Dual contractile effects of ATP released by field stimulation revealed by effects of alpha,beta-methylene ATP and suramin in rat tail artery.

1. The field stimulation-induced release of endogenous ATP and noradrenaline (NA) and contractile response in rat isolated tail artery were examined. The release of ATP was studied by extracellular electrophysiological recording and that of NA by a novel voltammetrical technique. The effects of the P2-purinceptor antagonist, suramin, on these parameters were compared with those of alpha,beta-methylene ATP, a P2X-purinoceptor desensitizing agent. 2. Neither alpha,beta-methylene ATP (10 microM) nor suramin (100-500 microM) had significant effects on the extracellularly recorded nerve terminal action potential but both abolished the ATP-induced excitatory junction current caused by stimulation at 0.1 Hz. Neither agent affected significantly the voltammetrically measured release of NA induced by 10 or 100 pulses at 20 Hz. 3. Combined blockade of both postjunctional alpha 1- and alpha 2-adrenoceptors by prazosin and yohimbine (both 0.1 microM) profoundly depressed the contractile response to 10 pulses at 20 Hz. The small and fast residual contraction in the presence of these agents was abolished by alpha,beta-methylene ATP (10 microM) and inhibited by suramin in a concentration-dependent manner (10-500 microM; IC50 75 microM) and was hence probably caused by ATP or a related nucleotide. 4. When added first, alpha,beta-methylene ATP (10 microM) or suramin (100-500 microM) delayed the onset and enhanced the amplitude of the neurogenic contraction. This enhanced response was abolished by further addition of prazosin and yohimbine (both 0.1 microM). 5. The K+ channel blocker, tetraethylammonium (10 mM), dramatically enhanced the contractile response to 100 pulses at 1 Hz and caused it to become diphasic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual regulation of pancreatic vascular tone by P2X and P2Y purinoceptor subtypes

The effects of ATP on the pancreatic vascular bed of the rat were studied under resting tone. ATP exerted two different effects depending on the concentration used: a slight vasodilatation in the 1.65-49.5 microM range which was statistically significant only at 16.5 microM and a concentration-related vasoconstriction in the 495-4 950 microM range. Theophylline, a P1 purinoceptor antagonist, did not modify the vasodilator effect of ATP. The existence of two P2 purinoceptor subtypes (P2y and P2x) in our preparation may be responsible for the dual effect of ATP. The P2y antagonist 2,2'pyridylisatogen (PIT) used at 5 microM, revealed a vasoconstrictor effect of ATP 165 microM, a concentration without effect per se. Furthermore, the transient vasoconstrictor effect of ATP 495 microM was changed into a long-lasting one in the presence of PIT. On the other hand, the blockade of P2x purinoceptors by the desensitizing agent, alpha,beta-methylene ATP, increased the vasodilator effect of ATP 16.5 microM. In conclusion, two subtypes of P2 purinoceptor do exist on the pancreatic vascular bed: P2y inducing vasodilatation and P2x inducing vasoconstriction. At vascular resting tone, the effect observed with ATP therefore depends on the concentration used and on the balance between P2y/P2x purinoceptors.     

Effects of acute administration of and tachyphylaxis to alpha,beta-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 possible role of ATP and PACAP as mediators of apaminsensitive NANC inhibitory junction potentials in circular muscle of guinea-pig colon.

