Using antibodies against P2Y and P2X receptors in purinergic signaling research

The broad expression pattern of the G protein-coupled P2Y receptors has demonstrated that these receptors are fundamental determinants in many physiological responses, including neuromodulation, vasodilation, inflammation, and cell migration. P2Y receptors couple either G_q or G_i upon activation, thereby activating different signaling pathways. Ionotropic ATP (P2X) receptors bind extracellular nucleotides, a signal which is transduced within the P2X protein complex into a cation channel opening, which usually leads to intracellular calcium concentration elevation. As such, this family of proteins initiates or shapes several cellular processes including synaptic transmission, gene expression, proliferation, migration, and apoptosis. The ever-growing range of applications for antibodies in the last 30 years attests to their major role in medicine and biological research. Antibodies have been used as therapeutic tools in cancer and inflammatory diseases, as diagnostic reagents (flow cytometry, ELISA, and immunohistochemistry, to name a few applications), and in widespread use in biological research, including Western blot, immunoprecipitation, and ELISPOT. In this article, we will showcase several of the advances that scientists around the world have achieved using the line of antibodies developed at Alomone Labs for P2Y and P2X receptors.     

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38- cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 +/- 5.2 and 50.5 +/- 8.0 pmol/mg protein). P2Y receptor stimulation of CD38- cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave.     

Akt and Ca2+ signaling in endothelial cells

The protein kinase Akt participates in such important functions of endothelial cells as nitric oxide production and angiogenesis, activities that involve changes in cytosolic Ca2+ concentration. However, it is not known if activation of Akt is itself involved in the regulation of Ca2+ signals produced in these cells. The objective of this study was to examine if Akt is involved in the regulation of Ca2+ signaling in endothelial cells. Agonist-stimulated Ca2+ signals, assessed using fura-2, were compared in porcine aortic endothelial cells under control conditions or conditions in which Akt was blocked either by different inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt or by transient expression of a dominant-negative form of Akt (dnAkt). We found that the release of intracellular Ca2+ stores stimulated by bradykinin or thapsigargin is not affected by the PI3 kinase inhibitors LY294002 and wortmannin, or by expression of dnAkt. LY294002 dose-dependently inhibits store-operated Ca2+ entry, an effect not seen with wortmannin. Expression of dnAkt has no effect on store-operated Ca2+ entry. We conclude that Akt is not involved in the regulation of agonist-stimulated Ca2+ signals in endothelial cells. The compound LY294002 inhibits store-operated Ca2+ entry in these cells by a mechanism independent of PI3 kinase/Akt inhibition.     

ANG II AT2 receptor modulates AT1 receptor-mediated descending vasa recta endothelial Ca2+ signaling

We tested whether the respective angiotensin type 1 (AT(1)) and 2 (AT(2)) receptor subtype antagonists losartan and PD-123319 could block the descending vasa recta (DVR) endothelial intracellular calcium concentration ([Ca(2+)](i)) suppression induced by ANG II. ANG II partially reversed the increase in [Ca(2+)](i) generated by cyclopiazonic acid (CPA; 10(-5) M), acetylcholine (ACh; 10(-5) M), or bradykinin (BK; 10(-7) M). Losartan (10(-5) M) blocked that effect. When vessels were treated with ANG II before stimulation with BK and ACh, concomitant AT(2) receptor blockade with PD-123319 (10(-8) M) augmented the suppression of endothelial [Ca(2+)](i) responses. Similarly, preactivation with the AT(2) receptor agonist CGP-42112A (10(-8) M) prevented AT(1) receptor stimulation with ANG II + PD-123319 from suppressing endothelial [Ca(2+)](i). In contrast to endothelial [Ca(2+)](i) suppression by ANG II, pericyte [Ca(2+)](i) exhibited typical peak and plateau [Ca(2+)](i) responses that were blocked by losartan but not PD-123319. DVR vasoconstriction by ANG II was augmented when AT(2) receptors were blocked with PD-123319. Similarly, AT(2) receptor stimulation with CGP-42112A delayed the onset of ANG II-induced constriction. PD-123319 alone (10(-5) M) showed no AT(1)-like action to constrict microperfused DVR or increase pericyte [Ca(2+)](i). We conclude that ANG II suppression of endothelial [Ca(2+)](i) and stimulation of pericyte [Ca(2+)](i) is mediated by AT(1) or AT(1)-like receptors. Furthermore, AT(2) receptor activation opposes ANG II-induced endothelial [Ca(2+)](i) suppression and abrogates ANG II-induced DVR vasoconstriction.     

