P2X Purinoceptors Research Articles

P2X purinoceptor-mediated excitation of trigeminal lingual nerve terminals in an in vitro intra-arterially perfused rat tongue preparation.

A novel in vitro intra-arterially perfused adult rat tongue-nerve preparation was used to explore the possible actions of P2X purinoceptor agonists (ATP and alpha,beta-methylene ATP (alpha, beta-meATP)) on sensory nerve terminals innervating the rat tongue. We made whole-nerve recordings of the trigeminal branch of the lingual nerve (LN), which conducts general sensory information (pain, temperature, touch, etc.), and the chorda tympani (CT), which conducts taste information. Changes in LN and CT activity following intra-arterial application of P2X agonists were compared. In seven preparations, bolus close-arterial injection of ATP (30-3000 microM, 0.1 ml) or alpha,beta-meATP (10-300 microM, 0.1 ml) induced a rapid (< 1 s after injection), dose-related increase in LN activity that decayed within a few seconds. The minimal concentration of ATP (100 microM) required to elicit a response was about 10-fold higher than that of alpha,beta-meATP (10 microM). Bolus injection of ATP or alpha,beta-meATP induced a moderate decrease in firing frequency in three of seven CT preparations. LN responses to P2X agonists showed signs of rapid desensitisation with the peak frequency of discharge being smaller when the agonists were applied at short intervals. Suramin (200 microM) or PPADS (200 microM) applied by intra-arterial perfusion each antagonised the rapid increase in LN activity following application of alpha,beta-meATP (100 microM). Capsaicin (10 microM, 0.1 ml, n = 5 preparations) was injected intra-arterially to desensitise nociceptive fibres. This was found to block (n = 2) or greatly reduce (n = 3) the excitatory effects of alpha,beta-meATP (100 microM, 0.1 ml) on LN activity, implying that only capsaicin-sensitive nociceptive fibres in LN were responsive to P2X agonists. In contrast to the consistent excitatory responses in LN activity following fast application of P2X agonists as bolus, a variable and moderate change in discharge rate of LN and no change in CT activity (n = 5) was observed after applying ATP (100-300 microM, n = 21) or alpha,beta-meATP (100-300 microM, n = 14) by intra-arterial perfusion. The variable responses in LN activity to slow perfusion in contrast to close-arterial bolus injection are consistent with activation of the rapidly desensitising P2X3 receptors. In summary, ATP and alpha,beta-meATP preferentially activate general sensory afferent fibres (LN) but not taste fibres (CT). We suggest that the increase in whole-nerve activity of LN following application of P2X agonists represents activation of nociceptive fibres which possess P2X3 receptors. Our data indicate that ATP and P2X3 receptors may play a role in nociception, rather than taste sensation in the tongue.

One-way cross-desensitization between P2X purinoceptors and vanilloid receptors in adult rat dorsal root ganglion neurones.

1. Capsaicin and ATP can activate ligand-gated cation channels in nociceptive rat dorsal root ganglion (DRG) neurones. We have studied cross-desensitization between these two agents in rat isolated DRG neurones using the whole-cell voltage-clamp technique. 2. ATP (10 microM) activated an inward current in DRG neurones at a holding potential of -60 mV. ATP evoked 'fast' responses that underwent rapid activation and desensitization, 'slow' responses that activated and desensitized more slowly, or responses that displayed a mixture of these two characteristics. The time course of the response to ATP was not related obviously to capsaicin sensitivity. 3. Prior application of capsaicin (0.5 microM) increased the proportion of cells displaying only fast responses to ATP (10 microM) suggesting that cross-desensitization had occurred between capsaicin and the slow component of the ATP response. Prior desensitization to ATP had no apparent effect on the inward current response to capsaicin (0.5 microM). 4. Cross-desensitization between capsaicin and ATP was Ca2+ dependent. 5. Changing the membrane holding potential (Vh) to +40 mV for brief period before applying ATP at -60 mV had a similar effect to capsaicin, i.e. the proportion of cells displaying only fast responses to ATP was increased significantly. This effect of depolarization was not Ca2+ dependent. 6. The heterogenity of responses to ATP is probably due to co-expression of homomeric P2X3 receptors and heteromeric receptors comprising P2X3 subunits with other P2X subunits. We propose that the change in time course of the ATP response produced by prior desensitization to capsaicin is due to selective cross-desensitization with the heteromeric P2X receptors.