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), nifedipine (1 microM), L-nitroarginine (L-NOARG, 100 microM), and the selective tachykinin NK1 and NK2 receptor antagonists, SR 140,333 and GR 94,800, respectively (0.1 microM each), a single pulse of electrical field stimulation (EFS) produced a monophasic non-adrenergic non-cholinergic (NANC) inhibitory junction potential (i.j.p., about 10 mV in amplitude) in the circular muscle of guinea-pig proximal colon, recorded by the modified single sucrose gap technique. 2. The P2 purinoceptor agonist, alpha, beta methylene ATP (alpha, beta mATP, 100 microM) and the pituitary adenylyl cyclase activating peptide (PACAP, 1 microM) both produced hyperpolarization (11 +/- 0.8 mV, n = 14 and 10.2 +/- 0.8 mV, n = 19, respectively) and relaxation (1.1 +/- 0.2 mV, n = 14 and 1.5 +/- 0.2 mN, n = 19, respectively) of the circular muscle. 3. Apamin (0.1 microM) nearly abolished (about 90% inhibition) the NANC i.j.p. and the alpha, beta mATP-induced hyperpolarization, markedly reduced the alpha, beta mATP-induced relaxation (73% inhibition) and the PACAP-induced hyperpolarization (65% inhibition), while the PACAP-induced relaxation was unaffected. 4. Tetraethylammonium (TEA, 10 mM) increased the EFS-evoked i.j.p. and revealed an excitatory junction potential (e.j.p.). In the presence of TEA, alpha, beta mATP induced a biphasic response: transient depolarization and contraction followed by hyperpolarization and relaxation. The hyperpolarization to PACAP was reduced by TEA (45% inhibition) but the relaxation was unaffected. 5. The combined application of apamin (0.1 microM) and TEA (10 mM) abolished the i.j.p. and single pulse EFS evoked a pure e.j.p. with latency three times longer than that of the i.j.p. In the majority of strips tested, alpha, beta mATP and PACAP elicited a biphasic response : depolarization and small contraction followed by hyperpolarization and relaxation. 6. The P2 purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) inhibited the NANC i.j.p. in concentration-dependent manner and inhibited the alpha, beta mATP-induced hyperpolarization and relaxation, without affecting the hyperpolarization and relaxation induced by PACAP. On the other hand, the P2 purinoceptor antagonist, suramin (100 microM) inhibited to a similar extent (60-80%) the NANC i.j.p. and the hyperpolarization and relaxation induced by alpha, beta mATP or PACAP. 7. PPADS and suramin reduced the NANC e.j.p. evoked by a single pulse EFS in the presence of apamin and TEA (100 microM of PPADS and 300 microM of suramin inhibited the e.j.p. by about 40%). 8. We conclude that ATP, but not PACAP, mediates the apamin-sensitive NANC i.j.p. in the circular muscle of the guinea-pig colon. After blockade of the NANC i.j.p., ATP may act as an excitatory transmitter by activating excitatory P2 purinoceptors. The subtypes of P2 purinoceptor involved in the inhibitory and excitatory responses remain to be established. The data suggest that excitatory P2 purinoceptors may be located extrajunctionally.     

Stimulation of lumbar sympathetic nerves evokes contractions of cat colon circular muscle mediated by ATP and noradrenaline.

1. The action of the lumbar sympathetic nerves to cat colon was studied in vitro using isolated muscle strips with attached lumbar colonic nerves (LCN) orientated in the axis of circular muscle layer. Electrical stimulation of LCN caused frequency-dependent increases in resting tension and in amplitude of spontaneous contractions. Contractile responses were abolished by tetrodotoxin (3 microM) and by guanethidine (30 microM), indicating that they were neurogenic, involving the release of neurotransmitter from sympathetic fibres. 2. Propranolol (1-9 microM), a beta-adrenoceptor antagonist, caused a concentration-dependent potentiation of LCN-evoked contractile responses. Propranolol (3 microM) potentiated contractile responses to exogenously applied noradrenaline but not to phenylephrine. 3. Phentolamine (1-9 microM), an alpha-adrenoceptor antagonist, and prazosin (1-9 microM), an alpha 1-adrenoceptor antagonist, caused a concentration-dependent reduction of amplitude but did not abolish LCN-evoked contractile responses. Prazosin (3 microM) or phentolamine (3 microM) antagonized contractile responses to noradrenaline and phenylephrine. 4. Desensitization of purinoceptors with the P2x-receptor agonist, alpha,beta-methylene ATP, caused a decrease in amplitude of LCN-evoked contractile responses and abolished contractile responses to ATP. In muscle strips where alpha 1-adrenoceptors were blocked with prazosin (3 microM) and P2-purinoceptors were desensitized with alpha,beta-methylene ATP, the amplitude of contractile responses was reduced by 82-100%. 5. The P2x-purinoceptor antagonists, arylazido amino propyl adenosine triphosphate (ANAPP3) and 5. The P2x-purinoceptor antagonists, arylazido amino propyl adenosine triphosphate (ANAPP3) and suramin, affected LCN-evoked contractile responses. ANAPP3 (50-100 microM) caused a concentration-dependent reduction in the amplitude of contractile response. Suramin (100 microM) caused a small reduction in amplitude of contractile responses but potentiated their amplitude at a concentration of 500 microM. 6. ANAPP3 (100 microM) irreversibly inhibited contractions to alpha,beta-methylene ATP or ATP. Suramin (100-500 microM) inhibited contractions to alpha,beta-methylene ATP (0.5-1 microM) or low concentrations of ATP (10-50 microM) but potentiated contractions at higher concentrations. ANAPP3 (100 microM) and suramin (100, 500 microM) had no effect on contractile responses to noradrenaline. 7. Clonidine (0.05-1 microM), a selective alpha 2-adrenoceptor agonist, caused a concentration-dependent reduction in amplitude of LCN-evoked contractile responses, at 10 Hz, while yohimbine (0.1-1 microM), a selective alpha 2-adrenoceptor antagonist, increased them. At 1 microM, both compounds affected LCN-evoked contractions at all frequencies. This suggests that prejunctional alpha 2-receptors are involved in autoinhibition at sympathetic terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Extracellular ATP-induced calcium channel inhibition mediated by P1/P2Y purinoceptors in hamster submandibular ganglion neurons

