P2Y Purinoceptors Research Articles

ATP-induced [Ca2+]i changes in the human corneal epithelium.

ATP, when leaked from damaged cells, is capable of eliciting responses in neighboring cells. A better understanding of the mechanism of this response is essential for designing therapeutic strategies for disease, there have been only a limited number of studies on the effect of ATP on the human cornea. We examined ATP-induced intracellular Ca2+ ([Ca2+]i) changes in the human corneal epithelia, cultured to near confluence. Cells were loaded with the Ca2+ indicators, Indo-1 or Fluo-4, and [Ca2+]i was monitored. ATP was found to induce an increase in [Ca2+]i, which was initiated from the perinuclear region and the nuclear envelope per se, and then propagated gradually towards the periphery. Intranuclear Ca2+ was momentarily increased. UTP elicited an identical response, but adenosine and alpha, beta-methylene ATP had no effect. Pretreatment with U73122 or thapsigargin inhibited the ATP-induced increase in [Ca2+]i. When a cell was topically stimulated with ATP, the [Ca2+]i increase spread beyond the cell boundary. The intercellular communications that accompanied the [Ca2+]i changes were inhibited by octanol. We conclude that extracellular ATP in the human cornea caused the mobilization of Ca2+ from intracellular Ca2+ stores (e.g. the endoplasmic reticulum and nuclear envelope) via P2Y purinoceptors of the epithelial cell. The response to ATP appears to spread to neighboring regions through gap junctions in the epithelium.

Neurotransmitters involved in the fast inhibitory junction potentials in mouse distal colon

We investigated, in murine colon circular muscle, the role of adenosine 5′-triphosphate (ATP) and pituitary adenylate cyclase activating peptide (PACAP) as inhibitory neurotransmitters of the fast component of nerve-evoked inhibitory junction potential (fast IJP). Fast IJP was antagonised by apamin or suramin, abolished by desensitisation with the P2Y receptor agonist, adenosine 5′- O-2-thiodiphosphate (ADPβS), unaffected by desensitisation with P2X receptor agonist, α,β-methylene ATP (α,β-meATP), and reduced by PACAP-(6–38), a PACAP receptor antagonist. ATP induced membrane hyperpolarization resistant to tetrodotoxin, N ω-nitro- l-arginine methyl ester ( l-NAME) or PACAP-(6–38), but antagonised by apamin, suramin, P2X and P2Y receptor desensitisation. PACAP-(1–27) caused membrane hyperpolarization antagonised by PACAP-(6–38), apamin and P2Y receptor desensitisation, reduced by tetrodotoxin, but not affected by l-NAME and by P2X receptor desensitisation. Therefore, in murine colon circular muscle, an ATP-like endogenous P2Y purinoceptor ligand is the final nonadrenergic, noncholinergic (NANC) inhibitory mediator involved in the generation of fast IJP. A neuromodulator role of PACAP in the inhibitory pathway is supposed.

P2Y Purinoceptors Are Responsible for Oscillatory Fluid Flow-induced Intracellular Calcium Mobilization in Osteoblastic Cells

We previously found that oscillatory fluid flow activated MC3T3-E1 osteoblastic cell Ca(2+)(i) mobilization via the inositol 1,4,5-trisphosphate pathway in the presence of 2% fetal bovine serum (FBS). However, the molecular mechanism of fluid flow-induced Ca(2+)(i) mobilization is unknown. In this study, we first demonstrated that oscillatory fluid flow in the absence of FBS failed to increase [Ca(2+)](i) in MC3T3-E1 cells. Apyrase (10 units/ml), which rapidly hydrolyzes 5' nucleotide triphosphates to monosphophates, prevented the fluid flow induced increases in [Ca(2+)](i) in the presence of FBS. Adding ATP or UTP to flow medium without FBS restored the ability of fluid flow to increase [Ca(2+)](i), suggesting that ATP or UTP may mediate the effect of fluid flow on [Ca(2+)](i). Furthermore, adenosine, ADP, UDP, or adenosine 5'-O-(3-thiotriphosphate) did not induce Ca(2+)(i) mobilization under oscillatory fluid flow without FBS. Pyridoxal phosphate 6-azophenyl-2,4'-disulfonic acid, an antagonist of P2X purinoceptors, did not alter the effect of fluid flow on the Ca(2+)(i) response, whereas pertussis toxin, a G(i/o)-protein inhibitor, inhibited fluid flow-induced increases in [Ca(2+)](i) in the presence of 2% FBS. Thus, by the process of elimination, our data suggest that P2Y purinoceptors (P2Y2 or P2Y4) are involved in the Ca(2+)(i) response to fluid flow. Finally, a decreased percentage of MC3T3-E1 osteoblastic cells treated with P2Y2 antisense oligodeoxynucleotides responded to fluid flow with an increase in [Ca(2+)](i), and an increased percentage of ROS 17/2.8 cells, which do not normally express P2Y2 purinoceptors, transfected with P2Y2 purinoceptors responded to fluid flow in the presence of 2% FBS, confirming that P2Y2 purinoceptors are responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization. Our findings shed new light of the molecular mechanisms responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization in osteoblastic cells.

