Beta meATP Research Articles

Activation of P2X‐purinergic receptors in the lateral parabrachial nucleus increases renal sympathetic nerve discharge and mean arterial pressure in anesthetized rats (686.13)

The lateral parabrachial nucleus (LPBN) is an important area involved in cardiorespiratory control. ATP is an important neurotransmitter and purinergic receptors are present into LPBN. The involvement of purinergic mechanisms into LPBN in the control of mean arterial pressure (MAP) and renal sympathetic nerve discharge (RSND) is unknown. We investigated the effect of alpha, beta‐me ATP (P2X agonist) alone or following pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulfonic acid (PPADS, P2 antagonist) injected into LPBN on MAP and RSND in rats. Anesthetized, vagotomized and mechanically ventilated male rats (n=3) were used. Unilateral injections of alpha, beta‐me ATP (2.0 nmol/50 nl) into LPBN increased MAP (Δ 12±2 mmHg, p<0.05) and RSND (Δ 32±8%), without changing heart rate (HR). Unilateral injection of PPADS (0.125 nmol/50 nl) into LPBN did not change MAP, HR and RSND, however, pre‐treatment with PPADS into LPBN (10 min before) abolished the pressor response (Δ 2±0.8 mmHg, p<0.05) and reduced the RSND increase (Δ 12±7%, p<0.05) produced by alpha, beta‐me ATP injected into LPBN. In 2 rats responses to alpha, beta‐me ATP recovered to control within 30 min while in the remaining rat the response had recovered 31% by 70 min. These results suggest that alpha, beta‐me ATP acting on P2X purinergic receptors into LPBN produces pressor and sympathoexcitatory responses in anesthetized rats.Grant Funding Source: CAPES (18906‐12‐5); FAPESP; CNPq

The effects of extracellular nucleotides on [Ca2+]i signalling in a human‐derived renal proximal tubular cell line (HKC‐8)

HKC-8 cells are a human-derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca(2+)](i) signalling mediated by P2 receptor in HKC-8. Fura-2 and a ratio imaging method were employed to measure [Ca(2+)](i) in HKC-8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca(2+)](i) that was dependent on an intracellular source of Ca(2+), while prolonged activation of P2Y receptors induced a rise in [Ca(2+)](i) that was dependent on intra- and extracellular sources of Ca(2+). Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca(2+) entry in coupling to activation of P2Y in HKC-8 cells. U73221, an inhibitor of PI-PLC, did not inhibit the initial ATP-induced response; whereas D609, an inhibitor of PC-PLC, caused a significant decrease in the initial ATP-induced response, suggesting that P2Y receptors are coupled to PC-PLC. Although P2X were present in HKC-8, The P2X agonist, alpha,beta me-ATP, failed to cause a rise in [Ca(2+)](i). However, PPADS at a concentration of 100 microM inhibits the ATP-induced rise in [Ca(2+)](i). Our results indicate the presence of functional P2Y receptors in HKC-8 cells. ATP-induced [Ca(2+)](i) elevation via P2Y is tightly associated with PC-PLC and TRP channel.

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.

Developmental downregulation of P2X3 receptors in motoneurons of the compact formation of the nucleus ambiguus

Motoneurons of the compact division of the nucleus ambiguus (cNA) are the final output neurons of the swallowing pattern generator. Thus, their normal function is critical to neonatal survival. To explore the role of purinergic signaling in modulating the excitability of these motoneurons during development, immunohistochemical and whole-cell recording techniques were used to characterize expression patterns of ionotropic P2X receptors and the effects of ATP on cNA motoneurons. Medullary slices containing the cNA were prepared from neonatal (P0-4) and juvenile (P15-21) rats. In neonatal cNA motoneurons, local application of 1 mM ATP produced a large (-133 +/- 17 pA; n = 78), desensitizing, inward current that was mimicked by 1 mM alpha,beta meATP and 2meSATP, and inhibited by the P2 antagonist, PPADS (5 microM), and the P2X3 antagonist, A-317481 (0.1-1 mM). In juvenile cNA motoneurons, 1 mM ATP produced negligible currents, while 10 mM ATP produced small (-59 +/- 14 pA; n = 42), primarily non-desensitizing currents. Immunohistochemistry demonstrated that in the neonate, the expression of P2X3 was robust, P2X2 and P2X5 moderate, P2X4 and P2X6 weak, and P2X1 absent. In the juvenile cNA, only low levels of P2X5 and P2X6 labeling were detected. These data indicate that P2X receptors in cNA motoneurons are profoundly downregulated during the first two postnatal weeks, and suggest a role for the purinoceptor system, particularly P2X3 receptors, in the control of esophageal motor networks during early postnatal periods.