1. The presence and profile of purinoceptors in neurons of the hamster submandibular ganglion (SMG) have been studied using the whole-cell configuration of the patch-clamp technique. 2. Extracellular application of adenosine 5'-triphosphate (ATP) reversibly inhibited voltage-dependent Ca(2+) channel (VDCC) currents (I(Ca)) via G(i/o)-protein in a voltage-dependent manner. 3. Extracellular application of uridine 5'-triphosphate (UTP), 2-methylthioATP (2-MeSATP), alpha,beta-methylene ATP (alpha,beta-MeATP) and adenosine 5'-diphosphate (ADP) also inhibited I(Ca). The rank order of potency was ATP=UTP>ADP>2-MeSATP=alpha,beta-MeATP. 4. The P2 purinoceptor antagonists, suramin and pyridoxal-5-phosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS), partially antagonized the ATP-induced inhibition of I(Ca), while coapplication of suramin and the P1 purinoceptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), virtually abolished I(Ca) inhibition. DPCPX alone partially antagonized I(Ca) inhibition. 5. Suramin antagonized the UTP-induced inhibition of I(Ca), while DPCPX had no effect. 6. Extracellular application of adenosine (ADO) also inhibited I(Ca) in a voltage-dependent manner via G(i/o)-protein activation. 7. Mainly N- and P/Q-type VDCCs were inhibited by both ATP and ADO via G(i/o)-protein betagamma subunits in seemingly convergence pathways.     

P2x-purinoceptors of myenteric neurones from the guinea-pig ileum and their unusual pharmacological properties.

1. Whole-cell and outside-out patch clamp recordings were used to characterize the physiological and pharmacological properties of the P2x-purinoceptors of myenteric neurones from the guinea-pig ileum. 2. Adenosine 5'-triphosphate (ATP) and analogues (1-3000 microM) evoked a rapid inward current in > 90% of all recorded neurones. The reversal potential of this current was dependent on the extracellular sodium concentration, at +14 +/- 1.9, 0 +/- 1.6 and -12 +/- 1 mV for 166, 83 and 42 mM of sodium, respectively. The fast activation and inactivation of this current occurred even when guanosine 5'-triphosphate (GTP) was omitted from the pipette solution or substituted with an equimolar concentration of guanosine 5'-o-[2-thiotriphosphate] (GTP-gamma-S). Single channel currents were observed when these outside-out membrane patches were exposed to ATP (10-30 microM). These channels have a unitary conductance of about 17 picosiemens. 3. The rank-order of potency of the agonists used to induce the whole-cell currents was: ATP-gamma-S = ATP = 2-methylthio-ATP (2-Me-S-ATP) > > alpha, beta-methylene ATP = beta, gamma-methylene ATP; adenosine and uridine 5'-triphosphate (UTP) (up to 1 mM) were inactive. 4. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1-30 microM) antagonized the effects of ATP (1 mM) with an IC50 of 4 microM. alpha, beta-Methylene ATP (100 microM) did not affect the ATP (30 microM)-induced current. Cibacron Blue 3GA increased the ATP activated cationic current whereas Basilen Blue E-3G had a very weak antagonistic effect (IC50 > or = 3 mM). Suramin potentiated the currents induced by ATP through a mechanism that was independent of its inhibitory effect on ectonucleotidase activity, as suramin also potentiated the effect of alpha, beta-methylene ATP (an ATP analogue that is resistant to nucleotidases). 5. In conclusion, the myenteric P2x-purinoceptor shares some properties with other purinoceptors in particular with the P2x4- and P2x6-purinoceptors. This receptor has also some unusual pharmacological properties suggesting that myenteric neurones express a novel subtype of P2x-purinoceptors. The properties of this receptor, however, might be a result of the combination of two or more of the homomeric purinoceptors so far characterized.     

Suramin and reactive blue 2 are antagonists for a newly identified purinoceptor on rat megakaryocyte.

1. The effects of purinoceptor antagonists on ATP-induced oscillatory K(+)-currents in rat isolated megakaryocytes were investigated. 2. Both reactive blue-2 (RB-2), a selective antagonist of the P2Y purinoceptor, purinoceptor, at concentrations of 0.3-10 microM and suramin, a non-selective P2 purinoceptor antagonist, at 1-30 microM blocked the ATP-induced oscillation in a concentration-dependent manner. 3. RB-2 and suramin also blocked the ADP-induced K(+)-current oscillation at the same concentration range as in the case of ATP. However, both suramin and RB-2 had no effect on thrombin- and inositol 1,4,5-trisphosphate (IP3)-induced K+ current oscillation, indicating that they act as specific purinoceptor antagonists. 4. Thus, the purinoceptors on megakaryocytes show the properties of the P2 subtype according to their blockade by antagonists.     