P2X1 receptors mediate sympathetic postjunctional Ca2+ transients in mesenteric small arteries

Brief, spatially localized Ca(2+) transients occur in the smooth muscle adjacent to perivascular nerves of small arteries during neurogenic contractions. We named these "junctional Ca(2+) transients" (jCaTs) and postulated that they arose from Ca(2+) entering smooth muscle cells through P2X(1) receptors activated by neurally released ATP. Nevertheless, the lack of potent, subtype-selective P2X-receptor antagonists made determining the exact molecular identity of the channels difficult. Here we used small, pressurized mesenteric arteries from P2X(1)-receptor-deficient mice (KO) to test the hypothesis that jCaTs arise from Ca(2+) entering the smooth muscle cell via P2X(1) receptors. In wild-type (WT) arteries, confocal microscopy of fluo-4 fluorescence during electrical field stimulation (EFS) of perivascular sympathetic nerves revealed jCaTs in the smooth muscle cells adjacent to the perivascular nerves, similar to those reported previously in rat arteries, and alpha-latrotoxin (2.5 nM) markedly increased the frequency of "spontaneous" jCaTs. In the KO arteries, however, neither EFS nor alpha-latrotoxin elicited any jCaTs. A potent P2X-receptor agonist, alpha,beta-methylene ATP (10.0 microM), elicited strong contractions and increased intracellular Ca(2+) concentration in WT arteries but elicited neither in KO arteries. A biphasic vasoconstriction in response to EFS was observed in WT arteries. In KO arteries, however, the initial rapid, transient component of the biphasic vasoconstriction was absent. The data support the hypothesis that jCaTs represent Ca(2+) that enters the smooth muscle cells through P2X(1) receptors activated by neurally released ATP and that this Ca(2+) is involved in the initial rapid component of the sympathetic neurogenic contraction.     

The role of P2Y1 purinergic receptors and cytosolic Ca2+ in hypotonically activated osmolyte efflux from a rat hepatoma cell line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca(2+) concentration ([Ca(2+)](i)) to increase transiently by approximately 90 nm. This rise in [Ca(2+)](i) was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca(2+)](i) increase. The hypotonically induced rise in [Ca(2+)](i) was prevented by the prior discharge of the intracellular Ca(2+) store of the cells by thapsigargin. Removal of extracellular Ca(2+) or inhibition of Ca(2+) influx by 1-10 microm Gd(3+) depleted the thapsigargin-sensitive Ca(2+) stores and thereby diminished the rise in [Ca(2+)](i). The hypotonically induced rise in [Ca(2+)](i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca(2+)](i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca(2+)](i) elicited by the addition of the P2Y(2) and P2Y(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y(1), P2Y(2), and P2Y(6) receptors. Inhibition of the hypotonically induced rise in [Ca(2+)](i) blocked hypotonically induced K(+) ((86)Rb(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl(-) ((125)I-) efflux. The interaction of extracellular ATP and/or ADP with P2Y(1) purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.     

Activation of purinergic P2X receptors inhibits P2Y-mediated Ca2+ influx in human microglia

Purinoceptor (P2X and P2Y) mediated Ca2+ signaling in cultured human microglia was studied using Ca2+ sensitive fluorescence microscopy. ATP (at 100 microM) induced a transient increase in [Ca2+]i in both normal and Ca(2+)-free solution suggesting a primary contribution by release from intracellular stores. This conclusion was further supported by the failure of ATP to cause a divalent cationic influx in Mn2+ quenching experiments. However, when fluorescence quenching was repeated after removal of extracellular Na+, ATP induced a large influx of Mn2+, indicating that inward Na+ current through a non-selective P2X-coupled channel may normally suppress divalent cation influx. Inhibition of Mn2+ entry was also found when microglia were depolarized using elevated external K+ in Na(+)-free solutions. The possibility of P2X inhibition of Ca2+ influx was then investigated by minimizing P2X contributions of purinergic responses using either the specific P2Y agonist, ADP-beta-S in the absence of ATP or using ATP combined with PPADS, a specific inhibitor of P2X receptors. In quenching studies both procedures resulted in large increases in Mn2+ influx in contrast to the lack of effect observed with ATP. In addition, perfusion of either ATP plus PPADS or ADP-beta-S alone caused a significantly enhanced duration (about 200%) of the [Ca2+]i response relative to that induced by ATP. These results show that depolarization induced by P2X-mediated Na+ influx inhibits store-operated Ca2+ entry resulting from P2Y activation, thereby modulating purinergic signaling in human microglia.     

