ATP facilitates spontaneous glycinergic IPSC frequency at dissociated rat dorsal horn interneuron synapses.
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1. The ATP action on spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) was investigated in rat substantia gelatinosa (SG) neurons mechanically dissociated from the 2nd layer of the dorsal horn in which their presynaptic glycinergic nerve terminals remained intact. 2. ATP reversibly facilitated the frequency of the mIPSCs in a concentration-dependent manner without affecting their amplitude distribution. The ATP agonist, 2-methylthioATP (2MeSATP), mimicked the ATP action, while another ATP receptor agonist, alphabeta-methylene-ATP (alpha,beta-meATP), had no effect on mIPSCs. 3. The ATP receptor antagonists, suramin (1 x 10-6 M) and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1 x 10-5 M), completely blocked the facilitatory effect of ATP on glycine release (102.0 +/- 11.2 % and 99.3 +/- 16.2 %, n = 6, respectively) without altering the current amplitude distributions. 4. N-Ethylmaleimide (NEM), a sulphydryl alkylating agent, suppressed the inhibitory effect of adenosine on mIPSC frequency (111.2 +/- 13. 3 %, n = 4) without altering the current amplitude distribution. However, ATP still facilitated the mIPSC frequency (693.3 +/- 245.2 %, n = 4) even in the presence of NEM. 5. The facilitatory effect of ATP (1 x 10-5 M) on mIPSC frequency was not affected by adding 1 x 10-4 M Cd2+ to normal external solution but was eliminated in a Ca2+-free external solution. 6. These results suggest that ATP enhances glycine release from nerve terminals, presumably resulting in the inhibition of SG neurons which conduct nociceptive signals to the CNS. This presynaptic P2X-type ATP receptor may function to prevent excess excitability in SG neurons, thus preventing an excessive pain signal and/or SG cell death. (+info)
ATP, an agonist at the rat P2Y(4) receptor, is an antagonist at the human P2Y(4) receptor.
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The nucleotide selectivities of the human P2Y(4) (hP2Y(4)) and rat P2Y(4) (rP2Y(4)) receptor stably expressed in 1321N1 human astrocytoma cells were determined by measuring increases in intracellular [Ca(2+)] under conditions that minimized metabolism, bioconversion, and endogenous nucleotide release. In cells expressing the hP2Y(4) receptor, UTP, GTP, and ITP all increased intracellular [Ca(2+)] with a rank order of potency of UTP (0.55) > GTP (6.59) = ITP (7.38), (EC(50), microM). ATP, CTP, xanthine 5'-triphosphate (XTP), and diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A), all at 100 microM, were inactive at the hP2Y(4) receptor. In cells expressing the rP2Y(4) receptor, all seven nucleotides increased intracellular [Ca(2+)] with similar maximal effects and a rank order of potency of UTP (0.20) > ATP (0. 51) > Ap(4)A (1.24) approximately ITP (1.82) approximately GTP (2. 28) > CTP (7.24) > XTP (22.9). Because ATP is inactive at the hP2Y(4) receptor, we assessed whether ATP displayed antagonist activity. When coapplied, ATP shifted the concentration-response curve to UTP rightward in a concentration-dependent manner, with no change in the maximal response. A Schild plot derived from these data gave a pA(2) value of 6.15 (K(B) = 708 nM) and a slope near unity. Additionally, CTP and Ap(4)A (each at 100 microM) inhibited the response to an EC(50) concentration of UTP by approximately 40 and approximately 50%, respectively, whereas XTP had no effect. The inhibitory effects of ATP, CTP, and Ap(4)A were reversible on washout. Thus, ATP is a potent agonist at the rP2Y(4) receptor but is a competitive antagonist with moderate potency at the hP2Y(4) receptor. (+info)
Ecto-nucleotide pyrophosphatase modulates the purinoceptor-mediated signal transduction and is inhibited by purinoceptor antagonists.
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1. The effect of ecto-nucleotide pyrophosphatase (ecto-NPPase; EC 3.6.1. 9) on the ATP- and ADP-mediated receptor activation was studied in rat C6 glioma cells. The P2-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC(50)=12+/-3 microM) of the latter enzyme. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS), 5'-phosphoadenosine 3'-phosphate (PAP) and suramin were less potent inhibitors with an IC(50) of 22+/-4, 36+/-7 and 72+/-11 microM respectively. 2. P1-purinoceptor antagonists CGS 15943, cyclo-pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto-NPPase. 3. ATP- and ADP-mediated P2Y(1)-like receptor activation inhibited the (-)-isoproterenol-induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y-antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto-NPPase. 4. We conclude that ecto-NPPase has a modulator effect on purinoceptor-mediated signalling in C6 glioma cell cultures. (+info)
Novel mechanism of inhibition by the P2 receptor antagonist PPADS of ATP-activated current in dorsal root ganglion neurons.