ATP as a mediator of mammalian central CO2 chemoreception.

1. A role for P2 purinoceptors in the chemosensory response of respiratory neurones localised in the ventrolateral medulla to changes in arterial CO2 levels was investigated in the anaesthetised rat. Extracellular recordings were made from different classes of respiratory neurone and the effects of P2 receptor blockade on CO2-evoked changes in activity investigated. 2. Increasing inspired CO2 excited 85 % of inspiratory neurones in the pre-Botzinger complex. In all cases, CO2-evoked excitation was blocked by ionophoretic application of the P2 receptor antagonists suramin (0.02 M) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 100 microM), but not the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 100 microM). Suramin and PPADS often reduced ongoing activity, and blocked the excitatory effects of ATP. Inspiratory neurones were also excited by the P2X receptor agonist alphabeta-methyleneATP, suggesting a specific role for P2X receptors. 3. Sixty-six per cent of pre-inspiratory neurones were also excited by CO2. This effect was reduced or abolished by prior application of P2 receptor antagonists. Although post-inspiratory and expiratory neurones were excited by increasing levels of CO2, and also by ionophoretically applied ATP, the CO2-evoked effects were unaffected by P2 receptor blockade. 4. We suggest that ATP, possibly acting via P2X purinoceptors localised within the ventral respiratory group, is involved in central chemoreception. Specifically, these distinctive CO2-P2X-mediated actions were observed only in inspiratory neurones (incrementing inspiratory neurones and pre-inspiratory neurones), which appear to have purinoceptors with pH sensitivity that can account for the actions of CO2 in modifying ventilatory activity.

P2X purinoceptor-induced sensitization of ferret vagal mechanoreceptors in oesophageal inflammation.

1. Using an in vitro single unit recording technique we studied the changes in mechanical and chemical sensitivity of vagal afferent fibres in acute oesophagitis, with particular attention to inflammatory products such as purines. 2. Histologically verified oesophagitis was induced by oesophageal perfusion of 1 mg ml-1 pepsin in 150 mM HCl in anaesthetized ferrets for 30 min on two consecutive days. Controls were infused with 154 mM NaCl. 3. The number of action potentials evoked in oesophageal mucosal afferents by mucosal stroking with calibrated von Frey hairs (10-1000 mg) was stimulus dependent. In oesophagitis responsiveness was reduced across the range of stimuli compared with controls. 4. Topical application of the P2X purinoceptor agonist alphabeta-methylene ATP had no direct excitatory effect on afferents. In oesophagitis, but not in controls, there was a significant increase in responses to stroking with von Frey hairs during superfusion with alphabeta-methylene ATP (1 microM). 5. Mucosal afferents responded directly to one or more chemical stimuli: 26 % (5/19 afferents) responded in controls, and 47 % (7/15 afferents) in oesophagitis. There were no differences in responsiveness to bradykinin (1 microM), prostaglandin E2 (100 microM), 5-hydroxytryptamine (100 microM), capsaicin (1 mM) or hydrochloric acid (150 mM) between control and oesophagitis groups. 6. We conclude that a sensitizing effect of a P2X purinoceptor agonist on mechanosensory function is induced in oesophagitis. This effect is offset by a decrease in basal mechanosensitivity.

P2y and P2x purinoceptors are respectively implicated in endothelium- dependent relaxation and endothelium independent contraction in human corpus cavernosum.

