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SIGNAL-TRANSDUCTION VIA P2-PURINERGIC RECEPTORS FOR EXTRACELLULAR ATP AND OTHER NUCLEOTIDES
Abstract
Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors t at stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.
... P2X receptors are ligand-gated ionotropic channel family, especially Ca 2+ channel, and P2Y receptors are involved in pertussis toxin-sensitive and -insensitive G proteins thatregulate diverse enzymes (Akbar et al., 1996; Chang et al., 1995; Cowen et al., 1990; Dubyak and el-Moatassim, 1993; Harden et al., 1995; Lazarowski and Harden, 1994). Dispersed smooth muscle cells were pretreated with Ca 2+ channel blocker nifedipine 1 PM for 10 min or with pertussis toxin PTX 400 ng/ml and GDP S 10 PM for 1 h respectively, and then treated with 10 PM or 0.1 PM ATP and UTP for 30 s. Contraction induced by 10 PM ATP were abolished by only the Ca 2+ channel blocker, nifedipine but were not affected by pretreatment of dispersed cells with PTX, and GDP S (Fig. 3A). ...
... P2Y receptors involve diverse enzymes including phospholipase C (Akbar et al.,1996; Dubyak and el-Moatassim, 1993; Harden et al., 1995; Lazarowski and Harden, 1994 ), and protein kinase C (van der Weydenet al., 2000), which can induce activation of MLC kinase and inhibition of MLC phosphatase (Ikebeet al., 1987 ). Dispersed smooth muscle cells were pretreated with PLC inhibitor (U73122) 1 PM, PKC inhibitor (chelerythrine) 10 PM, or MLC kinase inhibitor (ML-9) 10 PM for 10 min, respectively, and then treated with 0.1 PM ATP or UTP for 30 s. Contraction induced by 0.1 PM ATP and UTP were partially abolished by U73122, chelerythrine, and ML-9 (Figs. 5A and 5B). ...
P2 receptors are membrane-bound receptors for extracellular nucleotides such as ATP and UTP. P2 receptors have been classified as ligand-gated ion channels or P2X receptors and G protein-coupled P2Y receptors. Recently, purinergic signaling has begun to attract attention as a potential therapeutic target for a variety of diseases especially associated with gastroenterology. This study determined the ATP and UTP-induced receptor signaling mechanism in feline esophageal contraction. Contraction of dispersed feline esophageal smooth muscle cells was measured by scanning micrometry. Phosphorylation of MLC20 was determined by western blot analysis. ATP and UTP elicited maximum esophageal contraction at 30 s and 10 μM concentration. Contraction of dispersed cells treated with 10 μM ATP was inhibited by nifedipine. However, contraction induced by 0.1 μM ATP, 0.1 μM UTP and 10 μM UTP was decreased by U73122, chelerythrine, ML-9, PTX and GDPβS. Contraction induced by 0.1 μM ATP and UTP was inhibited by Gαi3 or Gαq antibodies and by PLCβ1 or PLCβ3 antibodies. Phosphorylated MLC20 was increased by ATP and UTP treatment. In conclusion, esophageal contraction induced by ATP and UTP was preferentially mediated by P2Y receptors coupled to Gαi3 and Gαq proteins, which activate PLCβ1 and PLCβ3. Subsequently, increased intracellular Ca2+ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC20 generated esophageal contraction.
... Ortinau et al. (2003) reported that extracellular ATP inhibits the function of N-methyl-Daspartate (NMDA) glutamate receptors by directly binding to the receptor, suggesting that extracellular ATP may function as an allosteric modulator for neurotransmitter receptors [17]. High levels of ATP also exist in the extracellular compartment under both normal physiological conditions (i.e. as result of synaptic release)181920, and pathological conditions such as traumatic and ischemic brain insults21222324. In addition, previous studies suggest that ATP and GABA are released at GABAergic synapses [18,19,25]. ...
... The fact that ATP potentiation of mIPSCs is primarily manifested as a specific increase in mIPSC amplitude, without altering its frequency, is in good agreement with the allosteric modulation of postsynaptic GABA A Rs. Thus, this mode of modulation may function at certain GABAergic synapses under physiological conditions. Similarly, the modulation may also occur under pathological conditions (including neuronal overexcitation, epileptic episodes, inflammation, traumatic insults, hypoxia/ischemia ), as ATP release from damaged neurons and astrocytes can rapidly increase extracellular ATP concentrations21222324. ATP may increase both phasic and tonic GABA currents by acting on both synaptic and extrasynaptic GABA A Rs. ...
The gamma-aminobutyric acid type A receptor (GABAAR) is the primary receptor mediating fast synaptic inhibition in the brain and plays a critical role in modulation of neuronal excitability and neural networks. Previous studies have demonstrated that ATP and its nucleotide analogs may regulate the function of GABAARs via Ca2+-dependent intracellular mechanisms, which require activation of purinergic 2 (P2) receptors or cross-talk between two receptors.
Here, we report a potentiation of GABAARs by extracellular ATP via a previously un-recognized allosteric mechanism. Using cultured hippocampal neurons as well as HEK293 cells transiently expressing GABAARs, we demonstrate that extracellular ATP potentiates GABAAR mediated currents in a dose-dependent manner with an EC50 of 2.1 +/- 0.2 mM. The potentiation was mediated by a postsynaptic mechanism that was not dependent on activation of either ecto-protein kinase or P2 receptors. Single channel recordings from cell-free excised membrane patches under outside-out mode or isolated membrane patches under cell-attached mode suggest that the ATP modulation of GABA currents is achieved through a direct action of ATP on the channels themselves and manifested by increasing the single channel open probability without alteration of its conductance. Moreover, this ATP potentiation of GABAAR could be reconstituted in HEK293 cells that transiently expressed recombinant rat GABAARs.
Our data strongly suggest that extracellular ATP allosterically potentiates GABAAR-gated chloride channels. This novel mode of ATP-mediated modulation of GABAARs may play an important role in regulating neuronal excitability and thereby in fine-tuning the excitation-inhibition balance under conditions where a high level of extracellular ATP is ensured.
... Multiple subtypes of P2-purinergic receptors exist, as was first proposed by Burnstock (Burnstock and Kennedy, 1985; reviewed by Burnstock, 1997). P2Y receptors function as G-protein coupled Ca 2þ -mobilizing ATP receptors, operating via stimulation of phospholipase C and formation of inositol trisphosphate (IP 3 ) and diacylglycerol (DAG), the former of which causes release of Ca 2þ from intracellular stores on the endoplasmic reticulum (see chapter by Scapagnini et al.); P2X-type receptors act as ligand-gated ion channels; and P2-Z receptors are associated with ATP-induced pore formation (Dubyak and el-Moatassim, 1993). Astrocytes express two subtypes of P2Y receptors, the P2Y1 and the P2Y2 receptor (Zhou and Kimelberg, 2001). ...
... It is in agreement with this suggestion that an ATP-induced increase in glial [Ca 2þ ] i in the acutely isolated rat optic nerve can be evoked not only by a P2Y-selective agonist but also by a P2X-selective agonist (James and Butt, 2001). Calcium waves in other cell types are also propagated using ATP as the extracellular messenger (Osipchuk and Cahalan, 1992; Dubyak and el-Moatassim, 1993; Schlosser et al., 1996; Jorgensen et al., 1997; Frame and deFeijter, 1997; Schneider et al., 1994; Klepeis et al., 2001). On the other hand, activation of P2Y receptors on astrocytes have additional effects besides the ability to elicit calcium waves. ...
This chapter reviews that intercellular calcium waves in astrocytes represent a phenomenon whereby a wave of increases in free cytosolic calcium concentration ([Ca2+]i) spreads from an initially stimulated cell across an astrocytic syncytium. Three key factors merged to trigger the discovery of calcium waves in the astrocyte syncytium. The first was finding that astrocytes possessed a full array of neurotransmitter receptors that operated on a millisecond time frame similar to that expected by neurons. The second contribution was the many technological advances made in time-lapse video microscopy, particularly involving the newly marketed confocal scanning laser microscope, which allowed longer recording periods free of phototoxicity. Lastly, most critical was the development of Ca2+ sensitive fluorescent probes that were ion-specific that allowed precise quantification of changes in intracellular Ca2+. The chapter also explores that nitric oxide (NO)-mediated signaling mechanisms are involved, but only in some types of calcium waves, especially those triggered by mechanical stimulation. Neuronal activity also induces astrocytic calcium waves by stimulation of metabotropic glutamate receptors on astrocytes. In turn, glutamate release from astrocytes sustaining a calcium wave is capable of triggering neuronal activity.
... Targeted disruption of the NTPDase1 gene resulted in severe alterations of hemostasis, underscoring an important role previously proposed for P2 receptor signaling in platelet aggregation (Enjyoji et al., 1999). Marked transient elevation of extracellular nucleotides occurs as a consequence of regulated release from both excitatory and nonexcitatory cells (Dubyak and El-Moatassim, 1993; Lazarowski et al., 1995 Lazarowski et al., , 1997 Schlosser et al., 1996; Grygorczyk and Hanrahan, 1997 ). In addition, basal constitutive release of nucleotides occurs from most if not all cell types (Lazarowski et al., 2000; Ostrom et al., 2000). ...
... These results indicate that we have engineered a modified P2Y receptor that, when expressed in a cellular context, is activated less favorably by hydrolyzable nucleotides but retains full responsiveness to exogenous agonists that are resistant to the action of the nucleotidase. The P2Y receptor family of signaling proteins is essentially ubiquitously expressed on nonexcitatory cells, such as fibroblasts and hepatocytes, as well as endothelial, epithelial, glial, and smooth muscle cells (Dubyak and El-Moatassim, 1993; Harden et al., 1995; Ralevic and Burnstock, 1998). The source of nucleotide regulating these receptors has not been unambiguously established. ...
To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y1 receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y1receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y1-NTPDase1 fusion protein. Stable expression of the P2Y1-NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca2+response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPβS to stimulate inositol phosphate accumulation was similar, and the EC50 of ADPβS was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y1 and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y1 receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y1 receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y1 receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.
... In the present study, the BzATP-stimulated increase in [Ca 2 ] i is 10 times greater than that of ATP. This result is consistent with a common notion that BzATP is much more potent than ATP in activation of the P2X 7 receptor (Dubyak and El-Moatassim, 1993). Pharmacological selectivity for the P2X 7 receptor is ATP 4 BzATP ATP (Harden et al., 1995), and therefore ATP is a partial agonist with lower efficiency. ...
... The amount of ATP released from synaptic terminals during neuronal transmission, which then diffuses to the astrocyte, probably falls below these high thresholds. Therefore, additional extracellular ATP would have to be provided by (a) the release of cytosolic ATP via intrinsic plasma channels or pores in the absence of irreversible cytolysis, or (b) stimulationinduced release of cytosolic ATP upon sudden breakage of intact cells (Dubyak and El-Moatassim, 1993 ). Recently , P2X 7 immunoreactivities were shown to appear in a necrotic area in the brain by prior occlusion of the middle cerebral artery (Collo et al., 1997). ...
This study characterizes and examines the P2 receptor-mediated signal transduction pathway of a rat brain-derived type 2 astrocyte cell line, RBA-2. ATP induced Ca2+ influx and activated phospholipase D (PLD). The ATP-stimulated Ca2+ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), in a concentration-dependent manner. The agonist 2′- and 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) stimulated the largest increases in intracellular Ca2+ concentrations ([Ca2+]i); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATPS were much less effective, whereas UTP, ADP, α,-methylene-ATP, and ,-methylene-ATP were ineffective. Furthermore, removal of extracellular Mg2+ enhanced the ATP- and BzATP-stimulated increases in [Ca2+]i. BzATP stimulated PLD in a concentration- and time-dependent manner that could be abolished by removal of extracellular Ca2+ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca2+ mobilization agent, ionomycin, in an extracellular Ca2+ concentration-dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate that activation of the P2X7 receptors induces Ca2+ influx and stimulates a Ca2+-dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD.
... Among the integral membrane proteins, ion channels are particularly important because of their role in maintaining the cellular homeostasis by acting like molecular switches operating under influences such as electrical potential [9], chemical mediators [10], mechanical [11], thermal [12], and even optical stimuli [13]. Nonetheless, their function merely depends on basic physical laws of nature [14,15]. ...
