelial cells (ECs) is still a matter of controversy. This review describes different types of ion channels in ECs and their role in electrogenesis, Ca2+ signaling, vessel permeability, cell-cell communication, mechano-sensor functions, and pH and volume regulation. One major function of ion channels in ECs is the control of Ca2+ influx either by a direct modulation of the Ca2+ influx pathway or by indirect modulation of K+ and Cl- channels, thereby clamping the membrane at a sufficiently negative potential to provide the necessary driving force for a sustained Ca2+ influx. We discuss various mechanisms of Ca2+ influx stimulation: those that activate nonselective, Ca(2+)-permeable cation channels or those that activate Ca(2+)-selective channels, exclusively or partially operated by the filling state of intracellular Ca2+ stores. We also describe the role of various Ca(2+)- and shear stress-activated K+ channels and different types of Cl- channels for the regulation of the membrane potential.
... The endothelium-dependent vasodilation is triggered by an increase in [Ca 2+ ] i , which has typically been shown to present 2 phases: an initial inositol 1,4,5-triphosphatemediated transient Ca 2+ release from the endoplasmic reticulum, and a subsequently sustained Ca 2+ influx from the extracellular space. Although Ca 2+ release from intracellular stores is a key event in the transduction pathway of the response, the development and stability of the vasodilation depends on the Ca 2+ influx phase, which has been mainly attributed to capacitative Ca 2+ entry (27,28). However, NCX has also been found to contribute to the rise of [Ca 2+ ] i in different cell types (9,17). ...
... However, NCX has also been found to contribute to the rise of [Ca 2+ ] i in different cell types (9,17). It is wellaccepted that NCX-mediated Ca 2+ extrusion has an important role in the control of [Ca 2+ ] i in endothelial cells (11,12,27), but the exchanger may also transport Ca 2+ into the cell by the activation of the reverse mode (1 Ca 2+ in/3 Na + out) (9). Consistent with that notion, Na + loading of endothelial cells using the Na + ionophore monensin was found to potentiate the NO-dependent vasodilation induced by ACh and bradykinin in rat aorta (13,14). ...
... SEA0400 has been recognized as a selective blocker of NCXrm (23), but several splice variants of NCX1 have been described, and the maximum inhibitory effect of SEA0400 was found in range between 0.3 mM and ;10 mM, depending on the spliced isoform evaluated (46,47), which is coherent with the concentration-dependent inhibitory effect that we observed between 1 and 10 mM of this blocker because the NCX1 spliced isoforms present in microvascular endothelial cells have not been determined. Although SEA0400 at a concentration of 10 mM may start to affect the function of L-type, voltage-dependent Ca 2+ channels (47,48), endothelial cells do not normally express L-type channels, and those channels are not involved in the response activated by ACh (27). In contrast to SEA0400, the specificity of KB-R7943 has been questioned. ...
Na+-Ca2+ exchanger (NCX) contributes to control the intracellular free Ca2+ concentration ([Ca2+]i), but the functional activation of NCX reverse mode (NCXrm) in endothelial cells is controversial. We evaluated the participation of NCXrm-mediated Ca2+ uptake in the endothelium-dependent vasodilation of rat isolated mesenteric arterial beds. In phenylephrine-contracted mesenteries, the acetylcholine (ACh)-induced vasodilation was abolished by treatment with the NCXrm blockers SEA0400, KB-R7943, or SN-6. Consistent with that, the ACh-induced hyperpolarization observed in primary cultures of mesenteric endothelial cells and in smooth muscle of isolated mesenteric resistance arteries was attenuated by KB-R7943 and SEA0400, respectively. In addition, both blockers abolished the NO production activated by ACh in intact mesenteric arteries. In contrast, the inhibition of NCXrm did not affect the vasodilator responses induced by the Ca2+ ionophore, ionomycin, and the NO donor, S-nitroso-N-acetylpenicillamine. Furthermore, SEA0400, KB-R7943, and a small interference RNA directed against NCX1 blunted the increase in [Ca2+]i induced by ACh or ATP in cultured endothelial cells. The analysis by proximity ligation assay showed that the NO-synthesizing enzyme, eNOS, and NCX1 were associated in endothelial cell caveolae of intact mesenteric resistance arteries. These results indicate that the activation of NCXrm has a central role in Ca2+-mediated vasodilation initiated by ACh in endothelial cells of resistance arteries.-Lillo, M. A., Gaete, P. S., Puebla, M., Ardiles, N. M., Poblete, I., Becerra, A., Simon, F., Figueroa, X. F. Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.
... The subsequent increase in cytosolic Ca 2+ can activate endothelial nitric oxide synthase [Fleming et al., 1997]. Thus, the modulation of BK Ca channels in endothelial cells would lead to the change in membrane potentials of endothelial cells and significantly contribute to the regulation of vascular tone [Nelson and Quayle, 1995;Nilius et al., 1997]. ...
... Previous observations at our laboratory showed that BK Ca channels can be expressed in human umbilical vascular endothelial cells [Wu et al., 1999]. The K + selectivity, unitary conductance, voltage-dependence, and pharmacological properties of these channels are similar to those of BK Ca channels, as reported previously in other endothelial cells [Rusko et al., 1992;Haburcák et al., 1997;Nilius et al., 1997]. Therefore, the purpose of this study was to determine whether pinacidil affects Ca 2+ -activated K + currents in cultured endothelial cells of human umbilical veins. ...
... The singlechannel conductance of BK Ca channels calculated from the linear I-V relationship in control (i.e., in the absence of pinacidil) was 166 ± 8 pS (n = 8). This value was found to be similar to that reported previously in endothelial cells [Rusko et al., 1992;Haburcák et al., 1997;Nilius et al., 1997;Wu et al., 1999], but not significantly different from that (164 ± 7 pS; P > 0.05, n = 8) measured in the presence of pinacidil (30 µM). These results thus indicate that pinacidil produced no significant change in the single-channel conductance of BK Ca channels, but stimulated the channel activity in these cells. ...
The effect of pinacidil, an opener of ATP-sensitive K+ (K-ATP) channels, on large-conductance Ca2+-activated K+ (BKCa) channels was investigated in cultured endothelial cells of human umbilical veins. In whole cell configuration, pinacidil (30 mu M) increased the amplitude of K+ outward currents (I-K). Charybdotoxin (100 nM), but not glibenclamide (10 mu M), suppressed pinacidil-induced increase in I-K Neither carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 10 mu M), an inhibitor of mitochondrial Ca2+-uniporter, nor cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore, affected pinacidil-induced increase in I-K. In inside-out patch configuration, bath application of pinacidil (30 mu M) did not change single channel conductance but increased the activity of BKCa channels. Pinacidil (30 mu M) shifted the activation curve of BKCa channels to less positive membrane potential by approximately 15 mV. Pinacidil stimulated the activity of these channels in a concentration-dependent manner. The EC50 value for pinacidil-induced channel activity was 20 mu M. After BKCa channels had been enhanced by Evans blue (100 mu M), subsequent application of pinacidil (100 mu M) did not further increase the channel activity. These results clearly indicate that in addition to the activation of K-ATP channels, pinacidil can also stimulate BKCa channels in endothelial cells. These effects could contribute to the regulation of vascular tone if similar results were found in endothelial cells in vivo. Drug Dev. Res. 48:6-16, 1999. (C) 1999 Wiley-Liss, Inc.
... This would have been important to determine whether the K channels affected by resveratrol belong to the endothelium or the smooth muscle of the vessel wall. Although not as well understood (in terms of their role), K channels do exist in endothelial cells (32,33) as well as in arterial smooth muscle cells (26,27). But since our results did not support our hypothesis, we will consider other possible mechanisms in the future that may be responsible for resveratrol's relaxation of the rat tail artery, such as again the endothelium (though not focusing on its K channels) but also smooth muscle voltage-gated calcium channels for which others have already provided initial evidence suggesting they play a partial role in the rat mesenteric artery (12). ...
... Less is known about the role of endothelial K channels in endothelial release of contracting factors compared to relaxing factors (32,33). However, it is known that vascular endothelial cells possess mechanosensitive ion channels which when stretched can directly alter the release of endothelial contracting factors (as well as relaxing factors) or indirectly alter the ability of various endogenous agonists like acetylcholine to release contracting factors (32,33,(43)(44)(45). ...
... Less is known about the role of endothelial K channels in endothelial release of contracting factors compared to relaxing factors (32,33). However, it is known that vascular endothelial cells possess mechanosensitive ion channels which when stretched can directly alter the release of endothelial contracting factors (as well as relaxing factors) or indirectly alter the ability of various endogenous agonists like acetylcholine to release contracting factors (32,33,(43)(44)(45). There are at least two reasons why we suspect that resveratrol may be acting as such an agonist; or more specifically, acting on such mechano-sensitive ion channels in the endothelium. ...
Our aims were to determine 1) if resveratrol’s vasorelaxant action is greater in the distal (resistance) versus proximal (conductance) portion of the rat tail artery, and 2) if it can be blocked by agents known to block different potassium (K) channels in arterial smooth muscle. We found that its half-maximally effective concentration values were essentially identical (25 ± 3 versus 27 ± 3 μM) for relaxing adrenergically-precontracted rings prepared from distal versus proximal tissues. This does not confirm a previous report of greater relaxation in resistance versus conductance arteries. We also found that its relaxation could not be blocked by any of seven different K channel blockers. However, we uncovered a novel unanticipated action not yet reported. In half our arterial ring preparations, resveratrol transiently enhanced adrenergically-induced precontractions beginning well before its sustained relaxant effect became apparent. This action provides the first reasonable explanation for previously unexplained increases in arterial pressures observed during acute intravenous administration of resveratrol to animal models of traumatic ischemic tissue injury, in which hypotension is often present and in need of correction. Also unanticipated, this same transient enhancement of adrenergic contraction was notably inhibited by some of the same K channel blockers (particularly tetraethylammonium and glibenclamide) that failed to influence its relaxant effect. Although we do not rule out smooth muscle as a possible site for such a paradoxical finding, we suspect resveratrol could also be acting on K-selective mechano-sensitive ion channels located in the endothelium where they may participate in release of contracting factors.
... The endothelium regulates vascular tone by releasing various vasoactive factors, including nitric oxide (NO), prostacyclin (PGI 2 ), and endothelium-derived hyperpolarizing (EDH) factors [15] most prominently in resistance arteries and arterioles. A common pathway responsible for the stimulus-induced release of endothelial factors involves an increase in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in endothelial cells (EC) [44]. Transient receptor potential (TRP) vanilloid 4 (TRPV4), the fourth member of the TRP vanilloid subfamily, is a non-selective Ca 2+ -permeable cation channel expressed in vascular ECs of multiple species [18,70]. ...
... Since TRPV4 is a Ca 2+ -permeable cation channel, the mechanism of TRPV4 activation by receptor agonists such as ACh likely involves receptor-operated Ca 2+ (ROC) entry following the binding of the agonist to the membrane receptor in EC [21,28,43,44]. To investigate the endothelial factors released in responses to ACh and TRPV4 agonist GSK1016790A, arterioles were pretreated with NO synthase and cyclooxygenase inhibitors, L-NAME, and indomethacin, respectively. ...
Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca²⁺ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca²⁺ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca²⁺ entry and subsequent endothelium-dependent dilation in human arterioles.
... This enhanced IRK conductance is sufficient to overcome the depolarizing action of the nonselective cation and Cl conductance (see also Fig. 11) and shifts the membrane potential toward more negative potentials. The conductance of single endothelial K ir channels ranges from 23 to 30 pS in symmetrical K solutions ( 84,156,180,290,295,325,372,373). Typical features of this channel are the increase in single-channel conductance with the square root of the extracellular K concentration (289, 373, 462) and a permeation profile of P K P Rb P Cs (325, 373). ...
... Synchronization of these Ca 2 oscillations in confluent EC depends on cell-cell coupling (268). These oscillations are frequently accompanied by oscillatory changes in membrane potential due to activation of BK Ca channels (272,295,296,314,415). These oscillations are mainly due to periodic discharges of intracellular Ca 2 stores, since they are sustained for a long time in cells exposed to Ca 2-free media (162, 272) and apparently not via activation of a Ca 2-influx pathway since they are not affected by changes in the driving force for Ca 2 (272). ...
Endothelial cells (EC) form a unique signal-transducing surface in the vascular system. The abundance of ion channels in the plasma membrane of these nonexcitable cells has raised questions about their functional role. This review presents evidence for the involvement of ion channels in endothelial cell functions controlled by intracellular Ca ²⁺ signals, such as the production and release of many vasoactive factors, e.g., nitric oxide and PGI 2 . In addition, ion channels may be involved in the regulation of the traffic of macromolecules by endocytosis, transcytosis, the biosynthetic-secretory pathway, and exocytosis, e.g., tissue factor pathway inhibitor, von Willebrand factor, and tissue plasminogen activator. Ion channels are also involved in controlling intercellular permeability, EC proliferation, and angiogenesis. These functions are supported or triggered via ion channels, which either provide Ca ²⁺ -entry pathways or stabilize the driving force for Ca ²⁺ influx through these pathways. These Ca ²⁺ -entry pathways comprise agonist-activated nonselective Ca ²⁺ -permeable cation channels, cyclic nucleotide-activated nonselective cation channels, and store-operated Ca ²⁺ channels or capacitative Ca ²⁺ entry. At least some of these channels appear to be expressed by genes of the trp family. The driving force for Ca ²⁺ entry is mainly controlled by large-conductance Ca ²⁺ -dependent BK Ca channels ( slo), inwardly rectifying K ⁺ channels (Kir2.1), and at least two types of Cl ⁻ channels, i.e., the Ca ²⁺ -activated Cl ⁻ channel and the housekeeping, volume-regulated anion channel (VRAC). In addition to their essential function in Ca ²⁺ signaling, VRAC channels are multifunctional, operate as a transport pathway for amino acids and organic osmolytes, and are possibly involved in endothelial cell proliferation and angiogenesis. Finally, we have also highlighted the role of ion channels as mechanosensors in EC. Plasmalemmal ion channels may signal rapid changes in hemodynamic forces, such as shear stress and biaxial tensile stress, but also changes in cell shape and cell volume to the cytoskeleton and the intracellular machinery for metabolite traffic and gene expression.
... Recently, endothelial K Ca channels have been used as new drug targets for cardiovascular diseases such as hypertension to stimulate EDHF and NO production to improve endothelial dysfunction [13,[15][16][17]. There are three types of K Ca channels based on their conductances, including IK Ca (intermediate conductance), SK Ca (small conductance), and BK Ca (large conductance) [18]. Although IK Ca and SK Ca are the major K Ca channels present in the endothelial cells of arteries, BK Ca channels have been identified in the endothelium of rat pulmonary and mesenteric arteries [19,20] and cultured endothelial cells [21,22]. ...
... ] i elevation has been implicated in endothelium-mediated vasodilation [18] and is also needed for K Ca activation [34]. Since calycosin and formononetin increased outward currents via endothelial K Ca channel, we next examined their effects on [Ca 2+ ] i in HUVEC. ...
Calycosin and formononetin are two structurally similar isoflavonoids that have been shown to induce vasodilation in aorta and conduit arteries, but study of their actions on endothelial functions is lacking. Here, we demonstrated that both isoflavonoids relaxed rat mesenteric resistance arteries in a concentration-dependent manner, which was reduced by endothelial disruption and nitric oxide synthase (NOS) inhibition, indicating the involvement of both endothelium and vascular smooth muscle. In addition, the endothelium-dependent vasodilation, but not the endothelium-independent vasodilation, was blocked by BKCa inhibitor iberiotoxin (IbTX). Using human umbilical vein endothelial cells (HUVECs) as a model, we showed calycosin and formononetin induced dose-dependent outwardly rectifying K ⁺ currents using whole cell patch clamp. These currents were blocked by tetraethylammonium chloride (TEACl), charybdotoxin (ChTX), or IbTX, but not apamin. We further demonstrated that both isoflavonoids significantly increased nitric oxide (NO) production and upregulated the activities and expressions of endothelial NOS (eNOS) and neuronal NOS (nNOS). These results suggested that calycosin and formononetin act as endothelial BKCa activators for mediating endothelium-dependent vasodilation through enhancing endothelium hyperpolarization and NO production. Since activation of BKCa plays a role in improving behavioral and cognitive disorders, we suggested that these two isoflavonoids could provide beneficial effects to cognitive disorders through vascular regulation.