The erectile function of the corpus cavernosum is a balance between smooth muscle contraction and relaxation modulated by nerve terminals and endothelial cells1. It is clearly demonstrated that the mechanisms of erection and detumescence are not exclusively modulated by cholinergic and adrenergic nerves but also by non-adrenergic, non-cholinergic (NANC) neuromodulators2. Purines (particularly ATP, ADP) are present in the environment of endothelial cells and smooth muscle cells of the corpus cavernosum. Nevertheless, the implication of P2y and P2x purinoceptors respectively in tumescence and detumescence have not studied before in man.

Effect of diadenosine tetraphosphate (AP4A) on coronary arterial microvessels in the beating canine heart.

Diadenosine tetraphosphate (AP4A) can be released from activated platelets and the present study examined its effect on coronary arterial microvessels. The role of purinoceptors in the coronary microcirculation in vivo was also investigated. In open chest dogs, coronary arterioles were observed using a microscope with a floating objective. In Protocol 1, AP4A (1, 10, 100 and 1,000 micromol/L) was superfused onto the heart surface before and during the superfusion of 10 micromol/L of 8-phenyltheophylline (8-PT), a P1 purinoceptor blocker. In Protocol 2, AP4A (0.1, 1, 10, and 100 nmol x kg(-1) x min(-1)) was infused into the left anterior descending coronary artery before and during the superfusion of 10 micromol/L of 8-PT. In addition to 8-PT, 30 micromol/L of pyridoxalphosphate-6-azophenyl 2',4'-disulphonic acid (PPADS), a P2X purinoceptor blocker in Protocol 3, or 300 micromol/L of N(omega)-nitro-L-arginine (LNNA) in Protocol 4, was continuously superfused, and 4 doses of AP4A were cumulatively superfused as in Protocol 1. In Protocol 5, 10 micromol/L of alpha,beta-methylene ATP, an agonist of P2X purinoceptors, was superfused for 60 min. Superfused AP4A dilated arterioles in a dose-dependent manner. The magnitude of dilatation was greater in smaller arterioles (small vessel < or = 150 microm: 24.5+/-2.2% vs large vessel > 150 microm: 10.6+/-1.5% at a dose of 1,000 micromol/L, p<0.001). On the other hand, intraluminally applied AP4A also dilated arterioles, but no size dependency was shown. In the presence of 8-PT, vasodilatory responses to superfused and intraluminally applied AP4A were attenuated and the lower doses of AP4A constricted arterioles. This vasoconstrictor effect was not affected by PPADS. The vasodilatory effect of the higher doses of AP4A was almost abolished in the presence of LNNA. Alpha,beta-methylene ATP had no effect on coronary microvascular diameters. AP4A has bidirectional effects on coronary arterial microvessels: vasodilatory effects mediated by P1 purinoceptors and NO, which might be mediated by P2Y purinoceptors, and a vasoconstrictor effect, which is not mediated by P2X purinoceptors.

Three different vasoactive responses of rat tail artery to nicotine.

The vasoactive effects of nicotine on isolated rat tail artery tissues were studied. Nicotine transiently contracted rat tail artery tissues (EC50, 55.6 +/- 2 microM) in an extracellular Ca2+ dependent and endothelium-independent fashion. The blockade of alpha1-adrenoceptors, but not alpha2-adrenoceptors or P2X purinoceptors, inhibited the nicotine-induced contraction by 38 +/- 7% (p < 0.05). Nicotine (1 mM) depolarized membrane by 13 +/- 3 mV, but did not affect L-type Ca2+ channel currents, of the isolated rat tail artery smooth muscle cells. The phenylephrine-precontracted tail artery tissues were relaxed by nicotine (EC50, 0.90 +/- 0.31 mM), which was significantly inhibited after the blockade of nicotinic receptors. Simultaneous removal of phenylephrine and nicotine, after a complete relaxation of the phenylephrine-precontracted tail artery strips was achieved by nicotine at accumulated concentrations (> or =10 mM), triggered a Ca2+-dependent rebound long-lasting vasoconstriction (n = 20). This rebound contraction was abolished in the absence of calcium or in the presence of tetracaine in the bath solution. Pretreatment of vascular tissues with a nicotinic receptor antagonist did not affect the nicotine-induced vasoconstriction or nicotine withdrawal induced rebound contraction. The elucidation of the triphasic vascular effects of nicotine and the underlying mechanisms is important for a better understanding of the complex vascular actions of nicotine.