Patch-clamp technique is the gold standard for cellular electrophysiological measurements, which is capable of measuring single ion transport events across the cell membrane. However, the measurement possesses significant complexity, and it requires a high level of expertise, while its experimental throughput is nevertheless considerably low. Here, we suggest and experimentally demonstrate a laser-assisted method for performing cellular electrophysiological measurements. Femtosecond laser pulses, coupled to an optical microscope, are used to form a sub-micrometer hole on a thin polymer membrane separating two electrodes, where a nearby cell is subsequently placed onto the hole by negative pressure. Afterwards, the cell is punctured using subsequent laser exposure, revealing the cell membrane over the hole for electrophysiological recording. This system could be used to increase the output amount of the electrophysiological measurements substantially.
... Under pathologic conditions, a large amount of ATP is released into the extracellular compartment by injured and stressed cells [45,46]. The released eATP exerts multifaceted effects on various patho-physiological responses, including immune responses and inflammation [47,48] . ...
Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases.
... During research of lacrimal gland functioning irritation effects of parasympathetic and sympathetic nerve fibers are often modulated by action of various cholinergic and adrenergic agonists. But, ATP is comediator of many cholinergic and adrenergic synapses, the release of which could significantly modulate the effect of primary mediator [7, 15]. [36] Extracellular ATP causes an increase in cytosolic Ca 2+ concentration in the acinar cells of various exocrine glands by interacting with P2-type of purinoreceptors [44, 51, 55]. ...
It is known that different Ca2+-transport systems make distinct contribution to the formation of Ca2+-signal in various secretory cells. In recent years there has been a significant increase in interest in study of the lacrimal glands functioning. However, the results of study of the Ca2+-signalling in lacrimal gland have not yet been summarized in the literature. This review is devoted to analysis of functioning of Ca2+-transport system of secretory cells in the rat exorbital lacrimal gland.
IP3Rs in exorbital lacrimal gland cells were effectively inhibited by 2-APB (10 μM) and activated by IP3 (2 μM), as well as by cholinomimetics, carbacholine (10 μM) and purine receptor agonists, ATP (1 mM). Signaling pathways activated by P2Y-receptors in the lacrimal gland secretory cells were partially mediated by IP3R activation. RyRs of lacrimal gland secretory cells were activated by Ca2+ and low concentrations of ryanodine (0,05–1 μM). Simultaneous activation of RyRs and IP3Rs in these cells caused Ca2+ release from the same store. Ca2+ mobilization from the intracellular stores induced by carbacholine (10 μM) or thapsigargin (1 μM) caused store-operated Ca2+ entry in secretory cells of the studied glands, which was partially inhibited by 2-APB.
SERCA of exorbital lacrimal gland cells was efficiently inhibited by eosin Y (5–10 μM) and by thapsigrgin (1 μM). In contrast to permeabilized cells, the Ca2+ content in intact cells did not change under the influence of eosin Y (5–20 μM), indicating the inhibition of PMCA. Ca2+-ATPase activity of permeabilized cells of studied glands depended on the incubation time, the substrate amount and Ca2+ concentration in the incubation medium.
Mitochondrial Ca2+-uniporter of lacrimal gland secretory cells was inhibited by ruthenium red (10 μM). Effects of ryanodine and ruthenium red on the Ca2+ content in cells were statistically significantly non-additive. Furthermore, ryanodine at concentrations of 1–3 μM caused a dose-dependent decrease in the respiration rate of the studied cells and this effect persisted after cells preincubation with ruthenium red or thapsigargin. This suggests that in addition to the endoplasmic reticulum RyRs activation, ryanodine inhibited the Ca2+ transport to the mitochondrial matrix, which was insensitive to the ruthenium red.
Keywords: lacrimal glands, IP3Rs, RyRs, SOCC, SERCA, Ca2+-uniporter.
... Extracellular nucleotides have been recognized as important mediators in many systems, where they trigger different responses via activation of plasma membrane receptors known as P2 receptors[1]. The different subclasses of P2 receptors have been identified on a wide variety of cell types: muscle, endothelial, endocrine and others; including cells of the immune system: lymphocytes, neutrophils, macrophages, mast cells[2]; as well as eosinophils [3,4]. ...
ATP and other nucleotides are released from cells through regulated pathways or following the loss of plasma membrane integrity. Once outside the cell, these compounds can activate P2 receptors: P2X ionotropic receptors and G protein-coupled P2Y receptors. Eosinophils represent major effector cells in the allergic inflammatory response and they are, in fact, associated with several physiological and pathological processes. Here we investigate the expression of P2 receptors and roles of those receptors in murine eosinophils. In this context, our first step was to investigate the expression and functionality of the P2X receptors by patch clamping, our results showed a potency ranking order of ATP>ATPγS> 2meSATP> ADP> αβmeATP> βγmeATP>BzATP> UTP> UDP>cAMP. This data suggest the presence of P2X1, P2X2 and P2X7. Next we evaluate by microfluorimetry the expression of P2Y receptors, our results based in the ranking order of potency (UTP>ATPγS> ATP > UDP> ADP >2meSATP > αβmeATP) suggests the presence of P2Y2, P2Y4, P2Y6 and P2Y11. Moreover, we confirmed our findings by immunofluorescence assays. We also did chemotaxis assays to verify whether nucleotides could induce migration. After 1 or 2 hours of incubation, ATP increased migration of eosinophils, as well as ATPγS, a less hydrolysable analogue of ATP, while suramin a P2 blocker abolished migration. In keeping with this idea, we tested whether these receptors are implicated in the migration of eosinophils to an inflammation site in vivo, using a model of rat allergic pleurisy. In fact, migration of eosinophils has increased when ATP or ATPγS were applied in the pleural cavity, and once more suramin blocked this effect. We have demonstrated that rat eosinophils express P2X and P2Y receptors. In addition, the activation of P2 receptors can increase migration of eosinophils in vitro and in vivo, an effect blocked by suramin.
... As now well documented, high levels (millimolar range) of extracellular nucleotides, including ATP, may be Time (min) leased in vivo under pathological conditions such as inflammation , trauma and stress (Di Virgilio et al., 1998; Nieber et al., 1999; Lazarowski et al., 2000). In these conditions, released ATP originates from activated microglia , endothelial cells, astrocytes, neurons and damaged cells (Dubyak and Moatassim, 1993; Ferrari et al., 1997; Bodin and Burnstock, 1998; Cotrina et al., 1998; Neary et al., 1999; James and Butt, 2002). Signaling via purinergic receptors may thus allow astrocytes to sense and respond to increase in [ATP] o , for instance by modulating the transcription of genes involved in cellular inflammatory responses and thereby regulating cytokine responses (Liu et al., 2000). ...
Nucleotides are signaling molecules involved in variety of interactions between neurons, between glial cells as well as between neurons and glial cells. In addition, ATP and other nucleotides are massively released following brain insults , including inflammation, and may thereby be involved in mechanisms of cerebral injury. Recent concepts have shown that in astrocytes intercellular communication through gap junctions may play an important role in neuroprotection. Therefore , we have studied the effects of nucleotides on gap junction communication in astrocytes. Based on measurement of inter-cellular dye coupling and recording of junctional currents, the present study shows that ATP (10 –100 M) induces a rapid and a concentration-dependent inhibition of gap junction communication in cultured cortical astrocytes from newborn mice. Effects of agonists and antagonists of purinergic receptors indicate that the inhibition of gap junctional communication by ATP mainly involves the stimulation of metabotropic purinergic 1 (P2Y 1) receptors. Pretreatment with the pro-inflammatory cyto-kine interleukin-1 (10 ng/ml, 24 h), which has no effect by itself on gap junctional communication, increases the inhibitory effect of ATP and astrocytes become sensitive to uridine 5-triphosphate (UTP). As indicated by the enhanced expression of P2Y 2 receptor mRNA, P2Y 2 receptors are responsible for the increased responses evoked by ATP and UTP in interleukin-1-treated cells. In addition, the effect of endothelin-1, a well-known inhibitor of gap junctional communication in astrocytes was also exacerbated following interleukin-1 treatment. We conclude that ATP decreases intercellular communication through gap junctions in astrocytes and that the increased sensitivity of gap junction channels to nucleotides and endothelin-1 is a characteristic feature of astrocytes exposed to pro-inflammatory treatments.
... In the case of cerebral arterioles, the reaction of smooth muscle cells is different from that of testicular arterioles. The P2Y purinoceptors are comprised of seven membrane-spanning receptors, which are coupled to G proteins and result in the mobilization of [Ca 2+ ] i from internal Ca 2+ stores (Dubyak 1991; Burnstock 1996; Fredholm et al. 1997). In the present study, the inhibition of Ca 2+ influx failed to abolish Ca 2+ -related responses to ATP, and the depletion of Ca 2+ stores failed to inhibit the response. ...
The regulation of cytosolic Ca(2+) homeostasis is essential for cells, including vascular smooth muscle cells. Arterial tone, which underlies the maintenance of peripheral resistance in the circulation, is a major contributor to the control of blood pressure. Confocal microscopy was employed to study the alteration in intracellular calcium ion concentration ([Ca(2+)](i)) in arterioles (external diameters <100 microm) with respect to selected modifying reagents. 5-Hydroxytryptamine (1 microM), ATP (10 microM), and endothelin 1-3 (5 nM) elicited an increase in [Ca(2+)](i) in most arteriole smooth muscle cells. The [Ca(2+)](i) increase sometimes propagated in an intercellular manner. When noradrenaline (10 microM) was used as a stimulant, [Ca(2+)](i) increase was observed only in a portion of the smooth muscle cells. It was also noted that the reaction of these cells with respect to ATP is different between testis and brain arterioles; the [Ca(2+)](i) increase in testicular arterioles is dependent on Ca(2+) influx from extracellular space, whereas in cerebral arterioles it plays a role in both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores (i.e., sarco/endoplasmic reticulum). These results indicate that arterioles in different tissues may differ greatly in their responses. Real-time confocal microscopy was found to be a useful tool for investigating the structural and functional changes in living tissues.
... It is now widely recognized that adenosine 5 -triphosphate (ATP), a ubiquitous compound, functions as a neurotransmitter or cotransmitter, when released from nerve endings that innervate a variety of tissues, including smooth muscles12345. In addition, it has been proposed that ATP is involved in specific extracellular signaling actions, thus, playing a role in the regulation of a variety of functions (e.g. ...
Adenosine 5'-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca(2+)](i)) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca(2+)](i) in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca(2+)](i) increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca(2+)](i) dynamics in both arteriole smooth muscle cells. In testicular arterioles, Ca(2+) channel blockers and the removal of extracellular Ca(2+), but not thapsigargin pretreatment, abolished the ATP-induced [Ca(2+)](i) dynamics. In contrast, Ca(2+) channel blockers and the removal of extracellular Ca(2+) did not completely inhibit ATP-induced [Ca(2+)](i) dynamics in cerebral arterioles. Uridine 5'-triphosphate caused an increase in [Ca(2+)](i) only in cerebral arterioles and alpha,beta-methylene ATP caused an increase in [Ca(2+)](i) in both testicular and cerebral arterioles. We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.
... For a positive control for [Ca 2 ] cyt in human granulosa cells, we used ATP, which regulates a variety of biological processes [33] and elicits [Ca 2 ] i changes in human granulosa-lutein cells [32] . ATP colocalizes with neurotransmitters at concentrations 100 mM and is coreleased with both adrenaline and acetylcholine [33, 34]. ExtracellularFIG. ...
... The amounts of the secreted enzymes appeared to correlate with the level of cytotoxicity exhibited by these cell-free supernatant samples, suggesting that the secreted ATPutilizing enzymes allowed biotransformation of ATP to various adenine nucleotides that in turn activated P2- purinergic receptors on the surface of the macrophage. Activation of P2-purinergic receptors (Dubyak & El-Moatassim, 1993), particularly the P2Z (P2X ( ...
A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium. These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium. Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5'-nucleotidase (ATPase and/or phosphatase), were detected in the medium. These ATP-utilizing enzymes are believed to convert external ATP, presumably effluxed from macrophages, to various adenine nucleotides, which then activate purinergic receptors such as P2Z, leading to enhanced macrophage cell death. Pretreatment of macrophages with periodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z receptor activation, prevented subsequent ATP-induced macrophage cell death. A second type of cytotoxic factor(s) operated in an ATP-independent manner such that it triggered activation of apoptotic processes in macrophages, leading to proteolytic conversion of procaspase-3 to active caspase-3. This cytotoxic factor(s) did not appear to act on procaspase-3 present in macrophage cytosolic extracts. Intact macrophages, when exposed to the cytotoxic factor(s) for 6–16 h, underwent apoptosis and demonstrated the presence of active caspase-3 in their cytosolic extracts. Interestingly, two redox proteins, azurin and cytochrome c 551 , were detected in the cytotoxic preparation. When cell-line-derived or peritoneal macrophages or mast cells were incubated overnight with Q-Sepharose column flow-through fraction or with a mixture of azurin and cytochrome c 551 , they underwent extensive cell death due to induction of apoptosis.