... Nevertheless, independent of 1g through gap junctions, the distance the vessel wall can be modulated by 4 Endothelial conduction 85 nPo [5) as discussed in the following section. An array of ion channels may be expressed in the plasma membrane of ECs [38,62). Here we focus on those concerned with the initiation ofhyperpolarization and its conduction along the vessel · wall leading to the coordinated relaxation of SMCs and vasodilation. ...
... For ECs that contain the inward-rectifying K+ channel (K1R; dominant isoforms are of the KIR2.x family [18,22,62)), KIR can serve as an extracellular K+ sensor to modulate K+ efflux [61]. It should also be recognized that, with electrical coupling between ECs and SMCs via myoendothelial gap junctions, activation of KIR in SMCs can contribute additional outward current flow and thereby enhance the conduction of hyperpolarization and vasodilation [40,70]. ...
The regulation of tissue blood flow in response to the metabolic demand of parenchymal cells is effected through changes in vascular resistance as governed by arteriolar networks and their proximal feed arteries. Vasodilation and vasoconstriction must be coordinated among downstream and upstream segments to optimize blood flow distribution within the tissue and to attain maximal perfusion of the vascular supply. Such coordinated vasomotor activity is promoted by the transmission of electrical signals (e.g., hyperpolarization and depolarization) through gap junctions from cell-to-cell along the vessel wall. Based upon underlying structural and functional relationships, we explore the biophysical basis of intercellular electrical signaling along the endothelium of resistance arteries. The endothelium is presented as a cable, whereby electrical signals decay passively with distance from the site of initiation. Key to our findings is how K+ channels expressed constitutively in endothelial cell membranes [e.g., KCa2.3 (SKCa) and KCa3.1 (IKCa)] regulate the spatial domain of electrical signal transmission and how this role is effected during advanced age through the actions of hydrogen peroxide. New insights into the regulation of electrical conduction along microvascular endothelium advance our understanding of how blood flow is governed by ion channels while providing mechanistic insight into how such processes can be affected during vascular disease.
... The modulation of potassium channel activity by selective activators and inhibitors can provide crucial information about the role of potassium channels in physiological processes. BK Ca channels are blocked by charybdotoxin, iberiotoxin, TEA (tetraethylammonium), D-tubocurarine , quinine (Nilius and Droogmans, 2001), nicotine (Kuhlmann et al., 2005), 2-methoxyestradiol (Chiang and Wu, 2001) and peroxynitrite (Liu et al., 2002) and are sensitive to activators such as sphingosine-1-phosphate (Kim et al., 2006), NO (Mistry and Garland, 1998), resveratrol (Li et al., 2000), NS1619 and NS004 (Gribkoff et al., 1996). The conductance of these channels ranges from 165 to 240 pS (Nilius and Droogmans, 2001). ...
... BK Ca channels are blocked by charybdotoxin, iberiotoxin, TEA (tetraethylammonium), D-tubocurarine , quinine (Nilius and Droogmans, 2001), nicotine (Kuhlmann et al., 2005), 2-methoxyestradiol (Chiang and Wu, 2001) and peroxynitrite (Liu et al., 2002) and are sensitive to activators such as sphingosine-1-phosphate (Kim et al., 2006), NO (Mistry and Garland, 1998), resveratrol (Li et al., 2000), NS1619 and NS004 (Gribkoff et al., 1996). The conductance of these channels ranges from 165 to 240 pS (Nilius and Droogmans, 2001). The BK Ca channel family is known for its Ca 2 þ -sensing abilities, which are attributed to the presence of two Ca-binding sites (Cox, 2005). ...
A large conductance potassium (BKCa) channel opener, NS1619 (1,3-dihydro-1- [2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one), is well known for its protective effects against ischemia-reperfusion injury; however, the exact mode of its action remains unclear. The aim of this study was to characterize the effect of NS1619 on endothelial cells. The endothelial cell line EA.hy926, guinea pig hearts and submitochondrial particles isolated from the heart were used. In the isolated guinea pig hearts, which were perfused using the Langendorff technique, NS1619 caused a dose-dependent increase in coronary flow that was inhibited by l-NAME. In EA.hy926 cells, NS1619 also caused a dose-dependent increase in the intracellular calcium ion concentration [Ca(2+)]i, as measured using the FURA-2 fluorescent probe. Moreover, NS1619 decreased the oxygen consumption rate in EA.hy926 cells, as assessed using a Clark-type oxygen electrode. However, when NS1619 was applied in the presence of oligomycin, the oxygen consumption increased. NS1619 also decreased the mitochondrial membrane potential, as measured using a JC-1 fluorescent probe in the presence and absence of oligomycin. Additionally, the application of NS1619 to submitochondrial particles inhibited ATP synthase. In summary, NS1619 has pleiotropic actions on EA.hy926 cells and acts not only as an opener of the BKCa channel in EA.hy926 cells but also as an inhibitor of the respiratory chain component, sarcoplasmic reticulum ATPase, which leads to the release of Ca(2+) from the endoplasmic reticulum. Furthermore, NS1619 has the oligomycin-like property of inhibiting mitochondrial ATP synthase.
... Mechanosensitive channels are widely distributed in the body regions such as the heart (Kim 1992;Friedrich et al. 2012), brain, colon, sensory nerves, lungs, urinary bladder, prostate glands, and vascular smooth muscles (Davis et al. 1992;Su et al. 2000;Choi et al. 2015). In the cardiovascular system, mechanosensitive channels are localized on vascular endothelial cells, aorta, atria, ventricles, coronary blood vessels (Craelius et al. 1988;Nilius et al. 1997;Lehoux and Tedgui 2003), and sensory nerves innervating the heart (Shenton and Pyner 2014). TRPV channels are also located on the heart, sensory nerve endings, sciatic nerve, and skeletal muscles (Schultz 2003;Fischer et al. 2003). ...
... The attenuation of remote hind limb preconditioning-induced cardioprotection in the presence of gadolinium suggests the important role of TRP (a type of mechanosensitive channels) in mediating remote hind limb preconditioning induced cardioprotection. Mechanosensitive channels are localized on different regions of the heart including vascular endothelial cells, aorta, atria, ventricles (Yao et al. 2003), coronary blood vessels (Nilius et al. 1997;Lehoux and Tedgui 2003), and sensory nerves innervating the heart (Xian Tao et al. 2006;Hayabuchi et al. 2011;Shenton and Pyner 2014). These channels are sensitive to mechanical stretch, and there have been studies suggesting the key role of these stretch activated channels in mediating ischemic preconditioning-induced cardioprotection (Ovize et al. 1994;Obadia et al. 1997;Nakagawa et al. 1997;Gysembergh et al. 1998). ...
Remote ischemic preconditioning is a well reported therapeutic strategy that induces cardioprotective effects but the underlying intracellular mechanisms have not been widely explored. The current study was designed to investigate the involvement of TRP and especially TRPV channels in remote hind limb preconditioning-induced cardioprotection. Remote hind limb preconditioning stimulus (4 alternate cycles of inflation and deflation of 5 min each) was delivered using a blood pressure cuff tied on the hind limb of the anesthetized rat. Using Langendorff's system, the heart was perfused and subjected to 30-min ischemia and 120-min reperfusion. The myocardial injury was assessed by measuring infarct size, lactate dehydrogenase (LDH), creatine kinase (CK), LVDP, +dp/dtmax, -dp/dtmin, heart rate, and coronary flow rate. Gadolinium, TRP blocker, and ruthenium red, TRPV channel blocker, were employed as pharmacological tools. Remote hind limb preconditioning significantly reduced the infarct size, LDH release, CK release and improved coronary flow rate, hemodynamic parameters including LVDP, +dp/dtmax, -dp/dtmin, and heart rate. However, gadolinium (7.5 and 15 mg kg(-1)) and ruthenium red (4 and 8 mg kg(-1)) significantly attenuated the cardioprotective effects suggesting the involvement of TRP especially TRPV channels in mediating remote hind limb preconditioning-induced cardioprotection. Remote hind limb preconditioning stimulus possibly activates TRPV channels on the heart or sensory nerve fibers innervating the heart to induce cardioprotective effects. Alternatively, remote hind limb preconditioning stimulus may also activate the mechanosensitive TRP and especially TRPV channels on the sensory nerve fibers innervating the skeletal muscles to trigger cardioprotective neurogenic signaling cascade. The cardioprotective effects of remote hind limb preconditioning may be mediated via activation of mechanosensitive TRP and especially TRPV channels.
... Several types of ion channels cooperate in elaborating the cell membrane potential, which regulates calcium entry into the endothelial cells (118). Among those, K ϩ channels, especially Ca 2ϩ -activated K ϩ channels (251), inwardly rectifying K ϩ channels (K ir ), and voltage-dependent K ϩ channels, are the major classes of ion channels involved in setting the membrane potential (165), which in turn influences the driving force for the Ca 2ϩ via SOCE. Indeed, blockers of K ϩ channels, as well as plasma membrane depolarization, stop the cell cycle in G 1 phase, thus reducing cell proliferation (153,165). ...
... Among those, K ϩ channels, especially Ca 2ϩ -activated K ϩ channels (251), inwardly rectifying K ϩ channels (K ir ), and voltage-dependent K ϩ channels, are the major classes of ion channels involved in setting the membrane potential (165), which in turn influences the driving force for the Ca 2ϩ via SOCE. Indeed, blockers of K ϩ channels, as well as plasma membrane depolarization, stop the cell cycle in G 1 phase, thus reducing cell proliferation (153,165). However, among the Ca 2ϩ -dependent K ϩ channels, the Ca 2ϩ -dependent intermediate conductance K ϩ channel IK1 (SK4, KCNN4) increases cell proliferation with a completely different mechanism, in a HEK-overexpressing system. ...
... 48,49 When the cells receive the FSS mechanical signals, several mechanosensors will be triggered, including integrins, the glycocalyx, primary cilia, Gprotein-coupled receptors, and ion channels (K + , Ca 2+ ). [50][51][52][53][54][55][56][57] Piezo channels are important sensors for mechanical stimulation. Piezo1 initially senses SS and transmits biomechanical signals to the nucleus to promote nuclear contraction. ...
Cellular mechanotransduction, a critical regulator of numerous biological processes, is the conversion from mechanical signals to biochemical signals regarding cell activities and metabolism. Typical mechanical cues in organisms include hydrostatic pressure, fluid shear stress, tensile force, extracellular matrix stiffness or tissue elasticity, and extracellular fluid viscosity. Mechanotransduction has been expected to trigger multiple biological processes, such as embryonic development, tissue repair and regeneration. However, prolonged excessive mechanical stimulation can result in pathological processes, such as multi-organ fibrosis, tumorigenesis, and cancer immunotherapy resistance. Although the associations between mechanical cues and normal tissue homeostasis or diseases have been identified, the regulatory mechanisms among different mechanical cues are not yet comprehensively illustrated, and no effective therapies are currently available targeting mechanical cue-related signaling. This review systematically summarizes the characteristics and regulatory mechanisms of typical mechanical cues in normal conditions and diseases with the updated evidence. The key effectors responding to mechanical stimulations are listed, such as Piezo channels, integrins, Yes-associated protein (YAP) /transcriptional coactivator with PDZ-binding motif (TAZ), and transient receptor potential vanilloid 4 (TRPV4). We also reviewed the key signaling pathways, therapeutic targets and cutting-edge clinical applications of diseases related to mechanical cues.
... Although several types of K þ channel have been identified in endothelial cells (20)(21)(22), exactly which K þ channels contribute to remifentanil-induced hyperpolarization in intact HUA rings has not been investigated previously. Ohashi et al. (7) showed that acetylcholine increases the extracellular K þ concentration in isolated rabbit aorta, an effect antagonized by both CTX and TEA. ...
... for both resveratroland vehicle-treated rings, but only when analyzed in combination not separately by Bonferroni's mean comparisons *p < .05 versus vehicle represents a statistically significant effect from two-factor ANOVA for comparisons of resveratrol versus vehicle in both endothelium-intact and endothelium-removed rings because vascular endothelial cells possess mechanosensitive ion channels which when stretched can directly alter the release of endothelial contracting factors or indirectly alter the ability of various endogenous agonists to release contracting factors (Harder, 1987;Hishikawa & Lüscher, 1997;Katusic et al., 1987;Nilius & Droogmans, 2001;Nilius et al., 1997;Sekiguchi et al., 1996;Vanhoutte, 1987;Yang et al., 2002). In our previous work, we found that the K + channel blocking agents TEA and glibenclamide notably inhibited the magnitude of the initial transient contractions caused by trans-resveratrol (Stom et al., 2016). ...
The health benefits of the natural polyphenol trans-resveratrol may play an important role in preventing a variety of diseases. Resveratrol has been shown to reduce blood pressure and improve metabolic diseases such as type 2 diabetes mellitus and obesity. Our previous studies examined the role of K+ channels in the vasorelaxation responses to trans-resveratrol in the rat tail artery. During these studies, we uncovered a novel transient contraction prior to the sustained relaxation effect of trans-resveratrol. Thus, the purpose of this study was to determine the role of the endothelium in these vascular contraction and relaxation responses to trans-resveratrol. We additionally sought to determine if the cis-isomer of resveratrol exerts any of the same vascular effects as the trans-isomer. The vascular responses to trans-resveratrol were examined in rat tail arteries with intact or denuded endothelium over a 2-hr period. Additionally, the vascular responses to trans- and cis-resveratrol were compared in rat tail arteries with intact endothelium. Both the transient contractile response and the persistent relaxation response to trans-resveratrol were similar in the arterial rings with intact or denuded endothelium. There was a significant correlation between the initial contraction-enhancing action of trans-resveratrol and the magnitude of the sustained relaxation for vessels with both intact and denuded endothelium. Moreover, we demonstrated that cis-resveratrol produced a significantly greater relaxation response as compared to trans-resveratrol without the initial contractile response. These data demonstrate the role of the vascular smooth muscle in the vascular responses to resveratrol and the potential clinical benefits of the cis-isomer of resveratrol as compared to the trans-isomer.
... The TRPV channels are mechanosensitive ion channels that conduct Na þ and Ca 2þ . 45 They are located on the sarcolemma of cardiomyocytes, 46,47 on endothelial cells of the aorta, atria, ventricles, and coronary arteries, [48][49][50] and on the endings of the afferent nerves in the heart, skeletal muscles, and other organs. [51][52][53] Randhawa and Jaggi 54 used four 5-minute cycles of inflation/deflation of a cuff located on a hind limb of a rat and then isolated its heart. ...
A humoral mechanism of cardioprotection by remote ischemic preconditioning (RIP) has been clearly demonstrated in various models of ischemia–reperfusion including upper and lower extremities, liver, and the mesenteric and renal arteries. A wide range of humoral factors for RIP have been proposed including hydrophobic peptides, opioid peptides, adenosine, prostanoids, endovanilloids, endocannabinoids, calcitonin gene-related peptide, leukotrienes, noradrenaline, adrenomedullin, erythropoietin, apolipoprotein, A-I glucagon-like peptide-1, interleukin 10, stromal cell-derived factor 1, and microRNAs. Virtually, all of the components of ischemic preconditioning’s signaling pathway such as nitric oxide synthase, protein kinase C, redox signaling, PI3-kinase/Akt, glycogen synthase kinase β, ERK1/2, mitoKATP channels, Connexin 43, and STAT were all found to play a role. The signaling pattern also depends on which remote vascular bed was subjected to ischemia and on the time between applying the rip and myocardial ischemia occurs. Because there is convincing evidence for many seemingly diverse humoral components in RIP, the most likely explanation is that the overall mechanism is complex like that seen in ischemic preconditioning where multiple components are both in series and in parallel and interact with each other. Inhibition of any single component in the right circumstance may block the resulting protective effect, and selectively activating that component may trigger the protection. Identifying the humoral factors responsible for RIP might be useful in developing drugs that confer RIP’s protection in a more comfortable and reliable manner.
... Thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca 2+ -ATPase, and tunicamycin-an inhibitor of protein glycosylation, both induce UPR by disrupting ER Ca 2+ homeostasis. Store-operated calcium channel (SOC) is the main regulator of Ca 2+ homeostasis in non-excitable cells, such as endothelial cells [10]. Orai1 and STIM1 were the molecular basis of store-operated Ca 2+ entry (SOCE) in endothelial cells and could mediate the proliferation of the cells [11]. ...
Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca2+ homeostasis. The store-operated calcium (SOC) channel is the primary Ca2+ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca2+ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca2+ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.
... Soluble klotho also prevents endothelial dysfunction by maintaining endothelial integrity and protecting against vascular permeability. In endothelial cells, calcium regulates numerous functions including proliferation, migration, and apoptosis (52). Studies report that sKl binds the transient receptor potential canonical 1 (TRPC1) calcium-permeable channel and vascular endothelial growth factor receptor 2 to strengthen their association and cause their cointernalization which regulates the expression level of TRPC1 on the plasma membrane (53). ...
The klotho gene encodes a type I single-pass transmembrane protein that contains a large extracellular domain, a membrane spanning segment, and a short intracellular domain. Klotho protein exists in several forms including the full-length membrane form (mKl) and a soluble circulating form [soluble klotho (sKl)]. mKl complexes with fibroblast growth factor receptors to form coreceptors for FGF23, which allows it to participate in FGF23-mediated signal transduction and regulation of phosphate and calcium homeostasis. sKl is present in the blood, urine, and cerebrospinal fluid where it performs a multitude of functions including regulation of ion channels/transporters and growth factor signaling. How sKl exerts these pleiotropic functions is poorly understood. One hurdle in understanding sKl’s mechanism of action as a “hormone” has been the inability to identify a receptor that mediates its effects. In the body, the kidneys are a major source of sKl and sKl levels decline during renal disease. sKl deficiency in chronic kidney disease makes the heart susceptible to stress-induced injury. Here, we summarize the current knowledge of mKl’s mechanism of action, the mechanistic basis of sKl’s protective, FGF23-independent effects on the heart, and provide new insights into the mechanism of action of sKl focusing on recent findings that sKl binds sialogangliosides in membrane lipid rafts to regulate growth factor signaling.
... Endothelial cells also express potassium channels 51 . The channels regulate the endothelial cell membrane potential and are, via Ca 2+ signalling, involved in the production and release of endothelial derived vasoactive factors, such as nitric oxide, prostaglandins and EDHF 52 . ...
Normal pregnancy requires adaptations of the maternal vasculature. During preeclampsia these adaptations are not well established, which may be related to maternal hypertension and proteinuria. The effects of preeclampsia on the maternal vasculature are not yet fully understood. We aimed to evaluate gene expression in aortas of pregnant rats with experimental preeclampsia using a genome wide microarray. Aortas were isolated from pregnant Wistar outbred rats with low-dose LPS-induced preeclampsia (ExpPE), healthy pregnant (Pr), non-pregnant and low-dose LPS-infused non-pregnant rats. Gene expression was measured by microarray and validated by real-time quantitative PCR. Gene Set Enrichment Analysis was performed to compare the groups. Functional analysis of the aorta was done by isotonic contraction measurements while stimulating aortic rings with potassium chloride. 526 genes were differentially expressed, and positive enrichment of “potassium channels”, “striated muscle contraction”, and “neuronal system” gene sets were found in ExpPE vs. Pr. The potassium chloride-induced contractile response of ExpPE aortic rings was significantly decreased compared to this response in Pr animals. Our data suggest that potassium channels, neuronal system and (striated) muscle contraction in the aorta may play a role in the pathophysiology of experimental preeclampsia. Whether these changes are also present in preeclamptic women needs further investigation.
... In whole cell voltage clamp experiments in the endothelial cells from CC, it appears from the reversal potentials that other channels, probably cationic non-selective channels from the Transient Receptor Potential (TRP) channels, also are expressed in CC endothelial cells shifting the reversal potential by cationic transport through the membrane toward depolarized membrane voltages (Figure 2). TRP channels have been reported to be expressed in endothelial cells (Nilius and Droogmans, 2001;Wandall-Frostholm et al., 2015), and further studies should address which particular TRP channels is/are expressed in primary culture of rat CC endothelial cells. However, whole cell voltage clamp experiments in the endothelial cells from CC, showed increased K + currents in response to both openers (1 µM). ...
Modulation of endothelial calcium-activated potassium (KCa) channels has been proposed as an approach to restore endothelial function. The present study investigated whether novel openers of KCa channels with small (KCa2.x) and intermediate (KCa3.1) conductance, NS309 and NS4591, improve endothelium-dependent relaxation and erectile function. Rat corpus cavernosum (CC) strips were mounted for isometric tension recording and processed for immunoblotting. Mean arterial pressure (MAP), intracavernosal pressure (ICP), and electrocardiographic (ECG) measurements were conducted in anesthetized rats. Immunoblotting revealed the presence of KCa2.3 and large KCa conductance (KCa1.1) channels in the corpus cavernosum. NS309 and NS4591 increased current in CC endothelial cells in whole cell patch clamp experiments. Relaxation induced by NS309 (<1 μM) was inhibited by endothelial cell removal and high extracellular potassium. An inhibitor of nitric oxide (NO) synthase, and blockers of KCa2.x and KCa1.1 channels, apamin and iberiotoxin also inhibited NS309 relaxation. Incubation with NS309 (0.5 μM) markedly enhanced acetylcholine relaxation. Basal erectile function (ICP/MAP) increased during administration of NS309. Increases in ICP/MAP after cavernous nerve stimulation with NS309 were unchanged, whereas NS4591 significantly improved erectile function. Administration of NS309 and NS4591 caused small changes in the electrocardiogram, but neither arrhythmic events nor prolongation of the QTc interval were observed. The present study suggests that openers of KCa2.x and KCa3.1 channels improve endothelial and erectile function. The effects of NS309 and NS4591 on heart rate and ECG are small, but will require additional safety studies before evaluating whether activation of KCa2.3 channels has a potential for treatment of erectile dysfunction.
... For example in pancreatic β-cells, they are responsible for controlled release of insulin[59]. Similarly, in endothelial cells (ECs), they are responsible for regulation of nitric oxide (NO) synthase[60]. G protein-coupled inwardly rectifying potassium channels (GIRKs), belong to family of IRKs. These channels are opened as a result of signal transduction that is initiated by ligand binding to G protein-coupled receptors (GPCRs). ...
Introduction: From therapeutic point of view, it is often beneficial to enhance the expression of certain enzymes whose low expression is responsible for the observed ailment. Small molecules as activators of several enzymes have great biological potential as anti-microbial and anti-cancer agents, for the treatment of diabetes, obesity, metabolic disorders, and for the treatment of neurological disorders including Alzheimer’s disease. This review covers patents describing small molecules as activators, and provides structural leads for the design of even more potent activators.
Area covered: This review is focused on small molecules that have been explored as activators of enzymes in the last and current decade (2000–2016).
Expert opinion: The ability to modulate activity of enzymes has long been a quest of medicinal chemistry. This has been the impetus behind the development of a plethora of drugs as enzyme inhibitors. However only a few enzyme activators as drugs have made it to the market. Disorders characterized by supressed enzyme activity can be treated by enhancing the activity of a specific enzyme.
... BK Ca channels are present in endothelial cell of some vascular beds. 143,144 Hyperpolarization of EC following activation of BK Ca channels can increase the electrochemical gradient for Ca 2+ , thus increasing the Ca 2+ influx to modulate NO production. [144][145][146] In addition, release of K + from endothelial BK Ca channels has been suggested to function as an endothelial-derived hyperpolarizing factor (EDHF) by activation of K IR channels or Na + /K + -ATPases on VSMC. ...
The control of renal vascular tone is important for the regulation of salt and water balance, blood pressure and the protection against damaging elevated glomerular pressure. The K(+) conductance is a major factor in the regulation of the membrane potential (Vm ) in vascular smooth muscle (VSMC) and endothelial cells (EC). The vascular tone is controlled by Vm via its effect on the opening probability of voltage operated Ca(2+) channels (VOCC) in VSMC. When K(+) conductance increases Vm becomes more negative and vasodilation follows, while deactivation of K(+) channels leads to depolarization and vasoconstriction. K(+) channels in EC indirectly participate in the control of vascular tone by endothelium derived vasodilation. Therefore, by regulating the tone of renal resistance vessels, K(+) channels have a potential role in the control of fluid homeostasis and blood pressure as well as in the protection of the renal parenchyma. The main classes of K(+) channels (calcium activated (KCa ), inward rectifier (Kir ), voltage activated (Kv ) and ATP sensitive (KATP )) have been found in the renal vessels. In this review, we summarize results available in the literature and our own studies in the field. We compare the ambiguous in vitro and in vivo results. We discuss the role of single types of K(+) channels and the integrated function of several classes. We also deal with the possible role of renal vascular K(+) channels in the pathophysiology of hypertension, diabetes mellitus and sepsis. This article is protected by copyright. All rights reserved.
... Ion channels are membrane bound proteins that act as gated pathways for the movement of ions across the cell membrane and they play important roles in the physiology of all cells[1][2][3][4]. A large number of inherited or drug-induced human diseases are associated with malfunction in ion transport across ion channels and generally known as channelopathy[5]. ...
The intra-cavitary drug blockade of hERG1 channel has been extensively studied, both experimentally and theoretically. Structurally diverse ligands inadvertently block the hERG1 K⁺ channel currents lead to drug induced Long QT Syndrome (LQTS). Accordingly, designing either hERG1 channel openers or current activators, with the potential to target other binding pockets of the channel, has been introduced as a viable approach in modern anti-arrhythmia drug development. However, reports and investigations on the molecular mechanisms underlying activators binding to the hERG1 channel remain sparse and the overall molecular design principles are largely unknown. Most of the hERG1 activators were discovered during mandatory screening for hERG1 blockade. To fill this apparent deficit, the first universal pharmacophore model for hERG1 K⁺ channel activators was developed using PHASE. 3D structures of 18 hERG1 K⁺ channel activators and their corresponding measured binding affinity values were used in the development of pharmacophore models. These compounds spanned a range of structurally different chemotypes with moderate variation in binding affinity. A five sites AAHRR (A, hydrogen-bond accepting, H, hydrophobic, R, aromatic) pharmacophore model has shown reasonable high statistical results compared to the other developed more than 1000 hypotheses. This model was used to construct steric and electrostatic contour maps. The predictive power of the model was tested with 3 external test set compounds as true unknowns. Finally, the pharmacophore model was combined with the previously developed receptor-based model of hERG1 K⁺ channel to develop and screen novel activators. The results are quite striking and it suggests a greater future role for pharmacophore modeling and virtual drug screening simulations in deciphering complex patterns of molecular mechanisms of hERG1 channel openers at the target sites. The developed model may serve as basis for the synthesis of novel potential therapeutic hERG1 activators.
... In the search for other pathways potentially involved in gastroprotection, we investigated the involvement of potassium channels, a 2 –noradrenergic receptors and capsaicin Àsensitive afferent neurons. The opening of potassium channels on endothelial cells contributes to protect the gastric mucosa from ulcerogenic agents [53]. Previous studies have demonstrated that some substances, including natural products that activate these channels contribute to protect the gastric mucosa [54]. ...
... First, endothelial derived EETs can act in a paracrine fashion to activate large conductance calcium-sensitive potassium channels in the underlying smooth muscle membrane, eliciting hyperpolarization, leading to relaxation. In MARK the intracellular calcium concentration ([Ca 2+ ] i ) from internal inositol 1,4,5-trisphosphate (IP 3 ) and/or ryanodine-sensitive stores [2,3] is followed by a sustained elevation of [Ca 2+ ] I , mediated by TRPV4 or TRPC3 and TRPC6 channels [4,5]. Increases in [Ca 2+ ] i activate small conductance (SKCa) and intermediate conductance (IKCa) calciumdependent potassium channels and a release of K + ions into the subendothelial space. ...
Objective:
Prominent among the endothelium-derived hyperpolarizing factors (EDHFs) are the Cytochrome P450 (CYP) epoxygenase-derived arachidonic acid metabolites-the epoxyeicosatrienoic acids (EETs), that are known as vasodilators in the microcirculation. Among the EET isomers, 5,6-EET undergoes rapid lactonization in aqueous solution to the more stable 5,6-δ DHTL (5,6-dihydroxytrienoic lactone) isomer. It is unclear whether this metabolic transformation maintains its vasodilator potential and what is the mechanism of action. Thus, the aim of this study was to investigate the capacity of the lactone isomer, 5,6- δ DHTL, to induce dilation of arterioles and explore the endothelial Ca(2+) response mechanism.
Approach and results:
In isolated human microvessels 5,6- δ DHTL induced a dose dependent vasodilation, that was inhibited by mechanical denudation of the endothelial layer. This 5,6- δ DHTL -dependent dilation was partially reduced in the presence of L-NAME (NOS inhibitor) or the NO-scavenger, cPTIO (by 19.7%, which was not statistically significantly). In human endothelial cells, 5,6- δ DHTL induced an increase in intracellular Ca(2+) ([Ca(2+)]i) in a dose dependent manner. This increase in [Ca(2+)]i was similar to that induced by the 5,6-EET isomer, and significantly higher than observed by administering the hydrolytic dihydroxy isomer, 5,6-DHET. Further experiments aimed to investigate the mechanism of action revealed, that the 5,6-δ DHTL-mediated ([Ca(2+)]i elevation was reduced by IP3 and ryanodine antagonists, but not by antagonists to the TRPV4 membrane channel. Similar to their effect on the dilation response in the arteries, NO inhibitors reduced the 5,6-δ DHTL-mediated ([Ca(2+)]i elevation by 20%. Subsequent 5,6-δ DHTL -dependent K(+) ion efflux from endothelial cells, was abolished by the inhibition of small and intermediate conductance KCa.
Conclusions:
The present study shows that 5,6-δ DHTL is a potential EDHF, that dilates microvessels through a mechanism that involves endothelial dependent Ca(2+) entry, requiring endothelial hyperpolarization. These results suggest the existence of additional lactone-containing metabolites that can be derived from the PUFA metabolism and which may function as novel EDHFs.
... Mechanical forces and calcium influx also open chloride channels which act as apoptotic agents through a delineated mechanism. few anion (Cl ? ) channels (Jackson, 2000; Nilius and Droogmans, 2001). The vast majority of channels open because of the changes in lipid bilayer, membrane fluidity or tension and are regulated by voltage, extracellular ligands, phosphorylation, influx of Ca 2+ and direct (physical interactions between G-protein subunits and the channel protein) or indirect (via second messengers and protein kinases) interaction with activated G proteins (Christensen, 1987; Maroto et al., 2005; Lumpkin and Caterina, 2007; Hahn and Schwartz, 2009). ...
The increased use of tissue expander in the past decades and its potential market values in near future give enough reasons to sum up the consequences of tissue expansion. Furthermore, the patients have the right to know underlying mechanisms of adaptation of inserted biomimetic, its bioinspired materials and probable complications. The mechanical strains during tissue expansion are related to several biological phenomena. Tissue remodeling during the expansion is highly regulated and depends on the signal transduction. Any alteration may lead to tumor formation, necrosis and/or apoptosis. In this review, stretch induced cell proliferation, apoptosis, the roles of growth factors, stretch induced ion channels, and roles of second messengers are organized. It is expected that readers from any background can understand and make a decision about tissue expansion.