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.

Algogen-specific pain processing in mouse spinal cord: differential involvement of voltage-dependent Ca(2+) channels in synaptic transmission.

1. The effects of intrathecal (i.t.) administration of N-, P/Q- or L-type voltage-dependent Ca(2+)-channel blockers were tested in two pain models involving bradykinin (BK)- and alpha,beta-methylene ATP (alpha,beta meATP)-induced activation of primary afferent neurons in mice. 2. The nociceptive response (amount of time spent licking and biting the hindpaw) induced by intraplantar injection of BK (500 pmol mouse(-1)) was significantly attenuated by both omega-conotoxin GVIA (N-type blocker) and calciseptine (L-type) but not by omega-agatoxin IVA (P/Q-type). 3. The nociceptive response induced in a similar way by alpha,beta meATP (100 nmol) was significantly inhibited by both the above N- and P/Q-type Ca(2+)-channel blockers but not by the L-type blocker. 4. The nociceptive responses elicited by BK and alpha,beta meATP were dose-dependently inhibited by a tachykinin-NK1-receptor antagonist (L-703,606) and an N-methyl-D-aspartate (NMDA)-receptor antagonist (D-AP5), respectively. 5. Intrathecal administration of substance P (SP) (1.8 nmol) or NMDA (350 pmol) elicited algesic responses, such as licking, biting and scratching of the hindquarters. The SP-induced algesic behaviour was significantly inhibited by the L-type blocker but not by the N-type. The NMDA-induced response was not affected by either the N- or the P/Q-type blocker. 6. These findings suggest that BK and ATP most likely excite different types of sensory neurons in the periphery and that within the spinal cord the former stimulates peptidergic transmission regulated by presynaptic N- and postsynaptic L-type Ca(2+) channels, while the latter stimulates glutamatergic transmission regulated by presynaptic N- and P/Q-type channels.

Modulation of cyclooxygenase-2 and brain reactive astrogliosis by purinergic P2 receptors.

Astroglial cells respond to trauma and ischemia with reactive gliosis, a reaction characterized by increased astrocytic proliferation and hypertrophy. Although beneficial to a certain extent, excessive gliosis may be detrimental, contributing to neuronal death in neurodegenerative diseases. We have tested the hypothesis that ATP may act as a trigger of reactive gliosis in an in vitro model (rat brain primary astrocytes) where reactive astrogliosis can be quantified as elongation of astrocytic processes. Challenge of cells with the ATP analog alpha,beta methyleneATP (alpha,beta meATP) resulted in concentration dependent elongation of astrocytic processes, an effect that was fully counteracted by the non-selective ATP/P2 receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). Signalling studies revealed that alpha,beta meATP-induced gliosis is mediated by a novel G-protein-coupled receptor (a P2Y receptor) coupled to an early release of arachidonic acid. Challenge of cells with alpha,beta meATP also resulted in an increase of inducible cyclooxygenase-2 (COX-2), the activity of which has been reported to be pathologically increased in a variety of neurodegenerative diseases characterized by inflammation and astrocytic activation. Induction of COX-2 by alpha,beta meATP was causally related to reactive astrogliosis, since the selective COX-2 inhibitor NS-398 prevented both the purine-induced elongation of astrocytic processes and the associated COX-2 increase. Preliminary data on the putative receptor-to-nucleus pathways responsible for purine-induced gliosis suggest that induction of the COX-2 gene may occur through the protein kinase C/mitogen activated protein kinase system, and may involve the formation of activated AP-1 transcription complexes. We speculate that antagonists selective at this novel P2Y receptor subtype may represent a novel class of neuroprotective agents able to slow down neurodegeneration by counteracting the inflammatory events contributing to neuronal cell death.