Functional antagonism between nitric oxide and ATP in the motor responses of guinea-pig ileum

1. The interaction of nitric oxide and ATP in the non-adrenergic, non-cholinergic (NANC) motor responses and the presence of NADPH-diaphorase and quinacrine-positive myenteric neurones were studied on guinea-pig ileum using mechanographic, histochemical and quinacrine-fluorescence techniques. In the presence of phentolamine, propranolol and atropine, the non-precontracted longitudinally oriented organ bath preparations responded to sodium nitroprusside (1-100 microM) or ATP (5-50 microM) with tetrodotoxin (0.1 microM)-resistant relaxation or contraction, respectively. The effects of ATP were suramin (50 microM)- and apamin (5 microM)-sensitive. 2. The NANC motor responses elicited by electrical stimulation (0.8 ms, 1-20 Hz, 20 s) consisted of tetrodotoxin-sensitive relaxation phase followed by a phase of twitch-like and tonic contractions. 3. NG-nitro-L-arginine (L-NNA, 0.1-0.5 mM) inhibited or abolished the relaxation phase. L-arginine (0.5 mM), but not D-arginine (0.5 mM), restored the relaxation phase in L-NNA-pretreated preparations. The relaxation phase increased after ATP-induced desensitization of purinoceptors and in the presence of suramin (50 mciroM) but was abolished by apamin (5 microM). 4. The phase of contractions was enhanced by L-NNA (0.1-0.5 mM) and restored by L-arginine (0.5 mM). The twitch-like and tonic contractions were decreased during ATP-induced purinoceptor desensitization and in the presence of suramin (50 microLM). Apamin (5 microM) inhibited the tonic contractions. 5. The desensitization of purinoceptors by ATP did not change the L-NNA-induced inhibition of the relaxation phase but decreased the L-NNA-increased phase of contractions. L-NNA reduced the relaxation phase and increased the phase of contractions during purinoceptor desensitization. 6. We conclude that in the longitudinal muscle layer of the guinea-pig ileum, nitric oxide mediates the relaxation phase while ATP contributes via smooth muscle P2 purinoceptors to the phase of contractions suggesting a postjunctional functional antagonism between nitric oxide and ATP. The presence of NADPH-diaphorase- and quinacrine-positive neuronal cells and processes running to the muscle cells confirms a physiological role of nitric oxide and ATP in the ileal neurotransmission.     

Effects of PPADS and suramin on contractions and cytoplasmic Ca2+ changes evoked by AP4A, ATP and alpha, beta-methylene ATP in guinea-pig urinary bladder.

1. The contraction and intracellular Ca2+ change evoked by diadenosine tetraphosphate (AP4A) were studied in the outer longitudinal muscle of the guinea-pig urinary bladder and compared with those evoked by ATP and alpha, beta-methylene ATP (a P2-purinoceptor agonist). 2. AP4A, ATP and alpha, beta-methylene ATP produced concentration-dependent transient contractions. These contractions were inhibited by PPADS (pyridoralphosphate-6-azophenyl- 2'-4'-disulphonic acid), 0.3- 30 microM, a P2x-purinoceptor antagonist, and suramin, 1-300 microM, a P2-purinoceptor antagonist in a concentration-dependent manner. From Schild plot analysis, the apparent pA2 values for PPADS for contractions evoked by AP4A, ATP and alpha, beta-methylene ATP were 6.86, 6.56, 6.74, and those for suramin were 6.01, 4.59 and 5.12, respectively; the Schild slopes for PPADS were 1.07, 1.14 and 1.06, and, those for suramin 0.75, 1.05 and 1.16, respectively. 3. AP4A (10 microM) and ATP (100 microM) failed to elicit any contraction of the tissue after a desensitization produced by repeated application of alpha, beta-methylene ATP (1 microM). 4. In fluorescence experiments with fura-2, the increases in [Ca2+]i and contraction evoked by AP4A were suppressed by suramin and nifedipine, an L-type Ca2+ channel blocker. 5. These findings suggest that P2x-purinoceptors, which are more sensitive to PPADS than suramin, exist on the outer longitudinal muscles of guinea-pig urinary bladder, and that the AP4A-evoked contraction results from Ca2+ influx.     