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia.

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were -4.4 +/- 1.3 mV (mean +/- SD, n = 12) and 517.7 +/- 39.4 Omega. cm(2), respectively. The apical membrane potential was -43.9 +/- 1.7 mV, and the ratio of apical to basolateral membrane resistance (R(A)/R(B)) was 3.5 +/- 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC(50) of approximately 24 microM (apical) and approximately 30 microM (basal). Apical ATP/UTP (100 microM) depolarized apical membrane potential by 17.6 +/- 0.8 mV (n = 7) and decreased R(A)/R(B) by a factor of approximately 3. The addition of adenosine to either side (100 microM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca(2+) concentration (427 +/- 206 nM; 15-25 microM ATP, apical; n = 6). This Ca(2+) increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 +/- 3 microl x cm(-2) x h(-1) (n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y(2) purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca(2+)-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.

Extracellular ATP Is an Autocrine/Paracrine Regulator of Hypoxia-induced Adventitial Fibroblast Growth: SIGNALING THROUGH EXTRACELLULAR SIGNAL-REGULATED KINASE-1/2 AND THE Egr-1 TRANSCRIPTION FACTOR

Important autocrine/paracrine functions for the adenine nucleotides have been proposed in several tissues. We addressed the possibility that extracellular ATP would modulate/mediate hypoxia-induced adventitial fibroblast growth. Acute hypoxia (3% O(2), 10-60 min) increased extracellular ATP concentrations in adventitial fibroblasts and in lung microvascular endothelial cells, and chronic hypoxia (3% O(2), 14-30 days) markedly attenuated the rate of extracellular ATP hydrolysis by ecto-nucleotidase(s). Exogenous ATP stimulated [(3)H]thymidine incorporation in fibroblasts as did UTP, ADPbeta, 2-methylthioadenosine triphosphate, adenosine 5'-(alpha,beta-methylene)triphosphate, and benzoylbenzoyl-ATP (2'-3'-O-(4-benzoylbenzoyl)-ATP), indicating that both P2Y and P2X purinoceptors can mediate mitogenic responses. Suramin (100 microm), Cibacron blue 3GA (100 microm), and pyridoxalphosphate-6-azophenyl-2',-4'-disulfonic acid (100 microm) as well as apyrase (5 units/ml) attenuated hypoxia- and ATP-induced and DNA synthesis, indicating activation and a functional role of purinoceptors under hypoxic conditions. ATP-induced DNA synthesis was augmented by hypoxia in an additive fashion, whereas ATP and hypoxia synergistically increased growth factor-induced DNA synthesis, again suggesting that ATP and hypoxia utilize similar signaling pathways to induce proliferation. Indeed, we found that ATP (100 microm) and hypoxia (3% O(2)) induced expression and activation of Egr-1 transcription factor, and both stimuli acted, in part, through a G(alpha)(i)/ERK1/2-dependent signaling pathway. Suramin, Cibacron blue 3GA, and apyrase attenuated hypoxia-induced ERK1/2 activation and Egr-1 expression. We conclude that hypoxia induces ATP release from endothelial cells and fibroblasts and that the activation of P2 purinoceptors is involved in the regulation of DNA synthesis by fibroblasts under hypoxic conditions.