Multiple P2X receptors on guinea-pig pelvic ganglion neurons exhibit novel pharmacological properties.

1. Application of ATP and alpha,beta-methylene ATP (alpha beta meATP) to voltage-clamped guinea-pig pelvic neurons produced three types of inward currents. A fast-desensitizing response was present in 5% (25/660) of neurons, 70% gave slowly-desensitizing currents, and the remainder had biphasic responses. 2. Slowly-desensitizing responses were characterized pharmacologically. The response to alpha beta meATP 100 microM was 46+/-27% (range 0--100%) of that evoked by ATP 100 microM in the same cell. Cross-desensitization indicated the presence of alpha beta meATP-sensitive and -insensitive receptors. 3. The concentration-response curve for alpha beta meATP had an EC(50) of 55 microM, and a Hill coefficient of 0.99, while at the alpha beta meATP-insensitive receptor, ATP had an EC(50) of 73 microM, with a Hill coefficient of 1.78. 4. The response to alpha beta meATP was blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin and Cibacron blue. However, the alpha beta meATP-insensitive receptor was inhibited by PPADS, but not by the other two antagonists. 5. 2'- (or 3'-) O-trinitrophenyl-ATP was 10 times more potent in inhibiting responses to alpha beta meATP than to ATP (at the alpha beta meATP-insensitive receptor). 6. Lowering extracellular pH potentiated responses to alpha beta meATP and ATP, while raising pH attenuated them. 7. Co-application of Zn(2+) (3--300 microM) inhibited the responses to alpha beta meATP and ATP, with IC(50) values of 286 and 60 microM, respectively. 8. In conclusion, unlike rat and mouse pelvic ganglion neurons, which only express P2X(2) homomers, at least three distinct P2X receptors are present in guinea-pig pelvic neurons, probably homomeric P2X(2), P2X(3) and heteromeric P2X(2/3) receptors. However, some of the novel pharmacological properties observed suggest that the guinea-pig P2X receptor subtypes may differ from their rat orthologues.

Extracellular ATP inhibits the small-conductance K channel on the apical membrane of the cortical collecting duct from mouse kidney.

We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (K(i) = 64 microM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P(2)) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-gamma-S, whereas alpha, beta-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P(2)Y(2) purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (N(G)-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P(2)Y(2) receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.

Functional and molecular diversity of purinergic ion channel receptors.

P2X receptors are membrane ion channels gated by extracellular adenosine 5'-triphosphate (ATP); nucleotides also activate a family of seven transmembrane G protein-coupled receptors (P2Y). P2X receptors are widely expressed on mammalian cells, where they can be broadly differentiated into three groups. The first group is almost equally well activated by ATP and its analog alpha beta methyleneATP (alpha beta meATP), whereas a second group is not activated by alpha beta meATP. A third-group type of receptor (termed P2Z) is distinguished by the fact that the channel opening is followed by cell permeabilization and lysis if the agonist application is continued for more than a few seconds. Seven cDNAs have been cloned that encode P2X receptor subunits. When expressed individually in heterologous systems, P2X1 and P2X3 subunits form channels activated by ATP or alpha beta meATP; whereas P2X2, P2X4, and P2X5 form channels activated by ATP but not alpha beta meATP. P2X6 receptors do not express readily, and P2X7 receptors correspond closely in their properties to P2Z. Further phenotypes can be produced when two subunits are coexpressed, indicating hetero-multimerization. This chapter compares the properties of the native P2X receptors with those of the cloned and expressed subunits.

Characterization of Ca2+ influx through recombinant P2X receptor in C6BU-1 cells.