... Additionally , extracellular signals such as ATP dynamically reorganize the cytoskeleton of each migrating neuroblast and regulate chemotaxis of microglia via Gi/o-coupled P2Y re- ceptors [67, 68] . Early studies showed that electrical stimulation of axons liberated ATP [69, 70] and that extracellular ATP induces excitation and increases in calcium in neurons [49, 50, 71, 72], Erk activation [73], and calcium wave propagation [74]. This calcium signalling is essential for directed cell migration induced by an applied EF [75]. ...
Neuroblasts migrate as directed chains of cells during development and following brain damage. A fuller understanding of the mechanisms driving this will help define its developmental significance and in the refinement of strategies for brain repair using transplanted stem cells. Recently, we reported that in adult mouse there are ionic gradients within the extracellular spaces that create an electrical field (EF) within the rostral migratory stream (RMS), and that this acts as a guidance cue for neuroblast migration. Here, we demonstrate an endogenous EF in brain slices and show that mimicking this by applying an EF of physiological strength, switches on chain migration in mouse neurospheres and in the SH-SY5Y neuroblastoma cell line. Firstly, we detected a substantial endogenous EF of 31.8 ± 4.5 mV/mm using microelectrode recordings from explants of the subventricular zone (SVZ). Pharmacological inhibition of this EF, effectively blocked chain migration in 3D cultures of SVZ explants. To mimic this EF, we applied a physiological EF and found that this increased the expression of N-cadherin and β-catenin, both of which promote cell-cell adhesion. Intriguingly, we found that the EF up-regulated P2Y purinoceptor 1 (P2Y1) to contribute to chain migration of neuroblasts through regulating the expression of N-cadherin, β-catenin and the activation of PKC. Our results indicate that the naturally occurring EF in brain serves as a novel stimulant and directional guidance cue for neuronal chain migration, via up-regulation of P2Y1.
Electronic supplementary material
The online version of this article (doi:10.1007/s12015-014-9524-1) contains supplementary material, which is available to authorized users.
... The metabotropic P2Y receptors belonging to the G-protein-coupled receptor (GPCR) family play important roles in several signaling pathways, and eight P2Y receptors have been cloned and identified as GPCRs in mammals [2,9]. Although P2Y receptors are distributed in a wide range of normal tissues, P2X receptors are mainly expressed in the nervous system, platelets, and smooth muscle cells (SMCs) [10,11]. P2Y2 has often been reported to be a functional receptor that transduces several biological signals induced by ATP and UTP, studies largely conducted in normal cells, such as epithelial cells, smooth muscle cells, leukocytes, and nerve cells. ...
Background
Nasopharyngeal carcinoma (NPC) is a common malignant tumor observed in the populations of southern China and Southeast Asia. However, little is known about the effects of purinergic signal on the behavior of NPC cells. This study analyzed the effects of ATP on the growth and migration of NPC cells, and further investigated the potential mechanisms during the effects.
Methods
Cell viability was estimated by MTT assay. Transwell assay was utilized to assess the motility of NPC cells. Cell cycle and apoptosis were detected by flow cytometry analysis. Changes in OPN, P2Y2 and p65 expression were assessed by western blotting analysis or immunofluorescence. The effects of ATP and P2Y2 on promoter activity of OPN were analyzed by luciferase activity assay. The binding of p65 to the promoter region of OPN was examined by ChIP assay.
Results
An MTT assay indicated that ATP inhibited the proliferation of NPC cells in time- and dose-dependent manners, and a Transwell assay showed that extracellular ATP inhibited the motility of NPC cells. We further investigated the potential mechanisms involved in the inhibitory effect of extracellular ATP on the growth of NPC cells and found that extracellular ATP could reduce Bcl-2 and p-AKT levels while elevating Bax and cleaved caspase-3 levels in NPC cells. Decreased levels of p65 and osteopontin were also detected in the ATP-treated NPC cells. We demonstrated that extracellular ATP inhibited the growth of NPC cells via p65 and osteopontin and verified that P2Y2 overexpression elevated the inhibitory effect of extracellular ATP on the proliferation of NPC cells. Moreover, a dual luciferase reporter assay showed that the level of osteopontin transcription was inhibited by extracellular ATP and P2Y2. ATP decreased the binding of p65 to potential sites in the OPN promoter region in NPC cells.
Conclusion
This study indicated that extracellular ATP inhibited the growth of NPC cells via P2Y2, p65 and OPN. ATP could be a promising agent serving as an adjuvant in the treatment of NPC.
... This cell permeabilization process was considered to be due to the opening of a non-selective " large pore " in contrast to the cationic channels that formed the P2X receptor subtype [15]. The P2Z receptor in macrophage and lymphocytes became a potential antiinflammatory drug target by the early 1990s with studies by the groups of Chaplin [16], Gabel171819, Di Virgilio2021222324, and Dubyak [12,2526272829 demonstrating that ATP, most likely acting on P2Z receptors, was the most potent physiological stimulus for the rapid release of the proinflammatory cytokine, interleukin-1β (IL-1β), from activated monocytes and macrophages. Soon after the Glaxo- Geneva group discovered the molecular identity of the P2Z receptor as the P2X 7 receptor (P2X 7 R) in 1996/1997 [30, 31], high throughput screening on heterologously expressed, or endogenous, P2X 7 receptors using dye uptake assays was begun by virtually all Big Pharma companies [32, 33]. ...
The P2X7 receptor (P2X7R) is uniquely associated with two distinct cellular responses: activation of a dye-permeable pathway allowing passage of molecules up to 900 Da and rapid release of the pro-inflammatory cytokine, interleukin-1β (IL-1β), from activated macrophage. How this dye uptake path forms and whether it is involved in IL-1β release has not been known. Pannexin-1 is a recently identified protein found to physically associate with the P2X7R. Inhibition of pannexin-1 does not alter P2X7R ion channel activation or associated calcium flux but blocks one component of P2X7R-induced dye uptake and unmasks a slower, previously undetected, dye uptake pathway. Inhibition of pannexin-1 blocks P2X7R-mediated IL-1β release from macrophage as well as release mediated by other stimuli which couple to activation of capase-1 and additionally inhibits the release of interleukin-1α, a member of the IL-1 family whose processing does not require caspase-1 activation. Thus, pannexin-1 is linked to both dye uptake and IL-1β release but via distinct mechanisms.
... Next, Ca 2+ homeostasis of LPS-stimulated EA.hy926 cells was studied using ATP or TG to elicit mobilization of intracellular and extracellular calcium pools. ATP releases Ca 2+ by activating inositol triphosphate receptors (IP 3 R) (Dubyak & el-Moatassim, 1993; Ralevic & Burnstock, 1998), while TG inhibits SERCA (Thastrup et al., 1990) and allows Ca 2+ to leak out of the endoplasmic reticulum. In the presence of extracellular Ca 2+ , the common cellular response to ATP or TG was biphasic with an initial rapid increase in [Ca 2+ ] i followed by a plateau phase of elevated [Ca 2+ ] i . ...
Recent evidence showed that 17 β-estradiol (E2) decreased cytokine-induced expression of cell adhesion molecules (CAM). Changes in intracellular Ca2+ concentration ([Ca2+]i) has been shown to be associated with CAM expression in endothelial cells. Here, the effects of E2 (1μM, 24h) on the expression of intracellular adhesion molecule-1 (ICAM-1) and [Ca2+]i were investigated in a lipopolysaccharide (LPS) (100ng/mL, 18h)-stimulated human endothelial cell line, EA.hy926, using real-time PCR and spectrofluorometry, respectively. PCR analysis revealed a significant increase in ICAM-1 expression in calcium ionophore A23187 (1nM)- or LPS-stimulated cells. Pretreatment of cells with E2 significantly inhibited LPS-induced ICAM-1 mRNA expression. [Ca2+]i was monitored in Fura-2AM-loaded cells in the presence and absence of extracellular Ca2+ with thapsigargin (TG, 1μM), a sarco/endoplasmic reticulum ATPase inhibitor or ATP (100μM). The extent of TG- or ATP-induced [Ca2+]i increase was significantly higher in LPS-stimulated cells than in control cells. Pre-treatment of LPS-stimulated cells with E2 limited the Ca2+ response to the same level as in control cells. Furthermore, ICI 182,780, an estrogen receptor antagonist, attenuated the inhibitory actions of E2 on ICAM-1 mRNA expression and Ca2+ responses, suggesting that estrogen receptors mediate, at least in part, the effects of estrogen. These data suggest a potential underlying mechanism for the protective effect of E2 against atherosclerosis.
... In the present work we have shown that ATP were able to inhibit the aggregation of platelets in response to thrombin. The concentration of ATP in platelet granules is very high (between 100 and 400 mM) [34,35] and the degranulation of platelets transiently increases the concentration of ATP in a range between 50 and 100 mM [36,37]. Thus, the local concentration of ATP is probably much higher than the plasma concentration following platelet activation by thrombin [38]. ...
In this study we have investigated the role of extracellular ATP on thrombin induced-platelet aggregation (TIPA) in washed human platelets. ATP inhibited TIPA in a dose-dependent manner and this inhibition was abolished by apyrase but not by adenosine deaminase (ADA) and it was reversed by extracellular magnesium. Antagonists of P2Y1 and P2Y12 receptors had no effect on this inhibition suggesting that a P2X receptor controlled ATP-mediated TIPA inhibition. ATP also blocked inositol phosphates (IP1, IP2, IP3) generation and [Ca(2+)]i mobilization induced by thrombin. Thrombin reduced cAMP levels which were restored in the presence of ATP. SQ-22536, an adenylate cyclase (AC) inhibitor, partially reduced the inhibition exerted by ATP on TIPA. 12-lipoxygenase (12-LO) inhibitors, nordihidroguaretic acid (NDGA) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE), strongly prevented ATP-mediated TIPA inhibition. Additionally, ATP inhibited the increase of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) induced by thrombin. Pretreatment with both SQ-22536 and NDGA almost completely abolished ATP-mediated TIPA inhibition. Our results describe for the first time that ATP implicates both AC and 12-LO pathways in the inhibition of human platelets aggregation in response to agonists.
Since their discovery in the 1970s, purinergic receptors have been shown to play key roles in a wide variety of biologic systems and cell types. In the immune system , purinergic receptors participate in innate immunity and in the modulation of the adaptive immune response. In particular, P2 receptors, which respond to extra-cellular nucleotides, are widely expressed on leukocytes, causing the release of cytokines and chemokines and the formation of inflammatory mediators, and inducing phagocytosis, degranulation, and cell death. The activity of these receptors is regulated by ectonucleotidases-expressed in these same cell types-which regulate the
Geoffrey Burnstock is a purinergic signalling legend who’s discoveries and conceptualisation created and shaped the field. His scientific achievements were extraordinary and sustained. They included his demonstration that ATP can act as a neurotransmitter and hence extracellular signalling molecule, which he championed despite considerable initial opposition to his proposal that ATP acts outside of its role as an energy source inside cells. He led on purine receptor classification: initially of the P1 and P2 receptor families, then the P2X and P2Y receptor families, and then subtypes of P2X and P2Y receptors. This was achieved across several decades as he conceptualised and made sense of the emerging and growing evidence that there were multiple receptor subtypes for ATP and other nucleotides. He made discoveries about short term and long term/trophic purinergic signalling. He was a leader in the field for over 50 years. He inspired many and was a great colleague and mentor. I had the privilege of spending over 10 years (from 1985) with Geoff at the Department of Anatomy and Developmental Biology, University College London. This review is a personal perspective of some of Geoff’s research on P2 receptors carried out during that time. It is a tribute to Geoff who I regarded with enormous respect and admiration.
Pannexin 1 (Panx1) channels are widely recognized for their role in ATP release, and as follows, their function is closely tied to that of ATP-activated P2X7 purinergic receptors (P2X7Rs). Our recent work has shown that extracellular ATP induces clustering of Panx1 with P2X7Rs and their subsequent internalization through a non-canonical cholesterol-dependent mechanism. In other words, we have demonstrated that extracellular ATP levels can regulate the cell surface expression of Panx1. Here we discuss two situations in which we hypothesize that ATP modulation of Panx1 surface expression could be relevant for central nervous system function. The first scenario involves the development of new neurons in the ventricular zone. We propose that ATP-induced Panx1 endocytosis could play an important role in regulating the balance of cell proliferation, survival, and differentiation within this neurogenic niche in the healthy brain. The second scenario relates to the spinal cord, in which we posit that an impairment of ATP-induced Panx1 endocytosis could contribute to pathological neuroplasticity. Together, the discussion of these hypotheses serves to highlight important outstanding questions regarding the interplay between extracellular ATP, Panx1, and P2X7Rs in the nervous system in health and disease.