Two-phase response of acid extrusion triggered by purinoceptor in Chinese hamster ovary cells

The functional characteristics of purinoceptors in Chinese hamster ovary (CHO) cells were investigated using a microphysiometer which detects small metabolic changes to living cells in real-time as variations of pH in the extracellular microenvironment. Uridine 5′-triphosphate (UTP) increased the extracellular acidification rate biphasically, namely a transient and a steady response were observed. The transient phase reached a peak (four- to fivefold the basal extracellular acidification rate in amplitude) within 20 s and was followed by the steady phase which was sustained for more than 1 min at an amplitude less than twofold the basal extracellular acidification rate. Both phases showed a concentration-dependent increase in response to UTP. However, there was a significant difference in the pEC 50 value for UTP between the transient (4.8) and steady phases (6.1). Like UTP, ATP increased the extracellular acidification rate, but α,β-methyleneATP (α,β-MeATP), 2-methylthioATP (2-MeSATP), ADP, UDP and adenosine did not. This result suggests that the acid is extruded through a P2Y 2 or P2Y 2-like purinoceptor. 5-( N-ethyl- N-isopropyl) amiloride (EIPA) and 4-isopropyl-3-methylsulphonylbenzoyl-guanidine methanesulphonate (HOE642) suppressed both phases of the UTP-stimulated extracellular acidification rate response with high affinity (pIC 50: approximately 7.0). This result suggests that the Na +/H + exchanger 1 (NHE-1) predominantly mediates the UTP-induced acid extrusion response in CHO cells. Elimination of extracellular Ca 2+ or treatment with thapsigargin diminished both phases of the UTP-stimulated extracellular acidification rate. In addition, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7) also abrogated the two phases. These results are consistent with the involvement of NHE-1 which is activated via Ca 2+/calmodulin. Persistent exposure to UTP reduced both extracellular acidification rate phases, causing desensitization of the P2Y purinoceptor. This desensitization did not affect the acid extrusion response mediated by the α 1-adrenoceptor.

Activation of P2Y2 purinoceptor inhibits the activity of the Na +/K +-ATPase in HeLa cells

The role of ATP on regulation of the Na +/K +-ATPase activity in the human cancerous HeLa cells was investigated. HeLa cells stimulated with increasing ATP concentrations showed a dose-dependent inhibition of the Na +/K +-ATPase activity. These effects were also obtained by UTP. ATP and UTP provoked a rise in intracellular calcium concentration ([Ca 2+] i) persisting for at least 4 min. The inhibitor of phospholipase C, U73122, blocked the elevation of [Ca 2+] i provoked by ATP/UTP. The expression of mRNA for P2Y2 and P2Y6 receptors was demonstrated by RT-PCR. ATP/UTP activated PKC-α, -βI and -ε isoforms, but not PKC-δ and -ζ. The inhibition of the Na +/K +-ATPase activity by ATP/UTP was blocked by Gö6976, a specific inhibitor of the calcium-dependent PKCs. In conclusion, our results suggest that ATP/UTP modulate Na +/K +-ATPase activity in HeLa cells through the P2Y2 purinoceptor via calcium mobilisation and activation of calcium-dependent PKCs.

Mesenteric and renal vascular effects of diadenosine polyphosphates (APnA).

Diadenosine polyphosphates (APnA) are endogenous dinucleoside molecules consisting of two adenosine moieties linked via their 5'-ribose positions by a variable number of phosphate groups. APnA have been shown to be present in different cell types and to be released from platelets as well as co-released with catecholamines and ATP from bovine adrenal medulla. Candidate metabolites of APnA are ATP, ADP, AMP and adenosine. Vascular effects induced by APnA and their metabolites in several models have been reported to be mediated by A1- and A2-adenosine receptors as well as P2-purinoceptors. APnA have been demonstrated to differentially affect regional perfusion, to influence cardiac output and blood pressure as well as the reactivity of isolated blood vessels and vascular beds. Vascular effects of APnA vary with the number of phosphate groups linking the adenosine molecules. This review outlines the effects of APnA on mesenteric and renal circulation. The effects of the antagonists varying with the type of vascular bed and the heterogeneous and dynamic vascular effects of diadenosine polyphosphates indicate a regionally different distribution of P2X and of P2Y purinoceptors in resistance arteries from different vascular beds. Although APnA have vasoconstrictor effects on the local level, it was repeatedly confirmed that systemically applied APnA induce hypotensive effects. The vasoconstrictor effects of APnA in isolated vessels are most prominent under resting tone conditions. In vivo, the vasculature exhibits a vasotone which makes dilatory effects more likely. Information on effects of APnA in vivo is still limited despite the fact that these compounds already have been used in man.

Altered endothelial Ca2+ regulation after ischemia/reperfusion produces potentiated endothelium-derived hyperpolarizing factor-mediated dilations.

Endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations are potentiated after several pathologies, including ischemia/reperfusion (I/R). However, no study to date has addressed the mechanism by which this potentiation occurs. This study tested the hypothesis that potentiated EDHF-mediated dilations are due to altered endothelial Ca2+ handling after I/R. Rat middle cerebral arteries (MCAs) were isolated after 2 hours of MCA occlusion and 24 hours of reperfusion (or sham surgery). This model has been previously demonstrated to produce potentiated EDHF-mediated dilations. MCAs were studied in a pressurized/perfused vessel chamber equipped for the simultaneous measurement of endothelial Ca2+ (with fura 2) and artery diameter. Measures were made after luminal administration of UTP (P2Y2 purinoceptor agonist), 2 MeS-ATP (P2Y1 purinoceptor agonist), and Br-A23187 (receptor-independent Ca2+ ionophore) for sham and I/R MCAs. I/R resulted in significantly potentiated UTP-mediated dilations (through a P2Y2 purinoceptor) and endothelial Ca2+ responses in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) and indomethacin. Endothelial Ca2+ and diameter responses were also significantly potentiated with 2 MeS-ATP (through a P2Y1 purinoceptor) when L-NAME and indomethacin were absent. Br-A23187, a receptor-independent Ca2+ ionophore, produced significantly potentiated endothelial Ca2+ responses after I/R in the presence of L-NAME/indomethacin. Evaluation of artery diameter as a function of endothelial Ca2+ demonstrated no differences between sham and I/R groups. These findings demonstrate that I/R results in augmented endothelial Ca2+ responses that appear to be downstream of the receptor level. Moreover, these data suggest that this augmented Ca2+ response contributes to the potentiated EDHF-mediated dilations after I/R.

Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

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 α,β-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.

Vasoactivity of diadenosine polyphosphates in human small mesenteric resistance arteries.

Diadenosine polyphosphates (ApnA) (n = 3-6) induced vasoconstrictions in isolated human mesenteric resistance arteries (hMRAs) mounted in a microvessel myograph (rank order of potency: Ap5A > Ap6A > Ap4A > Ap3A). The contractile effects of ApnA in hMRA were similar to their effects in rat MRA investigated previously. ATP, ADP, AMP, and adenosine had less contractile potency than ApnA, suggesting that the observed effects were not induced by the degradation products of ApnA. Ap4A- and Ap5A-induced vasoconstriction was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (P2X purinoceptor antagonist) but not by ADP3'5' (P2Y purinoceptor antagonist). Thus, this purinergic vasoconstriction of hMRA seems to be P2X but not P2Y purinoceptor-mediated. In precontracted hMRA all ApnA caused vasorelaxations but (in contrast to rat MRA) the potencies of the ApnA did not differ significantly from each other. The ApnA degradation products had less vasorelaxing potency than ApnA, demonstrating that the vasorelaxations can be ascribed to the ApnA themselves. Ap5A-induced vasorelaxation of hMRA could neither be inhibited with ADP3'5' nor with PPADS, which reveals a decisive difference to the rat MRA where the inhibitory profile demonstrated the importance of the P2Y purinoceptor for Ap5A-induced vasorelaxation. However, Ap4A-induced vasorelaxation in hMRA could be inhibited by ADP3'5'. These findings show that Ap4A-induced vasorelaxation in hMRA is due to P2Y purinoceptor activation, that Ap5A evokes vasorelaxation in hMRA via another mechanism than Ap4A, and that data derived from the animal model cannot be simply transferred to human conditions.

ADP and other metabolites released from Acanthamoeba castellanii lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines.

Monocytes/macrophages are thought to be involved in Acanthamoeba infections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released by Acanthamoeba castellanii trophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium by A. castellanii, interacting with specific P2y(2) purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca(2+)](i), morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-alpha). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-alpha release induced by amoebic ADP were blocked by the P2y(2) inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, >30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of <10 kDa caused the secretion of interleukin-1beta. These findings suggest that pathogenic free-living A. castellanii by release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines.