Differential effects of omega-conotoxin GVIA, tetrodotoxin and prolonged cold storage on purinergic and adrenergic transmission in isolated canine splenic artery.

Double-peaked vasoconstrictions (biphases of vasoconstrictions) were readily induced in the conditions of 30 s trains of pulses at 1 Hz in the isolated, perfused canine splenic artery. P2X purinoceptors have previously been shown to be involved mainfy in the first-peaked response and alpha1-adrenoceptors mostly in the second. The treatment with 10 nM omega-conotoxin GVIA (omega-CTX) produced a parallel inhibitory effect on the first- and second-peaked vasoconstrictor responses to nerve stimulation. A submaximal concentration of tetrodotoxin (TTX) (3 nM) did not affect the first peak of constriction, but strongly inhibited the second peak, although a larger dose of TTX (30 nM) abolished either the first- or second-peaked response. On the other hand, after cold storage at 4 degrees C for 7 days, the first-peaked vasoconstriction markedly decreased, whereas the second-peaked response was not significantly modified. (1) omega-CTX-sensitive calcium channels may produce a parallel modulation of purinergic and adrenergic components of sympathetic cotransmission; (2) TTX-sensitive sodium channels may have a more important role in controlling the adrenergic rather than purinergic transmission; and (3) the function of purinergic transmission of sympathetic nerve might be affected more strongly than that of adrenergic transmission in the cold-stored canine splenic artery.

Neurotransmitter ATP and cytokine release

Microglia, the resident macrophages in the central nervous system (CNS), are rapidly activated upon trauma or ischemic injury, releasing cytokines and undertaking tissue repair. Recent studies have indicated that CNS immune cells express ionotropic P2X and metabotropic P2Y purinoceptors and undergo functional changes in response to extracellular ATP. Non-stimulated cultured rat retinal microglia expressed metabotropic P2U(P2Y2, P2Y4) and ionotropic P2Z(P2X7) purinoceptors equally, whereas in LPS-stimulated microglia, P2Z and its CA2+ response became dominant. Upon hypoxia (1% oxygen) activation, the P2U response became dominant, and proliferation was induced possibly via intracellular Ca2+ mobilization and/or capacitative Ca2+ entry. TNF-alpha and IL-1 beta were released in both LPS- and hypoxia-activated states, enhanced by the P2Z agonist BzATP and suppressed by the antagonist oATP, indicating P2Z involvement in their release. P2Z activation was simultaneously anti-mitotic and induced apoptosis of microglia. Release of cytokines may be induced via Ca2+ influx and activation of NFAT, NF-kappa B or p44/42 and p38 MAP kinases, switching off the mitotic signal transduction pathway and triggering the apoptotic cascade at the same time.