... The inherent local liberation of calcium may also be of interest for creation of local calcium gradients around cells immobilized near the scaffolds surface, as calcium is thought to support endothelial structure reformation. Calcium ions contribute to the maintenance of endothelial cells and also the formation of vascularized tissue [22]. Calcium ion concentration has been shown to affect proliferation of adult rat hepatocytes directly in a tight compositional range [23]; their highest proliferation rates in vitro were observed at physiological concentrations of 0.4 mM while lower or higher concentrations resulted in lower proliferation rates. ...
Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.
... Alternatively, because protons can pass through some membranes they can deliver the acidic core by diffusion rather than by direct fusion with the cells [32]. The result is a marked increase in the local acidity at the sites where FA vesicles attach to the endothelium, which disrupts many important ion exchange processes, namely trafficking of Ca 2+ , K + , and Na + [33]. These ions control vital cell processes, and namely calcium ions control contractibility of smooth muscle in the arteries [6], and also of the myocardium as it is known that this muscle is also controlled by the endocardium [34]. ...
The interpretative framework of cardiovascular diseases presented here provides a rationale for many well-known features of cardiovascular diseases. Prolonged acidemia with high blood levels of free fatty acids is proposed to shape the basic context for formation of fatty acid micelles and vesicles with an acidic core that fuse with the endothelia, disrupt vital cell processes, and initiate atherosclerotic plaque formation. It offers an explanation for the distributed localization of atherosclerotic lesions, and how mild cases of occurrence of fatty acids vesicles formed within the heart and the arteries close to the heart may cause such lesions. It provides a rationale for how acute events, namely heart attacks and strokes, may arise from stormy development of fatty acid vesicles within the heart. Additionally, a process is proposed for clot development from the existing fatty acid vesicles.
... CaCC action is not limited to Cl − secretion in epithelia. CaCC activity was found in many excitable tissues such as smooth muscles, cardiac muscles, olfactory sensory neurons, and somatosensory neurons, too [6, 27, 53, 54, 60, 75, 76, 120] . CaCCs are activated by intracellular Ca 2+ exhibiting an outwardly rectifying currentvoltage relationship at relatively low Ca 2+ [57, 58]. ...
Ca2+-activated Cl− channels (CaCCs) are a class of Cl− channels activated by intracellular Ca2+ that are known to mediate numerous physiological functions. In 2008, the molecular identity of CaCCs was found to be anoctamin 1 (ANO1/TMEM16A). Its roles have been studied in electrophysiological, histological, and genetic aspects. ANO1 is known to mediate Cl− secretion in secretory epithelia such as airways, salivary glands, intestines, renal tubules, and sweat glands. ANO1 is a heat sensor activated by noxious heat in somatosensory neurons and mediates acute pain sensation as well as chronic pain. ANO1 is also observed in vascular as well as airway smooth muscles, controlling vascular tone as well as airway hypersensitivity. ANO1 is upregulated in numerous types of cancers and thus thought to be involved in tumorigenesis. ANO1 is also found in proliferating cells. In addition to ANO1, involvement of its paralogs in pathophysiological conditions was also reported. ANO2 is involved in olfaction, whereas ANO6 works as a scramblase whose mutation causes a rare bleeding disorder, the Scott syndrome. ANO5 is associated with muscle and bone diseases. Recently, an X-ray crystal structure of a fungal TMEM16 was reported, which explains a precise molecular gating mechanism as well as ion conduction or phospholipid transport across the plasma membrane.
... Several studies show that endothelium-dependent vasodilatation is sensitive to inhibition of the small and intermediate-conductance calcium-activated potassium channels SK Ca and IK Ca (Potocnik et al., 2009;Brahler et al., 2009;Parkington et al., 2002;Dora et al., 2008;Dora et al., 2000;Brandes et al., 2000;Takamura et al., 1999;Shimokawa et al., 1996). Thus, an increase in EC intracellular Ca 2+ concentration ([Ca 2+ ] i ) to shear stress (Nilius & Droogmans, 2001) may activate SK Ca and IK Ca channels leading to a hyperpolarization of VSMCs (endothelium-dependent hyperpolarization, EDH) and vasodilatation (Si et al., 2006;Miura et al., 2001;Brahler et al., 2009). To this end, it has been suggested that the EDH mechanism plays a more prominent role in small resistance vessels compared to the NO-mediated pathway (Scotland et al., 2005;Brahler et al., 2009;Brandes et al., 2000;Shimokawa et al., 1996). ...
Key points:
Blood pressure and flow exert mechanical forces on the walls of small arteries, which are detected by the endothelial and smooth muscle cells, and lead to regulation of the diameter (basal tone) of an artery. CaV 3.2 T-type calcium channels are expressed in the wall of small arteries, although their function remains poorly understood because of the low specificity of T-type blockers. We used mice deficient in CaV 3.2 channels to study their role in pressure- and flow-dependent tone regulation and the possible impact of ageing on this role. In young mice, CaV 3.2 channels oppose pressure-induced vasoconstriction and participate in endothelium-dependent, flow-mediated dilatation. These effects were not seen in mature adult mice. The results of the present study demonstrate an age-dependent impact of CaV 3.2 T-type calcium channel deletion in rodents and suggest that the loss of CaV 3.2 channel function leads to more constricted arteries, which is a risk factor for cardiovascular disease.
Abstract:
The myogenic response and flow-mediated vasodilatation are important regulators of local blood perfusion and total peripheral resistance, and are known to entail a calcium influx into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), respectively. CaV 3.2 T-type calcium channels are expressed in both VSMCs and ECs of small arteries. The T-type channels are important drug targets but, as a result of the lack of specific antagonists, our understanding of the role of CaV 3.2 channels in vasomotor tone at various ages is scarce. We evaluated the myogenic response, flow-mediated vasodilatation, structural remodelling and mRNA + protein expression in small mesenteric arteries from CaV 3.2 knockout (CaV 3.2KO) vs. wild-type mice at a young vs. mature adult age. In young mice only, deletion of CaV 3.2 led to an enhanced myogenic response and a ∼50% reduction of flow-mediated vasodilatation. Ni(2+) had both CaV 3.2-dependent and independent effects. No changes in mRNA expression of several important K(+) and Ca(2+) channel genes were induced by CaV 3.2KO However, the expression of the other T-type channel isoform (CaV 3.1) was reduced at the mRNA and protein level in mature adult compared to young wild-type arteries. The results of the present study demonstrate the important roles of the CaV 3.2 T-type calcium channels in myogenic tone and flow-mediated vasodilatation that disappear with ageing. Because increased arterial tone is a risk factor for cardiovascular disease, we conclude that CaV 3.2 channels, by modulating pressure- and flow-mediated vasomotor responses to prevent excess arterial tone, protect against cardiovascular disease.
Biological tissues are fed by arterial networks whose task is to set blood flow delivery in accordance with energetic demand. Coordinating vasomotor activity among hundreds of neighboring segments is an essential process, one dependent upon electrical information spreading among smooth muscle and endothelial cells. The "Conducted Vasomotor Response" is a functional expression of electrical spread and it's this process that lies at the heart of this critical review. Written in a narrative format, this review will first highlight historical manuscripts and then characterize the conducted response across a range of preparations. Trends will be highlighted and used to guide subsequent sections, focused on cellular foundations, biophysical underpinnings, and regulation in health and disease. Key information has been tabulated in table format; illustrative figures reinforce grounding concepts and reveal a framework within which theoretical and experimental work can be rationalized. This summative review highlights that despite thirty years of concerted experimentation, key aspects of the conducted response remain ill-defined. Of note is the need to rationalize the regulation and deterioration of conduction in pathobiological settings. New quantitative tools, along with transgenic technology, will be discussed as a means of propelling this investigative field forward.
In 1934, the discovery of angiotensin II opened an Eldorado in the field of the cardiovascular system. Until today, this Eldorado is still evolving due to the implication of this octapeptide, not only in normal physiology but also in its effect on the remodeling of the cardiovascular system. The intense scientific research led to the discovery of components that regulates the conversion of Angiotensin I to angiotensin II including angiotensin II converting enzyme and chymase-dependent production of angiotensin II. One important advancement is discovering an inhibitor of the angiotensin II converting enzyme, which is the most clinically used antihypertensive drug. Identifying the receptors of angiotensin II, AT1, and AT2, also led to determining the signaling pathways of these two receptors and their contribution to the regulation of the cardiovascular system in health and disease. This made it possible to develop two specific AT1 and AT2 receptor antagonists. Its is not until recently that the AT1 receptor antagonist, losartan, is used as an antihypertensive drug. The role of the AT2 receptor in the angiotensin II effect is still a matter of debate. These two receptors were also found to be localized at the nuclear envelope membranes, and the normal crosstalk between the plasma and the nuclear envelope membranes angiotensin II receptors seems to be an important factor in the angiotensin II effect. Remodeling this crosstalk may contribute to the angiotensin II effect in cardiovascular diseases. (Dedicated to Prof. Domenico Regoli, a pioneer in the pharmacology of the renin angiotensin system).KeywordsAngiotensin IIAT1 receptorAT2 receptorAngiotensin II signalingNuclear AT1And AT2 receptorsCalciumCardiomyocytesVascular endotheliumVascular smooth muscleEndocardial endothelial cells
Учебное пособие по современным проблемам биохимии для студентов биологического факультета включает теоретические основы предмета, лабораторные работы, контрольные вопросы и современные источники литературы. Учебное пособие составлено из трудов специалистов-биохимиков Республики Беларусь, института биологической и медицинской химии им. В. Н. Ореховича, ММА им. И. М. Сеченова (г. Москва) и призвано оказать помощь студентам биологических специальностей в изучении современных проблем биохимии в рамках специализации "Биохими".
The humoral pathway mediating the cardioprotective effect of remote ischemic preconditioning has been demonstrated in various models: ischemia-reperfusion of the fore- and hindlimbs and liver and occlusion-reperfusion of the mesenteric and renal arteries. This review assesses humoral components in the formation and realization of the cardioprotective actions of remote ischemic preconditioning of the heart. Endogenous agonists of opioid receptors, arachidonic acid derivatives (prostanoids), agonists of cannabinoid and vanilloid receptors, calcitonin gene-related peptide, leukotrienes, and microRNA are all regarded as humoral components. Adenosine, which also has a role in mediating the cardioprotective effects of remote ischemic preconditioning, is regarded as a mediator between the humoral factor and cardiomyocytes. Knowledge of the role of humoral factors in mediating cardioprotection may be useful for developing methods and therapeutic agents to increase the resistance of the myocardium to ischemic-reperfusion damage.
Comorbidities are a hallmark of stroke that both increase the incidence of stroke and worsen outcome. Hypertension is prevalent in the stroke population and the most important modifiable risk factor for stroke. Hypertensive disorders promote stroke through increased shear stress, endothelial dysfunction, and large artery stiffness that transmits pulsatile flow to the cerebral microcirculation. Hypertension also promotes cerebral small vessel disease through several mechanisms, including hypoperfusion, diminished autoregulatory capacity and localized increase in blood–brain barrier permeability. Preeclampsia, a hypertensive disorder of pregnancy, also increases the risk of stroke 4–5-fold compared to normal pregnancy that predisposes women to early-onset cognitive impairment. In this review, we highlight how comorbidities and concomitant disorders are not only risk factors for ischemic stroke, but alter the response to acute ischemia. We focus on hypertension as a comorbidity and its effects on the cerebral circulation that alters the pathophysiology of ischemic stroke and should be considered in guiding future therapeutic strategies.
Rat brown fat cells respond to the neurotransmitter norepinephrine in vivo through a number of signaling pathways including changes in intracellular calcium. This dissertation describes a previously unknown effect of β-adrenergic stimulation on release of calcium from endoplasmic reticulum (ER) stores. Namely, that β-adrenergic stimulation potentiates such release initiated by other receptor types such as α-adrenoceptors or P2 purinergic receptors. This finding suggests that norepinephrine elicits brown fat calcium signals in vivo through activation of both α1- and β-adrenoceptors. Simultaneous to ER calcium release, norepinephrine activates a calcium influx mechanism that contributes to intracellular calcium increases. Some have proposed that this influx is secondary to depletion of ER calcium stores – the “capacitative” calcium entry model. Pursuing this model, I have identified a store-operated calcium current (ISOC) in brown adipocytes activated by the fungal toxin cyclopiazonic acid. This current shares characteristics with the store-operated current ICRAC that has been described in other cells. The shared properties include inward rectification, voltage-dependent inactivation, blockage by lanthanides, and an apparent cation permeability sequence of Ca2+ > Na+ ≥ Cs+ based on membrane current magnitudes. Experiments characterizing ISOC also revealed two other cation currents that have not been previously described in brown fat. One current is regulated by intracellular MgATP and has characteristics nearly identical to a cation current known variously as IMIC or MagNuM and believed to be mediated by the TRPM7 gene product. The other current is a background cation conductance that is inhibited by increases in extracellular calcium, and enhanced by removal of extracellular divalents.
Two other accompanying studies are previously published reports associated with my work as a trainee on the NSF Training Grant “Nonlinear Dynamics in Biology” through the UC Davis Institute of Theoretical Dynamics. The first is an experimental project describing a chloride current in endothelial cells activated by mechanical shear stress (flow). The second project is a methodological review of a particular class of spatially explicit mathematical models known as “non-local” models. This usefulness of this theoretical approach is illustrated with problems from both ecology and cell biology.
Hyperglycaemia is a key factor that contributes to the development of diabetes-related microvascular disease and a major risk factor for endothelial dysfunction. In the current study, we have explored glucose-induced abnormal intracellular calcium (Ca 2+ i) homeostasis in mouse microvessel endothelial cells (MMECs) in high glucose (HG) (40mmol/L) versus control (low glucose, LG) (11 mmol/L). We demonstrated that the exposure of MMECs to HG for 3 days did not change basal Ca 2+ i , however, there was a significant increase of acetylcholine-induced Ca 2+ entry. Western blots illustrated that exposure to HG also increased STIM1 (Stromal Interaction Molecule 1), but not Orai1 (the pore forming subunit), protein expression levels. Although the link between HG-induced changes in STIM1 expression, enhanced Ca 2+ entry and endothelial dysfunction requires further study, the current data are suggestive that targeting these pathways may reduce the impact of HG on endothelial function.
Objective:
Electrical signaling along the endothelium underlies spreading vasodilation and blood flow control. We use mathematical modeling to determine the electrical properties of the endothelium and gain insight into the biophysical determinants of electrical conduction.
Methods:
Electrical conduction data along endothelial cell (EC) tubes (40 μm wide, 2.5 mm long) isolated from mouse skeletal muscle resistance arteries were analyzed using cable equations and a multicellular computational model.
Results:
Responses to intracellular current injection attenuate with an axial length constant (λ) of 1.2-1.4 mm. Data were fitted to estimate the axial (ra ; 10.7 MΩ/mm) and membrane (rm ; 14.5 MΩ∙mm) resistivities, EC membrane resistance (Rm ; 12 GΩ), EC-EC coupling resistance (Rgj ; 4.5 MΩ) and predict that stimulation of ≥30 neighboring ECs is required to elicit 1 mV of hyperpolarization at distance = 2.5 mm. Opening Ca(2+) -activated K(+) channels (KCa ) along the endothelium reduced λ by up to 55%.
Conclusions:
High Rm makes the endothelium sensitive to electrical stimuli and able to conduct these signals effectively. Whereas the activation of a group of ECs is required to initiate physiologically relevant hyperpolarization, this requirement is increased by myoendothelial coupling and KCa activation along the endothelium inhibits conduction by dissipating electrical signals. This article is protected by copyright. All rights reserved.
