alpha,beta-meATP Research Articles

Purinoreceptor-mediated current in myocytes from renal resistance arteries.

Ionotropic purinoreceptors (P2X) in renal vascular smooth muscle cells (RVSMCs) are involved in mediating the sympathetic control and paracrine regulation of renal blood flow (RBF). Activation of P2X receptors elevates [Ca(2+)](i) in RVSMCs triggering their contraction, leading to renal vasoconstriction and decrease of RBF. The goal of the present work was to characterize the P2X receptor-mediated ionic current (I(P2X)) and to identify the types of P2X receptors expressed in myocytes isolated from interlobar and arcuate arteries of rat kidney. The expression of P2X receptors in isolated RVSMCs was analysed by reverse transcription (RT)-PCR. I(P2X) and membrane potential were recorded using the amphotericin B-perforated patch method. RT-PCR analysis on single RVSMCs showed the presence of genes encoding P2X1 and P2X4 receptors. Under voltage clamp conditions, the selective P2X receptor agonist alphabeta-methylene ATP (alphabeta-meATP) evoked I(P2X) similar to that induced by ATP. Under current clamp conditions, both ATP and alphabeta-meATP evoked a spike-like membrane depolarization followed by a sustained depolarization, linking P2X receptors in RVSMCs to sympathetic control of renal vascular tone. A selective antagonist of P2X1 receptors, NF279, reduced I(P2X) amplitude by approximately 65% concentration-dependently manner within the nanomolar to sub-micromolar range. The residual current was resistant to micromolar concentrations of NF279, but was inhibited by sub-millimolar to millimolar concentrations of NF279. Two types of functional P2X receptors, monomeric P2X1 and heteromeric P2X1/4 receptors, are expressed in RVSMCs. Our study has identified important targets for possible pharmacological intervention in the sympathetic control of renal circulation.

A P2X Ion Channel–Triggered NF-κB Pathway Enhances TNF-α–Induced IL-8 Expression in Airway Epithelial Cells

Extracellular ATP, acting at P2Y and P2X receptors, has recently been shown to contribute to airway inflammation. The aim of our study was to investigate the molecular mechanisms involved in the ATP-dependent regulation of IL-8 production by airway epithelial cells. Treatment of human normal tracheal (NT)-1 cells with ATP or its two analogs, alpha,beta-methylene ATP (alpha,beta-meATP) and 2'- and 3'-O-(4-benzoyl-benzoyl)-ATP (BzATP) activated NF-kappaB through the IkappaB kinase (IKK) complex, a process requiring Ca(2+), calmodulin (CaM), and Ca(2+)/CaM-dependent kinase (CaMK), but independent from phospholipase C. alpha,beta-meATP-induced IKK activation also occurred in the alveolar A549 cell line. Real-time RT-PCR revealed that NT-1 and A549 cells expressed P2X(4), P2X(5),and P2X(6) subtype mRNAs, whereas P2X(7) mRNAs were only detected in NT-1 cells. Polarized human primary nasal epithelial cells expressed all four P2X subtypes. Both alpha,beta-meATP and BzATP caused Ca(2+)-dependent binding of phosphorylated p65 (S536) NF-kappaB subunit to the endogenous IL-8 gene promoter in NT-1 cells. Although these agonists did not induce significant IL-8 gene expression by these cells, they markedly enhanced TNF-alpha-induced NF-kappaB activation, resulting in increased IL-8 expression and release. Application of alpha,beta-meATP or BzATP at the apical side of polarized human primary nasal epithelial cells sufficed to cause CaMK-dependent IL-8 release by these cells. Thus, ATP promotes TNF-alpha-elicited IL-8 expression through P2X ion channel-triggered Ca(2+) entry, leading to CaMK-dependent IKK activation and binding of active p65 to IL-8 gene promoter.