Purinergic modulation of pacemaker Ca2+ activity in interstitial cells of Cajal

Purinoceptors are widely distributed throughout the body, and are thought to have important contributions to numerous functions. In this study, we characterised the contribution of purinoceptors to the mechanisms underlying spontaneous rhythmicity of the gastro-intestinal tracts. Using cell cluster preparations (100-200 microm diameter) obtained from murine ileum, we measured spontaneous intracellular Ca2+([Ca2+]i) oscillations in the presence of nifedipine, as an index of pacemaker [Ca2+]i activity in interstitial cells of Cajal (ICCs, c-Kit-immunopositive cells), the pacemaker cells for gastrointestinal motility. This small preparation also contained smooth muscle and enteric neurones. Using various purinoceptor agonists and an antagonist, we characterised both TTX-sensitive and insensitive modulations of pacemaker [Ca2+]i activity in ICCs. Continuous application of either ATP, ATPgammaS, suramin or alpha,beta-methylene ATP (alpha,beta-meATP) suppressed pacemaker [Ca2+]i activity. The inhibitory effect of alpha,beta-meATP was completely abolished by a prior application of TTX. On the other hand, even in the presence of TTX, continuous application of 2-methylthio ATP (2-MeSATP) at concentrations greater than 30 microM caused a prompt rise followed by a slow decline of the baseline [Ca2+]i, and pacemaker [Ca2+]i oscillations were gradually suppressed during the decline. Neither UTP nor alpha,beta-meATP at high concentrations (30-100 microM) produced a similar [Ca2+]i response. These results suggest that the TTX-resistant, direct purinergic modulation of pacemaker [Ca2+]i activity in ICCs is mediated via P2X purinoceptors distinct from those involved in TTX-sensitive modulation. The slow decline may be attributed to desensitisation of these purinoceptors. The possible involvement of other purinoceptors is also discussed.     

Suramin antagonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli.

1. Suramin, an inhibitor of several types of ATPase, was investigated for its ability to antagonize responses mediated via P2X-purinoceptors in the guinea-pig urinary bladder and P2Y-purinoceptors in the guinea-pig taenia coli. 2. In isolated strips of bladder detrusor muscle, suramin (100 microM-1 mM) caused a non-competitive antagonism of responses to alpha, beta-methylene ATP with an estimated pA2 of approximately 4.7, and inhibited responses to stimulation of the intramural purinergic nerves, with a similar pA2 value. At a concentration of 10 microM, suramin had little effect, but at a concentration of 1 microM, suramin potentiated responses to alpha,beta-methylene ATP, and potentiated responses to electrical stimulation of intramural purinergic nerves. 3. In isolated strips of taenia coli, in which a standard tone had been induced by carbachol (100 nM), suramin at 100 microM and 1 mM significantly antagonized relaxant responses to ATP (at an EC50 concentration) with an estimated pA2 of 5.0 +/- 0.82 and relaxant responses to electrical stimulation of the intramural non-adrenergic, non-cholinergic inhibitory nerves, either single pulses or trains of 8 Hz for 10 s, with estimated pA2 values of 4.9 +/- 0.93 and 4.6 +/- 1.01, respectively. Suramin had no significant effect at 1 or 10 microM. 4. Suramin, at any of the concentrations tested, did not affect contractile responses to histamine (10 microM) or carbachol (10 microM) in the bladder detrusor preparations. In the taenia coli, suramin did not affect either the relaxant responses to noradrenaline (at an EC50 concentration) or the contractile responses to carbachol (100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Different effect of antiulcer agents on rat cysteamine-induced duodenal ulcer after sialoadenectomy, but not gastrectomy

An attempt has been made to pharmacologically isolate cholinergic, P(2) purinoceptor-mediated and peptidergic (capsaicin-sensitive, tachykinin-mediated) contraction of the guanethidine-treated rat bladder detrusor preparation, in vitro. The effect of experimental diabetes was assessed on these types of contraction. Responses were evoked by electrical field stimulation (single shocks or 1 Hz for 30 s or 10 Hz for 40 s). Single shocks and 1-Hz stimulation were applied in the presence of (a). atropine (1 microM) or (b). P(2) purinoceptor antagonists (50 microM pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid) [PPADS] plus 100 microM suramin. Long-term electrical field stimulation (10 Hz for 40 s) (c). was applied with both atropine and the P(2) purinoceptor antagonists present in the organ bath. The effects of capsaicin (d). and ATP (e). were also studied. Three groups of experimental animals were used: streptozotocin-treated (50 mg.kg(-1) i.p., 8 weeks before the experiment), parallel solvent-treated and untreated rats. (a). Responses to electrical field stimulation in the presence of atropine were reduced by half by PPADS plus suramin, but were resistant to capsaicin tachyphylaxis. They were enhanced in preparations taken from diabetic rats. (b). Contractions to electrical field stimulation in the presence of PPADS plus suramin were reduced by 2/3 by atropine, but were left unchanged by capsaicin or diabetes. (c). Contractions to long-term stimulation had a quick and a sustained phase. Especially the latter was inhibited by capsaicin tachypyhlaxis; it was also strongly reduced in preparations taken from diabetic rats. (d). Contractions to capsaicin (30 nM and 1 microM) were resistant to tetrodotoxin, strongly reduced by a combination of tachykinin NK(1) and NK(2) receptor antagonists, and slightly reduced in preparations from diabetic animals. Capsaicin (1 microM) had no acute inhibitory action on cholinergic or purinergic responses, nor did it cause relaxation in precontracted preparations treated with tachykinin receptor antagonists. (e) ATP-induced contractions were strongly reduced by PPADS plus suramin (50 plus 100 microM) and to a similar degree by 100 plus 200 microM, respectively. It is concluded that experimental diabetes selectively impairs peptidergic, capsaicin-sensitive responses (especially those that involve impulse conduction) in the rat detrusor preparation. The contractile response to electrical field stimulation that remains after atropine plus the P(2) purinoceptor antagonists has a yet unknown transmitter background.     