Previous results link the mitochondrial potassium channel Kv1.3 (mitoKv1.3) to the regulation of apoptosis. By synthesizing new, mitochondria-targeted derivatives (PAPTP and PCARBTP) of PAP-1, a specific membrane-permeant Kv1.3 inhibitor, we have recently provided evidence that both drugs acting on mitoKv1.3 are able to induce apoptosis and reduce tumor growth in vivo without affecting healthy tissues and cells. In the present article, by exploiting these new drugs, we addressed the question whether mitoKv1.3 contributes to the regulation of cell proliferation as well. When used at low concentrations, which do not compromise cell survival, both drugs slightly increased the percentage of cells in S phase while decreased the population at G0/G1 stage of cells from two different pancreatic ductal adenocarcinoma lines. Our data suggest that the observed modulation is related to ROS levels within the cells, opening the way to link mitochondrial ion channel function to downstream, ROS-related signaling events that might be important for cell cycle progression.
The Epithelial Sodium Channel (ENaC) is a key player in renal sodium homeostasis. The expression of α β γ ENaC subunits has also been described in the endothelium and vascular smooth muscle, suggesting a role in vascular function. We recently demonstrated that endothelial ENaC is involved in aldosterone-modulated endothelial stiffness. Here we explore the functional role of the endothelial αENaC subunit in vascular function in vivo. Compared to littermates, mice with conditional αENaC subunit gene inactivation in the endothelium only (endo-αENaC Knock Out mice) had no difference in their physiological parameters such as systolic blood pressure or heart rate. Acute and long-term renal Na⁺ handlings were not affected, indicating that endothelial αENaC subunit is not involved in renal sodium balance. Pharmacological inhibition of ENaC with benzamil blunted acetylcholine-induced nitric oxide production in mesenteric arteries from wild type mice but not in endo-αENaC KO mice, suggesting a critical role of endothelial ENaC in agonist-induced nitric oxide production. In endo-αENaC KO mice, compensatory mechanisms occurred and steady state vascular function was not altered except for flow-mediated dilation. Our data suggest that endothelial αENaC contributes to vascular endothelial function in vivo.
Store‑operated Ca2+ entry (SOCE) via store‑operated Ca2+ channels (SOCC), encoded by transient receptor potential canonical (TRPC) channel proteins, is an important underlying mechanism regulating intracellular Ca2+ concentration ([Ca2+]i) and various intracellular functions in endothelial cells (ECs). TRPC1, the probable candidate for SOCC, is expressed in ECs. Ca2+‑sensing receptor (CaSR) is functionally expressed in vascular endothelium and is important in Ca2+ mobilization and cardiovascular functions. To date, there have been no reports demonstrating an association between CaSR and TRPC1 in ECs. The present study investigated the effects of TRPC1 on CaSR‑induced Ca2+ influx and nitric oxide (NO) production in human umbilical vein ECs (HUVECs). TRPC1 and CaSR proteins in HUVECs were measured by immunostaining and western blot analysis. [Ca2+]i levels were measured using the Fura‑2‑acetoxymethyl ester method. The indicator 3‑amino, 4‑aminomethyl‑2, 7‑difluorescein diacetate was used to measure NO production in HUVECs. The expression of TRPC1 protein in HUVECs was silenced by transfecting HUVECs with small interfering RNA (siRNA) against TRPC1. Although changes in extracellular Ca2+ failed to alter [Ca2+]i in HUVECs, the CaSR agonist spermine increased [Ca2+]i and NO production in HUVECs. NO production in HUVECs was diminished in Ca2+‑free medium or following treatment with a CaSR negative allosteric modulator (Calhex231), SOCC inhibitor (MRS1845) or TRPC inhibitor (SKF96365). The spermine‑induced increases in [Ca2+]i and NO production were reduced in HUVECs transfected with TRPC1 siRNA. These results suggested that TRPC1 is a primary candidate in forming SOCC that stimulates CaSR‑induced SOCE and NO production in HUVECs and is a potential therapeutic target for vascular diseases.
Vascular endothelium constitutes the interface between the flowing blood and the deformable solid wall. The endothelium is a thin layer of connected and anchorage-dependent cells that are subjected to chemical, physical, and mechanical stimuli. They are directly exposed to molecules that circulate in the blood stream.
Endothelial cells express a diverse array of ion channels including members of the strong inward rectifier family composed of KIR2 subunits. These two-membrane-spanning-domain channels are modulated by their lipid environment, and exist in macromolecular signaling complexes with receptors, protein kinases and other ion channels. Inward rectifier K+ channels (KIR) currents display a region of negative slope conductance at membrane potentials positive to the K+ equilibrium potential that allows outward current through the channels to be activated by membrane hyperpolarization, permitting KIR to amplify hyperpolarization induced by other K+ channels and ion transporters. Increases in extracellular K+ concentration activate KIR allowing them to sense extracellular K+ concentration and transduce this change into membrane hyperpolarization. These properties position KIR to participate in the mechanism of action of hyperpolarizing vasodilators and contribute to cell-cell conduction of hyperpolarization along the wall of microvessels. Expression of KIR in capillaries in electrically active tissues may allow KIR to sense extracellular K+, contributing to functional hyperemia. Understanding the regulation of expression and function of microvascular endothelial KIR will improve our understanding of the control of blood flow in the microcirculation in health and disease and may provide new targets for development of therapeutics in the future. This article is protected by copyright. All rights reserved.
Previous studies have documented that rapid juvenile growth is accompanied by functional changes in the arteriolar endothelium, but much less is known about functional changes in arteriolar smooth muscle over this period. In this study, we investigated the possible contribution of epithelial sodium channels (ENaC) to the myogenic behavior of arterioles at two stages of juvenile growth. The effects of the ENaC inhibitor benzamil on different levels of myogenic tone were studied in isolated gracilis muscle arterioles from rats aged 21-28 days (“weanlings”) and 42-49 days (“juveniles”). ENaC subunit expression in the arteriolar wall was also determined, and the interaction between ENaC and nitric oxide (NO) in regulating vascular tone was explored by combined use of benzamil and NG-monomethyl-L-arginine (L-NMMA). At physiological pressures, both steady-state myogenic tone and the dynamic adjustments in this tone triggered by acute pressure changes were less in juvenile arterioles than in weanling arterioles. α, β and γ ENaC protein was present in arterioles at both ages, but benzamil only had an effect on myogenic tone in weanling arterioles. In these vessels, benzamil increased, rather than decreased, myogenic tone, and this effect was prevented by L-NMMA or endothelial removal. These findings suggest that although ENaC is present in gracilis muscle arterioles of both weanling and juvenile rats, it is not obligatory for the genesis of myogenic activity in these vessels at either age. However, ENaC activity can significantly modulate the level of myogenic tone through stimulation of endothelial NO release at an early stage of growth. This article is protected by copyright. All rights reserved.
Ion channels importantly contribute to the function of endothelial cells. They serve as the major source of intracellular Ca2+, which, in turn, controls the production of endothelium-derived vasodilators, the permeability of the endothelium, gene expression, and other properties of endothelial cells. In addition, the activity of ion channels determines the membrane potential of endothelial cells that serves as an important signal for cell-cell communication between endothelial cells and between endothelial cells and overlying smooth muscle cells, and may feed-back to regulate the activity of the ion channels themselves. This review provides an overview of the expression and function of endothelial ion channels that contribute to Ca2+ and membrane potential signaling that is involved in the regulation and modulation of vasomotor tone of resistance arteries and arterioles. Channels discussed include inositol 1,4,5 trisphosphate receptors that mediate agonist-induced Ca2+ release from endoplasmic reticulum stores; members of the transient receptor potential family and other channels that mediate agonist-induced Ca2+ influx through the plasma membrane; Ca2+-activated K+ channels that mediate agonist-induced membrane hyperpolarization; and inward rectifier K+ channels that serve as sensors for changes in extracellular K+ and amplifiers of hyperpolarization induced by the activity of other ion channels. It is emphasized that all of these channels exist as members of macromolecular signaling complexes providing a rich environment for regulation of their activity and the function of endothelial cells in resistance arteries and arterioles.
Portal hypertension is associated with a chronic hyperkinetic syndrome1–3. This syndrome is characterized by elevated cardiac output, low arterial pressure and low systemic vascular resistance2, 3. Splanchnic circulation is also hyperdynamic; i.e. blood flow is elevated and vascular resistance is low in arteries that supply splanchnic organs1, 4. Systemic and splanchnic alterations are interrelated: decreased systemic vascular resistance (systemic vasodilation) is largely due to the decrease in splanchnic arterial resistance (splanchnic vasodilation)5. Finally, in portal hypertension, there is in-vivo and ex-vivo arterial hypo reactivity to different receptor-dependent and -independent vasoconstrictors6–14. A hyperkinetic syndrome also occurs in extrahepatic portal hypertension15, but it is less marked than that observed in cirrhosis.
The vascular endothelium performs a number of its functions through the release of several physiologically active mediators (Furchgott and Vanhoutte, 1989). These include nitric oxide (NO) and prostaglandin I2 (PGI2), which are involved in the regulation of vascular tone and the inhibition of platelet activity. The generation and release of both of these mediators is influenced by the cytosolic calcium concentration ([Ca2+]i
) in the endothelial cells (Moncada et al., 1991; Hallam et al., 1988). Thus, endothelium-dependent vasodilators act, at least in part, through the modulation of endothelial [Ca2+]i
. The [Ca2+]i
- also influences endothelial barrier properties (Rotrosen and Gallin, 1986).
Vascular endothelium appears to be a unique organ that not only responds to numerous hormonal and chemical signals but also senses changes in physical parameters, such as changes in blood flow through shear stress and changes in blood pressure through stretch. The endothelium integrates these signals and responds to them by regulating the production and release of vasoactive substances that play a role in blood pressure regulation and vascular growth. These regulatory substances include prostaglandins, endothelium-derived relaxing factor (EDRF or NO) and endotheliumderived hyperpolarizing factor (EDHF), endothelin, natriuretic peptides, small signaling molecules, such as substance P, ATP, growth factors, steroids, and even larger proteins, such as receptors and proteins involved in the blood clotting cascade (for reviews, see Inagami et al., 1995; Nilius and Casteels, 1996). In addition to this endocrine function, endothelial cells either prevent or trigger blood clotting in response to various signals and exert thrombolytic as well as thrombogenic activity. As antigen-presenting cells, they are also involved in immune responses. Their ability to modulate cell-cell contacts controls the permeability of the blood-tissue interface. Finally, they initiate angiogenesis and vessel repair.
Although acute hypoxic pulmonary vasoconstriction (HPV; see Table 1) has been studied intensively for over 50 years, its mechanisms remain unclear. Understanding these mechanisms is important because HPV plays significant roles in both normal and diseased lungs. In normal lungs, HPV maximizes systemic arterial oxygen tension by diverting pulmonary blood flow from poorly ventilated hypoxic lung regions to well ventilated normoxic lung regions. In diseased lungs, where hypoxia is diffuse, HPV occurs throughout the pulmonary vasculature, resulting in pulmonary hypertension, right ventricular failure, and increased morbidity and mortality.
Despite the surgical and other insertional interventions, the complete recuperation of myocardial disorders is still elusive due to the insufficiency of functioning myocardiocytes. Thus, the use of stem cells to regenerate the affected region of heart becomes a prime important. In line with this human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have gained considerable interest due to their potential use for mesodermal cell based replacement therapy and tissue engineering. Since MSCs are harvested from various organs and anatomical locations of same organism, thus the cardiac regenerative potential of human cardiac-derived MSCs (hC-MSCs) and human umbilical cord Wharton’s Jelly derived MSC (hUC-MSCs) were tested concurrently. At in vitro culture, both hUC-MSCs and hC-MSCs assumed spindle shape morphology with expression of typical MSC markers namely CD105, CD73, CD90 and CD44. Although, hUC-MSCs and hC-MSCs are identical in term of morphology and immunophenotype, yet hUC-MSCs harbored a higher cell growth as compared to the hC-MSCs. The inherent cardiac regenerative potential of both cells were further investigated with mRNA expression of ion channels. The RT-PCR results demonstrated that both MSCs were expressing a notable level of delayed rectifier-like K+ current (I
KDR
) ion channel, yet the relative expression level was considerably varied between hUC-MSCs and hC-MSCs that Kv1.1(39 ± 0.6 vs 31 ± 0.8), Kv2.1 (6 ± 0.2 vs 21 ± 0.12), Kv1.5 (7.4 ± 0.1 vs 6.8 ± 0.06) and Kv7.3 (27 ± 0.8 vs 13.8 ± 0.6). Similarly, the Ca2+-activated K+ current (I
KCa
) channel encoding gene, transient outward K+ current (I
to
) and TTX-sensitive transient inward sodium current (I
Na.TTX
) encoding gene (Kv4.2, Kv4.3 and hNE-Na) expressions were detected in both groups as well. Despite the morphological and phenotypical similarity, the present study also confirms the existence of multiple functional ion channel currents IKDR, IKCa, Ito, and INa.TTX in undifferentiated hUC-MSCs as of hC-MSCs. Thus, the hUC-MSCs can be exploited as a potential candidate for future cardiac regeneration.
Potassium channels play an important role in the regulation of the membrane potential (E
m
) of endothelial cells and thereby modulate the entry of extracellular Ca2+ (Adams, 1994; Himmel et al., 1993; Adams et al., 1989). Ca2+ entry in concert with intracellular Ca2+ release is important for the synthesis of a number of endothelium-derived vasoactive factors. Thus, the synthesis of the endothelium-derived relaxing factor (EDRF), nitric oxide (NO), and of prostacyclin (PGI2) requires, respectively, the Ca2+-calmodulin-dependent activation of the constitutive endothelial cell nitric oxide synthase (eNOS) and the Ca2+ -dependent activation of phospholipase A2 (Pollock et al., 1991; Bredt and Snyder, 1990; Carter et al., 1988; Hallam et al., 1988). Similarly, the synthesis of the vasoconstrictor peptide endothelin-1 (ET-1) requires the mobilization of intracellular Ca2+ and the activation of protein kinase C (Yanagisawa et al., 1989).
The distributions of connexin 43 (Cx43) and connexin 40 (Cx40) in smooth muscle and endothelium of resistance vessels were examined using indirect immunofluorescence techniques coupled with confocal microscopy. Cx43 and Cx40 were found in smooth muscle and endothelium. Similar staining patterns were found in microvessel samples from brain and cremaster of the rat and from arterioles of the hamster cheek pouch. Double-labeling studies showed a high degree of colocalization of Cx40 with Cx43, suggesting the presence of multiple connexins within a single junctional plaque. Quantitative comparisons were made of the fluorescent patterns in the endothelium and smooth muscle of rat brain arterioles. Cx43 and Cx40 plaque diameters were 0.9 +/- 0.1 and 0.8 +/- 0.1 (SE) microns, respectively, in the endothelial layer and 0.5 +/- 0.1 and 0.5 +/- 0.1 microns, respectively, in the smooth muscle. There was no difference between mean plaque diameters of Cx43 and Cx40 in endothelium or smooth muscle. However, plaques were significantly larger in endothelium than in smooth muscle (P < 0.05). These findings demonstrate the potential for cell-cell communication in both cell types of the wall of arterioles from three different tissues. The data also suggest a greater level of coupling within the endothelium.