Spatial and temporal aspects of Ca2+ signaling mediated by P2Y receptors in cultured rat hippocampal astrocytes

ATP produces a variety of Ca2+ responses in astrocytes. To address the complex spatio-temporal Ca2+ signals, we analyzed the ATP-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal astrocytes using fura-2 or fluo-3 based Ca2+ imaging techniques. ATP at less than 10 nM produced elementary Ca2+ release event "puffs" in a manner independent of extracellular Ca2+. Stimulation with higher ATP concentrations (3 or 10 micro M) resulted in global Ca2+ responses such as intercellular Ca2+ wave. These Ca2+ responses were mainly mediated by metabotropic P2Y receptors. ATP acting on both P2Y1 and P2Y2 receptors produced a transient Ca2+ release by inositol 1,4,5-trisphosphate (InsP3). When cells were stimulated with ATP much longer, the transient [Ca2+]i elevation was followed by sustained Ca2+ entry from the extracellular space. This sustained rise in [Ca2+]i was inhibited by Zn2+ (<10 micro M), an inhibitor of capacitative Ca2+ entry (CCE). CCE induced by cyclopiazonic acid or thapsigargin and Ca2+ entry evoked by ATP share the same pharmacological profile in astrocytes. Taken together, the hierarchical Ca2+ responses to ATP were observed in hippocampal astrocytes, i.e., puffs, global Ca2+ release by InsP3, and CCE in response to depletion of InsP3-sensitive Ca2+ stores. It should be noted that these Ca2+ signals and their modulation by Zn2+ could occur in the hippocampus in situ since both ATP and Zn2+ are rich in the hippocampus and could be released by excitatory stimulation.     

P2Y1, P2Y6, and P2Y12 receptors in rat splenic sinus endothelial cells: an immunohistochemical and ultrastructural study

Localization of three P2X and six P2Y receptors in sinus endothelial cells of the rat spleen was examined by immunofluorescent microscopy, and ultrastructural localization of the detected receptors was examined by immunogold electron microscopy. In immunofluorescent microscopy, labeling for anti-P2Y1, P2Y6, and P2Y12 receptors was detected in endothelial cells, but P2X1, P2X2, P2X4, P2Y2, P2Y4, and P2Y13 receptors was not detected. P2Y1 and P2Y12 receptors were prominently localized in the basal parts of endothelial cells. P2Y6 receptor was not only predominantly localized in the basal parts of endothelial cells, but also in the superficial layer. Triple immunofluorescent staining for a combination of two P2Y receptors and actin filaments showed that P2Y1, P2Y6, and P2Y12 receptors were individually localized in endothelial cells. Phospholipase C-β3, phospholipase C- γ2, and inositol-1,4,5-trisphosphate receptors, related to the release of the intracellular Ca²⁺ from the endoplasmic reticulum, were also predominantly localized in the basal parts of endothelial cells. In immunogold electron microscopy, labeling for P2Y1, P2Y6, and P2Y12 receptors were predominantly localized in the basal part of endothelial cells and, in addition, in the junctional membrane, basal plasma membrane, and caveolae in the basal part of endothelial cells. Labeling for phospholipase C-β3 and phospholipase C-γ2 was dominantly localized in the basal parts and in close proximity to the plasma membranes of endothelial cells. The possible functional roles of these P2Y receptors in splenic sinus endothelial cells are discussed.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

Endogenously released ATP mediates shear stress-induced Ca2+ influx into pulmonary artery endothelial cells