Regulation of the pH sensitivity of human P2X3 receptors by N‐linked glycosylation

The human (h) P2X(3) receptor and its mutants deficient in one out of four N-glycosylation sites were expressed in HEK293 cells. Concentration-response curves were generated by whole-cell recordings of alpha,beta-methylene ATP (alpha,beta-meATP)-induced currents. A gradual change of external pH from the alkaline 8.0 to the acidic 5.0 successively decreased the maximum current amplitude (E(max)) without affecting the EC(50) value. The replacement of Asn-139 and -170 by Asp (N139D, N170D) abolished the pH sensitivity of the wild-type (WT) hP2X(3) receptor. In the case of N194D, the E(max) was again the highest at the alkaline pH value with no change from 7.4 to 6.5, whereas in the case of N290D, there was an inverse pH sensitivity, with an increase of E(max) in the acidic range. However, this effect appeared to be due to enhanced protonation by the insertion of Asp into the receptor, because replacement of Asn by the neutral Thr resulted in a comparable potency of alpha,beta-meATP at any of the pH values investigated. In accordance with the reported finding that His-206 is involved in the modulation of WT P2X(3) receptors by protons, we showed that the normal change of E(max) by an acidic, but not alkaline pH was abolished after substitution of this His by Ala. However, the double mutant H206A + N290D did not react to acidification or alkalinization with any change in E(max). In conclusion, only fully N-glycosylated P2X(3) receptors recognize external pH with a modified sensitivity towards alpha,beta-meATP.

Cross talk between P2 purinergic receptors modulates extracellular ATP-mediated interleukin-10 production in rat microglial cells

Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 microM) and bell-shaped. The concentrations of ATP, ATP-gammaS, ADP, and ADP-betaS that showed maximal IL-10 release were 100, 10, 100, and 100 microM respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP)=dATP>2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), alpha,beta-methylene ATP (alpha,beta-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca(2+) release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down-regulated by either MRS2179 (a P2Y(1) antagonist) or 5'-AMPS (a P2Y(11) antagonist), indicating that both the P2Y(1) and P2Y(11) receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-gammaS (100 microM) was restored by TNP-ATP (an antagonist of the P2X(1), P2X(3), and P2X(4) receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y(12) receptor antagonist) or pertussis toxin (PTX; a Gi protein inhibitor), indicating that the P2X(1), P2X(3), P2X(4)receptor group, or the P2Y(12) receptor, negatively modulate the P2Y(11) receptor or the P2Y(1) receptor, respectively.

P2X1 and P2X4 receptor currents in mouse macrophages

Activation of P2X receptors on macrophages is an important stimulus for cytokine release. This study seeks evidence for functional expression of P2X receptors in macrophages that had been only minimally activated. Whole-cell recordings were made from macrophages isolated 2-6 h before by lavage from mouse peritoneum, without further experimental activation. ATP (1-1000 muM) elicited inward currents in all cells (holding potential -60 mV). The properties of this current were compared among cells from wild type, P2X1 (-/-) and P2X4 (-/-) mice. Immunoreactivity for P2X1 and P2X4 receptors was observed in wild type macrophages but was absent from the respective knock-out mice. In cells from wild type mice, ATP and alpha beta methyleneATP (alpha beta meATP) evoked inward currents rising in 10-30 ms and declining in 100-300 ms: these were blocked by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM). ATP also elicited a second, smaller ( approximately 10% peak amplitude), more slowly decaying (1-3 s) at concentrations > or =10 microM: this was resistant to PPADS and prolonged by ivermectin. Macrophages from P2X1 (-/-) mice responded to ATP (>100 microM) but not alpha beta meATP: these small currents were prolonged by ivermectin. Macrophages from P2X4 (-/-) mice responded to ATP and alpha beta meATP as cells from wild type mice, except that ATP did not evoke the small, slowly decaying component: these currents were blocked by PPADS. Mouse peritoneal macrophages that are minimally activated demonstrate membrane currents in response to ATP and alpha beta meATP that have the predominate features of P2X1 receptors.