The flow of intracellular calcium (Ca²⁺) is critical for the activation and regulation of important biological events that are required in living organisms. As the major Ca²⁺ repositories inside the cell, the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of muscle cells are central in maintaining and amplifying the intracellular Ca²⁺ signal. The morphology of these organelles, along with the distribution of key calcium-binding proteins (CaBPs), regulatory proteins, pumps, and receptors fundamentally impact the local and global differences in Ca²⁺ release kinetics. In this review, we will discuss the structural and morphological differences between the ER and SR and how they influence localized Ca2⁺ release, related diseases, and the need for targeted genetically encoded calcium indicators (GECIs) to study these events.
The P2X7 receptor is expressed in both anterior and posterior segments of the eyeball. In the ocular surface, the P2X7 receptor is activated in case of external aggressions: preservatives and surfactants induce the activation of P2X7 receptors, leading to either apoptosis, inflammation, or cell proliferation. In the retina, the key endogenous actors of age-related macular degeneration, diabetic retinopathy, and glaucoma act through P2X7 receptors' activation and/or upregulation of P2X7 receptors' expression. Different therapeutic strategies aimed at the P2X7 receptor exist. P2X7 receptor antagonists, such as divalent cations and Brilliant Blue G (BBG) could be used to target either the ocular surface or the retina, as long as polyunsaturated fatty acids may exert their effects through the disruption of plasma membrane lipid rafts or saffron that reduces the response evoked by P2X7 receptor stimulation. Treatments against P2X7 receptor activation are proposed by using either eye drops or food supplements.
Background:
The mechanisms underlying the onset of labor are not fully understood. Extracellular adenosine 5'-triphosphate (ATP) is known to cause uterine contractions in different species but the exact underlying mechanisms are poorly investigated to date. The aims of this study were to investigate the effect of extracellular ATP on spontaneous uterine contractions from different gestational stages and to elucidate its possible underlying mechanisms.
Methods:
Longitudinal uterine strips were obtained from rats in different gestational stages (nonpregnant, late-pregnant, and term-pregnant). The effects of 1 mM ATP were examined on uterine contractions generated spontaneously, depolarized by high-KCl (60 mM), induced by oxytocin (5 nM), in the presence of high external Ca2+, or in the absence of external Ca2+.
Results:
Application of 1 mM extracellular ATP significantly increased the force of spontaneous contraction in uterine strips obtained from all gestational stages with prominent increase in term-pregnant rats compared to other gestations. ATP significantly increased the force induced by depolarization (122%, p=0.010, n=6), oxytocin (129%, p=0.001, n=7), high-Ca2+ (145%, p=0.005, n=6) and it was able to cause transient contraction in the absence of external Ca2+ (33%, p<0.01).
Conclusions:
Extracellular ATP is able to increase the force and frequency of uterine contractions and its effect increases with the progression of pregnancy and it involves Ca2+ influx and release. These findings open a new window for clinicians to consider ATP as a therapeutic target to control the uterine activity during difficult labors.
The control of venous smooth muscle tone and venous function is dominated by the sympathetic nervous system, its activity being coordinated in the vasomotor center in the brainstem. The degree of adrenergic innervation of the veins varies widely; cutaneous and splachnic veins are most densely innervated, while large conduit veins and the deep limb veins receive only scarce adrenergic innervation (396).
Osteoarthritis (OA) is a degenerative and progressive disease characterized by cartilage breakdown and by synovial membrane inflammation, which results in disability, joint swelling, and pain. The purinergic P2X3 and P2X2/3 receptors contribute to development of inflammatory hyperalgesia, participate in arthritis processes in the knee joint, and are expressed in chondrocytes and nociceptive afferent fibers innervating the knee joint. In this study, we hypothesized that P2X3 and P2X2/3 receptors activation by endogenous ATP (adenosine 5′-triphosphate) induces articular hyperalgesia in the knee joint of male and female rats through an indirect sensitization of primary afferent nociceptors dependent on the previous release of pro-inflammatory cytokines and/or on neutrophil migration. We found that the blockade of articular P2X3 and P2X2/3 receptors significantly attenuated carrageenan-induced hyperalgesia in the knee joint of male and estrus female rats in a similar manner. The carrageenan-induced knee joint inflammation increased the expression of P2X3 receptors in chondrocytes of articular cartilage. Further, the blockade of articular P2X3 and P2X2/3 receptors significantly reduced the increased concentration of TNF-α, IL-6, and CINC-1 and the neutrophil migration induced by carrageenan. These findings indicate that P2X3 and P2X2/3 receptors activation by endogenous ATP is essential to hyperalgesia development in the knee joint through an indirect sensitization of primary afferent nociceptors dependent on the previous release of pro-inflammatory cytokines and/or on neutrophil migration.
In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 10(7) cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl2 stimulated the ATP hydrolysis to a greater extent compared with CaCl2 and ZnCl2. This activity was also observed when replaced by MnCl2. The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 10(7) cells/h. The apparent K m for ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg(2+)-dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.
Background:
Enhanced expression of the purinergic P2X7 receptor (P2X7R) occurs in several neuroinflammatory conditions where increased microglial activation is a co-existing feature. P2X7 receptors can function either as a cation channel or, upon continued stimulation, a large pore. P2X7R-over-expression alone is sufficient to drive microglial activation and proliferation in a process that is P2X7R pore dependent, although the biological signaling pathway through which this occurs remains unclear. Once activated, microglia are known to release a number of bioactive substances that include the proinflammatory cytokine interleukin-1β (IL-1β). Previous studies have linked P2X7R stimulation to the processing and release of IL-1β, but whether the channel or pore state of P2X7R is predominant in driving IL-1β release is unknown and is a major aim of this study. In addition, we will determine whether IL-1β has trophic effects on surrounding microglia.
Methods:
Electron microscopy and immunohistochemistry were used to delineate the sub-cellular localization of P2X7R and IL-1β in primary hippocampal rat cultures. FM1-43 fluorescent dye and confocal microscopy were used to quantify vesicular exocytosis from microglia expressing the pore-forming P2X7R versus a non-pore-forming point mutant, P2X7RG345Y. IL-1β in culture was quantified with an enzyme-linked immunosorbent assay (ELISA). IL-1β intracellular processing was blocked with inhibition of caspase 1 (with a synthetic peptide antagonist), and its extracellular form was neutralized with an IL-1β neutralizing antibody. Microglial activation and proliferation was quantified immunohistochemically with confocal microscopy.
Results:
P2X7R and IL-1β were co-localized in lysosomes. Vesicular exocytosis was higher in microglia expressing the pore-forming P2X7R compared to those expressing the non-pore-forming mutant. There was increased IL-1β in cultures expressing the pore-forming P2X7R, and this proinflammatory cytokine was found to mediate the trophic effects of P2X7R pore in microglia. Inhibition of IL-1β production and function resulted in a significant decrease in P2X7R-mediated microglial activation and proliferation.
Conclusions:
IL-1β is a mediator of microglial activation and proliferation, and its release/production is P2X7R pore dependent. Blockade of P2X7R pore could serve as a therapeutic target in alleviating the degree of inflammation seen in neurodegenerative and neoplastic conditions.
With the unequivocal demonstration that purine and pyrimidine nucleosides and nucleotides function as key extracellular messengers in all mammalian tissue systems (Ralevic and Burnstock 1998), a major challenge has been to understand the factors that govern purine availability in the extracellular space and the dynamics of the process. The focus of the present chapter is to summarize recent knowledge on purine release under various physiological and pathophysiological conditions.
The existence of cell surface receptors for extracellular adenine nucleotides in mammalian cells has been postulated for decades (Burn-stock, 1972), based on scores of reports on diverse responses to these molecules in a wide variety of tissues and cell types (Dubyak and El-Moatassim, 1993). Pharmacological studies have provided evidence for the expression of two major types of nucleotide receptors: those belonging to the G protein-coupled receptor family and those belonging to the ligand-gated ion channel family. Nucleotide receptors (P2 purinergic receptors) are distinct from P1 receptors for adenosine, and, from results of earlier studies, were thought to be selective for purine nucleotides, in particular adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP) and various analogs, including 2MeSATP, α,β-MeATP, and β,γ-MeATP. Recent studies have demonstrated that uridine nucleotides are equally or more effective than adenine nucleotides in activating several G protein-coupled P2 receptor subtypes, raising a question as to the appropriateness of referring to this family of receptors as “purinergic.” Nonetheless, nucleotide receptors have retained the designation “P2,” which now refers to both their purine- and/or pyrimidine-based nucleotide agonists.
Purines are substantially involved in postjunctional and prejunctional neuromodulation in the cardiovascular system. Co-stored with noradrenaline and acetylcholine in sympathetic and parasympathetic nerves, ATP may be differentially released in response to nerve stimulation. ATP released from the nerve terminals of sympathetic neurones may contribute to neurogenic smooth muscle contraction through activation of P2X1 receptors, or alternatively induce blood vessel relaxation through endothelial P2Y1 receptors. ATP also induces ATP liberation from smooth muscle and endothelial cells, possibly reflecting a feedforward mechanism. Presynaptically, ATP may modulate transmitter release via P2 autoreceptors. Adenosine generated by the breakdown of ATP or arising from other sources has direct vasodilator effects in all vascular beds except in the kidney and the placenta. Adenosine is able to inhibit transmitter release via prejunctional A1 receptors thereby diminishing α1 adrenoceptor-mediated vasoconstriction. Presumably, not only adenosine but also ATP inhibits transmitter release through activation of presynaptic A1 receptors which might explain various observations previously ascribed to the involvement of a hybrid P3 receptor.
Adenosine 5'-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺](i)) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺](i) in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺](i) increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺](i) increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺](i), whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺](i). A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺](i), although UTP (a P2Y₂,₄,₆ receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP > UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺](i), but α,β-meATP (a P2X₁,₃ receptor agonist) had no effect. RT-PCR indicated that P2X₂,₃,₄,₅,₆,₇ and P2Y₁,₂,₄,₁₂,₁₄ are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.
Gentherapie und Zellersatz im zentralen Nervensystem (ZNS) werden durch die Blut-Hirn-Schranke behindert, die den Übertritt von Plasmabestandteilen und Zellen in das ZNS limitiert. Genetisch modifizierte Knochenmarkszellen können jedoch ungeachtet der intakten Blut-Hirn-Schranke in das ZNS einwandern und dort zu Mikroglia differenzieren. Neurone signalisieren eine Schädigung an benachbarte Glia und rekrutieren Zellen hämatopoetischen Ursprungs gezielt an den Ort der Läsion. Aus adulten Stammzellen des Knochenmarks können schliesslich Nervenzellen entstehen, die in die komplexe Architektur des ZNS integriert werden. Die Befunde liefern neue Ansätze für die Therapie von ZNS-Erkrankungen.
Cells of the macrophage lineage express a peculiar surface receptor for extracellular ATP, designated P2Z/P2X7 purinergic receptor, that induces pore formation and collapse of the plasma membrane potential. Although the function of the P2Z receptor is largely unknown, accumulating evidence implicates its role in cell signaling and immune reactions. Here, we investigated the effect of P2Z receptor ligation on the activation of NF-κB, a transcription factor controlling cytokine expression and apoptosis. Exposure of microglial cells to ATP but not other nucleotides resulted in potent NF-κB activation. This effect was specifically mediated by the P2Z receptor, because selective receptor antagonists prevented NF-κB activation. NF-κB activation required reactive oxygen intermediates and proteases of the caspase family, because it was abolished by antioxidants and specific protease inhibitors. The subunit composition of the ATP-induced NF- κB–DNA complex was rather unusual. Whereas exposure to LPS-induced prototypical NF-κB p50 homo- and p65 (RelA)/p50 heterodimers, ATP stimulation resulted in the sole appearance of a p65 homodimer. This is the first demonstration that a certain stimulus activates a particular NF-κB subunit. Because different NF-κB complexes exhibit distinct transcriptional and DNA-binding activities, ATP may control the expression of a subset of NF-κB target genes distinct from those activated by classical proinflammatory mediators.
To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl⁻ channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl⁻ currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56–80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl⁻ channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 μM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl⁻ channel blocker, 4-acetamido-4′-isothiocyanostilbene at 100 μM. Glibenclamide (500 μM) and arachidonic acid (100 μM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd³⁺, a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl⁻ currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach ∼13 μM by a biosensor technique using P2X2 receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl⁻ channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 μM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl⁻ channel; and third, the ATP release is not a prerequisite to activation of the Cl⁻ channel.