The mechanisms by which flow-imposed shear stress elevates intracellular Ca2+ in cultured endothelial cells (ECs) are not fully understood. Here we report finding that endogenously released ATP contributes to shear stress-induced Ca2+ responses. Application of flow of Hanks' balanced solution to human pulmonary artery ECs (HPAECs) elicited shear stress-dependent increases in Ca2+ concentrations. Chelation of extracellular Ca2+ with EGTA completely abolished the Ca2+ responses, whereas the phospholipase C inhibitor U-73122 or the Ca2+-ATPase inhibitor thapsigargin had no effect, which thereby indicates that the response was due to the influx of extracellular Ca2+. The Ca2+ influx was significantly suppressed by apyrase, which degrades ATP, or antisense oligonucleotide targeted to P2X4 purinoceptors. A luciferase luminometric assay showed that shear stress induced dose-dependent release of ATP. When the ATP release was inhibited by the ATP synthase inhibitors angiostatin or oligomycin, the Ca2+ influx was markedly suppressed but was restored by removal of these inhibitors or addition of extracellular ATP. These results suggest that shear stress stimulates HPAECs to release ATP, which activates Ca2+ influx via P2X4 receptors.     

Stimulation of Cl- secretion via membrane-restricted Ca2+ signaling mediated by P2Y receptors in polarized epithelia.

Extracellular nucleotides such as ATP have been shown to regulate ion transport processes in a variety of epithelia. This effect is mediated by the activation of plasma membrane P2Y receptors, which leads to Ca(2+) signaling cascade. Ion transport processes (e.g. activation of apical calcium-dependent Cl(-) channels) are then stimulated via an increase in [Ca(2+)](i). Many polarized epithelia express apical and/or basolateral P2Y receptors. To test whether apical and basolateral stimulation of P2Y receptors elicit polarized Ca(2+) signaling and anion secretion, we simultaneously measured the two parameters in polarized epithelia. Although activation of P2Y receptors located at both apical and basolateral membranes evoked an increase in [Ca(2+)](i), only apical P2Y receptors-coupled Ca(2+) release stimulated an increase in anion secretion. Moreover, the calcium influx evoked by apical and basolateral P2Y receptor stimulation is predominately via the basolateral membrane domain. It appears that the apical P2Y receptor-regulated Ca(2+) release and activation of apical Cl(-) channels is compartmentalized in polarized epithelia with basolateral P2Y-stimulated Ca(2+) release failing to activate anion secretion. These data suggest that there may be two distinct ATP-releasable Ca(2+) pools, each coupled to apical and basolateral membrane receptor but linked to the same calcium influx pathway located at the basolateral membrane.     

Autocrine stimulation of P2Y1 receptors is part of the purinergic signaling mechanism that regulates T cell activation

Purinergic Signalling - Previous studies have shown that T cell receptor (TCR) and CD28 coreceptor stimulation involves rapid ATP release, autocrine purinergic feedback via P2X receptors, and...     

Ionotropic NMDA and P2X1/5 receptors mediate synaptically induced Ca2+ signalling in cortical astrocytes

Local, global and propagating calcium (Ca(2+)) signals provide the substrate for glial excitability. Here we analyse Ca(2+) permeability of NMDA and P2X(1/5) receptors expressed in cortical astrocytes and provide evidence that activation of these receptors trigger astroglial Ca(2+) signals when stimulated by either endogenous agonists or by synaptic release of neurotransmitters. The Ca(2+) permeability of the ionotropic receptors was determined by reversal potential shift analysis; the permeability ratio P(Ca)/P(K) was 3.1 for NMDA receptors and 2.2 for P2X(1/5) receptors. Selective stimulation of ionotropic receptors (with NMDA and α,β-methyleneATP) in freshly isolated cortical astrocytes induced ion currents associated with transient increases in cytosolic Ca(2+) concentration ([Ca(2+)](i)). Stimulation of neuronal afferents in cortical slices triggered glial synaptic currents and [Ca(2+)](i) responses, which were partially blocked by selective antagonists of NMDA (D-AP5 and UBP141) and P2X(1/5) (NF449) receptors. We conclude that ionotropic receptors contribute to astroglial Ca(2+) signalling and may provide a specific mechanism for fast neuronal-glial signalling at the synaptic level.     