1. The effects of exogenous adenosine 5'-triphosphate (ATP) and alpha,beta-methylene ATP (alpha,beta meATP) on C6BU-1 cells transfected with P2X2 and P2X3 subtypes, separately or together (P2X2+3), were investigated using fura-2 fluorescence recording and whole-cell patch clamp recording methods. 2. Untransfected C6BU-1 cells showed no intracellular Ca2+ ([Ca2+]i) increase in response to depolarizing stimulation with high K+ or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca2+ influx only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca2+ influx through the P2X subtypes. 3. In C6BU-1 cells transfected with the P2X2 subtype, ATP (more than 10 microM) but not alpha,beta meATP (up to 100 microM) evoked a rise in [Ca2+]i. 4. In the cells transfected with the P2X3 subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 microM of alpha,beta meATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca2+]i rise and the current response was seen only with the P2X3 subtype. In addition, the agonist-induced rise in [Ca2+]i was observed only after the first application because of desensitization of this subtype. 5. In C6BU-1 cells co-transfected with P2X2 and P2X3, ATP at 1 microM evoked a [Ca2+]i rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co-transfection with P2X2 and P2X3, when compared to transfection with the P2X3 subtype alone. The desensitization of the P2X2+3 was apparently slower than that of the P2X3 subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca2+]i rise. 6. These results suggest that the P2X2 and P2X3 subtypes assemble a heteromultimer and that this heterogeneous expression acquires more effective Ca2+ dynamics than that by homogeneously expressed P2X2 or P2X3.

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase.

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK). 2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+-response, but resulted in a more rapid decline to basal. There was no response to alpha,beta-MethylATP (alpha,beta MeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3. ATP (log EC50 -5.1+/-0.2) also caused an increase in the total [3H]-inositol (poly)phosphates ([3H]-InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and alpha,beta MeATP giving no detectable response. 4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P3. 5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPgammaS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 microM forskolin. 6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with activation at P2Y2 receptors. 2MeSATP gave a much smaller response with a lower potency than UTP. 7. These results are consistent with brain endothelial regulation by P2Y2 receptors coupled to phospholipase C, Ca2+ and MAPK; and by P2Y1-like (2MeSATP-sensitive) receptors which are linked to Ca2+ mobilization by a mechanism apparently independent of agonist stimulated Ins(1,4,5)P3 levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK coupling of these receptors suggests that they exert fundamentally distinct influences over brain endothelial function.

ATP-gated cation channels (P2X purinoceptors) in trigeminal mesencephalic nucleus neurons of the rat.

1. We have investigated whether receptors for ATP exist on neurones of the trigeminal mesencephalic nucleus (MNV) of the rat using whole-cell and outside-out patch-clamp recording in coronal brainstem slices. 2. With whole-cell recording, the batch application of ATP, adenosine 5'-O-[3-thiotriphosphate] (ATP gamma S) and alpha,beta methylene adenosine triphosphate (alpha beta meATP) caused concentration-dependent inward currents in all cells tested (holding potential, -62 mV), with EC50 values of 437, 15 and 254 microM, respectively. All three agonist-evoked currents developed rapidly (rise time, approximately 10-25 s), desensitized slowly (over approximately 20-50 s), cross-desensitized with each other, were associated with an increase in membrane conductance and were attenuated by the application of suramin (30 microM). 3. The inward current evoked by ATP decreased as the membrane potential was made less negative and had a zero current potential of +1.0 +/- 3.7 mV. The current-voltage relationship showed marked inward rectification. 4. Brief flickery single-channel openings could be resolved in response to ATP (3 microM) in outside-out membrane patches. Unitary current at -82 mV was -1.81 +/- 0.2 pA, which corresponds to a unitary conductance of 22 pS. 5. We conclude that proprioceptive MNV neurones contain ATP-gated cation channels. Such P2X purinoceptors may be involved in the processing of proprioceptive information, thus suggesting a potentially important physiological role of ATP.

The effect of extracellular Ca2+ on responses to purinoceptor agonists in cultured swine tracheal smooth muscle cells.