Identification of a G protein-coupled receptor activated by UDP-glucose led us to develop a sensitive and specific assay for UDP-glucose mass and to test whether this sugar nucleotide is released as an extracellular signaling molecule. Mechanical stimulation of 1321N1 human astrocytoma cells by a change of medium resulted in an increase in extracellular levels of both ATP and UDP-glucose. Whereas ATP levels peaked within 10 min and subsequently returned to resting extracellular levels of 3 nM, UDP-glucose levels attained a steady state that exceeded that of resting ATP levels by 3- to 5-fold for at least 3 h. Similar rates of basal release of UDP-glucose and ATP (72 and 81 fmol/min/10⁶ cells) combined with a rate of UDP-glucose metabolism approximately three times lower than ATP hydrolysis account for the elevated extracellular UDP-glucose levels on resting cells. A medium change also resulted in rapid appearance of UDP-glucose on the luminal surface of highly differentiated polarized human airway epithelial cells but at levels 2- to 3-fold lower than ATP. However, nucleotide sugar levels increased 3- to 5-fold over the ensuing 2 h, whereas ATP levels decayed to a resting level; consequently, resting extracellular UDP-glucose levels exceeded those of ATP by 5- to 10-fold. UDP-glucose also was observed at levels that equaled or exceeded those of ATP in the extracellular medium of Calu-3 airway epithelial, COS-7, CHO-K1, and C6 glioma cells. Consistent with the observation of significant extracellular UDP-glucose levels, expression of the UDP-glucose-activated P2Y14 receptor in COS-7 cells resulted in G protein-promoted inositol phosphate accumulation that was partially reversed by enzymatic removal of UDP-glucose from the medium. Taken together, these results indicate constitutive release of UDP-glucose from physiologically relevant tissues and suggest that UDP-glucose acts as an autocrine activator of the P2Y14receptor. Because cellular UDP-glucose is concentrated in the lumen of the endoplasmic reticulum, we speculate that UDP-glucose release may occur as a result of vesicle transport during trafficking of glycoproteins to the plasma membrane.
The cystic fibrosis gene product (CFTR) is a chloride channel which, once phosphorylated, is regulated by nucleotide phosphates (Anderson, M. P., and M. J. Welsh. 1992. Science. 257:1701-1704; Venglarik, C. J., B. D. Schultz, R. A. Frizzell, and R. J. Bridges. 1994. Journal of General Physiology. 104:123-146). Nucleotide triphosphates initiate channel activity, while nucleotide diphosphates and nonhydrolyzable ATP analogues do not. To further characterize the role of these compounds on CFTR channel activity we examined their effects on chloride channel currents in excised inside-out membrane patches from CFTR transfected mouse L cells. ADP competitively inhibited ATP-dependent CFTR channel gating with a Ki of 16 +/- 9 microM. AMP neither initiated CFTR channel gating nor inhibited ATP-dependent CFTR channel gating. Similarly, ATP analogues with substitutions in the phosphate chain, including AMPCPP, AMPPCP, AMPPNP, and ATP gamma S failed to support CFTR channel activity when present at the cytoplasmic face of the membrane and none of these analogues, when present at three to 10-fold excess of ATP, detectably altered ATP-dependent CFTR channel gating. These data suggest that none of these ATP analogues interact with the ATP regulatory site of CFTR which we previously characterized and, therefore, no inference regarding a requirement for ATP hydrolysis in CFTR channel gating can be made from their failure to support channel activity. Furthermore, the data indicate that this nucleotide regulatory site is exquisitely sensitive to alterations in the phosphate chain of the nucleotide; only a nonsubstituted nucleotide di- or triphosphate interacts with this regulatory site. Alternative recording conditions, such as the presence of kinase and a reduction in temperature to 25 degrees C, result in a previously uncharacterized kinetic state of CFTR which may exhibit distinctly different nucleotide dependencies.
ATP in quantity co-stored with neurotransmitters in the secretory vesicles of neurons, by being co-released with the neurotransmitters, takes an important role to modulate the stimulus-secretion response of neurotransmitters. Here, in this study, the modulatory effect of ATP was studied in Ca2+ channels of cultured rat adrenal chromaffin cells to investigate the physiological role of ATP in neurons. The Ca2+ channel current was recorded in a whole-cell patch clamp configuration, which was modulated by ATP. In 10 mM Ba2+ bath solution, ATP treatment (0.1 mM) decreased the Ba2+ current by an average of 36±6% (n=8), showing a dose-dependency within the range of 10-4~10-1 mM. The current was recovered by ATP washout, demonstrating its reversible pattern. This current blockade effect of ATP was disinhibited by a large prepulse up to +80 mV, since the Ba2+ current increment was larger when treated with ATP (37±5%, n=11) compared to the control (25±3%, n=12, without ATP). The Ba2+ current was recorded with GTPγS, the non-hydrolyzable GTP analogue, to determine if the blocking effect of ATP was mediated by G-protein. The Ba2+ current decreased down to 45% of control with GTPγS. With a large prepulse (+80 mV), the current increment was 34±4% (n=19), which 25±3% (n=12) under control condition (without GTPγS). The Ba2+ current waveform was well fitted to a single-exponential curve for the control, while a double-exponential curve best fitted the current signal with ATP or GTPγS. In other words, a slow activation component appeared with ATP or GTPγS, which suggested that both ATP and GTPγS caused slower activation of Ca2+ channels via the same mechanism. The results suggest that ATP may block the Ca2+ channels by G-protein and this Ca2+ channel blocking effect of ATP is important in autocrine (or paracrine) inhibition of adrenaline secretion in chromaffin cell.
We investigated the secretory profiles of prostanoids in two types of nonparenchymal cell from the rat liver, sinusoidal endothelial cells and Kupffer cells, in primary culture both under basal conditions and after stimulation with adenine nucleotides. Prostaglandin (PG) E2 was the main prostanoid secreted by both types of hepatic nonparenchymal cell in the basal and adenosine triphosphate (ATP)-stimulated states. Time- and concentration-dependent effects of ATP-mediated PGE2 secretion were noted in sinusoidal endothelial cells, whereas the profile of the relative potencies of individual nucleotides was consistent with the presence of P2y and P1 purinergic receptors. In Kupffer cells, the regulation of prostanoid secretion by adenine nucleotides was essentially the same as that in sinusoidal endothelial cells except that adenosine did not stimulate prostanoid secretion and that prostanoid secretion differed somewhat; Kupffer cells secreted relatively more PGF2α and less 6-keto-PGF1α than sinusoidal endothelial cells in the presence of ATP, suggesting the presence of only P2y receptors. In summary, PGE2 is the main prostanoid secreted by hepatic nonparenchymal cells and its secretion may be stimulated by adenine nucleotides and adenosine.
Extracellular adenosine-5′-triphosphate (ATP) triggers biological responses in a wide variety of cells and tissues and activates signaling cascades that affect cell membrane potential and excitability. It has been demonstrated that compressive loading promotes ATP production and release by intervertebral disc (IVD) cells, while a high level of extracellular ATP accumulates in the nucleus pulposus (NP) of the IVD. In this study, a noninvasive system was developed to measure ATP-induced changes in the membrane potential of porcine IVD cells using the potential sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS).The responses of NP and annulus fibrosus cells to ATP were examined in monolayer and 3-dimensional cultures. It was found that the pattern and magnitude of membrane potential change in IVD cells induced by extracellular ATP depended on cell type, culture condition, and ATP dose. In addition, gene expression of P2X4 purinergic receptor was found in both cell types. Inhibition of the ATP-induced response by pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonate, a non-competitive inhibitor of P2 receptors, suggests that ATP may modulate the biological activities of IVD cells via P2 purinergic receptors.
Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal (GI) motility. Purinergic neurotransmission, via P2Y1 receptors, mediates one phase of inhibitory neural control. For decades ATP has been assumed to be the purinergic neurotransmitter and smooth muscle cells (SMCs) have been considered the primary targets for inhibitory neurotransmission. Recent experiments have cast doubt upon both of these assumptions and suggested another cell type, PDGFRα(+) cells, as the target for purinergic neurotransmission. We compared responses of PDGFRα(+) cells and SMCs to several purine compounds to determine if these cells responded in a manner consistent with enteric inhibitory neurotransmission. ATP hyperpolarized PDGFRα(+) cells but depolarized SMCs. Only part of the ATP response in PDGFRα(+) cells was blocked by MRS2500, a P2Y1 antagonist. ADP, MRS2365, β-NAD and ADPR (P2Y1 agonists) hyperpolarized PDGFRα(+) cells, and these responses were blocked by MRS2500. ADPR was more potent in eliciting hyperpolarization responses than β-NAD. P2Y1 agonists failed to elicit responses in SMCs. Small hyperpolarization responses were elicited in SMCs by an SK channel agonist, CyPPA, consistent with the low expression and current density of SK channels in these cells. Large amplitude hyperpolarization responses, elicited in PDGFRα(+) cells but not SMCs by P2Y1 agonists, are consistent with the generation of inhibitory junction potentials in intact muscles in response to purinergic neurotransmission. The responses of PDGFRα(+) cells and SMCs to purines suggest that SMCs are unlikely targets for purinergic neurotransmission in colonic muscles.
Purinergic signalling plays major roles in the physiology and pathophysiology of digestive organs. Adenosine 5'-triphosphate (ATP), together with nitric oxide and vasoactive intestinal peptide, is a cotransmitter in non-adrenergic, non-cholinergic inhibitory neuromuscular transmission. P2X and P2Y receptors are widely expressed in myenteric and submucous enteric plexuses and participate in sympathetic transmission and neuromodulation involved in enteric reflex activities, as well as influencing gastric and intestinal epithelial secretion and vascular activities. Involvement of purinergic signalling has been identified in a variety of diseases, including inflammatory bowel disease, ischaemia, diabetes and cancer. Purinergic mechanosensory transduction forms the basis of enteric nociception, where ATP released from mucosal epithelial cells by distension activates nociceptive subepithelial primary afferent sensory fibres expressing P2X3 receptors to send messages to the pain centres in the central nervous system via interneurons in the spinal cord. Purinergic signalling is also involved in salivary gland and bile duct secretion.
Cystic fibrosis (CF), is an autosomal recessive disease frequently seen in the Caucasian population. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF is characterized by enhanced airway Na+ absorption, mediated by epithelial Na+ channels (ENaC), and deficient Cl− transport. In addition, other mechanisms may contribute to the pathophysiological changes in the CF lung, such as defective regulation of HCO3− secretion. In other epithelial tissues, epithelial Na+ conductance is either increased (intestine) or decreased (sweat duct) in CF. CFTR is a cyclic AMP-regulated epithelial Cl− channel, and appears to control the activity of several other transport proteins. Accordingly, defective epithelial ion transport in CF is likely to be a combination of defective Cl− channel function and impaired regulator function of CFTR, which in turn is linked to impaired mucociliary clearance and development of chronic lung disease. As the clinical course of CF is determined primarily by progressive lung disease, novel pharmacological strategies for the treatment of CF focus on correction of the ion transport defect in the airways.
In recent years, it has been demonstrated that activation of purinergic receptors in airway epithelia by extracellular nucleotides (adenosine triphosphate/uridine triphosphate) has beneficial effects on mucus clearance in CF. Activation of the dominant class of metabotropic purinergic receptors, P2Y2 receptors, appears to have a 2-fold benefit on ion transport in CF airways; excessive Na+ absorption is attenuated, most likely by inhibition of the ENaC and, simultaneously, an alternative Ca2+-dependent Cl− channel is activated that may compensate for the CFTR Cl− channel defect. Thus activation of P2Y2 receptors is expected to lead to improved hydration of the airway surface liquid in CF. Furthermore, purinergic activation has been shown to promote other components of mucociliary clearance such as ciliary beat frequency and mucus secretion. Clinical trials are under way to test the effect of synthetic purinergic compounds, such as the P2Y2 receptor agonist INS37217, on the progression of lung disease in patients with CF. Administration of these compounds alone, or in combination with other drugs that inhibit accelerated Na+ transport and help recover or increase residual activity of mutant CFTR, is most promising as successful therapy to counteract the ion transport defect in the airways of CF patients.
Among the variety of potential P2Y receptor subtypes that may be expressed in the nervous system, P2Y1 and P2Y2 receptors are the first to be cloned. A P2Y2 receptor is cloned from a NG108-15 neuroblastoma x glioma cell cDNA library by expression cloning in Xenopus luevis oocytes and functionally expressed in mammalian cells. The cloning and expression of P2Y receptors have provided the means to address the relevance of these receptors in neurological function. There is also a need to determine P2Y receptor subtype expression patterns in vivo under a variety of physiological and pathological conditions, including cell growth and differentiation, tissue damage and neurological disorders. Ultimately, P2Y receptors may prove to be ideal targets for drug therapies in the nervous system once a fuller understanding of the structural basis for receptor function has evolved. The delineation of P2Y receptor interactions leading to receptor desensitization, sequestration, and downregulation may help control these processes to increase the effectiveness of receptor agonists. The potential that P2Y2 receptors, in particular, may be localized to focal adhesions to participate in cell-to-cell communication suggests that these receptors may have functions yet to be recognized.