The purpose of the present study was to determine the mechanism by which bradykinin activates the small conductance, inwardly rectifying, Ca(2+)-activated K+ channel (KCa) found in cultured bovine aortic endothelial cells. Channel activity was studied using the patch-clamp technique in whole-cell, cell-attached, inside-out and outside-out configurations. Channel conductance at potentials positive to 0 mV was 10 +/- 2 pS and at potentials negative to 0 mV 30 +/- 3 pS (n = 7) when examined in symmetrical K+ (150 mmol/l) solutions. The channel open probability (P(o)) was only weakly voltage dependent changing approximately 0.2 units over 160 mV. In contrast, raising the intracellular Ca2+ concentration from 100 nmol/l to 10 mumol/l at -60 mV produced a graded increase in channel P(o) from 0.15 to 0.96; the concentration required for half-maximum response (apparent K0.5) was 719 nmol/l. At a constant Ca2+ concentration, application of guanosine triphosphate (GTP) to the cytoplasmic surface of the patch increased channel P(o). This effect was dependent upon the simultaneous presence of both GTP and Mg2+, and was reversed by the subsequent application of the guanosine diphosphate (GDP) analogue, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). The hydrolysis-resistant GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), induced a long-lasting increase in channel P(o). In the presence of Mg(2+)-GTP, the apparent K0.5 for Ca2+ decreased from a control value of 722 nmol/l to 231 nmol/l. Addition of bradykinin to outside-out patches previously exposed to intracellular Mg(2+)-GTP further enhanced KCa activity, shifting the apparent K0.5 for Ca2+ from 228 nmol/l to 107 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)
Receptor-mediated increases in the cytosolic free calcium ion concentration in most mammalian cells result from mobilization of Ca2+ from intracellular stores as well as transmembrane Ca2+ influx. Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores by opening a Ca(2+)-permeable channel in the endoplasmic reticulum. But the mechanism and regulation of Ca2+ entry into nonexcitable cells has remained elusive because the entry pathway has not been defined. Here we characterize a novel inositol 1,3,4,5-tetrakisphosphate (InsP4) and Ca(2+)-sensitive Ca(2+)-permeable channel in endothelial cells. We find that InsP4, which induces Ca2+ influx into acinar cells, enhances the activity of the Ca(2+)-permeable channel when exposed to the intracellular surface of endothelial cell inside-out patches. Our results suggest a molecular mechanism which is likely to be important for receptor-mediated Ca2+ entry.
Chloride channels have several functions, including the regulation of cell volume, stabilizing membrane potential, signal transduction and transepithelial transport. The plasma membrane Cl- channels already cloned belong to different structural classes: ligand-gated channels, voltage-gated channels, and possibly transporters of the ATP-binding-cassette type (if the cystic fibrosis transmembrane regulator is a Cl- channel). The importance of chloride channels is illustrated by the phenotypes that can result from their malfunction: cystic fibrosis, in which transepithelial transport is impaired, and myotonia, in which ClC-1, the principal skeletal muscle Cl- channel, is defective. Here we report the properties of ClC-2, a new member of the voltage-gated Cl- channel family. Its sequence is approximately 50% identical to either the Torpedo electroplax Cl- channel, ClC-0 (ref. 8), or the rat muscle Cl- channel, ClC-1 (ref. 9). Isolated initially from rat heart and brain, it is also expressed in pancreas, lung and liver, for example, and in pure cell lines of fibroblastic, neuronal, and epithelial origin, including tissues and cells affected by cystic fibrosis. Expression in Xenopus oocytes induces Cl- currents that activate slowly upon hyperpolarization and display a linear instantaneous current-voltage relationship. The conductivity sequence is Cl- greater than or equal to Br- greater than I-. The presence of ClC-2 in such different cell types contrasts with the highly specialized expression of ClC-1 (ref. 9) and also with the cloned cation channels, and suggests that its function is important for most cells.
Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.
Previous studies in non-excitable cells have suggested that depletion of internal Ca2+ stores activates Ca2+ influx from the extracellular space via a mechanism that does not require stimulation of phosphoinositide hydrolysis. To test this hypothesis in vascular endothelial cells, the effect of the Ca(2+)-ATPase/pump inhibitor 2,5-di-t-butylhydroquinone (BHQ) on cytosolic free Ca2+ concentration ([Ca2+]i) was examined. BHQ produced a dose-dependent increase in [Ca2+]i, which remained elevated over basal values for several minutes and was substantially inhibited in the absence of extracellular Ca2+. Application of bradykinin after BHQ demonstrated that the BHQ-sensitive compartment partially overlapped the bradykinin-sensitive store. Similar results were obtained with thapsigargin and cyclopiazonic acid, two other Ca(2+)-ATPase inhibitors. Although BHQ had no effect on phosphoinositide hydrolysis, both 45Ca2+ influx and efflux were stimulated by this agent. These results suggest that depletion of the agonist-sensitive Ca2+ store is sufficient for activation of Ca2+ influx. Several characteristics of the Ca(2+)-influx pathway activated by internal store depletion were compared with those of the agonist-activated pathway. Bradykinin-stimulated Ca2+ influx was increased at alkaline extracellular pH (pHo), and was inhibited by extracellular La3+, by depolarization of the membrane, and by the novel Ca(2+)-influx blocker 1-(beta-[3-(4-methoxyphenyl)propoxy]-4- methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365). Additionally, bradykinin stimulated influx of both 45Ca2+ and 133Ba2+, consistent with the hypothesis that the agonist-activated influx pathway is permeable to both of these bivalent cations. Likewise, activation of Ca2+ influx by BHQ, thapsigargin and cyclopiazonic acid was blocked by La3+, membrane depolarization and SKF 96365, but was unaffected by nitrendipine or BAY K 8644. Furthermore, Ca2+ influx stimulated by BHQ was increased at alkaline pHo and BHQ stimulated the influx of both 45Ca2+ and 133Ba2+ to the same extent. These results demonstrate that the agonist-activated Ca(2+)-influx pathway and the pathway activated by depletion of the agonist-sensitive internal Ca2+ store are indistinguishable.
Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.
Regulation of cell volume is essential for every cell and is accomplished by the regulated loss or gain of intracellular ions or other osmolytes. Regulatory volume decrease often involves the parallel activation of potassium and chloride channels. Overexpression of P-glycoprotein leads to volume-activated Cl- currents but its physiological importance for volume regulation is unclear. CIC-2 is a ubiquitously expressed Cl- channel activatable by non-physiologically strong hyperpolarization. We now show that CIC-2 can be activated by extracellular hypotonicity, which suggests that it has a widespread role in volume regulation. Domains necessary for activation by both voltage and volume are localized to the amino terminus. Mutations in an 'essential' region lead to constitutively open channels unresponsive to medium tonicity, whereas deletions in a 'modulating' region produce partially opened channels responsive to both hypo- and hypertonicity. These domains can be transplanted to different regions of the protein without loss of function.
Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.
Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.
The vascular wall must be one of the largest “organs” in the body with complex interactions in terms of signalling between various cell types. The interaction between the different cells is either by humoral factors or by direct cell-to-cell coupling; moreover, signal transduction can be integrated at the surface of the different cell types involved. A great variety of completely different functions are coordinated by these cell interactions, such as control of vasomotor activity, cell proliferation, Chemotaxis, immunological responses, and control of thrombogenic and thrombolytic activity and of the coagulation potential in the blood [2,12,25,54,62]. Communication between different cells is essential for the normal biology of the vessel wall, because the vascular system has to respond immediately to changed functional demands and priorities.
Endothelial cells form and secrete mediators that modulate tone of underlying smooth muscle. The signal for their release or synthesis is increased intracellular Ca activity resulting from intracellular Ca release and transmembrane Ca fluxes. Ca influx can occur via different Ca-permeable ion channels, including Ca-activated K channels.
Endothelial cells serve both autocrine and paracrine functions within the cardiovascular system to modulate blood pressure and maintain tissue perfusion. Endothelial cells respond to a variety of humoral and physical stimuli to release endothelium-dependent vasodilators, such as prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF), and vasoconstrictors such as endothelin (Furchgott and Vanhoutte, 1989). The synthesis and release of endothelium-derived vasodilators have been shown to be Ca2+-dependent whereby the production of PGI2 and EDRF is attenuated by the removal of extracellular Ca2+ (Singer and Peach, 1982; Long and Stone, 1985; Griffith et al., 1986; Lückhoff et al., 1988). It is now well established that an EDRF released from endothelial cells, both in situ and in culture, is nitric oxide (NO) or a nitroso compound that readily releases NO, and that NO is synthesized in endothelial cells by the oxidation of one of the two equivalent guanidino nitrogens of L-arginine via a cytoplasmic NADPH-and Ca2+-dependent enzyme, termed NO synthase (see Moncada et al., 1991). Changes in the extracellular Ca2+ concentration around the physiological range have been shown to modulate the synthesis/release of NO by the vascular endothelium and consequently, vascular tone (Lopez-Jaramillo et al., 1990).
Inward rectifier (IR) K+ channels of bovine pulmonary artery endothelial cells were studied using the whole-cell, cell-attached, and outside-out patch-clamp configurations. The effects of Rb+ on the voltage dependence and kinetics of IR gating were explored, with [Rb+]o + [K+]o = 160 mM. Partial substitution of Rb+ for K+ resulted in voltage-dependent reduction of inward currents, consistent with Rb+ being a weakly permeant blocker of the IR. In cells studied with a K(+)-free pipette solution, external Rb+ reduced inward IR currents to a similar extent at large negative potentials but block at more positive potentials was enhanced. In outside-out patches, the single-channel i-V relationship was approximately linear in symmetrical K+, but rectified strongly outwardly in high [Rb+]o due to a reduced conductance for inward current. The permeability of Rb+ based on reversal potential, Vrev, was 0.45 that of K+, whereas the Rb+ conductance was much lower, 0.034 that of K+, measured at Vrev-80 mV. The steady state voltage-dependence of IR gating was determined in Rb(+)-containing solutions by applying variable prepulses, followed by a test pulse to a potential at which outward current deactivation was observed. As [Rb+]o was increased, the half-activation potential, V1/2, changed less than Vrev. In high [K+]o solutions V1/2 was Vrev-6 mV, while in high [Rb+]o V1/2 was Vrev + 7 mV. This behavior contrasts with the classical parallel shift of V1/2 with Vrev in K+ solutions. Steady state IR gating was less steeply voltage-dependent in high [Rb+]o than in K+ solutions, with Boltzmann slope factors of 6.4 and 4.4 mV, respectively. Rb+ decreased (slowed) both activation and deactivation rate constants defined at V1/2, and decreased the steepness of the voltage dependence of the activation rate constant by 42%. Deactivation of IR channels in outside-out patches was also slowed by Rb+. In summary, Rb+ can replace K+ in setting the voltage-dependence of IR gating, but in doing so alters the kinetics.
Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.
Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo-3-labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino-phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion-dependent EC signals. mAb studies indicate that the beta 2 integrin-intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1-mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a role in EC alteration by lymphocyte adhesion.
We have used whole-cell and perforated patches to study ionic currents induced by hypotonic extracellular solutions (HTS, 185 mOsm instead of 290 mOsm) in endothelial cells from human umbilical veins. These currents activated within 30-50 s after application of HTS, reached a maximum value after approximately 50-150 s and recovered completely after re-exposing the cells to normal osmolarity. They slowly inactivated at potentials positive to +50 mV. The same current was also activated by breaking into endothelial cells with a hypertonic pipette solution (377 mOsm instead of 290 mOsm). The reversal potential of these volume-induced currents using different extracellular and intracellular Cl- concentrations was always close to the Cl(-)-equilibrium potential. These currents are therefore mainly carried by Cl-. DIDS only weakly blocked the current (KI = 120 microM), while another Cl(-)-channel blocker, DCDPC (20 microM) was ineffective. We were unable to record single channel activity in cell-attached patches but we always observed an increased current variance during HTS. From the mean current-variance relation of the whole-cell current records, we determined a single channel conductance of 1.1 pS. The size and kinetics of the current were not correlated with the concomitant changes in intracellular calcium. Furthermore, the currents could still be activated in the presence of 10 mmol/liter intracellular EGTA and are thus Ca2+ independent. A similar current was also activated with iso-osmotic pipette solutions containing 300 mumol/liter GTP gamma S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 mumol/liter) was also ineffective in modulation of this current. The HTS-induced current was completely blocked by 10 mumol/liter pBPB, a PLA2 inhibitor. NDGA (4 mumol/liter) and indomethacin (5 mumol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 mumol/liter) were variable. In 12 out of 40 cells it either directly activated a Cl- current or potentiated the current activated by HTS. The membrane current was decreased at all potentials in 18 cells, and was not affected in 10 cells. The HTS-induced currents may therefore be modulated by cleavage products of PLA2, but not by messengers downstream of arachidonic acid. Loading the cells with a segment of the heat stable protein kinase A inhibitor PKI (5-24) did not prevent activation of the HTS-induced current.(ABSTRACT TRUNCATED AT 400 WORDS)
Capacitative Ca2+ entry produces the very rapid light response in Drosophila photoreceptors; studies using this amenable system may help unravel the machinery and mechanisms that underlie this Ca2+-entry pathway.
To clarify the agonist-induced Ca2+ entry mechanism, effects of thapsigargin and cyclopiazonic acid, selective inhibitors of endoplasmic reticulum Ca2+-ATPase, on intracellular free Ca2+ concentration ([Ca2+]i) were studied in cultured human aortic endothelial cells loaded with the fluorescent Ca2+ indicator fura-2. Thapsigargin (1–1000 nM) and cyclopiazonic acid (0.1–100 μM) produced a biphasic change in [Ca2+]i, which consisted of a transient peak elevation followed by a long-lasting decline of [Ca2+]i in a concentration-dependent manner. In the presence of thapsigargin or cyclopiazonic acid, the rapid transient elevation of [Ca2+]i elicited by histamine was attenuated in a time-dependent manner. The slow declining phase of the response to thapsigargin and cyclopiazonic acid was completely eliminated by removal of extracellular Ca2+, and it was also prevented by reduction of the extracellular Cl− concentration to 40 mM or by the Cl− channel blocker N-phenylanthranilic acid. These findings suggest that the initial transient rising phase and the slow declining phase of [Ca2+]i in response to thapsigargin and cyclopiazonic acid reflect a blockade of Ca2+ uptake into the endoplasmic reticulum and the Cl−-sensitive Ca2+ entry activated by the depletion of agonist-sensitive intracellular Ca2+ stores, respectively, in human aortic endothelial cells.
A charybdotoxin-sensitive, Ca2+-activated K+ channel was identified in cultured rat brain capillary endothelial cells by using conventional single-channel recording techniques and 86Rb+-influx and efflux experiments. Channel activity was dependent on the presence of Ca2+ on the cytosolic face of the membrane with a threshold concentration of 100 nM. It was inhibited by charybdotoxin (IC50 30 nM) and quinine (IC50 0.1 mM) but not by apamin. K(Ca) channels showed unusual inward rectifying properties under asymmetrical ionic conditions. They were activated by endothelin-1 (EC50 0.7 nM) and endothelin-3 (EC50 7–10 nM). The actions of endothelins were prevented by BQ-123 (Ki = 8 nM) in a competitive fashion, hence suggesting the involvement of an ETA-receptor subtype. The channel activity was unaffected by cyclic AMP- or cyclic GMP-elevating agents. The possible role of the intermediate conductance, Ca2+-activated K+ channels for mediating K+ movements across the blood-brain barrier is discussed.
Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca2+ concentration, [Ca2+]
i
, accompanied by an increase in surface membrane K+ permeability. In this study, electrophysiological measurement of K+ current was combined with86Rb+ efflux measurements to characterize the K+ flux pathway in BAECs. Bradykinin- and Ca2+-activated K+ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca2+ activation is in the range of 10 to 100nm [Ca2+]
i
. The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K+ current present in control conditions, an inwardly rectifying K+ current, was blocked by Ba2+ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of86Rb– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,86Rb+ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca2+-activated membrane currents. The results confirm that bradykinin-stimulated86Rb+ efflux occurs via Ca2+-activated K+ channels. The blocking agents identified may provide a means for interpreting the role of the Ca2+-activated K+ current in the response of BAECs to bradykinin.
Recent cloning of a family of genes encoding inwardly rectifying K+ channels has provided the opportunity to explain some venerable problems in membrane biology. An expanding number of novel inwardly rectifying K+ channel clones has revealed multiple channel subfamilies that have specialized roles in cell function. The molecular determinants of inward rectification have been largely elucidated with the discovery of endogenous polyamines that act as voltage-dependent intracellular channel blockers, and with the identification of a critical site in the channel that mediates high-affinity block by both polyamines and Mg2+.