The effects of extracellular Ca2+ ions on purinergic responses were examined in swine tracheal smooth muscle cells (TSMCs) in primary culture. ATP (1 microM to 1 mM) and alpha, beta-methylene adenosine 5' triphosphate (alpha, beta-Me ATP) (100 microM and 1 mM) concentration-dependently increased [Ca2+]i in the presence and the absence of extracellular Ca2+. Responses to ATP (10 microM to 1 mM) in the presence of extracellular Ca2+ were significantly larger than those in its absence (n=8), whereas those to alpha, beta-Me ATP were not significantly different between the presence (n=7) and the absence (n=8) of extracellular Ca2+. Responses to ATP (1 mM) at extracellular Ca2+ concentration ([Ca2+]o) of 10 and 5 mM were significantly larger than that at extracellular EGTA concentration ([EGTA]o) of 1 mM (p< 0.01, n=5), whereas the responses to alpha, beta-Me ATP (1 mM) at 10 and 5 mM [Ca2+]o were significantly smaller than that at [EGTA]o of 1 mM (p<0.05, n=5). Increasing [Ca2+]o to 5 mM after the application of either 1 mM ATP (n=4) or 1 mM alpha, beta-Me ATP (n=4) in the absence of extracellular Ca2+ (1 mM [EGTA]o) further increased [Ca2+]i, though the increases in [Ca2+]i by agonists in 1 mM [EGTA]o had been already maximal. Incubating cells for 300 s in 5 mM [Ca2+]o before the application of ATP (1 mM) significantly increased the response to the drug than that obtained by incubating cells for 6 sec in 5 mM [Ca2+]o before the drug application (p< 0.01, n=4). However, alpha, beta-Me ATP (1 mM) induced similar responses by incubating cells for 30 or 300 s in 5 mM [Ca2+]o to that by incubating them for 6 s. These results suggest that the effect of alpha, beta-Me ATP in swine TSMCs in primary culture is mainly through Ca2+ release and that its effect on Ca2+ entry is smaller than other nucleotides.

Modulation by extracellular ATP of L-type calcium channels in guinea-pig single sinoatrial nodal cell.

1. The effects of extracellular adenosine 5'-triphosphate ([ATP]zero) on the L-type Ca2+ channel currents in guinea-pig single sinoatrial nodal (SAN) cells, isolated by enzymatic dissociation, were investigated by use of whole-cell patch-clamp techniques. 2. The application of [ATP]zero (2 microM-1 mM) produced an inhibitory effect on the L-type Ca2+ channel current peak amplitude (10 mM Ba2+ as charge carrier) in a concentration-dependent and reversible manner with an IC50 of 100 microM and a Hill coefficient of 1.83. 3. The presence of the adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM) and 8-phenyltheophylline (10 microM) did not affect the [ATP]zero-induced inhibition of the Ca2+ channel currents. Adenosine (100 microM) had little effect on the basal Ca2+ channel currents. Adenosine 500 microM, caused 23% inhibition of the Ca2+ channel current, which was abolished by 0.1 microM DPCPX. 4. The presence of the P2-purinoceptor antagonists, suramin (1, 10 and 100 microM), reactive blue 2 (1 and 10 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 50 and 100 microM) failed to affect the inhibitory action of [ATP]zero on Ca2+ channel currents. 5. The relative rank order of potency of different nucleotides and nucleosides, at a concentration of 100 microM, on the inhibition of the Ca2+ channel currents is as follows: adenosine 5'-triphosphate (ATP) = alpha,beta-methylene-ATP (alpha,beta MeATP) > > 2-methylthioATP (2-MeSATP) > or = adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > > uridine 5'-triphosphate (UTP) = adenosine 5'-diphosphate (ADP) > adenosine 5'-monophosphate (AMP) > or = adenosine. 6. These results suggest that [ATP]zero may play an important role in the heart beat by inhibiting the L-type Ca2+ channel currents in single SAN cells. This inhibitory effect is not due to the formation of adenosine resulting from the enzymatic degradation of [ATP]zero. Based on the relative order of inhibitory potency of different nucleotides and nucleosides on the L-type Ca2+ channel currents and the ineffectiveness of the purinoceptor antagonists tested, a novel type of purinoceptor may be involved.

P2X purinoceptors in cultured myenteric neurons of guinea-pig small intestine.