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The antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) has been proposed to selectively antagonize the actions of ATP at P2X receptors. Whole cell patch-clamp recording techniques therefore were used to characterize PPADS inhibition of ATP-activated current in bullfrog dorsal root ganglion (DRG) neurons. PPADS, 0.5-10 microM, inhibited ATP-activated current in a concentration-dependent manner with an IC(50) of 2.5 +/- 0.03 microM. PPADS produced a gradual decline of ATP-activated current to a steady state, but this was not an indication of use dependence as the gradual declining component could be eliminated by exposure to PPADS before ATP application. In addition, ATP-activated current recovered completely from inhibition by PPADS in the absence of agonist. The slow onset of inhibition by PPADS was not apparently due to an action at an intracellular site as inclusion of 10 microM PPADS in the recording pipette neither affected the ATP response nor did it alter inhibition of the ATP response when 2.5 microM PPADS was applied externally. PPADS, 2.5 microM, decreased the maximal response to ATP by 51% without changing its EC(50). PPADS inhibition of ATP-activated current was independent of membrane potential between -80 and +40 mV and did not involve a shift in the reversal potential of the current. The magnitude of PPADS inhibition of ATP-activated current was dependent on the duration of the prior exposure to PPADS. The time constants of both onset and offset of PPADS inhibition of ATP-activated current did not differ significantly with changes in ATP concentration from 1 to 5 microM. Recovery of ATP-activated current from PPADS inhibition also exhibited a slow phase that was not accelerated by the presence of agonist and was dependent on the concentration of PPADS. The apparent dissociation rate of PPADS from unliganded ATP-gated ion channels was much greater than the rate of the slow phase of recovery of ATP-activated current from PPADS inhibition. The results suggest that PPADS can inhibit P2X receptor function in a complex noncompetitive manner. PPADS produces a long-lasting inhibition that does not appear to result from open channel block but rather from an action at an allosteric site apparently accessible from the extracellular environment that involves a greatly reduced rate of dissociation from liganded versus unliganded ATP-gated ion channels. (+info)
Co-release of ATP and ACh mediates hypoxic signalling at rat carotid body chemoreceptors.
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Using functional co-cultures of rat carotid body (CB) O2 chemoreceptors and 'juxtaposed' petrosal neurones (JPNs), we tested whether ATP and ACh acted as co-transmitters. Perforated-patch recordings from JPNs often revealed spontaneous and hypoxia-evoked (PO2 approximately 5 mmHg) excitatory postsynaptic responses. The P2X purinoceptor blocker, suramin (50 microM) or a nicotinic ACh receptor (nAChR) blocker (hexamethonium, 100 microM; mecamylamine, 1 microM) only partially inhibited these responses, but together, blocked almost all activity. Under voltage clamp (-60 mV), fast perfusion of 100 microM ATP over hypoxia-responsive JPNs induced suramin-sensitive (IC50 = 73 microM), slowly-desensitizing, inward currents (IATP) with time constant of activation tauon = 30.6 +/- 4. 8 ms (n = 7). IATP reversed at 0.33 +/- 3.7 mV (n = 4), and the dose-response curve was fitted by the Hill equation (EC50 = 2.7 microM; Hill coefficient approximately 0.9). These purinoceptors contained immunoreactive P2X2 subunits, but their activation by alpha,beta-methylene ATP (alpha,beta-meATP; EC50 = 2.1 microM) suggests they are P2X2/P2X3 heteromultimers. Suramin and nAChR blockers inhibited the extracellular chemosensory discharge in the intact rat carotid body-sinus nerve preparation in vitro. Further, P2X2 immunoreactivity was widespread in rat petrosal ganglia in situ, and co-localized in neurones expressing the CB chemo-afferent marker, tyrosine hydroxylase (TH). P2X2 labelling in the CB co-localized with nerve-terminal markers, and was intimately associated with TH-positive type 1 cells. Thus ATP and ACh are co-transmitters during chemotransduction in the rat carotid body. (+info)
Antagonist effects on human P2X(7) receptor-mediated cellular accumulation of YO-PRO-1.