Metabotropic P2Y receptors inhibit P2X3 receptor‐channels via G protein‐dependent facilitation of their desensitization

The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X(3) receptor-channels. Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X(3) receptors (HEK293-hP2X(3) cells) and rat dorsal root ganglion (DRG) neurons. In both cell types, the P2Y(1,12,13) receptor agonist, ADP-beta-S, inhibited P2X(3) currents evoked by the selective agonist, alpha,beta-methylene ATP (alpha,beta-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-beta-S, a procedure known to block all G protein heterotrimers. P2X(3) currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X(3) receptor agonist alpha,beta-meATP. In the presence of intracellular GDP-beta-S, ATP- and alpha,beta-meATP-induced currents were identical. Recovery from P2X(3) receptor desensitization induced by repetitive ATP application was slower than the recovery from alpha,beta-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-beta-S, the recovery from the ATP- and alpha,beta-meATP-induced desensitization were of comparable speed. Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X(3) receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X(3) and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity.

Evidence that ATP or a related purine is an excitatory neurotransmitter in the longitudinal muscle of mouse distal colon

This study analysed the contribution of the purinergic system to enteric neurotransmission in the longitudinal muscle of mouse distal colon. Motor responses to exogenous ATP and to nerve stimulation in vitro were assessed as changes in isometric tension. ATP induced a concentration-dependent contraction, reduced by 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzene disulphonic acid (PPADS), suramin, P2Y purinoreceptor desensitisation with adenosine 5'-O-2-thiodiphosphate (ADPbetaS), and atropine, but unaffected by P2X purinoceptor desensitisation with alpha,beta-methylene ATP (alpha,beta-meATP) and by 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl ester (MRS 2395), a P2Y(12) selective antagonist. The response to ATP was increased by 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS 2179), a P2Y(1) selective antagonist, tetrodotoxin (TTX) or N(omega)-nitro-L-arginine methyl ester (L-NAME). ADPbetaS, a P2Y-purinergic agonist, induced muscular contraction, with the same pharmacological profile as the ATP-induced contraction. ADP, a natural ligand for P2Y(1) receptors, induced muscular relaxation, antagonized by MRS 2179 and by TTX or L-NAME. Nerve stimulation elicited a transient nitrergic relaxation, followed by contraction. Contractile responses was reduced by atropine, PPADS, suramin, P2Y purinoceptor desensitisation, but not by P2X purinoceptor desensitisation, MRS 2179 or MRS 2395. None of the purinergic antagonists modified the nerve-evoked relaxation. In the longitudinal muscle of mouse distal colon, ATP, through ADPbetaS-sensitive P2Y purinoceptors, contributed to the excitatory neurotransmission acting directly on smooth muscle and indirectly via activation of cholinergic neurons. Moreover, P2Y1 purinoceptors appear to be located on nitrergic inhibitory neurons. This study provides new insights into the role of purines in the mechanism inducing intestinal transit in mouse colon.

Long‐term potentiation of neuronal excitation by neuron–glia interactions in the rat spinal dorsal horn

By imaging neuronal excitation in rat spinal cord slices with a voltage-sensitive dye, we examined the role of glial cells in the P2X receptor agonist alphabeta-methylene ATP (alphabetameATP)-triggered long-term potentiation (LTP) in the dorsal horn. Bath application of alphabetameATP potentiated neuronal excitation in the superficial dorsal horn. The potentiation was inhibited in the presence of the P2X receptor antagonists TNP-ATP, PPADS and A-317491, and was not induced in slices taken from rats neonatally treated with capsaicin. These results suggest that alphabetameATP acts on P2X receptors, possibly P2X(3) and/or P2X(2/3), in capsaicin-sensitive primary afferent terminals. Furthermore, the potentiation was inhibited by treatment with the glial metabolism inhibitor monofluoroacetic acid. Results obtained with the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6, and antibodies to TNF-alpha and IL-6, as well as by double immunolabelling of activated p38 MAPK with markers of astrocytes and microglia, demonstrated that alphabetameATP activated p38 MAPK in astrocytes, and that the presence of proinflammatory cytokines and p38 MAPK activation were necessary for the induction of alphabetameATP-triggered LTP. These findings indicate that glial cells contribute to the alphabetameATP-induced LTP, which might be part of a cellular mechanism for the induction of persistent pain.