1. In the cell-attached and inside-out patch-clamp experiments using undispersed endothelial cells of the rat intrapulmonary artery, the majority of channels were cation selective. 2. Under physiological ionic conditions, the I-V relationship for the inward currents fell to -80 mV and the slope conductance was 22.5 pS. There was an inward rectification and the outward currents were smaller than the inward currents. 3. Under symmetric high-K+ conditions, the slope conductance for the inward currents was 26.4 pS and the inward rectification was observed when the high-K+ solution contained 1 mM-Mg2+. The channel activity was weakly voltage dependent at negative membrane potentials, while it was much enhanced at positive potentials. 4. The channel activity did not depend on intracellular Ca2+ concentrations. 5. Mg2+ was not only impermeant, it also blocked this channel in a voltage-dependent manner and rectifications appeared in the I-V relationship. Mg2+ blocked the channel from both sides of the membrane. 6. Ca2+ permeated this channel and the permeability ratios calculated from the reversal potentials using the constant-field theory were; PK:PNa:PCa = 1:1:15.7. 7. Histamine but not acetylcholine applied to the pipette activated this channel. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) applied to the intracellular surface of the patch did not mimic the effect of histamine. 8. Thus, in the endothelial cell membrane of the rat intrapulmonary artery, there exists a cation channel which is selective to Ca2+ but also permeable to Na+ and K+. This channel has inward rectifying properties, possibly due to intracellular Mg2+. Histamine, but not acetylcholine, activates this cation channel to elevate endothelial [Ca2+]i.
It is becoming clear that endothelial cells in the vascular system have important functions. In the microvessels they play an active role in regulating vascular permeability, while in large vessels, endothelial cells contribute to the control of smooth muscle tone. Control of both permeability and tone involve a range of mechanisms, in which changes in [Ca2+]i appear to play a major role. As elevation of [Ca2+]i can be caused by either release from intracellular stores or increased entry across the plasmalemma, and as the latter will be modulated by the resting membrane potential, the ion channels controlling the membrane potential are critical to an understanding of endothelial function. Patricia Revest and Joan Abbott summarize the properties of endothelial ion channels, and explore the ways in which the channels could control permeability, secretion and smooth muscle tone.
1. Single stretch-activated (SA) channels have been studied in isolated brain capillary endothelial cells as well as in the antiluminal membrane of intact porcine cerebral capillaries using the patch-clamp recording technique. 2. The SA channels were found to be cation selective and permeable to Na+, K+, Ba2+ and Ca2+. 3. With monovalent cations in the patch pipette, the channels showed inward rectification in cell-attached patches with a single-channel conductance of 37 pS at negative and 24 pS at positive clamp potentials. 4. With either 70 mM-Ca2+ or Ba2+ in the patch pipette, the current-voltage relation was linear with slope conductances of 16 and 19 pS, respectively. 5. Mean channel open probability increased with increasing pressure and with depolarizing clamp potentials. 6. Cell swelling induced by hypotonic shock activated the SA channels in cell-attached experiments. 7. The SA channel may be involved in cell volume or blood flow regulation. The contribution of these channels to the regulation of cerebrospinal salt and water content, especially in brain oedema, is discussed.
Electrophysiological experiments on cultured monolayers of guinea pig coronary endothelial cells were performed to substantiate the hypothesis that variation of the endothelial membrane potential may be a functionally important mechanism that contributes to the regulation of coronary blood flow. The endothelial cells were loaded with sodium by superfusion with K(+)-free solution at 37 degrees C for 5-30 min. Readmission of external K+ produced a transient hyperpolarization of up to 85 mV, which was due to stimulation of the electrogenic pump current. In most but not all of the monolayers, superfusion with 2 microM adenosine elicited a transient or a sustained hyperpolarization. The transient hyperpolarization had an amplitude of 15 +/- 6 mV. The sustained hyperpolarization had an amplitude of 11 +/- 3 mV. Our results are in line with the hypothesis that the hyperpolarization of the endothelium induced by release of adenosine into the perivascular space of the capillaries may be conducted electronically to the terminal arterioles and may cause vasodilation via current flow through myoendothelial gap junctions.
We investigated whether osmotic stress would activate specific ion channels in bovine aortic endothelial cells (BAECs). In isotonic medium (290 mosmol/kgH2O), cell-attached patch recordings contained both 165-pS K+ channels activated by depolarization and 40-pS K+ channels activated by 200 nM bradykinin. These inwardly rectifying K+ channels were activated by raising "cytoplasmic" Ca2+ in inside-out patches. BAEC exposed to hypotonic bath (220 mosmol/kg) exhibited a 20% decrease in intracellular K+ content within 5 min. Cell-attached patches revealed biphasic K+ channel activation with hypotonic exposure; initial activation of 165- and 40-pS K+ channels (1-3 min) was followed by a delayed but sustained reactivation of both K+ channels (> 5 min). The delayed reactivation phase was dependent on the presence of external Ca2+ and was attenuated by 10 microM gadolinium. A 28-pS nonselective cation channel (NSCC), which conducted inward Ca2+ current, was also detected during hypotonic exposure. This NSCC was stimulated by hyperpolarization and was blocked by 10 microM gadolinium. In BAEC 1) hypotonic exposure activates Ca(2+)-dependent, 165- and 40-pS K+ channels biphasically; 2) the initial phase is independent of external Ca2+, while the delayed phase requires external Ca2+; and 3) Ca(2+)-permeable, 28-pS NSCCs stimulated by membrane hyperpolarization provide a pathway for external Ca2+ influx.
Confluent bovine capillary endothelial cells display, when examined for voltage-dependent calcium entries using cell-attached channel recordings, two types of Ca2+ channels (4 and 23.5 pS in 110 mM Ba2+) both sensitive to the dihydropyridine Ca agonist BAY K 8644. In contrast to isolated cells, confluent cells display no T-type, low threshold activity, and Ca currents were typically only elicited at very depolarized potentials. In these cells, voltage-dependent calcium entries will only be made operative by substances able to shift their activation towards the resting potential.
Bovine endothelial cells cultured from pulmonary artery (ATCC cell line No. 209) were found to contain a high density of 125I-VIP (vasoactive intestinal polypeptide) binding sites. These were found to be saturable and to be fit by a single binding site model (Kd 1.8 nM; Bmax 534 fmol/mg protein). Studies of association and dissociation of 125I-VIP to this site revealed that binding was fully reversible and yielded a Kd value similar to that from equilibrium binding. However competition studies showed that VIP competed for binding at two sites (Ki1 1.2 x 10(-11) M, Ki2 4.7 x 10(-9) M; N1 = 21%, N2 = 77%; Ki a dissociation constant for inhibitor; N percentage of occupied receptors). [Phe1]VIP also competed at two sites, but VIP-(10-28), PHM, [4-Cl-D-Phe6,Leu17]VIP and [D-Ala4]VIP displaced all specific VIP binding in a simple competitive manner. These VIP binding sites were shown to be functional. In patch clamp studies VIP 10(-8)-10(-7) M inhibited opening of inwardly rectifying K+ channels on hyperpolarization. These channels were affected appropriately by alteration in the K(+)-gradient and by Ba2+ or Cs+. The VIP antagonist [4-Cl-D-Phe6, Leu17]VIP prevented or reversed the effects of VIP. These results show that functional VIP receptors are present in high density in a endothelial cell line and provide a possible model for analysis of the molecular biology of these receptors.
New methods are described to detect subconductance levels and to analyse ion channel gating. These methods are applied to simulated and experimental data. Single chloride channel records from inside-out membrane patches excised from human umbilical venous endothelial cells (HUVEC) exhibit, in addition to the full closed and full open configurations, intermediate subconductance levels which are multiple of an elementary conductance of 112.5 pS. Analysis of transitions from one state to another and the comparison of real data with simulated data leads to the proposal of a cooperative model of gating for the observed subunits of a chloride channel.
We have analyzed the effect of basic fibroblast growth factor (bFGF) on junctional communication (coupling) and connexin 43 (Cx43) expression in bovine microvascular endothelial (BME) cells. In control confluent cultures, the incidence of coupling, as assessed by the intercellular transfer of microinjected Lucifer Yellow, was limited to 13% of injected cells, and decreased to 0% with time in culture. After exposure to bFGF (3ng/ml), the incidence of coupling was increased in a time-dependent manner, reaching a maximum of 38% of microinjected cells after 10-12 hours. The extent of coupling, as assessed by scrape loading, was maximally increased 2.1-fold 8-9 hours after addition of bFGF. bFGF also induced a 2-fold increase in Cx43 as assessed by Western blotting, and increased Cx43 immunolabelling at contacting interfaces of adjacent BME cells. Cx43 mRNA was likewise increased after exposure to bFGF in a time- and dose-dependent manner, with a maximal 6-7-fold increase after a 4 hour exposure to 3-10ng/ml. Finally, the increase in coupling and Cx43 mRNA expression observed after mechanically wounding a confluent monolayer of BME cells was markedly reduced by antibodies to bFGF, which have previously been shown to inhibit migration. Taken together, these results indicate that exogenous and endogenous bFGF increase intercellular communication and Cx43 expression in microvascular endothelial cells. We propose that the bFGF-mediated increase in coupling is necessary for the coordination of endothelial cells during angiogenesis and other vessel wall functions.
We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca2+]o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca2+ ([Ca2+]i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential Erev shifted from -2.3 +/- 0.8 mV (n = 4) in a nominally Ca2+ free solution to +1.5 +/- 1.6 mV at 1.5 mM [Ca2+]o (n = 4) and to +21.9 +/- 4.4 mV (n = 7) at 10 mM [Ca2+]o. From our data we conclude that shear stress opens an ion channel that is 12.5 +/- 2.9 (n = 7) times more permeable for calcium than for sodium or cesium.
Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.
We recently discovered that the endothelium of skeletal muscle capillaries swells in the low-flow ischemia induced by hemorrhagic shock. The present study was undertaken to determine the Na+ transmembrane pathways involved in this swelling, since hypoxic cell swelling is attributed to an influx of Na+ and water. In an initial series of experiments, amiloride (5 mg/kg body wt), which blocks multiple Na+ pathways, was infused intravenously into anesthetized rabbits 30 min prior to shock (40% single-withdrawal hemorrhage). Intravital microscopy of treated capillaries in the rabbit tenuissimus muscle showed that after a 1-h shock period, there was no endothelial cell swelling, as evidenced by no measurable change in the width of red blood cells traversing the capillary. In contrast, the swollen endothelium of untreated capillaries reduced the luminal diameter by 20-25% with a preserved stationary abluminal membrane. The specific effects of amiloride on Na+ transport were investigated with amiloride analogues. Animal pretreatment with 5-(N,N-hexamethylene)amiloride, a selective inhibitor of Na(+)-H+ activity, in a dose of 0.5 mg/kg did not significantly mitigate shock-induced swelling; however, a dose of 1 mg/kg completely prevented it. Phenamil, a selective inhibitor of Na+ channel conductance, even at a potent dosage of 0.5 mg/kg, did not affect swelling. These results suggest a primary role for Na(+)-H+ exchange in endothelial cell swelling during hemorrhagic shock, possibly as a means to regulate cellular pH, which may become acidic during ischemia. Narrowed capillaries with elevated hydraulic resistances could delay and diminish resumption of microcirculatory flow on shock resuscitation.
1. Changes of the free cytosolic Ca2+ concentration induced by shear stress were measured in Fura-2 acetoxymethyl ester-loaded endothelial cells from human umbilical cord veins. 2. We were able to induce Ca2+ transients in almost every cell by blowing a stream of physiological solution onto a single endothelial cell thereby inducing shear stress between 0 and 50 dyn cm-2. The Ca2+ response could be graded by varying the shear stress, and reached a half-maximal value at a shear stress of 30 dyn cm-2. 3. The shear stress responses critically depended on the extracellular Ca2+ concentration and were absent in a Ca(2+)-free solution. Repetitive application of short pulses of shear stress induced cumulative effects because of the slow decay of the shear stress Ca2+ responses (time constants 82.3 +/- 17.8 s from twenty-five cells). Application of a depolarizing high potassium solution to reduce the driving force for Ca2+ entry decreased the Ca2+ transients in some of the cells. 4. Application of shear stress in the presence of other divalent cations, such as nickel, cobalt or barium, always produced substantial changes in the ratio of the 390/360 nm fluorescence signal, indicating influx of these cations and subsequent quenching of the Fura-2 fluorescence. 5. Shear stress responses in the presence of 10 mM Ca2+ were completely blocked by application of 1 mM La3+. 6. Incubation of the cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) did not alter the shear stress response, but completely blocked histamine-induced Ca2+ transients. 7. Small submaximal shear stress potentiated the Ca2+ transients induced by histamine. 8. We conclude that shear stress-dependent Ca2+ signals are induced by an influx of calcium that is not modulated via protein kinase C and not activated by membrane depolarization. The influx pathway is also permeable to divalent cations such as Ni2+, Co2+ and Ba2+, but is blocked by La3+.
Expression of P-glycoprotein, the product of the MDR1 gene, confers multidrug resistance on cell lines and human tumours (reviewed in refs 1,2). P-glycoprotein (relative molecular mass 170,000) is an ATP-dependent, active transporter which pumps hydrophobic drugs out of cells, but its normal physiological role is unknown. It is a member of the ABC (ATP-binding cassette) superfamily of transporters, which includes many bacterial transport systems, the putative peptide transporter from the major histocompatibility locus, and the product of the cystic fibrosis gene (the cystic fibrosis transmembrane regulator, CFTR). CFTR is located in the apical membranes of many secretory epithelia and is associated with a cyclic AMP-regulated chloride channel. At least two other chloride channels are present in epithelial cells, regulated by cell volume and by intracellular Ca2+, respectively. Because of the structural and sequence similarities between P-glycoprotein and CFTR, and because P-glycoprotein is abundant in many secretory epithelia, we examined whether P-glycoprotein might be associated with one or other of these channels. We report here that expression of P-glycoprotein generates volume-regulated, ATP-dependent, chloride-selective channels, with properties similar to channels characterized previously in epithelial cells.
Single-channel currents of an anionic channel in the plasma membrane of cultured bovine aortic endothelial cells have been recorded with the patch-clamp technique. The channel is selective for chloride over cations, and has an average single channel conductance of 382 picosiemens in symmetric 140 millimoles of chloride. In addition to the main conductance state it shows well-defined subconductance states of about 50, 100, 150 and 200 picosiemens. The channel is very active at membrane potentials close to 0 mV, but steps to either positive or negative membrane potentials above +/- 20 millivolt lead to a rapid inactivation of the channel. Changes in the concentrations of free calcium or adenosine tri-phosphate on the cytosolic surface do not influence channel activity. The chloride channel rarely opens at resting membrane potential, but it may help repolarize endothelial cells following depolarizing stimuli.
Isolated bovine capillary endothelial cells have been examined for voltage-dependent Ca entry. All cells displayed a low threshold activity, with the main characteristics of a T-type transient current, when examined using whole-cell recording for activation and inactivation and cell-attached conditions or inside-out patches for the elementary conductance (8 pS). 25% of the cells displayed an additional sustained current in 5 mM CaCl2 above -40 mV, which was enhanced by application of BAY K 8644, but almost insensitive to superfusion with nicardipine. Two types of channels (2.8 and 21 pS, in 110 mM BaCl2) were shown to have a BAY K 8644 sensitivity. The large conductance channels were L-type channels. The smaller events were elicited at more hyperpolarized potentials (by some 30 mV). Their mean open time was 16 ms in control conditions. In presence of BAY K 8644, additional long open times were observed (up to 100 ms as compared to 7.8 ms for the time constants of the slow mode of the L-type channel). We refer to these channels as SB channels: of small conductance and sensitive to BAY K 8644. In the presence of nicardipine, SB channels are not noticeably modified, in contrast to the L-type openings which are abolished. Also, SB open times are close to control values when nicardipine is added after a BAY K 8644 application. We suggest that, at physiological concentrations of divalent ions, an SB-type activity is elicited above -40 mV which generates the low threshold sustained current.