Purinergic and adrenergic cotransmission in canine isolated and perfused gastroepiploic arteries

1. The vasoconstrictor responses of canine gastroepiploic artery to periarterial electrical nerve stimulation (PNS; 30 s trains of pulses at a frequency of 2, 4 or 8 Hz) were observed in a frequency dependent manner. The PNS-induced vasoconstrictions were abolished by tetrodotoxin (1 micromol/L) and mostly depressed but not completely by guanethidine (10 micromol/L). 2. Vasoconstrictor responses to administered noradrenaline were antagonized significantly by prazosin (0.1 micromol/L), an alpha1-adrenoceptor antagonist, but were not significantly affected by suramin (100 micromol/L), a P2 purinoceptor antagonist, or alpha,beta-methylene ATP (1 micromol/L), a P2X receptor desensitizing agent. Exogenous ATP-induced responses were clearly depressed by suramin or alpha,beta-methylene ATP, but were not significantly affected by prazosin. 3. The vasoconstrictor responses to PNS at a low frequency (2 and 4 Hz) of stimulation were markedly inhibited by suramin (100 micromol/L) and by alpha,beta-methylene ATP (1 micromol/L). The remaining responses after suramin or alpha,beta-methylene ATP were abolished by subsequent application of prazosin (0.1 micromol/L). At a high frequency (8 Hz) of stimulation, the vascular response was not significantly inhibited by suramin or alpha,beta-methylene ATP, but it was abolished by prazosin. 4. Injection of xylazine (0.3-30 nmol/L), an alpha2-adrenoceptor agonist, did not induce any clear vasoconstriction. The exposure of tissues to rauwolscine (0.1-0.3 micromol/L), an alpha2-adrenoceptor antagonist, dose-dependently increased PNS-induced vasoconstrictions at all frequencies tested. 5. The present results indicate that ATP acts as a cotransmitter with noradrenaline and is responsible for post-junctional vasoconstrictor responses at low frequencies of sitmulation, whereas the effect of noradrenaline is dominant at high-frequency stimulation in canine gastroepiploic artery. Prejunctional alpha2-adrenoceptor autoinhibition may modulate the release of either noradrenaline or ATP from sympathetic nerve terminals.     

ATP modulates the efferent function of capsaicin-sensitive neurones in guinea-pig isolated atria.

1. The effect of adenosine triphosphate (ATP) and its stable analogues, alpha, beta-methylene-ATP and beta, gamma-methylene-ATP, on the efferent function of capsaicin-sensitive non-adrenergic, non-cholinergic (NANC) nerves was tested in guinea-pig isolated atria. 2. Transmural nerve stimulation of atria isolated from reserpine-pretreated guinea-pigs, in the presence of 1 microM atropine and 0.3 microM CGP 20712A, induced a transient positive inotropic effect attributable to calcitonin gene-related peptide (CGRP) release from NANC nerve endings. 3. ATP (1-30 microM) concentration-dependently reduced the cardiac response to transmural nerve stimulation, without affecting the inotropic response to 10 nM exogenous CGRP. The inhibitory effect of ATP was competitively antagonized by the P1-purinoceptor antagonist, 8-phenyltheophylline (8-PT, 1 microM), but was unaffected by the P2-purinoceptor antagonist, suramin (100 microM). 4. beta, gamma-methylene-ATP in the same concentration range as ATP, inhibited the cardiac response to transmural nerve stimulation. The inhibitory effect of beta, gamma-methylene ATP was antagonized by 1 microM 8-PT. The desensitizing agonist for P2-purinoceptors, alpha, beta-methylene ATP did not induce any inhibitory effect either on the cardiac response to transmural nerve stimulation or on the inhibitory effect curve for ATP. 5. The inhibitory effect of ATP on the NANC neurotransmission was inconsistently modified in the presence of 10 microM alpha, beta-methylene-adenosine diphosphate, an inhibitor of the 5'-ectonucleotidases. 6. These results demonstrate that ATP modulates the efferent function of cardiac NANC nerve endings through prejunctional inhibitory receptors belonging to the P1 type.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the binding characteristics of recombinant P2X1 and P2X2 purinoceptors.