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We have examined the interaction of P2 antagonists with the human P2X(7) receptor by studying their effect on 2' and 3'-O-benzoyl-benzoyl-ATP (DbATP) stimulated cellular accumulation of the fluorescent, DNA binding dye, YO-PRO-1 (MW=375Da). In suspensions of HEK293 cells expressing human recombinant P2X(7) receptors, DbATP produced time and concentration-dependent increases in YO-PRO-1 fluorescence. This response presumably reflects YO-PRO-1 entry through P2X(7) receptor channels and binding to nucleic acids. When studies were performed in a NaCl-free, sucrose-containing buffer, full concentration-effect curves to DbATP could be constructed. The P2 antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) and periodate oxidized ATP (oATP), reduced the potency of DbATP and decreased its maximum response. 1-[N,O-bis(1, 5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62) and its analogue, KN04, reduced the potency of DbATP. Schild slopes for KN62 and KN04 were shallow and exhibited a plateau at concentrations of compound greater than 1 microM, indicating that these compounds were not competitive antagonists. Calmidazolium and a monoclonal antibody to human P2X(7) receptors attenuated DbATP-stimulated YO-PRO-1 accumulation but they were not competitive antagonists and only produced 2 - 3 fold decreases in the potency of DbATP. The effects of PPADS and KN62 were partially reversible whereas those of oATP were not. PPADS protected cells against the irreversible antagonist effects of oATP suggesting a common site of action. In contrast KN62 was not effective suggesting that it may bind at a different site to oATP and PPADS. This study has demonstrated that P2X(7) receptor function can be quantified by measuring DbATP stimulated YO-PRO-1 accumulation and has provided additional information about the interaction of P2 receptor antagonists with the human P2X(7) receptor. (+info)
Evidence that 2-methylthioATP and 2-methylthioADP are both agonists at the rat hepatocyte P2Y(1) receptor.
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In the absence of selective antagonists, pharmacological characterization of P2Y receptor subtypes has relied heavily upon their distinct agonist profiles. 2-methylthioADP (2-MeSADP) is a selective agonist for the P2Y(1) receptor. The agonist action of 2-MeSATP at the P2Y(1) receptor has recently been questioned. The effects of both 2-MeSADP and 2-MeSATP have been studied on rat hepatocytes injected with the bioluminescent Ca(2+) indicator, aequorin. Single hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca(2+)](i)) when stimulated with agonists acting through the phosphoinositide signalling pathway. The transients induced by 2-MeSADP and 2-MeSATP in the same cell were indistinguishable, indicating that they act at a common receptor. In contrast the transients evoked by ATP and UTP had very different profiles. Treatment of 2-MeSATP with an ATP-regenerating system to remove contaminating 2-MeSADP did not abolish its agonist activity. Application of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) inhibited the transients induced by both 2-MeSADP and 2-MeSATP. In contrast the transients induced by ATP and UTP were enhanced by the addition of A3P5P. These results indicate that both 2-MeSADP and 2-MeSATP are agonists at the rat hepatocyte P2Y(1) receptor. (+info)
Brilliant blue G selectively blocks ATP-gated rat P2X(7) receptors.
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There are few antagonists selective for subtypes of the several P2X receptors, but these are needed to identify the receptors expressed on native cells and tissues. In particular, P2X(4) and P2X(7) receptor subunits are colocalized on immune, epithelial, and exocrine gland cells, but both are relatively insensitive to suramin and pyridoxal-5-phosphate-6-azo-2',4'-disulfonic acid derivative. In this article, we show that Coomassie Brilliant Blue G selectively inhibits P2X(7) receptors with nanomolar affinity. We measured currents in response to P2X receptor activation in HEK293 cells heterologously expressing human or rat P2X(1), P2X(2), P2X(3), P2X(2/3), P2X(4), P2X(1/5), and P2X(7) receptors. Brilliant Blue G produced a noncompetitive inhibition of rat and human P2X(7) receptors with IC(50) values of 10 and 200 nM, respectively. IC(50) values for inhibition of the other receptors ranged from 2 to >30 microM; the rat and human P2X(4) receptors showed IC(50) values of >10 and 3.2 microM. Coomassie Blue G also blocked YO-PRO1 uptake and membrane blebbing, which are uniquely associated with activation of P2X(7) receptors. Thus, Brilliant Blue G is at least 1000-fold more potent at rat P2X(7) receptors than at rat P2X(4) receptors. (+info)