Vascular Reactivity by Purinoceptor Activation in Rat Inferior Vena Cava

Background: : : :Extracellular ATP, released from platelets and nerve endings, plays significant roles in the regula- tion of circulation. The effects of ATP depend on the location of the vessels and the species of experimental animals. Until now, studies were limited to arteries, so we compared the effects of ATP in rat vena cava with those in the aorta and attempted to identify the characteristics of their receptors. Methods:Vascular rings were isolated from the rat inferior vena cava and descending thoracic aorta. Endothelial cells were preserved or removed by gentle rubbing. The isometric contractions were recorded on polygraph using a force transducer. Results:In the vena cava ring precontracted by 100 nM norepinephrine (NE, ATP elicited relaxations in a dose-dependent manner. These effects were abolished by removal of the endothelium or pretreatment with a nitric oxide synthase inhibitor. Relaxations to ATP in the vena cava (EC50:9.9 μ M were less potent than those in the aorta (1.7 μ M. The relative order of potencies was ADP> ATP >AMP>adenosine, but the maximal relaxation to ADP was smaller than to ATP. ATP-induced vasorelaxation was blocked by suramin, a nonselective antagonist for P2 purinoceptor and reactive blue-2, a P2Y blocker. At basal tension, ATP contracted the vena cava dose-dependently and these effects were potentiated by endothelium-removal. Contractions in the vena cava were also less potent than in the aorta, and the order of potencies was α , β -MeATP>UTP> ATP>ADP>AMP=adenosine. ATP-induced vasoconstriction was blocked by suramin and α , β-MeATP, a desensitizing antagonist of P2X purinoceptor, and potentiated by pretreatment with UTP. Conclusion:These results suggest that ADP and ATP acts on P2Y1- and P2Y2-purinoceptor in the endothelium, respectively and induces vasorelaxation of the vena cava, which is mediated by nitric oxide. Since ATP and UTP induced vasoconstriction in endothelium-denuded condition, it may be mediated by the activation of the P2X and P2Y4,6 purinoceptor on smooth muscles, respectively. (Korean Circulation J 2000;30(9 :1156-1164

Perivascular Purinergic Nerve-Induced Vasoconstrictions in Canine Isolated Splenic Arteries

We tried to induce selective perivascular purinergic nerve stimulation in isolated canine splenic arterial preparations, using the cannula insertion method. Under the conditions of periarterial electrical stimulation (ES), i.e., trains of 1, 3 and 10 pulses, 1-ms pulse duration and 10-V amplitude at 1 Hz, monophasic vasoconstriction was consistently induced. The ES-induced vasoconstriction was not influenced by prazosin in doses that completely inhibited noradrenaline-induced vasoconstrictions, but it was suppressed by alpha,beta-methylene ATP, a P2X purinoceptor desensitizer. Thus, it is indicated that a selective purinergic transmitter release is readily obtained in the isolated splenic arterial preparation.

Three different vasoactive responses of rat tail artery to nicotine

The vasoactive effects of nicotine on isolated rat tail artery tissues were studied. Nicotine transiently contracted rat tail artery tissues (EC50, 55.6 ± 2 µM) in an extracellular Ca2+ dependent and endothelium-independent fashion. The blockade of alpha1-adrenoceptors, but not alpha2-adrenoceptors or P2X purinoceptors, inhibited the nicotine-induced contraction by 38 ± 7% (p &lt; 0.05). Nicotine (1 mM) depolarized membrane by 13 ± 3 mV, but did not affect L-type Ca2+ channel currents, of the isolated rat tail artery smooth muscle cells. The phenylephrine-precontracted tail artery tissues were relaxed by nicotine (EC50, 0.90 ± 0.31 mM), which was significantly inhibited after the blockade of nicotinic receptors. Simultaneous removal of phenylephrine and nicotine, after a complete relaxation of the phenylephrine-precontracted tail artery strips was achieved by nicotine at accumulated concentrations (&gt;=10 mM), triggered a Ca2+-dependent rebound long-lasting vasoconstriction (n = 20). This rebound contraction was abolished in the absence of calcium or in the presence of tetracaine in the bath solution. Pretreatment of vascular tissues with a nicotinic receptor antagonist did not affect the nicotine-induced vasoconstriction or nicotine withdrawal induced rebound contraction. The elucidation of the triphasic vascular effects of nicotine and the underlying mechanisms is important for a better understanding of the complex vascular actions of nicotine.Key words: nicotine, smokeless tobacco, vascular smooth muscles, contraction, relaxation.

P2X(4) purinoceptors mediate an ATP-activated, non-selective cation current in rabbit osteoclasts.