1. We have recently provided evidence that [35S]-adenosine 5'-O-[3-thiotriphosphate] ([35S]-ATP gamma S) can label the human bladder recombinant P2X1 purinoceptor (human P2X1 purinoceptor). In this study we have characterized the binding of [35S]-ATP gamma S to a second P2X purinoceptor subtype, the rat PC12 phaeochromocytoma cell recombinant P2X2 purinoceptor (rat P2X2 purinoceptor), and compared its binding properties with those of both endogenous and recombinant P2X1 purinoceptors. 2. Infection of CHO-K1 cells with the rat P2X2 purinoceptor using Semliki forest virus (SFV) resulted in the expression of high affinity (pKd = 9.3; Bmax = 18.1 pmol mg-1 protein) binding sites for [35S]-ATP gamma S but not for [3H]-alpha, beta-methylene ATP ([3H]-alpha beta meATP). Since functional P2X purinoceptors could be detected electrophysiologically in these cells, but not in non-infected or CHO-K1 cells infected with SFV containing the LacZ gene, these results suggest that the rat P2X2 purinoceptor can be labelled using [35S]-ATP gamma S. 3. The binding characteristics of the rat P2X2 purinoceptor were compared with those of the human P2X1 purinoceptor, which was also expressed in the CHO-K1 cells using SFV. A major difference between the two recombinant P2X purinoceptor types was in the binding characteristics of alpha, beta-methylene ATP (alpha beta meATP). Thus, in the absence of divalent cations, alpha beta meATP possessed low affinity for both the human P2X1 purinoceptor (pIC50 = 7.2) and rat P2X2 purinoceptor (pIC50 = 7.1) labelled using [35S]-ATP gamma S. However, when the recombinant P2X purinoceptors were labelled with [3H]-alpha beta meATP in the presence of 4 mM CaCl2, the affinity of alpha beta meATP for the human P2X1 purinoceptor increased (pIC50 for alpha beta meATP = 8.2), while the affinity of the rat P2X2 purinoceptor for alpha beta meATP did not change (pIC50 for alpha beta meATP = 6.8). 4. Affinity estimates of 15 other nucleotide analogues for the [35S]-ATP gamma S binding sites on the two recombinant P2X purinoceptor subtypes were surprisingly similar (less than 5 fold difference), the only exception being 2'-deoxy ATP which possessed 8 fold higher affinity for rat P2X2 than for human P2X1 purinoceptors. In contrast dextran sulphate and the P2 purinoceptor antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid and 4,4'-diisothiocyanatostilbene-2,2' disulphonic acid, possessed 7 to 33 fold higher affinity for the human P2X1 than for the rat P2X2 purinoceptor. These data provide a correlation coefficient (r) of 0.894. 5. There was some evidence for species differences in the P2X1 purinoceptor. Thus, most nucleotides possessed slightly greater (up to 9-10 fold), while the P2 purinoceptor antagonists possessed slightly lower (up to 7-16 fold), affinity for the endogenous rat vas deferens and rat bladder P2X1 purinoceptors than for the human recombinant P2X1 purinoceptor. These differences were reflected in a slightly lower correlation coefficient, when comparing across species between the human recombinant P2X1 purinoceptor and the endogenous P2X1 purinoceptors labelled in either the rat deferens (r = 0.915) or the rat bladder (r = 0.932), than when comparing within species between the endogenous rat vas deferens and rat bladder P2X1 purinoceptors (r = 0.995). 6. In summary, [35S]-ATP gamma S can be used to label the recombinant P2X1 and P2X2 purinoceptors. Despite the marked differences reported between these two forms of P2X purinoceptor in functional studies, the differences in binding studies were more limited. However, a number of antagonists could discriminate between the P2X purinoceptor subtypes in the binding studies raising expectations that selective antagonists for these receptors can be developed.     

Suramin: a selective inhibitor of purinergic neurotransmission in the rat isolated vas deferens.