The patch-clamp technique was applied to the antiluminal membrane of freshly isolated capillaries of rat brain (blood-brain barrier). With 1.3 mM Ca2+ in the bath, excision of membrane patches evoked ion channels, which could not be observed in cell-attached mode. The channel was about equally permeable to Na+ and K+ ions, but not measurable permeable to Cl- and the divalent ions Ca2+ and Ba2+. The current-voltage curve was linear in the investigated voltage range (-80 mV to +80 mV), and the single-channel conductance was 31 +/- 2 pS (n = 22). The channel open probability was not dependent on the applied potential. Lowering of Ca2+ to 1 microM or below on the cytosolic side inactivated the channels, whereas addition of cytosolic ATP (1 mM) inhibited channel activity completely and reversibly. The channel was blocked by the inhibitor of nonselective cation channels in rat exocrine pancreas 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10 microM) and by the antiinflammatory drugs flufenamic acid (greater than 10 microM) and tenidap (100 microM), as well as by gadolinium (10 microM). Thus, these nonselective cation channels have many properties in common with similar channels observed in fluid secreting epithelia. The channel could be involved in the transport of K+ ions from brain to blood side.
The contraction of hepatic endothelial cell fenestrae after exposure to serotonin is associated with an increase in intracellular Ca2+ which is derived from extracellular Ca2+, is inhibited by pertussis toxin and is not associated with activation of phosphoinositol turnover or cAMP. Using cell-attached and excised patches in primary cultures of rat hepatic endothelial cells, we identified a serotonin-activated channel with conductance of 26.4+/-2.3 pS. The channel was also permeant to Na+, K+ and Ca++ but not to anions. In cell-attached patch recordings, addition of serotonin to the bath significantly increased channel activity with Ca2+ or Na+ as the charge-carrying ions. This channel provides a mechanism whereby serotonin can raise the cytosolic Ca2+ concentration in hepatic endothelial cells.
Single, large-conductance chloride-selective channels were studied in the membrane of pig aortic endothelial cells. These channels were usually inactive in cell-attached recordings and activated spontaneously upon formation of inside-out patches or amphotericin B-perforated vesicles. Channel activity was voltage dependent, with a maximum open probability within the range of -20 mV to + 20 mV. Addition of 1 mM Zn2+ to either the cytoplasmic or extracellular side blocked channel activity reversibly. Extracellular 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) blocked the channels; the concentration necessary for half-maximum blockade was 100 mumol/l. The frequency of observing channels in cell-attached patches increased from less than 5% to 27% when cells were treated for several minutes with 1 mumol/l bradykinin and to 80% in the presence of the calcium ionophore A23187 (1 mumol/l). Both agents increase the cytoplasmic Ca2+ concentration, thereby stimulating nitric oxide (NO) synthesis and cGMP formation in endothelial cells. Sodium nitroprusside (100 mumol/l), which spontaneously releases NO, did not increase Cl- channel activity in intact cells. Polymyxin B (100 mumol/l), an inhibitor of protein kinase C, clearly enhanced Cl- channel activity in intact cells, resulting in the observation of Cl- channels in 70% of cell-attached patches. Our results demonstrate the existence of a large-conductance (LC-type) Cl- channel in vascular endothelium which is subject to a complex cellular regulation, possibly involving inhibition via phosphorylation by protein kinase C, and activation by a Ca2(+)-dependent process which is different from the NO/cGMP pathway.
The distribution of inwardly rectifying (Ki) and calcium-activated (KCa) potassium channels on the apical and basal surfaces of bovine aortic endothelial cells (BAECs) was examined by inverting BAEC monolayers onto polylysine-coated cover slips. To monitor cellular polarity, we examined human red blood cell adherence (hemadsorption) to the influenza virus protein, hemagglutinin (HA), and virus budding on the surface of infected BAECs. Hemadsorption and virus budding occurred on the apical surface but were not apparent on the basal surface of monolayers 1 and 5 h after inversion, although cellular HA antigen localization confirmed that all monolayers were infected. In contrast, by 9.5 and 24 h after inversion, hemadsorption was evident on the "new" apical surface. Single-channel patch-clamp analysis revealed the presence of both Ki and KCa channels on the apical surface and basal surface of BAEC monolayers 2-5 h after inversion. K channel conductance and kinetics were similar regardless of the surface monitored. This nonenzymatic mechanical technique of exposing the basal surface of endothelium provides a useful tool to study the distribution of ion channels in endothelium and in other polarized cell types grown in tissue culture.
1. Isolated native endothelial cells, obtained by treatment of rabbit aortic endothelium with papain and dithiothreitol, were voltage clamped, and single channel (unitary) and spontaneous transient outward currents (STOCs) were recorded from both whole cells and excised membrane patches. 2. In inside-out patches, the reversal potential of unitary currents was dependent on the extracellular K+ concentration and had a single-channel slope conductance of 220 pS in symmetrical 140 mM-K+ solutions. The open-state probability (Po) of the unitary K+ currents was sensitive to the intracellular Ca2+ concentration with half-maximal activation at approximately 1 microM at +20 mV. The ionic selectivity and Ca2+ sensitivity indicate that a large conductance, Ca(2+)-activated K+ channel is present in freshly dissociated rabbit aortic endothelial cells. 3. The frequency and amplitude of whole-cell unitary currents and amplitude of spontaneous transient outward currents were voltage-dependent. Whole-cell outward K+ currents evoked by depolarizing voltage ramps had amplitudes often corresponding to the simultaneous opening of more than five single Ca(2+)-activated K+ channels. Lowering the intracellular EGTA concentration tenfold, and hence the Ca2+ buffering capacity of the cell, increased unitary K+ current activity and shifted the relationship between Po and membrane potential by approximately -20 mV. 4. Bradykinin (1 microM), adenosine 5'-triphosphate (3 microM) and acetylcholine (3 microM) applied extracellularly evoked a biphasic increase in N Po (where N is number of channels activated) of the Ca(2+)-activated K+ channel studied in the whole-cell recording configuration. The development of a biphasic response to agonist stimulation requires a source of extracellular Ca2+. The sustained increase in N Po of the Ca(2+)-activated K+ channel was attenuated upon the removal of external Ca2+ (Mg2+ replacement) or in the presence of the Ca2+ entry blocker, Ni2+, and the potassium channel blockers tetrabutylammonium (TBA) or tetraethylammonium (TEA). 5. Unitary and spontaneous transient outward currents were inhibited by extracellularly applied TEA (0.5 mM), TBA (0.5-5 mM) and charybdotoxin (100 nM). Ca(2+)-activated K+ currents were blocked completely by 5 mM-TEA, whereas 3,4-diaminopyridine (1 mM), Ba2+ (10 mM) and apamin (0.1-1 microM) did not abolish these K+ currents. 6. The K+ channel opener cromakalim (10 microM) evoked a sustained increase in N Po of the Ca(2+)-activated K+ channels which was not potentiated by the addition of bradykinin. Glibenclamide (10 microM) alone increased N Po and partially inhibited the cromakalim-induced increase in N Po with respect to control.(ABSTRACT TRUNCATED AT 400 WORDS)
Formation of endothelium-derived relaxing factor (EDRF) strictly correlates with the intracellular free Ca2+ ([Ca2+]i) concentration. We now demonstrate that the histamine-induced rise in [Ca2+]i of human umbilical vein endothelial cells is mostly due to activation of a membrane current which allows Ca2+ entry. This membrane current is sensitive to the novel inhibitor of agonist-induced Ca2+ entry, SK&F 96365, which blocked the histamine-induced sustained rise in [Ca2+]i, as well as 45Ca2+ uptake and membrane currents. Inhibition of the above cellular responses to histamine was accompanied by a considerable reduction of EDRF formation and release. Thus biosynthesis and release of EDRF from human umbilical vein endothelial cells significantly depend on agonist-induced Ca2+ entry involving receptor-operated Ca(2+)-permeable channels which can be blocked by SK&F 96365.
We have investigated the role of the intracellular Ca2+ pool in regulating Ca2+ entry into vascular endothelial cells. The intracellular Ca2+ pool was mobilized using either thapsigargin (TG) or 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibitors of the endoplasmic reticulum Ca(2+)-adenosinetriphosphatase (ATPase). Mobilization of intracellular Ca2+ stores with either inhibitor depleted intracellular Ca2+ and greatly reduced subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ pool by bradykinin. However, bradykinin-induced mobilization of the IP3-sensitive intracellular Ca2+ pool only partially reduced the subsequent response of cells to TG and BHQ. Mobilization of the intracellular Ca2+ pool by either TG or BHQ led to a concentration-dependent elevation of cytosolic Ca2+ concentrations ([Ca2+]i) without initiating inositol polyphosphate formation. In contrast to the rapidly developing, transient rise in Ca2+ concentration initiated by bradykinin, maximal concentrations of TG and BHQ stimulated a slowly developing, prolonged elevation of [Ca2+]i that required extracellular Ca2+ and could be blocked by extracellular Ni2+. Extracellular Ca2+ entered the cell through an activated cation entry pathway, since bradykinin, TG, and BHQ stimulated Mn2+ and 45Ca2+ entry. Bradykinin-stimulated 45Ca2+ uptake reached a peak within 2 min, whereas 45Ca2+ influx initiated by TG or BHQ continued for at least 8 min. Importantly, the [Ca2+]i response after low concentrations of BHQ was more transient than that seen after TG. The return of [Ca2+]i to basal values after low concentrations of BHQ was associated with reversal of Ca(2+)-ATPase inhibition and refilling of the IP3-sensitive Ca2+ pool. The continued elevation of [Ca2+]i and prolonged Ca2+ entry seen with TG was associated with continued Ca(2+)-ATPase inhibition and an empty IP3-sensitive Ca2+ pool. We conclude that mobilization of intracellular Ca2+ stores induces Ca2+ entry in endothelial cells which continues until the intracellular Ca2+ pool is refilled.
Clusters of electrically coupled endothelial cells were used to characterize a bradykinin (BK)-activated Ca2+ influx pathway. Spatial voltage control of clusters containing three to eight cells, evaluated as the ratio of the voltage response in one cell to a voltage pulse in the most distant cell of the cluster, was 0.96 at a holding potential of 0 mV in normal saline bath and 0.88 in the presence of BK. BK activated an inward current that was carried by either Na+ or Ca2+ when the membrane potential was held at -60 mV. Current was activated within 3 s of application of BK and peaked within 1 min. With Ca2+ as the permeable extracellular ion the current was stable for 1-3 min and then declined over a period of 5-8 min in the continued presence of BK. However, when Na+ carried the current it was sustained over a 10-min test period. The reversal potential of the BK-activated current was near 0 mV, suggesting activation of a nonspecific cation channel(s). The inward current at -60 mV averaged 13 +/- 4.5 pA (n = 9)/cell in Ca2+ and 12.2 +/- 9.3 pA (n = 5)/cell in Na+. Both Na+ and Ca2+ currents were blocked by 200 microM lanthanum.
Elevation of cellular cyclic AMP by agents such as isoproterenol plus 3-isobutyl-1-methylxanthine produced rapid and reversible dendritic formation of bovine pulmonary artery endothelial cells in the monolayer. The effect did not occur with exposure of the cells to a variety of other vasoactive agents, calcium ionophore, phorbol ester, or cyclic GMP. The cyclic AMP-induced configurational change was completely inhibited by 2.5 mM N-phenylanthranilic acid or 145 mM sodium gluconate (Cl- channel inhibitors) and was partially inhibited by 2.5 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), but it was not affected by deprivation of Ca2+ or Na+ ion, 1 mM bumetanide (Cl- cotransport inhibitor), 1 mM amiloride (Na+/H+ exchange inhibitor), 0.1 mM verapamil (Ca2+ channel inhibitor), or 5 mM BaCl2 (K+ channel inhibitor), by change in cellular pH, or by pertussis toxin. Trifluoperazine (calmodulin inhibitor, 50 microM), 1 mM EGTA plus 100 microM 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8, intracellular Ca2+ antagonist), and 5 microM cytochalasin B also produced cellular retraction, but these changes were not blocked by chloride channel inhibition. In the presence of 0.1 mM ouabain plus 0.1 mM bumetanide, 36Cl- uptake was decreased by isoproterenol plus isobutylmethylxanthine while its efflux was enhanced. N-Phenylanthranilic acid inhibited the stimulated efflux. We conclude that cyclic AMP induces a configurational change of endothelial cells that is related to Cl- efflux from the cells; the cellular effects may play a role in vascular function.
Ca2+ influx into stimulated endothelial cells is attenuated by depolarization. We hypothesized that Ca2+ influx is driven by the membrane potential and may be enhanced by hyperpolarizing drugs like activators of K+ channels. Therefore we studied the effects of pinacidil, cromakalim, and cicletanine on membrane currents and on the intracellular free calcium concentration ([Ca2+]i) in cultured endothelial cells from porcine aorta. In patch-clamped cells, pinacidil (1 mumol/l) and cromakalim (1 mumol/l) elicited outward currents carried by K+ and significantly prolonged the Ca2(+)-dependent K+ currents induced by bradykinin and ATP. Peak currents in response to bradykinin were not affected. In cells loaded with the fluorescent Ca2+ indicator indo-1 and prestimulated with thimerosal, pinacidil (0.1-1 mumol/l elicited long-lasting increases in [Ca2+]i from 100 +/- 10 to 550 +/- 110 nmol/l. These effects were completely abolished in a medium containing 90 mmol/l K+. Similar results were obtained with cromakalim. Likewise, in cells stimulated with bradykinin, pinacidil raised [Ca2+]i when applied during the decline of [Ca2+]i after the initial peak. Cicletanine elicited K+ currents in resting and attenuated K+ currents in bradykinin-stimulated cells. It elevated [Ca2+]i even in the absence of extracellular Ca2+ and in K(+)-rich medium. Hence, the effects of cicletanine cannot be explained by direct actions on K+ channels. However, our studies demonstrate that pinacidil and cromakalim elevate [Ca2+]i secondary to their activation of K+ channels by inducing hyperpolarization and augmenting the driving force for potential-dependent Ca2+ influx. In this way, the two drugs may promote Ca2(+)-dependent formation of endothelium-derived relaxing factor.
Endothelial cells obtained from human umbilical chord have been studied by the patch clamp method. An ion channel is described that is activated by microM concentrations of histamine and shows a slow run-down in cell-attached patches. After excision, channel activity quickly runs down to zero open probability. In symmetrical potassium concentrations (140 mM K in the bath and the pipette), the single channel conductance is 28 +/- 2 pS and the reversal potential is 0.3 +/- 0.8 mV (mean +/- SEM, n = 4). With 140 mM Na in the pipette, the conductance is 26 +/- 2 pS. A reversal potential of -1.5 +/- 0.9 mV (n = 7) was measured. With 60 mM Ca and 70 mM Na in the pipette, 140 mM K in the bath, the reversal potential was -11 +/- 3 mV, the single channel conductance in 16 +/- 3 pS (n = 5). The single channel conductance in 110 mM Ca (pipette) and 140 mM K (bath) is 8 +/- 2 pS and the reversal potential is -18 +/- 6 mV (n = 3). From analysis of the reversal potentials, a permeation ratio of K:Na:Ca = 1:0.9:0.2 was calculated. This ligand-gated non-selective cation channel in human endothelial cells is Ca permeable and could induce a sustained agonist mediated Ca influx.
Vascular endothelial cells produce a variety of substances known to modulate the tone of surrounding smooth muscle, but the initial steps involved in receptor-response coupling are poorly characterized in these cells. Because the stimulated release of endothelium-derived relaxing factor depends on the presence of external calcium, ion channel-mediated calcium influx might represent an essential first link. Furthermore, agonist-induced endothelial cell hyperpolarization has been widely described, although the ion channels involved and the functional significance of this response remain uncertain. A review of the available literature to date concerning voltage-dependent and agonist-activated ionic currents obtained using patch clamp techniques in vascular endothelial cells is presented here. A discussion of the possible functional roles of the underlying ion channels is included.