Extracellular nucleotides act as signaling molecules in numerous tissues. In bone, nucleotides stimulate osteoclast formation and activity; however, the receptors and signaling mechanisms underlying these effects have yet to be identified. To identify specific P2X purinoceptor subtypes in osteoclasts, degenerate oligonucleotide primers were used to PCR-amplify DNA fragments from a rabbit osteoclast cDNA library. A 372-base-pair fragment was obtained that encoded an amino acid sequence with 88% identity to the rat P2X(4) purinoceptor. The presence of P2X(4) mRNA in purified osteoclasts was confirmed by reverse transcription-PCR. Endogenous purinoceptors were functionally characterized in isolated rabbit osteoclasts by patch-clamp recording in whole-cell configuration. At negative membrane potentials, application of ATP or ADP rapidly activated an inward current followed by an outward current. In contrast, UTP or ADPbetaS elicited only an outward current, due to activation of a Ca(2+)-dependent K(+) conductance. The initial inward current was non-selective for cations and inactivated during agonist application. Furthermore, the inward current was insensitive to suramin and Cibacron blue, and was potentiated by Zn(2+). These characteristics are consistent with properties of P2X(4) purinoceptors. Activation of P2X(4) purinoceptors leads to cation influx and depolarization. Nucleotides, released at sites of trauma or inflammation, may act through these receptors on osteoclasts to stimulate bone resorption.

Ion Permeation and Block of P2X 2 Purinoceptors: Single Channel Recordings

P2X(2) purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG(+), Tris(+), TMA(+), and TEA(+). The selectivity sequence for currents carried by alkali metal ions is: K(+) > Rb(+) > Cs(+) > Na(+) > Li(+), which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG(+), Tris(+), TMA(+) and TEA(+) were virtually impermeant. The divalent ions (Mn(2+), Mg(2+), Ca(2+) and Ba(2+)) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn(2+) > Mg(2+) > Ca(2+) > Ba(2+).

Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells.

1. We used whole-cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5'-triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 microM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. 2. The ATP-activated inward current was a non-selective cation (NSC) current that showed inward rectification, reversed at -1.5 +/- 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 3. The outward current activated by ATP reversed at -68 +/- 3 mV (equilibrium potential for potassium (EK) = -84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi-KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+-activated K+ channel in rat RPE cells. The Ca2+-activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 4. In fluo-3 or fluo-4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+-ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. 5. These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non-selective cation conductance and G protein-coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium-activated K+ current.

Purinoceptors Evoke Different Electrophysiological Responses in Pancreatic Ducts: P2Y INHIBITS K+ CONDUCTANCE, AND P2X STIMULATES CATION CONDUCTANCE

In epithelia, extracellular nucleotides are often associated with regulation of ion transporters, especially Cl(-) channels. In this study, we investigated which purinoceptors are present in native pancreatic ducts and how they regulate ion transport. We applied whole-cell patch-clamp recordings, intracellular Ca(2+) and pH measurements, and reverse transcription-polymerase chain reaction (RT-PCR) analysis. The data show two types of purinoceptors and cellular responses. UTP and ATP produced large Ca(2+) transients, a decrease in intracellular pH, 8-10-mV depolarization of the membrane voltage, and a decrease in the whole-cell conductance. The membrane effects were due to closure of K(+) channels, as confirmed by dependence on extracellular K(+). UTP/ATP effects could be associated with P2Y(2) purinoceptors, and RT-PCR revealed mRNAs for P2Y(2) and P2Y(4) receptors. On the other hand, 2', 3'-O-4-benzoylbenzoyl-ATP induced Ca(2+) influx and approximately 20-mV depolarization of the membrane voltage with a concomitant increase in the whole-cell conductance. These effects were dependent on extracellular Na(+), not Cl(-), indicating opening of cation channels associated with P2X(7) purinoceptors. RT-PCR showed mRNAs for P2X(7) and P2X(4) receptors. In microperfused ducts, luminal (but not basolateral) ATP caused large depolarizations of membrane voltages recorded with microelectrodes, consistent with luminal localization of P2X(7) receptors. Thus, P2Y(2) (and possibly P2Y(4)) purinoceptors inhibit K(+) channels and may not support secretion in native ducts. P2X(7) (and possibly P2X(4)) receptors are associated with cation channels and may contribute to regulation of secretion.

Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors.

Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na(+) absorption [measured via Na(+) short-circuit current (I(Na)(sc))] and stimulated Cl(-) secretion [measured via Cl(-) short-circuit current (I(Cl)(sc))]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate I(Na)(sc) and I(Cl)(sc). By RT-PCR, two P2X receptor channels (P2X(3), P2X(4)) and two P2Y G protein-coupled receptors (P2Y(1), P2Y(2)) were identified. Functional localization of P2 purinoceptors suggest that I(Cl)(sc) is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas I(Na)(sc) is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit I(Na)(sc) across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate I(Cl)(sc) through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

Excitatory effect of P2X receptor activation on mesenteric afferent nerves in the anaesthetised rat.

1. We examined the effects of P2X purinoceptor agonists and P2 purinoceptor antagonists on mesenteric afferent nerves supplying the jejunum in the pentobarbitone sodium-anaesthetised rat. 2. ATP (0. 01-10 mg kg-1, i.a.) and alpha,beta-methylene-ATP (1-30 microg kg-1, i.a.) each induced dose-dependent increases in afferent nerve discharge and intrajejunal pressure. The effect on afferent nerves comprised an early (< 2 s after administration) intense burst of activity followed by a later increase (> 2 s after administration), less pronounced in comparison, which coincided with elevated intrajejunal pressure. 3. Pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (20 mg kg-1, i.v.) and suramin (80 mg kg-1, i.v. ) each antagonised both the early and later increases in afferent nerve discharge elicited by alpha,beta-methylene-ATP (30 microg kg-1, i.a.). 4. Co-administration of omega-conotoxin MVIIA and omega-conotoxin SVIB (each at 25 microg kg-1, i.v.), or treatment with the selective 5-HT3 receptor antagonist alosetron (30 microg kg-1, i.v.), did not affect the rapid burst of afferent nerve activity elicited by alpha,beta-methylene-ATP (30 microg kg-1, i.a.). However, toxin treatment did attenuate the elevations in intrajejunal pressure and the corresponding later phases of evoked afferent discharge, while alosetron inhibited basal afferent nerve activity. 5. In summary, ATP and alpha,beta-methylene-ATP each evoke excitation of mesenteric afferent nerves in the anaesthetised rat. We propose that the early increase in mesenteric afferent nerve activity represents a direct effect on the nerve ending, mediated by P2X receptors, whereas the later increase reflects activation of mechanosensitive fibres secondary to elevated intrajejunal pressure.

Involvement of endogenous tachykinins and CGRP in the motor responses produced by capsaicin in the guinea-pig common bile duct.