Cooperativity between extracellular adenosine 5′-triphosphate and activation of N-methyl- D-aspartate receptors in long-term potentiation induction in hippocampal CA1 neurons

The mechanism of ATP-induced long-term potentiation (LTP) was studied pharmacologically using guinea-pig hippocampal slices. LTP, induced in CA1 neurons by 10 min application of 10 μM ATP, was blocked by co-application of the N-methyl- D-aspartate (NMDA) receptor antagonist, D, L-2-amino-5-phosphonovalerate (5 or 50 μM). In ATP-induced LTP, the delivery of test synaptic inputs (once every 20 s) to CA1 neurons could be replaced by co-application of NMDA (100 nM) during ATP perfusion. These results suggest that, in CA1 neurons, a co-operative effect between extracellular ATP and activation of NMDA receptors is required to trigger the process involved in ATP-induced LTP. In addition, ATP-induced LTP was blocked by co-application of an ecto-protein kinase inhibitor, K-252b (40 or 200 nM), whereas a P2X purinoceptor antagonist, pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid 4-sodium (50 μM), or a P2Y purinoceptor antagonist, basilen blue (10 μM), had no effect. The results of the present study, therefore, indicate that the mechanisms of ATP-induced LTP involve the modulation of NMDA receptors/Ca 2+ channels and the phosphorylation of extracellular domains of synaptic membrane proteins, one of which could be the NMDA receptor/Ca 2+ channel.

Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain.

Phenotypic modification of dorsal root ganglion (DRG) neurons represents an important mechanism underlying neuropathic pain. However, the nerve injury-induced molecular changes are not fully identified. To determine the molecular alterations in a broader way, we have carried out cDNA array on the genes mainly made from the cDNA libraries of lumbar DRGs of normal rats and of rats 14 days after peripheral axotomy. Of the 7,523 examined genes and expressed sequence tags (ESTs), the expression of 122 genes and 51 expressed sequence tags is strongly changed. These genes encompass a large number of members of distinct families, including neuropeptides, receptors, ion channels, signal transduction molecules, synaptic vesicle proteins, and others. Of particular interest is the up-regulation of gamma-aminobutyric acid(A) receptor alpha5 subunit, peripheral benzodiazepine receptor, nicotinic acetylcholine receptor alpha7 subunit, P2Y1 purinoceptor, Na(+) channel beta2 subunit, and L-type Ca(2+) channel alpha2delta-1 subunit. Our findings therefore reveal dynamic and complex changes in molecular diversity among DRG neurons after axotomy. Sequences reported in this paper have been deposited in the GenBank database (accession numbers BG 662484-BG 673712)

P2Y and P2X purinoceptor mediated Ca 2+ signalling in glial cell pathology in the central nervous system

Activation of purinoceptors by extracellular ATP is an important component of the glial response to injury in the central nervous system (CNS). ATP has been shown to evoke raised cytosolic [Ca 2+] in astrocytes, oligodendrocytes, and microglia, the three major glial cell types in the CNS. Glial cells express a heterogenous collection of metabotropic P2Y and ionotropic P2X purinoceptors, which respectively mobilise Ca 2+ from intracellular stores and trigger Ca 2+ influx across the plasmalemma. It is likely that different receptors have distinct roles in glial cell physiology and pathology. Our studies on optic nerve glia in situ indicate that P2Y 1 and P2Y 2/4 receptors are activated at low ATP concentrations, suggesting they are the predominant purinoceptors mediating physiological Ca 2+ signalling. Glia also express P2X 1 and P2X 3 purinoceptors, which mediate fast, rapidly desensitising current and may also be important in signalling. At high concentrations, such as occur in CNS injury, ATP induces large and prolonged increases in glial [Ca 2+] i with a primary role for P2Y purinoceptors and inositol trisphosphate (IP 3)-dependent release of Ca 2+ from intracellular stores. In addition, we found that high concentrations of ATP activated a significant P2X component that did not desensitise or saturate and was dependent on extracellular Ca 2+. These are characteristic properties of the P2X 7 subtype, and we provide in situ evidence that application of the P2X 7 receptor agonist benzoyl-benzoyl ATP (BzATP) evokes raised [Ca 2+] i in optic nerve glia, and that the dye YO-PRO-1, which passes through pore-forming P2X 7 receptors, is taken up by astrocytes, oligodendrocytes and microglia. Glia also express P2X 2 and P2X 4 receptors that are also pore-forming in the presence of sustained high ATP concentrations and which may also be important in the glial injury response. There is evidence that activation of P2 purinoceptors is a key step in triggering reactive changes in glial cells, including expression of immediate early genes, induction of extracellular signal regulated kinase and cyclooxygenase-2, synthesis of phospholipase A 2, release of arachidonic acid, production of prostaglandins and release of interleukins. We show that the ATP-mediated increase in glial [Ca 2+] i is potentiated by arachidonic acid and reduced by the inhibition of phospholipase A 2 inhibition. Together, the results implicate ATP as a primary signalling molecule in glial cells and indicate specific roles for P2Y and P2X purinoceptors in glial cell pathology.