P2 receptor regulation of [Ca2+]i in cultured mouse mesangial cells.

Experiments were performed to establish the pharmacological profile of purinoceptors and to identify the signal transduction pathways responsible for increases in intracellular calcium concentration ([Ca(2+)](i)) for cultured mouse mesangial cells. Mouse mesangial cells were loaded with fura 2 and examined using fluorescent spectrophotometry. Basal [Ca(2+)](i) averaged 102 +/- 2 nM (n = 346). One hundred micromolar concentrations of ATP, ADP, 2',3'-(benzoyl-4-benzoyl)-ATP (BzATP), ATP-gamma-S, and UTP in normal Ca(2+) medium evoked peak increases in [Ca(2+)](i) of 866 +/- 111, 236 +/- 18, 316 +/- 26, 427 +/- 37, and 808 +/- 73 nM, respectively. UDP or 2-methylthio-ATP (2MeSATP) failed to elicit significant increases in [Ca(2+)](i), whereas identical concentrations of adenosine, AMP, and alpha,beta-methylene ATP (alpha,beta-MeATP) had no detectable effect on [Ca(2+)](i). Removal of Ca(2+) from the extracellular medium had no significant effect on the peak increase in [Ca(2+)](i) induced by ATP, ADP, BzATP, ATP-gamma-S, or UTP compared with normal Ca(2+); however, Ca(2+)-free conditions did accelerate the rate of decline in [Ca(2+)](i) in cells treated with ATP and UTP. [Ca(2+)](i) was unaffected by membrane depolarization with 143 mM KCl. Western blot analysis for P2 receptors revealed expression of P2X(2), P2X(4), P2X(7), P2Y(2), and P2Y(4) receptors. No evidence of P2X(1) and P2X(3) receptor expression was detected, whereas RT-PCR analysis reveals mRNA expression for P2X(1), P2X(2), P2X(3), P2X(4), P2X(7), P2Y(2), and P2Y(4) receptors. These data indicate that receptor-specific P2 receptor activation increases [Ca(2+)](i) by stimulating calcium influx from the extracellular medium and through mobilization of Ca(2+) from intracellular stores in cultured mouse mesangial cells.

Increased 5-HT3-mediated signalling in pelvic afferent neurons from mice deficient in P2X2 and/or P2X3 receptor subunits

Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT3 receptors, respectively. We have investigated the changes in P2X and 5-HT3-mediated signalling in pelvic afferent neurons in mice deficient in P2X2 and/or P2X3 subunits by whole-cell recording of L6–S2 dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X3−/− neurons, whereas sustained currents were absent in P2X2−/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X2/P2X3Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT3 receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X 2−/−, P2X3−/− and P2X2/P2X3Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X2/P2X3Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X2/P2X3Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X2/P2X3Dbl−/− preparations. These data suggest that P2X receptors containing the P2X2 and/or P2X3 subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X2 or P2X3 receptor genes. This effect is more pronounced when both subunits are absent.

Effects of Acute Administration of and Tachyphylaxis to α,β‐Methylene ATP in the Guinea‐Pig Small Intestine