The effects of the putative P2 purinoceptor antagonist suramin on contractile responses of the rat isolated vas deferens to electrical field stimulation and exogenously applied drugs (alpha,beta-methylene ATP and noradrenaline) were investigated. Suramin was devoid of agonist activity in the rat vas deferens. The response of the vas deferens to single pulse field stimulation was characteristically biphasic with a large first component peaking between 260-300 ms after the stimulus followed by a second smaller component peaking at about 650 ms post-stimulus. Suramin (100 nM-1 mM) selectively impaired the first, purinergic phase of the response to single pulse field stimulation but was without effect on the second, noradrenergic component. The response of the vas deferens to trains of electrical field stimuli (10 Hz for 10 s) was also biphasic. Suramin (1 microM-1 mM) reduced the first (less than 1 s) phase of the response by 30%, the second (greater than 5 s) plateau phase by 50% and inhibited the intermediate (2-4 s) phase by 80%. Dose-contact periods of 20 or 30 min respectively were sufficient to achieve equilibration of the inhibitory effects of suramin against the responses to single pulse field stimulation or trains of pulses. Following 30 min incubation with 1 mM suramin, the remaining first and second phase components of the response to trains of pulses were impaired and subsequently abolished by the alpha 1-adrenoceptor antagonist WB4101 establishing their noradrenergic origin. Suramin (300 microM) abolished responses of the vas deferens to alpha,beta-methylene ATP but was without effect on those to noradrenaline. Suramin (30 microM) induced a rightward shift in the concentration-response relationship to alpha,beta-methylene ATP that was associated with a significant 40% increase in the maximum response, but did not modify responses to noradrenaline. The inhibitory effects of suramin (3-300 microM) on responses of the vas deferens to approximate EC50 concentrations of alpha,beta-methylene ATP were reversible on repeated washout for 40-60 min. These results reveal suramin to be a useful pharmacological tool for the study of purinergic neurotransmission in rodent vasa deferentia.     

Mechanisms underlying contraction and relaxation induced by nerve stimulation in monkey uterine arteries

We investigated the mechanisms of contractile and relaxant responses to nerve stimulation by electrical pulses and nicotine in isolated monkey uterine artery strips denuded of the endothelium. In the strips contracted with prostaglandin F(2alpha), transmural electrical stimulation (5 Hz, 40 s) produced a contraction which was partially attenuated by prazosin and abolished or reversed to a relaxation by additional treatment with alpha,beta-methylene ATP. The relaxation was abolished by N(G)-nitro-L-arginine (L-NA) and restored by L-arginine but not by D-arginine. Atropine, D-NA, aminophylline and suramin, an inhibitor of P(2Y) purinoceptors, were without effect. The neurogenic relaxation was abolished by 1H-(1,2, 4)oxadiazolo(4,3)quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. Nicotine (10(-4) mol/l) elicited contraction or relaxation of uterine arteries; the contraction was reversed by combined treatment with prazosin and alpha,beta-methylene ATP. Nicotine-induced relaxations were abolished by L-NA and restored by L-arginine. The relaxation induced by exogenously applied NO (acidified NaNO(2) solution) was not influenced by L-NA but abolished by ODQ. It is concluded that contractions induced by nerve stimulation are mediated by norepinephrine and ATP liberated from sympathetic nerves that stimulate alpha(1)-adrenoceptors and P(2x) purinoceptors, respectively. The neurogenic relaxation seems to be mediated exclusively by nitric oxide synthesized from L-arginine in perivascular nerves that activates guanylate cyclase and produces cyclic GMP in smooth muscle.     

Different receptors mediating the inhibitory action of exogenous ATP and endogenously released purines on guinea-pig intestinal peristalsis.

1 Adenosine 5'-triphosphate (ATP) is an enteric neurotransmitter which acts at purine receptors on intestinal nerve and muscle. This study set out to shed light on the receptor mechanisms by which exogenous and endogenous ATP influences intestinal peristalsis. 2 Peristalsis in isolated segments of the guinea-pig small intestine was triggered by a perfusion-induced rise of the intraluminal pressure. Motor changes were quantified by alterations of the peristaltic pressure threshold (PPT) at which propulsive muscle contractions were elicited. 3 ATP (>/= 3 microM) increased PPT and abolished peristalsis at concentrations of 100-300 microM. Adenosine 5'-O-2-thiodiphosphate (ADPbetaS, 3-100 microM) was more potent, whereas alpha,beta-methylene ATP (alpha,beta-meATP, 3-100 microM) was less potent, than ATP in depressing peristalsis. 4 8-Phenyltheophylline (10 microM) attenuated the anti-peristaltic effect of 10 and 30 microM ATP but not that of higher ATP concentrations. Apamin (0.5 microM) counteracted the ability of ATP, ADPbetaS and alpha,beta-meATP to enhance PPT. Suramin (300 microM) and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 150 microM) antagonized the inhibitory effect of alpha,beta-meATP on peristalsis but did not alter the effect of ATP and ADPbetaS. 5 PPADS (50-150 microM) reduced PPT by as much as 50%. This stimulant effect on peristalsis was prevented by suramin (300 microM) but left unaltered by apamin (0.5 microM) and NG-nitro-L-arginine methyl ester (300 microM). 6 These data show that exogenous and endogenous ATP inhibits intestinal peristalsis via different apamin-sensitive purinoceptor mechanisms. Exogenous ATP depresses peristalsis mostly via suramin- and PPADS-insensitive P2 receptors, whereas endogenous purines act via P2 receptors sensitive to both suramin and PPADS.     

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.