Complementary role of extracellular ATP and adenosine in ischemic preconditioning in the rat heart.

Although adenosine is an important mediator of ischemic preconditioning (IPC), its relative contribution to IPC remains unknown. Because adenosine is formed through the hydrolysis of ATP, the present study investigated the role of ATP and adenosine in IPC. Isolated and buffer-perfused rat hearts underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. The rate-pressure product (RPP) 30 min after reperfusion was taken as an endpoint of functional protection. Interstitial fluid (ISF) adenine nucleotides and adenosine were measured by cardiac microdialysis techniques. Inhibition of IPC-induced recovery of RPP was partial by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (SPT; 100 microM) or by the structurally distinct P2Y purinoceptor antagonists suramin (300 microM) or reactive blue (RB; 10 microM) but was additive when SPT was given with suramin or RB. The P2X antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (50 microM) had no effect on functional protection. The improved functional recovery was not significantly affected by an ecto-5'-nucleotidase inhibitor, alpha,beta-methylene adenosine diphosphate (AMP-CP; 100 microM), alone but was inhibited by AMP-CP plus SPT, suramin, or RB. ISF ATP and adenosine increased temporarily by 10-fold during IPC. AMP-CP augmented the increase in ISF ATP associated with the decrease in ISF adenosine. There was a reciprocal correlation between the ISF concentration of ATP and adenosine in preconditioned hearts. In addition, there was a significant correlation between ISF adenosine and ATP and the inhibitory potency of SPT and suramin or RB against functional protection conferred by IPC. These results suggest that extracellular ATP and adenosine play a complementary role in IPC through P2Y purinoceptors and adenosine receptors, respectively.

Modulation of K(+) currents in Xenopus spinal neurons by p2y receptors: a role for ATP and ADP in motor pattern generation.

We have investigated the pharmacological properties and targets of p2y purinoceptors in Xenopus embryo spinal neurons. ATP reversibly inhibited the voltage-gated K(+) currents by 10 +/- 3 %. UTP and the analogues alpha,beta-methylene-ATP and 2-methylthio-ATP also inhibited K(+) currents. This agonist profile is similar to that reported for a p2y receptor cloned from Xenopus embryos. Voltage-gated K(+) currents could be inhibited by ADP (9 +/- 0.8 %) suggesting that a further p2y1-like receptor is also present in the embryo spinal cord. Unexpectedly we found that alpha,beta-methylene-ADP, often used to block the ecto-5'-nucleotidase, also inhibited voltage-gated K(+) currents (7 +/- 2.3 %). This inhibition was occluded by ADP, suggesting that alpha,beta-methylene-ADP is an agonist at p2y1 receptors. We have directly studied the properties of the ecto-5'-nucleotidase in Xenopus embryo spinal cord. Although ADP inhibited this enzyme, alpha,beta-methylene-ADP had no action. Caution therefore needs to be used when interpreting the actions of alpha,beta-methylene-ADP as it has previously unreported agonist activity at P2 receptors. Xenopus spinal neurons possess fast and slow voltage-gated K(+) currents. By using catechol to selectively block the fast current, we completely occluded the actions of ATP and ADP. Furthermore, the purines appeared to block only the fast relaxation component of the tail currents. We therefore conclude that the p2y receptors target only the fast component of the delayed rectifier. As ATP breakdown to ADP is rapid and ADP may accumulate at higher levels than ATP, the contribution of ADP acting through p2y1-like receptors may be an important additional mechanism for the control of spinal motor pattern generation.