The aim of the present study was to assess the acute motility effects and desensitizing activity of the stable ATP analogue and P(2X) purinoceptor agonist alpha,beta-methylene ATP (alpha,beta-meATP) and the effect of alpha,beta-meATP desensitization on nerve-mediated cholinergic responses in the guinea-pig ileum in vitro. It was confirmed that alpha,beta-meATP (1-30 microM) causes neurally-mediated, cholinergic (tetrodotoxin- and atropine-sensitive) longitudinal contractions. These responses were not influenced by the ganglionic blocking drug hexamethonium (50 microM), or a combination of the adrenergic neurone blocking drug guanethidine (3 microM), the opioid receptor antagonist naloxone (0.5 microM) and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 100 microM), but were strongly reduced or abolished by the P2 purinoceptor antagonist PPADS (30 microM) or by tachyphylaxis evoked by 10 microM alpha,beta-meATP. The contractile effect of alpha,beta-meATP (3 microM) was moderately inhibited by 10 microM and strongly suppressed by 30 microM of NF 279, an antagonist predominantly affecting P2X1 purinoceptors, but left uninfluenced by the P2X(5,7) receptor antagonist Brilliant blue G. No relaxant effect of alpha,beta-meATP was detected in the concentration range of 1-30 microM. Tachyphylaxis to alpha,beta-meATP (1-10 microM) caused a moderate inhibition of the cholinergic (atropine-sensitive) contractile response of the ileum to electrical field stimulation (5 Hz for 5 sec.). This reduction was unaltered in the presence of guanethidine, naloxone and L-NOARG. Responses to nicotine (1 or 2 microM) were not reduced by alpha,beta-meATP tachyphylaxis. It is suggested that alpha,beta-meATP-sensitive P(2X) purinoceptors are involved in the prejunctional modulation of cholinergic neurotransmission between the myenteric plexus and longitudinal smooth muscle in the guinea-pig small intestine.

The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range

The role of ATP-stimulated P2X1 receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y1 or P2Y12 receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X1 receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X1 antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on alphabeta meATP (P2X1 receptors)-induced or ADP (P2Y1 receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y1 & P2Y12 receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8',8'',8'''-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X1 antagonist reported to date, blocking alphabeta meATP-induced Ca2+ increases and shape change in a concentration-dependent manner, with a pA2 of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X1 receptor displayed the following order : NF864 > NF449 > or = NF110 > NF023 = MK-HU1 = suramin. A different antagonistic profile was observed for ADP-induced Ca2+ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X1 responses, with the order NF110 > NF449 > or = NF864 > or = MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X1 receptors in thrombosis and haemostasis, NF864 being the most promising compound.

Characterization of Multiple P2X Receptors in Cultured Normal Human Epidermal Keratinocytes

ATP-gated ion channels (P2X) are expressed in human epidermis and cultured keratinocytes. The aim of this study was to characterize native P2X receptors in normal human epidermal keratinocytes (NHEK) using whole-cell patch clamp technique, RT-PCR, and determination of intracellular Ca(2+) concentration ([Ca(2+)](i)). Application of ATP resulted in an inward current with a reversal potential of 0 mV. Response to ATP showed two types of currents: the slowly desensitizing response and the rapidly desensitizing response. The slowly desensitizing response was blocked by iso-pyridocaphosphate-6-azophenyl-2', 5' disulfonic acid (PPADS), a P2X receptor antagonist. We found that the expression of multiple P2X(2), P2X(3), P2X(5), and P2X(7) receptor subtype mRNA was increased in differentiated cells. On the other hand, the expression of G-protein-coupled P2Y(2) mRNA was downregulated in differentiated cells. Increases in [Ca(2+)](i) evoked by alphabeta-methylene ATP (alphabeta-meATP) and 2', 3'-O-(4-benzoylbenzoyl) ATP (BzATP) were elevated, whereas elevation of [Ca(2+)](i) evoked by uridine 5'-triphosphate (UTP) was decreased in differentiated cells. Application of ATP or UVB radiation increased the expression of P2X(1), P2X(2), P2X(3), and P2X(7) receptors in NHEK. Changes in the expression levels and cation influx via multiple P2X receptors might be involved in the regulation of differentiation and one of the epidermal external sensors.

Heterogeneity of P2X receptors in sympathetic neurons: contribution of neuronal P2X1 receptors revealed using knockout mice.