In functional experiments, we have investigated the effect exerted by neurotransmitters released from capsaicin-sensitive primary afferent nerve terminals in the isolated guinea-pig common bile duct. In resting preparations, capsaicin (0.1 microM) produced a quick contraction (45.1+/-4% of KCl 80mM) which was abolished by either atropine (1 microM) or tetrodotoxin (0.5 microM). The tachykinin receptor-selective antagonists GR 82334 (NK1 receptor-selective; 3 microM), MEN 11420 (NK2 receptor-selective; 1 microM) and SR 142801 (NK3 receptor-selective; 0.1 microM) administered separately failed to reduce the capsaicin-evoked contraction, whereas any combination of the three antagonists was effective: GR 82334 plus MEN 11420, 36+/-7% reduction; GR 82334 plus SR 142801, 48+/-4% reduction; MEN 11420 plus SR 142801, 55+/-3% reduction; GR 82334 plus MEN 11420 plus SR 142801, 57+/-5% reduction. Neither the CGRP1 receptor antagonist h-CGRP (8-37) (1.5 microM) nor the P2X purinoceptor antagonist PPADS (50 microM) affected the contractile response to capsaicin. The effect of capsaicin (0.1 microM) was abolished by pretreatment with capsaicin itself (10 microM for 15 min). Human calcitonin gene-related peptide (h-CGRP; 0.1 microM) mimicked the effect of capsaicin on resting preparations (contractile response =28% of KCl 80 mM). In preparations precontracted with a submaximal concentration of KCl (24 mM), and in the presence of atropine (1 microM), GR 82334 (3 microM) and MEN 11420 (3 microM), capsaicin (1 microM) produced a tetrodotoxin-insensitive long-lasting relaxation (45+/-3% reduction of tone, at 4min from administration), which was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM). h-CGRP (10-50 nM) produced a similar sustained relaxation of precontracted preparations (59+/-4% reduction of tone). h-CGRP (8-37) (1.5 microM) almost completely reversed the relaxations produced by both capsaicin and h-CGRP. Application of electrical field stimulation (EFS: trains of stimuli of 10Hz; 0.25ms pulse width; supramaximal voltage; for 60s) to precontracted preparations produced a sustained, tetrodotoxin (1 microM)-sensitive relaxation (32+/-4% reduction of tone). L-NOARG (100 microM) greatly reduced (69+/-5% inhibition) the EFS-elicited relaxation. A complete reversal of the relaxant response to EFS into a contraction was obtained by administering L-NOARG to preparations in which a functional blockade of capsaicin-sensitive primary afferent neurons had been achieved by incubating the tissue with capsaicin (10 microM) for 15 min. At immunohistochemistry, tachykinin- and CGRP-immunoreactivities (TK-IR/CGRP-IR) were detected in varicose nerve fibers throughout the common bile duct, while TK-IR cell bodies were observed in the terminal portion (ampulla) only. In vivo pretreatment with capsaicin (50 mg/kg; 6-7 days before) decreased the number of CGRP-IR nerves, whereas the TK-IR neural network was apparently unchanged. In conclusion, our data provide functional evidence for the presence of capsaicin-sensitive primary afferent nerve endings in the guinea-pig terminal biliary tract, whose stimulation by capsaicin or EFS produces the release of tachykinins and CGRP. In addition, morphological evidence is provided that the bulk of TK-IR material in the biliary tract is contained in intrinsic neuronal elements, while CGRP in this tissue is of extrinsic origin only. Tachykinins, probably released in small amounts by capsaicin, act by activating receptors of the NK1, NK2 and NK3 type, most probably located on intrinsic cholinergic neurons, which in turn release ACh to produce the final excitatory motor response. The contractile response to capsaicin obtained in the presence of the three tachykinin receptor antagonists could be due to the co-released CGRP and/or to other unknown neurotransmitters. CGRP produces either indirect excitatory or direct inhibitory responses by stimulation of CGRP2 and CGRP1 receptors, respectively.

Central CO2 chemoreception: a mechanism involving P2 purinoceptors localized in the ventrolateral medulla of the anaesthetized rat.

1. The involvement of P2 purinoceptors in chemosensory function in the ventrolateral regions of the medulla oblongata was investigated in the anaesthetized rat. We have investigated the effect of antagonizing, or desensitizing, P2 receptors in the retrofacial area of the ventrolateral medulla on factors modifying respiratory activity. 2. Bilateral microinjection of suramin (50 nl, 0.02 M), a P2 purinoceptor antagonist, into the retrofacial area in the artificially ventilated rat reduced resting phrenic nerve discharge. It also markedly affected the response of the phrenic nerve to increases in arterial CO2. Under conditions of hyperoxic, hypocapnic apnoea, the mean threshold for inducing phrenic nerve activity was raised significantly (from an end-tidal CO2 of 2.5 % to 4.5 %, n = 9). 3. In addition, the slope of the respiratory response curve to increases in CO2 was reduced after suramin. A similar effect was observed after desensitization of certain P2X receptors with alphabeta-methyleneATP. As arterial levels of O2 were greater than 100 mmHg, and an equivalent pattern of response was observed in sino-aortically denervated and vagotomized animals, we believe any contribution of the peripheral chemoreceptors to be minimal. 4. Our data suggest that respiratory neurones within the retrofacial area (Botzinger complex) represent part of the central site of action of CO2 on respiration. Moreover, our observations lead us to suggest that CO2-evoked changes in respiration are mediated at least in part by P2X purinoceptors.