Adenosine 5′-triphosphate-evoked calcium responses in terminal Schwann cells of lanceolate sensory endings isolated from rat vibrissae

Extracellular adenosine 5′-triphosphate (ATP) has been known to mediate and modulate cutaneous sensations. We examined the effect of this substance on isolated terminal Schwann cells associating with lanceolate endings, the mechanoreceptors of rat vibrissae. The free intracellular calcium concentration ([Ca 2+] i) of the sensory device was monitored by digital image microscopy in combination with a calcium-sensitive fluorescent probe, Fura-2. Application of ATP in concentrations raging from 10 μM to 1 mM evoked an increase in [Ca 2+] i in Schwann cell processes covering the lancet-like axon terminals as well as in round perikarya of the cells protruding from the terminals. In both portions, the ATP-evoked [Ca 2+] i elevations were slowly oscillatory at 10 and 20 μM, and continuous at concentrations higher than 50 μM. Suramin 100 μM blocked the effect of ATP. Uridine 5′-triphosphate was equipotent with ATP, while α,β-methylene ATP was ineffective. These data indicate that the terminal Schwann cells express P2Y purinoceptors linked with the intracellular Ca 2+ signaling, and that this phenomenon is involved in the ATP-mediated sensory modulation.

Invited commentary

It is a privilege to review and comment on this interesting article. The experimental study presented by Dr Munakata and coworkers demonstrates the role of phosphatidyl-inositol specific phospholipase C (PI-PLC) in the myocardium exposed to a short period of normothermic ischemia using an isolated rabbit heart model. The authors used a specific inhibitor of PI-PLC, U73122 and its inactive analogue U73343 to modify the effect of PI-PLC to activate protein kinase C (PKC). The authors also were able to show reversal of the U73122 induced post-ischemic cardiac dysfunction by direct PKC activation with phorbol myristate acetate (PMA). This suggests that the myocardial protective effect of PI-PLC after ischemia occurs through PKC activation. Many studies have shown a significant myocardial protective effect of ischemic preconditioning [1Murry C.E. Richard V.J. Reimer K.A. Jennings R.B. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode.Circ Res. 1990; 66: 913-931Crossref PubMed Scopus (746) Google Scholar]. However, the mechanism and its clinical significance have been discussed for more than 10 years without a definitive answer. One promising candidate for the mechanism of ischemic preconditioning is activation of PKC by PI-PLC [2De Jonge H.W. Van Heugten H.A. Lamers J.M. Signal transduction by the phosphatidylinositol cycle in myocardium.J Mol Cell Cardiol. 1995; 27: 93-106Abstract Full Text PDF PubMed Scopus (62) Google Scholar] to protect the heart against impairment in intracellular calcium flux. The authors’ observation in this article that PI-PLC inhibition impairs diastolic calcium flux without a significant decrease in cellular ATP level is new and important. Further investigation using modifications of the cellular calcium transport system, eg, sarcoplasmic reticulum inhibition and membrane L-type calcium channel inhibition would be interesting and may clarify the mechanism underlying the intrinsic, antiischemic property of myocardium [3Liu D.M. Katnik C. Stafford M. Adams D.J. P2Y purinoceptor activation mobilizes intracellular Ca2+ and induces a membrane current in rat intracardiac neurones.J Physiol. 2000; 526: 287-298Crossref PubMed Scopus (25) Google Scholar]. Although the myocardial protective effect related to PKC activation during ischemia attracts much interest, many controversies remain. These controversies may due to the difference between species, experimental models, ischemic durations, and degree of ischemic insult. In addition, differences in the role of PKC-ε and PKC-α during short periods of ischemia is not fully understood. Researchers and clinicians must resolve these problems before this mechanism can be used to improve clinical myocardial protection. Further investigation is required. I congratulate these investigators for stimulating our thinking about the mechanism of the intrinsic protective property of the myocardium against ischemic reperfusion damage.

Adenosine 5′-triphosphate inhibits slow depolarization induced by repetitive dorsal root stimulation via P2Y purinoceptors in substantia gelatinosa neurons of the adult rat spinal cord slices with the dorsal root attached

We previously reported that slow depolarization of substantia gelatinosa neurons is evoked by repetitive stimulation of C-fibers of dorsal root in adult rat spinal cord transverse slices with the dorsal root attached, which was considered to be an event relevant to spinal nociception. In the present study, we investigated the effects of adenosine 5′-triphosphate (ATP) and its analogs on the slow depolarization. ATP (10–100 μM) significantly inhibited the amplitude of slow depolarization in a concentration-dependent manner. The inhibitory effect of ATP was not reversed by suramin, an antagonist for some P2-purinoceptors, and was mimicked by a P2Y selective agonist uridine 5′-triphosphate, but not a P2X selective agonist α,β-methylene ATP. These results suggest that ATP inhibits the slow depolarization of substantia gelatinosa neurons relevant to nociceptive transmission in the spinal dorsal horn, via suramin-insensitive P2Y purinoceptors.