The effects of a hydroalcoholic extract of Agaricus brasiliensis (A. blazei) on functional parameters in the perfused rat liver were examined with emphasis on its content of nucleotides and nucleosides. Several nucleosides and nucleotides were identified in the A. brasiliensis extract, which was active on several liver functions. A significant part of the effects is the result of the purinergic action of nucleosides and nucleotides: pressure increment, glycogenolysis stimulation, transient inhibition of oxygen consumption, and redox state changes. Other phenomena such as the stimulation of gluco-neogenesis, ureogenesis, and oxygen consumption are more likely consequences of the metabolic transformation of substrates contained within the extract, especially amino acids. It seems apparent that consumption of A. brasiliensis represents not only the ingestion of metabolic precursors but also the ingestion of substances that, even at low concentrations, can exert important signaling functions in the liver as well as in the organism as a whole.
In the CNS, nucleotide receptors termed P2 receptors are identified on neurons and glial cells, mediating neuron–neuron, glia–glia and glia–neuron communication. In the present work, we qualify in vivo in the adult rat CNS the cellular/subcellular distribution of P2Y12 receptor protein in cerebral cortex, white matter and subcortical nuclei (striatum and substantia nigra), by means of immunofluorescence-confocal, electron microscopy and Western blot analysis. P2Y12 receptor immunoreactivity colocalizes neither with markers such as neuronal nuclei, neurofilament light chain, calbindin and tyrosine hydroxylase, nor with glial fibrillary acidic protein and isolectin B4, but with myelin basic protein and the oligodendrocyte marker RIP, in both cell bodies and processes, indicating therefore oligodendrocyte localization. Electron microscopy identifies P2Y12 receptors in both the perikaryon and under the plasmalemma of oligodendrocyte cell bodies and radiating processes, until the paranodal region of fibers. By Western blot analysis, P2Y12 receptor shows a specific band of 42–44 kDa, matching the molecular mass predicted from amino acid sequencing. Since in platelets P2Y12 receptor is known to regulate adhesion/activation and thrombus growth/stability, from our results we could speculate by analogy that, in oligodendrocytes, P2Y12 receptor signaling might contribute to the migration and adhesion of the glial processes to axons to be myelinated.
ATP can activate both G-protein-coupled signaling systems or ATP-gated channels by stimulating either P2Y-family or P2X-family receptors. Our studies indicate that different P2Y- and P2X-family receptors are expressed in mature human phagocytic leukocytes and in myeloid progenitor cells. The P2Y1 receptor is functionally expressed only in early myeloid progenitor cells, whereas the P2U/P2Y2 receptor is expressed in late-stage progenitor cells and in mature monocytes and neutrophils. The P2X1 receptor is not expressed in early myeloid progenitors, but it is abundantly expressed when these cells commit to monocytic differentiation. However, the expression of P2X1 and P2U/P2Y2 receptors is significantly down-regulated when myeloid leukocytes are activated with proinflammatory cytokines. In contrast, these same inflammatory stimuli induce a large up-regulation of P2Z/P2X7 receptor expression. We propose that the differential expression of ATP receptor subtypes in various myeloid leukocyte populations indicates that these cells utilize locally released ATP and particular ATP receptors for distinct types of autocrine and paracrine communication. Drug Dev. Res. 39:269–278, 1996. © 1997 Wiley-Liss, Inc.
The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
To develop a new approach to the treatment of advanced, hormone-refractory prostate cancer, the signal transductions regulating the growth of human androgen-independent prostate carcinoma cell lines were studied. Agonist-stimulated Ca2+ mobilization, a critical regulatory event in other secretory cell types, was studied as a means of identifying previously undescribed plasma membrane receptors that may transduce a growth inhibitory signal. In all of the cell lines tested, P2-purinergic receptor agonists, including ATP and certain hydrolysis-resistant adenine nucleotides, induced a rapid, transient increase in cytoplasmic free Ca2+ that was detectable at 50 to 100 nM ATP, was maximal at 100 microM ATP, and was inhibited approximately 50% by chelation of extracellular Ca2+. Within 8 s after addition, ATP stimulated accumulation of the polyphosphatidylinositol products inositol (1, 4, 5) trisphosphate, inositol (1, 3, 4) trisphosphate, and inositol tetrakisphosphate. In addition to stimulating phosphatidylinositol turnover and Ca2+ mobilization, ATP and hydrolysis-resistant ATP analogues induced greater than 90% inhibition of the growth of all lines tested. These data demonstrate that human androgen-independent prostate carcinoma cells express functional P2-purinergic receptors linked to phospholipase C, and that agonists of this receptor are markedly growth inhibitory, suggesting a novel therapeutic approach to this common adult neoplasm.
The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia.
Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS.
The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaffin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3T3t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells. In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2+]i was followed by a secretory response which originated from the entire surface of the cell. In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (greater than 200 nM), transient elevation in [Ca2+]i and no detectable secretory response. The rise in [Ca2+]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, approximately 25% of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest.(ABSTRACT TRUNCATED AT 250 WORDS)
Extracellular ATP is shown here to induce programmed cell death (or apoptosis) in thymocytes and certain tumor cell lines. EM studies indicate that the ATP-induced death of thymocytes and susceptible tumor cells follows morphological changes usually associated with glucocorticoid-induced apoptosis of thymocytes. These changes include condensation of chromatin, blebbing of the cell surface, and breakdown of the nucleus. Cytotoxicity assays using double-labeled cells show that ATP-mediated cell lysis is accompanied by fragmentation of the target cell DNA. DNA fragmentation can be set off by ATP but not the nonhydrolysable analogue ATP gamma S nor other nucleoside-5'-triphosphates. ATP-induced DNA fragmentation but not ATP-induced 51Cr release can be blocked in cells pretreated with inhibitors of protein or RNA synthesis or the endonuclease inhibitor, zinc; whereas pretreatment with calmidazolium, a potent calmodulin antagonist, blocks both DNA fragmentation and 51Cr release. The biochemical and morphological changes caused by ATP are preceded by a rapid increase in the cytoplasmic calcium of the susceptible cell. Calcium fluxes by themselves, however, are not sufficient to cause apoptosis, as the pore-forming protein, perforin, causes cell lysis without DNA fragmentation or the morphological changes associated with apoptosis. Taken together, these results indicate that ATP can cause cell death through two independent mechanisms, one of which, requiring an active participation on the part of the cell, takes place through apoptosis.
Platelets are rapidly activated by several agonists. When phorbol 12-myristate 13-acetate (PMA) was added to washed platelet suspensions 10 s prior to either thrombin or ADP, it caused a dose-dependent inhibition of shape change correlated with decreased myosin association with the cytoskeleton and with inhibition of the calcium transient measured in fura-2-loaded platelets. PMA added 5–10 s after agonists did not reverse shape change or the association of myosin with the cytoskeleton, but markedly increased the rate at which the calcium signal returned to the baseline. The analogue, 4α-phorbol didecanoate did not cause these effects. Our results suggest that one effect of C-kinase activation is to provide negative feedback in sequential responses.
1.A distinct population of ATP-sensitive cells, with response characteristics indicative of P2-type purinoceptors found in internal tissues of vertebrates, was identified among the antennular olfactory cells of the spiny lobster,Panulirus argus.2.Extracellular recordings from single cells showed that the ATP-sensitive cells had the following properties in common with P2 purinoceptors: a) potency sequence of ATP>ADP>AMP and adenosine; b) broad sensitivity to nucleotide triphosphates including those with modifications in both the purine and ribose moieties; c) stimulated by slowly degradable analogs of ATP, namely,ß, ?-imido ATP (AMPPNP),ß, ?-methylene ATP (AMPPCP), and a,ß-methylene ATP (AMPCPP).3.The activity sequence of the ATP-sensitive cells for nucleotides and related substances was ATP=2'-deoxyATP>GTP>CTP=XTP=ITP> 8-bromo-ATP=ADP, with pyrophosphate, AMP, and tripolyphosphate being virtually inactive.4.The potency sequence for the slowly degradable analogs was AMPPNP>ATP = AMPPCP> AMPCPP.5.Differences in structure-activity relationships, response duration, and response magnitude clearly distinguished the ATP-sensitive cells from another type of olfactory purinoceptor, the AMP-sensitive cells, also occurring in the antennules of the lobster.6.Comparisons between the ATP-sensitive chemoreceptors of the lobster and of certain insects revealed similarities in the activity sequence of ATP, ADP, AMP, and certain other nucleotides. However differences existed in the relative potencies of ATP, AMPPNP, and AMPPCP, and in the relative inactivity of inorganic pyrophosphate in the lobster.7.The findings of this study lend additional credence to the earlier hypothesis that receptors for transmitters and modulators existing in internal tissues may have evolved from external chemoreceptors of primitive unicellular organisms.
Techniques for cell permeabilization and control of cytosol composition include high-voltage electric discharge and the use of Sendai virus, which can both give long-term permeability to cell membranes, and application of ATP to susceptible cells, which allows controlled resealing. Non-lytic methods for introducing exogenous proteins into cells are also available, and recently electrophysiological patch pipettes have provided a novel approach to controlling cytosol composition and monitoring cellular processes. Here we describe the application of these techniques to the investigation of stimulus-secretion coupling in rat mast cells.
We have studied the apparent kinetic parameters of the ecto-nucleotide triphosphatase from CLL B lymphocytes and compared them to blood and tonsillar B and T cells. The Vmax of the ecto-ATPase activity in CLL B lymphocytes, was 65 ± 10 fmol Pi/cell per 30 min compared to 37 ± 2.1 in blood B lymphocytes, and 8.5 ± 1.7 in blood T lymphocytes. The ATPase of membranes prepared from CLL, tonsillar B and T, and blood T lymphocytes had a relationship among the cell types similar to that seen in intact cells. However, no difference in the km for ATP, .17 mM, or the km for magnesium, .15 mM was found in the ecto-ATPase of CLL lymphocytes as compared to blood or tonsillar B cells. The ectoenzyme of CLL cells hydrolyzed GTP, ITP, CTP, and UTP as well as ATP. Further, ATP added to an enzyme assay containing an alternative nucleotide did not result in increased phosphate release. Nucleotide acceptance of blood B and T lymphocytes was very similar to that of CLL B cells. ATP inhibited phosphate release when present in excess of magnesium in both CLL and blood B lymphocytes. These data indicate that there is greater ectonucleotide triphosphatase activity in tonsillar and blood B lymphocytes, including CLL, as compared either to blood or tonsillar T lymphocytes. However, CLL cells showed no qualitative difference from blood or tonsillar B cells in ectonucleotidase activity. Thus, the higher activity in CLL cells is “B cell-like” and might reflect, also, their maturation stage or monoclonal origin.
Astrocyte cultures prelabelled with either [3H]-inositol or 45Ca2+ were exposed to ATP and its hydrolysis products. ATP and ADP, but not AMP and adenosine, produced increases in the accumulation of intracellular 3H-labelled inositol phosphates (IP), efflux of 45Ca2+, and release of thromboxane A2 (TXA2). Whereas ATP-stimulated 3H-IP accumulation was unaffected, its ability to promote TXA2 release was markedly reduced by mepacrine, an inhibitor of phospholipase A2 (PLA2). ATP-evoked 3H-IP production was also spared following treatment with the cyclooxygenase inhibitor, indomethacin. We conclude that ATP-induced phosphoinositide (PPI) breakdown and 45Ca2+ mobilisation occurred in parallel with, if not preceded, the release of TXA2. Following depletion of intracellular Ca2+ with a brief preexposure to ATP in the absence of extracellular Ca2+, the release of TXA2 in response to a subsequent ATP challenge was greatly reduced when compared with control. These results suggest that mobilisation of cytosolic Ca2+ may be the stimulus for PLA2 activation and, thus, TXA2 release. Stimulation of -adrenoceptors also caused PPI breakdown and 45Ca2+ efflux but not TXA2 release. The effects of ATP and noradrenaline (NA) on 3H-IP accumulation were additive, but their combined ability to increase 45Ca2+ efflux was not. Interestingly, in the presence of NA, ATP-stimulated TXA2 release was reduced. Our data provide evidence that functional P2-purinergic receptors are present on astrocytes and that ATP is the first physiologically relevant stimulus found to initiate prostanoid release from these cells.
Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [3H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanidem-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Nonelectrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (). The data could be explaine by a threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K+, influx of Na+ and reduction of preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP actsvia specific sites on the surface of transformed cells.
procedures include the use of ionophores, manipulation of osmotic conditions, treatment with ouabain or with agents that reduce the level of intracellular ATP, and also the use of inhibitors of Ca ++calmodulin systems.
Using 3T3 and 3T6 mouse fibroblasts and A431 epidermoid carcinoma cells, we previously observed that extracellular ATP and ADP were mitogens and they synergized with other growth factors (Huang, N., Wang, D. and Heppel, L.A. (1989) Proc. Natl. Acad. Sci. USA , 7904–7908). We now report that ATP and ADP stimulated Na+ entry, intracellular alkalinization and pump activity, which are early events that had been proposed to play a central role in DNA synthesis. In addition, ATP, ADP and AMPPNP stimulated uridine uptake by a pathway involving arachidonic acid metabolism. In A431 cells, activation of protein kinase C also contributed to ATP-dependent stimulation of uridine uptake. Concentrations of indomethacin and pertussis toxin which inhibited uridine uptake also blocked arachidonic acid metabolism and DNA synthesis. ATP acted as a competence factor. Interestingly, ATP did not have to be continuously present to stimulate uridine uptake. It was equally effective even when it was washed away after brief treatment of cells.
The polypeptides PDGF, TGFα, and EGF have previously been shown by others to stimulate proliferation of fibroblasts and keratinocytes in the process of wound healing. Here we demonstrate that extracellular ATP, ADP or AMPPNP caused synergistic enhancement of DNA synthesis in 3T6 mouse fibroblasts and keratinocytes when combined with any of the above polypeptides. TGFβ showed synergistic stimulation with ATP in fibroblasts but it inhibited keratinocytes. ATP acted as a mitogen for NIE-115 neuroblastoma cultures. In 3T6 cells, ATP stimulated thymidine incorporation in combination with carbachol or norepinephrine. The effect of carbachol was sensitive to atropine. We suggest that extracellular ATP and ADP may play a physiological role in wound healing and as a mitogenic neurotransmitter in the nervous system.
ATP (as the tetrabasic acid, ATP4−) applied externally to rat mast cells causes the formation of lesions which permit influx and efflux of low molecular weight, normally impermeant aqueous solutes. To monitor membrane permeabilisation we have used two fluorescent dyes, ethidium which stains the nucleus, and TMA-DPH which stains the cytosolic surfaces of intracellular membranes following entry into the cells. Permeabilisation by ATP is not affected by the metabolic status of the cells, and is maintained at temperatures as low as 8°C. We have tested the ability of 30 structural analogues of ATP to effect mast cell permeabilisation. The analogues include those having substituents in the 2- and 8-positions of the purine ring, structural and optical isomers of the ribose sugar, and variations in the triphosphate chain. The pattern of selectivity displayed by the rat mast cell ATP4− receptor is distinct from those characteristic of the P1 purinoceptor for adenosine and the P2X and P2Y purinoceptors for adenine nucleotides.
It is proposed that the biochemical mechanisms which lead to the pathology of irreversible traumatic shock are based on the vasodilatory action of adenylates and failure of ecto-enzymes to clear these substances from the vascular bed. Following massive injury, or another assault which produces significant cytolysis, cytoplasmic ATP is released into the circulation. Ecto-phosphoesterhydrolases on erythrocytes normally control plasma adenylate concentrations to be maintained below 10−7 M. If a critical ATP (or ADP) concentration between 10−5 and 10−4 M is reached, erythrocytes become semi-permeable and themselves release additional ATP into the plasma compartment. When ecto-ATPase activity becomes insufficient in metabolizing plasma ATP at rates which are needed to compensate for the release of cytoplasmic ATP, the vasodilatory effects of adenylates will manifest themselves in a sustained manner. It is suggested that ecto-phosphoesterhydrolases on blood formed elements and endothelial cells serve a protective function. Circulatory shock ensues under conditions when these ecto-enzymes are rendered incompetent or when plasma adenylate concentrations exceed their catabolic capacity.
Exogeneous nucleotides or nucleosides may influence lymphocyte functions such as proliferation and cytotoxicity. We report that ATP, and to a lesser extent ADP, at concentrations as low as 0.3 mM, are highly mitogenic for medullary mature thymocytes, when added in combination with phorbol myristate acetate (PMA), which is only weakly mitogenic by itself. Under the same conditions, the other nucleotides (AMP; GTP, ITP, 2′d-deoxyATP), the non-hydrolysable ATP analogs (p[NH]ppA, pp[CH2]pA) and adenosine are unable to trigger thymocyte blastogenesis. p[NH]ppA, a potent inhibitor of ATP hydrolysis, potentiates the ATP mitogenic effect. In contrast, T-cell-enriched splenocytes do not proliferate in response to ATP + PMA. These data and measurements of interleukin 2 synthesis suggest that ATP may efficiently deliver in thymocytes the calcium signal necessary for the initiation of blastogenesis (in medullary cells). Indeed, among all nucleotides tested, only ATP or ADP were able to increase the intracellular free calcium level in thymocytes, but not in splenocytes. Our results led us to suggest that thymocytes express on their surface receptors specific for ATP, which might be P2 type nucleotide receptors and could be involved in the lymphocyte response through the regulation of intracellular free calcium levels.
ATP and other purine nucleotides and nucleosides have potent regulatory (neurotransmitter-like) actions which have been attributed to interaction with a specific plasma membrane receptor1. To date the receptor mechanisms underlying purinergic activation have been poorly characterized. One problem has been the variability of the evoked effects in different tissues. Burnstock2,3 proposed that much of the variability could be explained if the effects were mediated by two separate receptors, termed P1 and P2, with different specificities for agonists and antagonists. Receptor mechanisms have been extensively investigated in the parotid gland4. I now report that in that gland, ATP evokes a marked increase in membrane conductance, radioactive Rb efflux and amylase secretion. The effects of ATP are similar to those evoked by acetylcholine (ACh) and alpha-adrenergic agonists but are still present when cholinergic and adrenergic blocking agents are used. The latency and reversal potential of the ATP-evoked effects are comparable with those of the autonomic agonists. The ATP receptor on parotid acinar cells is of the P2 type2,3, since the order of potency of the nucleotide series was ATP>ADP>>AMP, adenosine had no effect, and the response could be blocked by quinidine but not by theophylline.
Mouse thymocyte populations composed principally of θ-bearing cells exhibited a fourfold or greated enhancement in DNA synthesis when cultured in the presence of adenosine or adenine nucleotides. In contrast θ-bearing cells derived from spleen were markedly inhibited under the same circumstances. The effects of a variety of other nucleosides and nucleotides on DNA synthesis by spleen and thymus cells are also presented.
SEVERAL groups have shown that ATP is released from neural tissues
during depolarisation, where it or its metabolites might function as
neurotransmitters or neuromodulators1. They measured
radiolabelled adenosine derivatives in perfusates following
depolarisation of their preparations. Recently, Israel et
al.2 described a method for continuously and directly
detecting the release of ATP from the electric organ of Torpedo
marmorata after stimulation of its motor nerve. I have used a
modification of this technique to study the depolarisation-induced
release of ATP from a purified synaptosomal fraction prepared from rat
brain. I report here that depolarisation of synaptosomes elicits a
release of ATP which can be directly and continuously detected by this
technique.
Endothelial cells in culture can modulate platelet aggregation and vascular tone, in part by producing prostacyclin (PGI2), a powerful vasodilator and inhibitor of platelet aggregation, but also by their ecto-ADPase activity, which initiates the conversion of pro-aggregating ADP to adenosine, a potent vasodilator and platelet inhibitor. We have now demonstrated that cultured aortic endothelial cells exposed to trypsin, thrombin or other stimuli can liberate a high proportion of their adenine nucleotides without substantial loss of lactate dehydrogenase. ADP rapidly accumulates extracellularly, reaching biologically active concentrations before there is further breakdown to adenosine. Whether this selective release of nucleotides is a response to damage, or whether it represents a specific secretory mechanism remains to be resolved. Cultured aortic smooth muscle cells can secrete adenine nucleotides in a similar manner, but extracellular conversion to adenosine occurs much faster.
Demonstration of release of ATP from smooth muscle preparations during stimulation of purinergic nerves is complicated by the difficulty in showing whether it comes from nerve or muscle. ATP released during relaxation of the guinea-pig taenia coli and contraction of bladder strips in response to purinergic nerve stimulation was measured in the superfusate using the luciferin-luciferase ATP assay method. The amount of ATP increased 2-6 fold during isometric responses to purinergic nerve stimulation. This release was blocked by tetrodotoxin but not by adrenergic nerve destruction with 6-hydroxydopamine. No significant release of ATP was detected during comparable responses elicited by direct muscle stimulation. These results provide further support for the purinergic nerve hypothesis.
The effect of exogenous adenosine triphosphate (ATP) and other nucleotides on the transport of Na in various mammalian red cells has been studied. While they have no effect on the transport of Na in human and cat red cells, in dog red cells adenosine and its mono-, di- and triphosphorylated forms were found to increase Na-influx. Of these, ATP has the most striking effect, causing a more than 8-fold increase at a concentration of 0.6 mM and exerting this effect at a dose range of 10(-5) to 10(-3) M. The effect of ATP is rapid (less than 5 minutes) and can be reversed by washing or the addition of calcium or magnesium. In contrast to the adenosine series other phosphorylated nucleotides (GTP, CTP, UDP, GDP and cAMP) have no effect. The well known volume dependent Na-transport in these cells is reversed in the presence of 0.6 mM ATP. It is suggested that ATP acts on passive cation movements either by chelation of membrane charge or by a direct interaction with membrane proteins and may be involved in the volume regulation of cation transport in the dog erythrocyte.
In the five years since the discovery that nitric oxide is produced as a signal in blood vessels, a great deal has been discovered about the processes involved. This article reviews current knowledge about the vascular cell synthesis, effects and subsequent destruction of this messenger molecule.
Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.
During wound healing, release of ATP from platelets potentially exposes the epidermis to concentrations of ATP known to alter cellular functions mediated via changes in inositol trisphosphate (IP3) and intracellular calcium (Cai) levels. Therefore, we determined whether keratinocytes respond to ATP with a rise in Cai and IP3 and whether such increases are accompanied by a change in their proliferation and differentiation. Changes in Cai were measured in Indo-1-loaded neonatal human foreskin keratinocytes after stimulation with extracellular ATP. Extracellular ATP evoked a transient and acute increase in Cai of keratinocytes both in the presence and in the absence of extracellular calcium. ATP also induced the phosphoinositide turnover of keratinocytes, consistent with its effect in releasing calcium from intracellular sources. ATP did not permeabilize keratinocytes, nor did it promote Ca influx into the cells. The half-maximal effect of ATP was at 10 microM, and saturation was observed at 30-100 microM. UTP, ITP, and ATP gamma S were as effective as ATP in releasing Cai from intracellular stores and competed with ATP for their response, whereas AMP and adenosine were ineffective, suggesting the specificity of P2 purinergic receptors in mediating the ATP response in keratinocytes. Single cell measurements revealed heterogeneity in the calcium response to ATP. This heterogeneity did not appear to be due to differences in the initial Cai response but to subsequent removal of increased Cai by these cells. ATP inhibited terminal differentiation of keratinocytes as measured by [35S]methionine incorporation into cornified envelopes and modestly stimulated incorporation of [3H]thymidine into DNA. Chelation of Cai by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid reduced basal Cai, blocked the Cai response to ATP, inhibited the basal rate of DNA synthesis, and blocked the ATP-induced increase in DNA synthesis. We conclude that extracellular ATP may be an important physiological regulator of epidermal growth and differentiation acting via IP3 and Cai.
PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.
The patch-clamp technique was used to study mechanisms of ATP-induced Ca2+ influx in rat peritoneal macrophages. The experiments on whole-cell and patch membranes have shown that extracellular ATP activates channels permeable to di- and monovalent inorganic cations. Ratios of unitary channel conductances in 105 mM Ca2+, Sr2+, Mn2+, Ba2+ and normal sodium solutions were 1.0, 0.95, 0.75, 0.55 and 0.85, respectively. The channels could open in the presence of non-hydrolyzable GTP analogues in artificial intracellular solution. The data are consistent with the hypothesis that a GTP-binding protein is involved in receptor-to-channel coupling.