1. Fast excitatory postsynaptic currents (fEPSCs) and responses to exogenously applied purinoceptor agonists were studied in primary cultures of myenteric neurons from guinea-pig small intestine. Whole-cell and outside-out configurations of the patch clamp technique were used. Hexamethonium (100 microM) partly inhibited fEPSCs in 28% of neurons. Hexamethonium-resistant fEPSCs were inhibited by 97 +/- 2% by the P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM). 2. ATP caused two types of inward currents. In 92% of neurons (n = 123), ATP caused a slowly desensitizing current that declined with a double exponential time course (tau 1 = 7.1 +/- 2.0 s; tau 2 = 57 +/- 7.4 s, n = 4). The rank order potency for purinoceptor agonists in these neurons was ATP > 2-methylthio-ATP (2-MeSATP) > > alpha, beta-methylene ATP (alpha, beta-me ATP) > beta, gamma-meATP > ADP. The EC50 values for ATP and 2-MeSATP were 40 and 65 microM, respectively. alpha, beta-MeATP acted as a partial agonist at these receptors. In 8% of neurons (n = 11), ATP-induced currents desensitized rapidly with a double exponential time course (tau 1 = 0.13 +/- 0.015 s; tau 2 = 2.2 +/- 1.3 s, n = 4); alpha, beta-meATP caused similar responses in these cells. Both types of ATP-induced current were associated with an increased conductance and an inwardly rectifying I-V relationship (Erev = 10 mV). Halving [Na+]o shifted the reversal potential of ATP currents by -22 +/- 6 mV. 3. ATP activated single channel currents in outside-out patches. The single channel I-V relationship was linear between -120 and 60 mV (Erev approximately 0 mV). Single channel conductance between -100 and -60 mV was 25 +/- 2 pS. Single channel open probability was voltage dependent and decreased from 0.05 +/- 0.01 at -100 mV to 0.007 +/- 0.002 at +40 mV. 4. These data show that P2X purinoceptors mediate some fEPSCs in cultured myenteric neurons. Myenteric neurons express the fast-desensitizing alpha, beta-me ATP-sensitive subtype of P2X receptor that has the properties of cloned P2X1 receptors and is similar to native receptors in smooth muscle cells. Myenteric neurons also express a P2X receptor that desensitized slowly and was alpha, beta-meATP-insensitive. This receptor has the properties of cloned P2X2 or P2X5 receptors and is similar to native receptors found in PC-12 cells and superior cervical ganglion neurons. The known distribution of P2X2 and P2X5 receptors suggests that myenteric neurons are likely to express predominantly P2X2 receptors.

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx.

The aim of this study was to determine whether 45Ca2+ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC50 = 45 microM), ATP gamma S (EC50 = 50 microM) and 2-meSATP (EC50 = 81 microM) but not alpha beta meATP (1 mM) stimulated 45Ca2+ influx 2-5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADP beta S did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC50 = 191 microM) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells. ATP-stimulated 45Ca2+ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated 45Ca2+ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency:- guanidinium (EC50 = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose). A number of P2 purinoceptor antagonists inhibited ATP-stimulated 45Ca2+ influx. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3-300 microM), pyridoxal 5-phosphate (3-300 microM) and d-tubocurarine (30-300 microM) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC50. Suramin (100-300 microM) and cibacron blue (30-300 microM) produced a surmountable antagonism while DIDS (4,4'-diisothiocyanatostilbene-2,2'disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 microM. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X2 purinoceptors. These findings suggest that ATP-stimulated 45Ca2+ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.

Effects of purinoceptor agonists on cytosolic Ca2+ concentration in swine tracheal smooth muscle cells in culture.

1. The effects of various purinoceptor agonists on intracellular Ca2+ concentration ([Ca2+]i) in swine tracheal smooth muscle cells in primary culture were examined to investigate the subtype of purinoceptors in these cells. 2. ATP (1 microM to 1 mM) concentration-dependently increased [Ca2+]i which was measured by monitoring the fluorescence signal of fura2. 3. alpha, beta-Me ATP at concentrations higher than 10 microM increased [ca2+]i in the presence of extracellular Ca2+. Responses to the drug were 12 +/- 5 and 61 +/- 4% of responses to ATP (100 microM) at 100 microM and 1 mM, respectively (n = 7). The response to 100 microM ATP was inhibited by 62% in the presence of 1 mM alpha, beta-Me ATP (n = 8), though the drug at concentrations lower than that did not affect the response to ATP. 4. ATP increased [Ca2+]i in the absence of extracellular Ca2+. The response to ATP in this condition was 40% of that in the presence of extracellular Ca2+ (n = 8). 5. Neither cibacron blue 3GA (10 microM) (n = 8) nor suramin (10 and 100 microM) (n = 10) affected the response to ATP (1 microM to 100 microM). 6. The rank order of potency in the absence of extracellular Ca2+ was UTP > ATP > adenosine 5'-o-(3-thiotriphosphate) > > ADP = alpha, beta-methylene adenosine 5'-triphosphate > 2-(methylthio)-adenosine 5'-(tetrahydrogen triphosphate). 7. UTP (1 microM to 100 microM) concentration-dependently increased inositol 1,4,5-triphosphate (IP3) production. 8. These results suggest that the increase in [Ca2+]i induced by purinoceptor agonists is mediated mainly via a nucleotide receptor in swine tracheal smooth muscle cells in primary culture.