P2X receptors are highly expressed throughout the nervous system, where ATP has been shown to be a neurotransmitter. The aim of this study was to characterize P2X receptor expression within sympathetic postganglionic neurons from the superior cervical ganglia. Reverse transcription-polymerase chain reaction showed the presence of mRNA for all P2X receptors, raising the possibility of multiple subunit expression within these ganglia. Whole-cell patch-clamp and calcium imaging studies revealed a heterogeneous population of P2X receptors in approximately 70% of neurons. We propose that the heterogeneity in properties could be caused by differential expression and/or subunit composition of the P2X receptor. The dominant phenotype was P2X2-like; neurons showed slow desensitization, sensitivity to antagonists, and a profile of ionic modulation that is characteristic of P2X2 receptors: potentiation by acidification and extracellular Zn2+ and attenuation by high extracellular Ca2+ and pH. A subpopulation of neurons (10-15%) were alpha,beta-methylene ATP (alpha,beta-meATP) sensitive, and in neurons from P2X1 receptor-deficient mice the alpha,beta-meATP response was reduced to 2% of all neurons, demonstrating a direct role for P2X1 subunits. Control alpha,beta-meATP responses were eliminated by high extracellular Ca(2+) and pH, indicating the presence of heteromeric channels incorporating the properties of P2X1 and P2X2 receptors. This study demonstrates that in neurons, the P2X1 receptor can contribute to the properties of heteromeric P2X receptors. Given the expression of P2X1 receptors in a range of neurons, it seems likely that regulation of the properties of P2X receptors by this subunit is more widespread.

A COMPARATIVE STUDY ON CONTRACTILE RESPONSES OF RABBIT AND GUINEA PIG VASA DEFERENTIA TO ELECTRICAL FIELD STIMULATION

Contractile responses of rabbit and guinea pig vasa deferentia to electrical field stimulation (EFS) are compared. A muscarinic receptor blocking agent, 1 microM atropine markedly reduced phasic and tonic contraction induced by EFS (20 Hz, 0.5 msec, 30 V, for 30 sec) in rabbit vas deferens, while it only slightly depressed those in guinea pig vas deferens. Further addition of an adrenergic alpha1 receptor blocking agent, 1 microM prazosin markedly depressed the second tonic contraction in both rabbit and guinea pig vasa deferentia. In the presence of atropine and prazosin, further addition of a P2X purinoceptor desensitizing agent, 10 microM alpha,beta-methylene ATP (alpha, beta-MeATP) abolished the residual phasic contractile response in guinea pig vas deferens, while it partially depressed that in rabbit vas deferens. The administration of 10 microM alpha,beta-MeATP in the absence of atropine and prazosin markedly potentiated the phasic contractile response of rabbit vas deferens to EFS, while it depressed that of guinea pig vas deferens. Contractile response of rabbit vas deferens to alpha,beta-MeATP was more potent than those of ATP and 2-methyl-thioATP (2-Me-thioATP), while these nucleotides had almost same potency in guinea pig vas deferens. These findings may indicate that contribution of cholinergic, adrenergic and purinergic neurotransmission to the contractile response of rabbit vas deferens to EFS is different from that of guinea pig vas deferens.

P2X1-mediated ERK2 Activation Amplifies the Collagen-induced Platelet Secretion by Enhancing Myosin Light Chain Kinase Activation