Specific phospholipase C enzymes can hydrolyse phosphatidylinositol 4,5-bisphosphate into two products: inositol 1,4,5-trisphosphate, which regulates the release of intracellular calcium stores, and diacylglycerol, which can stimulate protein kinase C. A new group of G proteins, the Gq subfamily, have recently been shown to mediate the regulation of this activity by a variety of hormones. How do different members of this family modulate unique phospholipase C isozymes? What is the mechanism of this regulation? How might the Gq subfamily act to modulate other important second messenger pathways? The tools to answer these questions are being rapidly developed.
Characteristics of extracellular ATP-evoked electrical responses in rat hippocampal neurons were investigated. Extracellular ATP (100 microM) induced a rapid depolarization followed by repetitive firings of spikes in these cells under whole-cell current-clamp. In whole-cell voltage-clamp experiments, ATP activated 2 types of inward currents that were inhibited by P2-purinoceptor blocker suramin (300 microM). One is a small (about -20 pA) sustained current which is insensitive to tetrodotoxin (TTX), and the other is a large (-100 to -300 pA) transient current which abolished in the presence of 3 microM TTX. The ATP-induced transient current was blocked by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 30 microM), a non-N-methyl-D-aspartate (non-NMDA) receptor antagonist. ATP failed to induce the transient current in the cell which showed the desensitization to quisqualic acid (QA; 10 microM), a non-NMDA receptor agonist. These findings suggest that ATP directly activates small sustained currents, and indirectly induces the transient currents by evoking glutamate release.
In addition to its diverse functions inside cells, ATP can act at several types of cell-surface receptor. One of these (P2X-purinoceptor) is believed to be a ligand-gated cation channel. The presence of P2X receptors on autonomic, sensory and central neurons suggests that ATP might be released to act as a fast excitatory synaptic transmitter. Here we record excitatory synaptic potentials and currents from cultured coeliac ganglion neurons which are mimicked by ATP, blocked by the P2-purinoceptor antagonist suramin, desensitized by alpha,beta-methylene-ATP and unaffected by antagonists acting at nicotine, 5-hydroxytryptamine, N-methyl-D-aspartate (NMDA), non-NMDA glutamate, gamma-aminobutyric acid (GABA), noradrenaline or adenosine receptors. We conclude that ATP is the neurotransmitter at this neuroneuronal synapse.
Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs.
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The effects of Leiurus quinquestriatus hebraeus (LQH) venom, mamba venom, Buthus tamulus (BT) venom, purified apamin and synthetic charybdotoxin on the membrane hyperpolarization induced by extracellular ATP were examined in Madin-Darby canine kidney cells. For this we used a membrane potential probe (bisoxonol) to determine the potential variations. The relation between bisoxonal fluorescence and membrane potential was established by treating Madin-Darby canine kidney cells suspended in solutions containing various external sodium concentrations with gramicidin. Extracellular ATP induced a rapid hyperpolarization that was blocked by LQH venom and synthetic charybdotoxin. BT venom also blocked the response but at a much higher concentration than that of LQH. Mamba venom (Dendroaspis polylepis) and apamin did not modify the ATP-induced hyperpolarization. We concluded that the ATP induced hyperpolarization was due to the augmentation of the potassium conductance probably through Ca(2+)-activated K+ channels sensitive to charybdotoxin but not to mamba venom. The interaction previously described between charybdotoxin and dendrotoxin (the main toxin of mamba venom) was not observed in our case.
Until now, the only well documented, fast excitatory neurotransmitter in the brain has been glutamate. Although there is evidence for adenosine 5'-triphosphate (ATP) acting as a transmitter in the peripheral nervous system, suggestions for such a role in the central nervous system have so far not been supported by any direct evidence. Here we report the recording of evoked and miniature synaptic currents in the rat medial habenula. The fast rise time of the currents showed that they were mediated by a ligand-activated ion channel rather than a second messenger system, thus limiting the known transmitter candidates. Evidence was found for the presence on the cells of glutamate, gamma-aminobutyric acid, acetylcholine and ATP receptors, but not for 5-hydroxytryptamine (5HT3) or glycine receptors. The evoked currents were unaffected by blockers of glutamate, gamma-aminobutyric acid or acetylcholine receptors but were blocked by the ATP receptor-blocker, suramin and the desensitizing ATP receptor-agonist alpha,beta-methylene-ATP. Our evidence identifies for the first time synaptic currents in the brain, mediated directly by ATP receptors.
The activation of heterotrimeric G proteins results in the exchange of GDP bound to the alpha-subunit for GTP and the subsequent dissociation of a complex of the beta- and gamma-subunits (G beta gamma). The alpha-subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free G beta gamma complex. G beta gamma has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the G beta gamma complex can act with G alpha subunit to modulate the activity of different isoforms of adenylyl cyclase. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified G beta gamma is found to activate one of them. Here we report that in co-transfection assays G beta gamma subunits specifically activate the beta 2 and not the beta 1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of G beta gamma from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).
Hydrolysis by phospholipase C (PLC) of phosphatidylinositol 4,5-bisphosphate is a key mechanism by which many extracellular signalling molecules regulate functions of their target cells. At least eight distinct isozymes of PLC are recognized in mammalian cells. Receptor-controlled PLC is often regulated by G proteins, which can be modified by pertussis toxin in some cells but not in others. In the latter cells, PLC-beta 1, but not PLC-gamma 1 or PLC-delta 1, may be activated by members of the alpha q-subfamily of the G protein alpha-subunits. An unidentified PLC in soluble fractions of cultured human HL-60 granulocytes is specifically stimulated by G protein beta gamma subunits purified from retina and brain. Identification of a second PLC-beta complementary DNA (PLC-beta 2) in an HL-60 cell cDNA library prompted us to investigate the effect of purified G protein beta gamma subunits on the activities of PLC-beta 1 and PLC-beta 2 transiently expressed in cultured mammalian cells. We report here that PLC-beta 1 and PLC-beta 2 were stimulated by free beta gamma subunits and that PLC-beta 2 was the most sensitive to beta gamma stimulation. Thus stimulation of PLC by beta gamma subunits is isozyme-selective and PLC-beta 2 is a prime target of beta gamma stimulation. Activation of PLC-beta 2 by beta gamma subunits may be an important mechanism by which pertussis toxin-sensitive G proteins stimulate PLC.
Extracellular ATP receptors in rat ventricular myocytes were investigated through intact cell photolabelling followed by protein isolation. 8-Azido-ATP (8Az-ATP) was used for labelling under specific conditions determined by parallel functional studies. In those studies ATP-induced cytosolic Ca2+ transients were irreversibly and specifically inhibited by UV-photolyzed 8Az-ATP, but not by 2-azido-ATP (2Az-ATP), even in the presence of high concentrations of phosphonucleotides not affecting myocardial ATP receptors. Under those conditions background labelling is minimized and radioactive 8Az-ATP specifically labels a band of 45-48 kDa on a SDS gel. Labelling under the above conditions in the presence of ATP gamma S or 2-methylthio-ATP (2-meSATP), which are distinct for two functionally different cardiac ATP receptors, shows two different proteins within the same band consistent with the possible labelling of these two receptors.
The membrane potential of cultured mouse astrocytes was recorded to assess the effects of extracellular adenosine 5'-triphosphate (ATP) and related H purines on astrocyte electrophysiology. The purines were applied with or without the presence of barium, which blocks the high resting K+ conductance in astrocytes. The response to ATP alone was a moderate depolarization; however, the response to ATP in the presence of barium was a large, dose dependent depolarization. The ED50 was approximately 10 microM. The effect of adenosine 5'-diphosphate (ADP) or adenosine 5'-monophosphate (AMP), in the presence of barium, on membrane potential was less than that of ATP. Adenosine, with or without barium, had no effect on membrane potential; furthermore, adenosine agonists in barium produced no response. The results of applying various ATP analogues indicate that the response is mediated via a P2-purinoceptor. Ion replacement studies reveal a complicated response to ATP that has several components and involves Na+ and K+. These results show that astrocytes respond with ionic changes to very small, physiological concentrations of extracellular ATP. We suggest that ATP plays a role in interactions between neurons/endothelial cells and glial cells.
P2-purinoceptor agonists stimulated the DNA synthesis of Jurkat cells via a pathway independent of cAMP and intracellular free calcium. The response was greatly enhanced by the synergistic action between adenine and guanine nucleotides, suggesting that binding sites of these nucleotides are different from each other, and the proliferation is stimulated by a novel interaction between adenine and guanine nucleotide receptors. The stimulatory effects of P2-agonists on proliferation was completely abolished by cholera toxin and attenuated by pertussis toxin, which suggests that substrates for cholera toxin and pertussis toxin are involved in the proliferative pathways associated with P2-purinoceptors.
Xenopus oocytes injected with embryonic guinea-pig brain mRNA expressed functional P2Y purinoceptors. Extracellular ATP stimulated in a dose-dependent manner a delayed Ca(2+)-dependent Cl- current component. Analysis of the interactions of ATP with compounds that affect Ca2+ fluxes through the plasma membrane or Ca2+ release from internal stores indicates that ATP raises [Ca2+]i by a mechanism that involves activation of voltage-dependent Ca2+ channels, which leads to influx of extracellular Ca2+ into the cells, as well as release of Ca2+ from intracellular stores. Since this phenomenon was not found in control oocytes, it is suggested that brain mRNA encoded for a newly synthesized Ca(2+)-release process stimulated by purinoceptor activation. This mechanism could be largely involved in the short-term regulation of intracellular Ca2+ level involved in ATP neuromodulation functions.
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In exocrine acinar cells a variety of neurotransmitters (e.g. acetylcholine) stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis elevating intracellular calcium to activate calcium-dependent membrane currents (outward K+ and inward Cl-). This study shows that in lacrimal acinar cells extracellular application of ATP is also associated with outward and inward current responses; these, however, are not the result of phosphoinositide metabolism. ATP directly activates receptor-operated cation channels which permit influx of Na+ and Ca+ (the inward current). The elevation in [Ca2+]i which results is sufficient to activate the outward K+ current. ATP thus promotes Ca+ influx in the absence of phosphoinositide metabolism.
The effect of suramin on the ATP-induced response in vas deferens DDT1 MF-2 smooth muscle cells was studied. Stimulation of P2-purinoceptors by ATP caused a change in membrane currents, measured by using the whole-cell patch-clamp configuration, and enhanced the formation of inositol phosphates, as analysed by high performance liquid chromatography. The ATP-induced membrane current consisted of a triphasic response, carried by a fast inward current, followed by a transient outward current and a sustained inward current. Inositol tetrakisphosphate (InsP4) formation increased in the presence of ATP. The formation of the isomers Ins(1,3,4,5)P4, Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4 increased significantly after 5 min stimulation with ATP. Suramin inhibited the ATP-evoked membrane currents and the ATP-induced formation of inositol tetrakisphosphate isomers concentration dependently, but did not affect the basal inositol phosphate levels in the absence of ATP. These results indicate that suramin inhibits ATP-activated cellular processes in DDT1 MF-2 vas deferens cells, most likely by acting on P2-purinoceptors.
Single-channel recordings were made using cell-free membrane patches (outside-out configuration) isolated from pheochromocytoma PC12 cells. ATP (50 microM) activated single channel currents in the isolated patches and the currents inactivated with a half-decay time of about 5s. The single channel conductance was about 13 pS in external solution with 140 mM Na. The amplitudes of the single-channel currents were decreased when external Ca was increased from 1.8 to 16.2 mM, suggesting that Ca blocks ion permeation through the channel. These properties of single-channel currents may underlie those of the macroscopic current.
The effects of suramin, reactive blue 2 (RB2) and d-tubocurarine (d-TC) were investigated electrophysiologically to elucidate the mechanisms underlying their antagonism of P2 purinoceptor-mediated responses. All three compounds inhibited an adenosine triphosphate (ATP)-activated inward current in rat phaeochromocytoma PC12 cells in a concentration-dependent manner. The order of potency was RB2 greater than suramin greater than d-TC. The inhibition induced by suramin or RB2 was reversible, whereas that induced by d-TC was not reversed after a 5-min rinse. The inactivation of the ATP-activated current was accelerated by d-TC but not by suramin or RB2. RB2 administered simultaneously with ATP exerted much weaker inhibition compared to that induced by prior administration, suggesting that RB2 is a slowly acting antagonist. This was not observed for suramin or d-TC. Suramin and RB2 caused a parallel shift in the concentration/response curve for the ATP-activated current. With d-TC the maximal response of ATP was decreased but the concentration producing half-maximal response was unchanged. The voltage dependency of the ATP-activated current showed less inward rectification in the presence of d-TC. Suramin or RB2 did not affect the voltage dependency. These results suggest that suramin and RB2 reversibly block binding of ATP to receptors, whereas d-TC blocks ion permeability through the ATP-activated channel.