Mechanisms of endothelial P2Y(1)- and P2Y(2)-mediated vasodilatation involve differential [Ca2+]i responses

The present study was designed to evaluate the role of endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) in the difference between P2Y(1)- and P2Y(2)-mediated vasodilatations in cerebral arteries. Rat middle cerebral arteries were cannulated, pressurized, and luminally perfused. The endothelium was selectively loaded with fura 2, a fluorescent Ca(2+) indicator, for simultaneous measurement of endothelial [Ca(2+)](i) and diameter. Luminal administration of 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP), an endothelial P2Y(1) agonist, resulted in purely nitric oxide (NO)-dependent dilation and [Ca(2+)](i) increases up to approximately 300 nM (resting [Ca(2+)](i) = 145 nM). UTP, an endothelial P2Y(2) agonist, resulted in dilations that were both endothelium-derived hyperpolarizing factor (EDHF)- and NO-dependent with [Ca(2+)](i) increases to >400 nM. In the presence of N(G)-nitro-L-arginine-indomethacin to inhibit NO synthase and cyclooxygenase, UTP resulted in an EDHF-dependent dilation alone. The [Ca(2+)](i) threshold for NO-dependent dilation was 220 vs. 340 nM for EDHF. In summary, the differences in the mechanism of vasodilatation resulting from stimulation of endothelial P2Y(1) and P2Y(2) purinoceptors result in part from differential [Ca(2+)](i) responses. Consistent with this finding, these studies also demonstrate a higher [Ca(2+)](i) threshold for EDHF-dependent responses compared with NO.     

Inhibition of Ca2+ signaling and glucagon secretion in mouse pancreatic alpha-cells by extracellular ATP and purinergic receptors

Glucagon secreted from pancreatic alpha-cells plays a critical role in glycemia, mainly by hepatic glucose mobilization. In diabetic patients, an impaired control of glucagon release can worsen glucose homeostasis. Despite its importance, the mechanisms that regulate its secretion are still poorly understood. Since alpha-cells are particularly sensitive to neural and paracrine factors, in this report we studied the role of purinergic receptors and extracellular ATP, which can be released from nerve terminals and beta-cell secretory granules. Using immunocytochemistry, we identified in alpha-cells the P2 receptor subtype P2Y1, as well as the P1 receptors A1 and A2A. In contrast, only P2Y1 and A1 receptors were localized in beta-cells. To analyze the role of purinergic receptors in alpha-cell function, we studied their participation in Ca2+ signaling. At low glucose concentrations, mouse alpha-cells exhibited the characteristic oscillatory Ca2+ signals that lead to secretion. Application of ATP (1-10 microM) abolished these oscillations or reduced their frequency in alpha-cells within intact islets and isolated in culture. ATPgammaS, a nonhydrolyzable ATP derivative, indicated that the ATP effect was mainly direct rather than through ATP-hydrolytic products. Additionally, adenosine (1-10 microM) was also found to reduce Ca2+ signals. ATP-mediated inhibition of Ca2+ signaling was accompanied by a decrease in glucagon release from intact islets in contrast to the adenosine effect. Using pharmacological agonists, we found that only P2Y1 and A2A were likely involved in the inhibitory effect on Ca2+ signaling. All these findings indicate that extracellular ATP and purinergic stimulation are effective regulators of the alpha-cell function.     

Effect of shear stress on cytosolic Ca2+ of calf pulmonary artery endothelial cells.

The purpose of the present study was to determine if hemodynamic shear stress increases free cytosolic Ca2+ concentration ([Ca2+]i) of cultured pulmonary artery endothelial cells exposed to steady laminar fluid flow in a parallel plate chamber. Average [Ca2+]i was estimated by measuring cell-associated fura-2 fluorescence using microfluorimetric analysis. To determine [Ca2+]i close to the membrane surface, 86Rb+ efflux via Ca(2+)-dependent K+ channels was measured. Upon initiation of flow or upon step increases in flow, no change in [Ca2+]i was observed using fura-2. However, increases in shear stress produced a large, transient increase in 86Rb+ efflux. The shear stress-dependent increase in 86Rb+ efflux was not blocked by either tetrabutylammonium ions (20 mM) or by charybdotoxin (10 nM), two specific inhibitors of the Ca(2+)-dependent K+ channel of vascular endothelial cells. These results demonstrate that shear stress per se has little effect on either the average cytosolic [Ca2+]i as measured by fura-2 or on [Ca2+]i close to the cytoplasmic surface of the plasmalemma as measured by the activity of Ca(2+)-dependent K+ channels.