Contribution of P2‐purinoceptors to neurogenic contraction of rat urinary bladder smooth muscle

1. The contribution of P2-purinoceptors to neurogenic contraction was investigated in rat urinary bladder smooth muscle by measurement of isotonic tension. 2. Contraction of rat urinary bladder smooth muscle induced by electrical stimulation was decreased to 84.19 +/- 3.90% of the control (n = 16) in the presence of atropine (1 microM), which was further decreased to 38.80 +/- 2.75% of the control (n = 49) in the presence of both atropine and 10 microM alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-Me ATP). 3. The contractile response induced by electrical stimulation in the presence of atropine and alpha, beta-Me ATP was decreased to 27.81 +/- 4.07% (n = 23) and 26.63 +/- 5.01% (n = 15) of the control, by the addition of 100 microM cibacron blue 3GA and 100 microM suramin, respectively. The application of 100 microM adenosine 5'-o-2-thiodiphosphate (ADP beta S) in the presence of atropine and alpha, beta-Me ATP decreased the contractile response induced by electrical stimulations to 17.15 +/- 3.71% (n = 15) of the control. 4. Pretreatment of muscle strips with 100 microM ADP beta S significantly reduced the response to either 200 microM alpha, beta-methylene adenosine 5'-diphosphate or 200 microM ADP beta S. 5. Uridine 5'-triphosphate (100 microM to 1 mM) concentration-dependently contracted muscle strips, and this contraction was significantly antagonized by desensitization of P2-receptors with alpha, beta-Me ATP (10 microM), and completely antagonized by pretreatment of muscle strips with both alpha, beta-Me ATP and ADP beta S (100 microM). 6. Di(adenosine-5') tetraphosphate (30 and 100 microM) contracted muscle strips, whereas it failed to contract after desensitization of P2-receptors.7. It is suggested that about 20% of the neurogenic contraction of rat urinary bladder smooth muscle is mediated via ADP beta S-sensitive purinoceptors.

The selective P2X purinoceptor agonist, beta,gamma-methylene-L-adenosine 5'-triphosphate, discriminates between smooth muscle and neuronal P2X purinoceptors.

The effects of the putative selective P2X purinoceptor agonist, beta,gamma-methylene-L-adenosine 5'-triphosphate (beta gamma me-L-ATP), were determined at rat neuronal and smooth muscle P2X purinoceptors. beta gamma Me-L-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 microM. In contrast, the archetypal P2X purinoceptor agonist, alpha,beta-methylene ATP (alpha beta meATP; 1-100 microM), produced concentration-related depolarisation responses with a mean EC50 value of 10.8 microM. The depolarising effects of alpha beta meATP were not attenuated by beta gamma me-L-ATP (100 microM). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 microM), but not beta gamma me-L-ATP (1-300 microM), evoked rapid (< 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, beta gamma me-D-ATP and alpha beta meATP competed with high affinity for [3H]alpha beta meATP binding sites, with mean pIC50 values of 7.7 and 8.3, respectively. However, beta gamma me-L-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 microM (mean pIC50 value 4.1). In prostatic segments of the rat vas deferens, beta gamma me-L-ATP (1-100 microM) and alpha beta meATP (0.3-100 microM) each produced concentration-related contractile responses with mean EC50 values of 17.1 and 3.6 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)