The ATP-gated P2X1 ion channel is the only P2X subtype expressed in human platelets. Via transmission electron microscopy, we found that P2X1 mediates fast, reversible platelet shape change, secretory granule centralization, and pseudopodia formation. In washed human platelets, the stable P2X1 agonist alpha,beta-methylene ATP (alpha,beta-meATP) causes rapid, transient (2-5 s), and dose-dependent myosin light chain (MLC) phosphorylation, requiring extracellular Ca2+. Phosphorylation was inhibited by the calmodulin (CaM) inhibitor W-7, but not by the Rho kinase inhibitor HA-1077, i.e. it is exclusively regulated by Ca2+/CaM-dependent MLC kinase. Correspondingly, the P2X1-induced platelet shape change was inhibited by W-7 and by the MLC kinase inhibitor ML-7 but not by HA-1077. W-7, ML-7, the protein kinase C inhibitor GF109203-X, and the Src family kinase inhibitor PP1 inhibited the collagen and convulxin-induced early platelet degranulation, shape change, and subsequent aggregation, indicating a role for Ca2+/CaM and MLC kinase in these glycoprotein VI-related platelet responses. The secreted ATP-mediated P2X1-dependent ERK2 activation induced by low collagen concentrations contributes to MLC kinase activation since P2X1 desensitization or blockade of ERK2 phosphorylation by U0126 strongly attenuated MLC phosphorylation, degranulation, and aggregation. We therefore conclude that at low doses of collagen, glycoprotein VI activation leads to early protein kinase C- and MLC kinase-dependent degranulation. Rapidly released ATP triggers P2X1 -mediated Ca2+ influx, activating ERK2, in turn amplifying platelet secretion by reinforcing the early MLC kinase phosphorylation. Hence, the P2X1-ERK2-MLC axis contributes to collagen-induced platelet activation by enhancing platelet degranulation.

TNP-ATP-resistant P2X ionic current on the central terminals and somata of rat primary sensory neurons.

P2X receptors have been suggested to be expressed on the central terminals of A delta-afferent fibers innervating dorsal horn lamina V and play a role in modulating sensory synaptic transmission. These P2X receptors have been widely thought to be P2X2+3 receptors. However, we have recently found that P2X receptor-mediated modulation of sensory transmission in lamina V is not inhibited by trinitrophenyl-adenosine triphosphate (TNP-ATP), a potent antagonist of P2X1, P2X3 homomers, and P2X2+3 heteromers. To provide direct evidence for the presence of TNP-ATP-resistant P2X receptors on primary afferent fibers, we examined alpha,beta-methylene-ATP (alpha beta meATP)-evoked currents and their sensitivity to TNP-ATP in rat dorsal root ganglion (DRG) neurons. alpha beta meATP evoked fast currents, slow currents, and mixed currents that contained both fast and slow current-components. Fast currents and fast current components in the mixed currents were both completely inhibited by 0.1 microM TNP-ATP (n = 14). Both slow currents and slow-current components in the mixed currents showed broad spectrum of sensitivity to 1 microM TNP-ATP, ranging from complete block (TNP-ATP-sensitive) to little block (TNP-ATP-resistant). TNP-ATP-resistant currents evoked by 10 microM alpha beta meATP could be largely inhibited by 10 microM iso-pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Cells with P2X currents that were highly resistant to TNP-ATP were found to be insensitive to capsaicin. These results suggest that TNP-ATP-resistant P2X receptor subtypes are expressed on capsaicin-insensitive A delta-afferent fibers and play a role in modulating sensory transmission to lamina V neurons.

P2X(1) receptor-deficient mice establish the native P2X receptor and a P2Y6-like receptor in arteries.

The contribution of P2 receptors to vasoconstriction of mouse mesenteric arteries was determined using wild-type (WT) and P2X(1) receptor-deficient (KO) animals. alpha,beta-methylene ATP (alpha,beta-meATP) and ATP evoked transient inward currents and constrictions of WT mesenteric arteries. In contrast, alpha,beta-meATP (100 microM) and ATP (100 microM) failed to evoke responses in KO arteries from a range of vascular beds. Nerve stimulation (100 pulses at 10 Hz) evoked constrictions of mesenteric arteries. For WT arteries, the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2'-5'-disulfonate (PPADS) (30 microM) reduced the amplitude of response by approximately 50%; the residual constriction was abolished by prazosin (0.1 microM). In KO mice, vasoconstriction induced by nerve stimulation was reduced in amplitude by approximately 50%, unaffected by PPADS, but was abolished by prazosin. ADP (1 mM) (a P2Y(1), P2Y(12), and P2Y(13) receptor agonist) was ineffective. Because ATP had no effect on mesenteric artery tone from KO mice, this rules out the contribution of P2Y(2) receptors. The P2Y(4) receptor agonist ITP also failed to contract mesenteric arteries. However, UTP and UDP evoked sustained contractions of mesenteric arteries with similar potency (EC(50) approximately 10 microM). Complementary studies using reverse-transcriptase polymerase chain reaction showed that mesenteric arteries express P2Y(1), P2Y(2), and P2Y(6) receptors. These results demonstrate that homomeric P2X(1) receptors underlie the artery smooth muscle P2X receptor phenotype and contribute approximately 50% to sympathetic neurogenic vasoconstriction and indicate the presence of a UTP- and UDP-sensitive P2Y(6)-like receptor, but not vasoconstrictor P2Y(2) or P2Y(4) receptors, on mouse mesenteric arteries.

ATP-induced in vivo neurotoxicity in the rat striatum via P2 receptors.

The present study examined the effects of ATP on the striatum of Sprague-Dawley rats. Intrastriatal administration of ATP produced dose-dependent striatal lesions as confirmed by cresyl violet staining. Additional immunostaining using neuronal nuclear protein (NeuN), OX-42 and GFAP antibodies revealed that ATP caused death of both neurons and glial cells. The nonmetabolizable ATP analogue ATPgammaS and P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-MeATP) mimicked ATP effects, whereas either P2Y receptor agonist ADP or P1 receptor agonist adenosine did not. The P2 receptor antagonist reactive blue 2, but not pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) attenuated ATP-induced striatal injury. These results suggest that intrastriatal administration of ATP causes P2X receptor-mediated cell death in the striatum and support the hypothesis that extracellular ATP can be an important mediator of neuropathological events of brain injuries.

Stimulation of P2Y1 Receptors Causes Anxiolytic-like Effects in the Rat Elevated Plus-maze: Implications for the Involvement of P2Y1 Receptor-Mediated Nitric Oxide Production

The widespread and abundant distribution of P2Y receptors in the mammalian brain suggests important functions for these receptors in the CNS. To study a possible involvement of the P2Y receptors in the regulation of fear and anxiety, the influences of the P2Y(1,11,12) receptor-specific agonist adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS), the P2X(1,3) receptor agonist alpha,beta-methylene ATP (alpha,betameATP), the unspecific P2 receptor antagonist pyridoxalphosphate-6-azopheny l-2',4'-disulfonic acid (PPADS), and the specific P2Y(1) receptor antagonist N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS 2179) on the elevated plus-maze behavior of the rat were investigated. All tested compounds were given intracerebroventricularly (0.5 microl). ADPbetaS (50 and 500 fmol) produced an anxiolytic-like behavioral profile reflected by an increase of the open arm exploration. The anxiolytic-like effects were antagonized by pretreatment with PPADS (5 pmol) or MRS 2179 (5 pmol). Both compounds caused anxiogenic-like effects when given alone. Furthermore, the anxiolytic-like effects of ADPbetaS could be antagonized by pretreatment with the nitric oxide synthase (NOS) inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME). In addition, the anxiogenic-like effects of PPADS were reversed by the pretreatment with L-arginine (500 pmol), which is the natural substrate for NOS, but not by D-arginine (500 pmol), which is not. Immunofluorescence staining revealed the presence of P2Y(1) receptors on neurons in different brain regions such as hypothalamus, amygdala, hippocampus and the periaqueductal gray. Furthermore, the colocalization of P2Y(1) receptors and neuronal NOS (nNOS) on some neurons in these regions could be demonstrated. The highest density of P2Y(1)- and nNOS-immunoreactivity was detected in the dorsomedial hypothalamic nucleus. Taken together, the present results suggest that P2Y(1) receptors are involved in the modulation of anxiety in the rat. The anxiolytic-like effects after stimulation of P2Y(1) receptors seem to be in close connection with the related nitric oxide production.