Identification of potent, selective P2Y-purinoceptor agonists: structure-activity relationships for 2-thioether derivatives of adenosine 5'-triphosphate.

Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonist, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coli, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5-770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6-8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coil P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP.

Activation of P1- and P2Y-purinoceptors by ADP-ribose in the guinea-pig taenia coli, but not of P2X-purinoceptors in the vas deferens.

1. The activity of adenosine 5'-diphosphoribose (ADP-ribose), a ribosylated purine nucleotide, was investigated on the carbachol-contracted taenia coli, a tissue possessing P1- (A2) and P2Y-purinoceptors and on the guinea-pig vas deferens which possesses P2X-purinoceptors. 2. In the vas deferens, where ATP (1 microM-1 mM) produced concentration-dependent contractions, ADP-ribose was without effect at concentrations up to 1 mM. 3. In the taenia coli, ADP-ribose (0.1 microM-1 mM) produced concentration-dependent relaxations with a potency similar to that of adenosine, but less than that of ATP. The pD2 values for ADP-ribose, adenosine and ATP were 4.5 +/- 0.07 (27), 4.4 +/- 0.10 (9) and 5.5 +/- 0.14 (21), respectively. The time-course of the relaxations elicited by ADP-ribose was found to be significantly longer than that for ATP and significantly shorter than that for adenosine. 4. The P1-purinoceptor antagonist, 8-phenyltheophylline (5 microM), produced parallel rightward shifts in the concentration-response curves of the relaxations of the taenia coli elicited by ADP-ribose and adenosine but not ATP. 5. Dipyridamole (0.3 microM), a purine nucleoside uptake inhibitor, potentiated the responses to adenosine and ADP-ribose in the taenia coli. These potentiations were sensitive to 8-phenyltheophylline (5 microM). 6. Reactive blue 2, a P2Y-purinoceptor antagonist, antagonized the inhibitory responses of ADP-ribose and ATP in the taenia coli, without significantly altering the inhibitory responses of either adenosine or noradrenaline.7. In the presence of the potassium channel blocker, apamin (0.3 microM), the inhibitory responses of ADP-ribose were severely attenuated, and the inhibitory responses of ATP in the taenia coli were converted to transient contractions. Further addition of 8-PT blocked the residual responses of ADPribose.8. The P2-purinoceptor antagonist, suramin (500 microM), antagonized responses to ATP and ADP-ribose,but not adenosine. Further addition of 8-PT antagonized the residual responses to ADP-ribose, but not to ATP.9. It is concluded that ADP-ribose has a mixed pharmacological profile, evoking both PI (A2)-purinoceptor-mediated responses and P2Y-purinoceptor-mediated responses, while being inert at P2Xpurinoceptors.It is suggested that ADP-ribose may provide a useful starting point for the generation of structural analogues which have specific activity at the P2Y-purinoceptor.

Actions of adenine dinucleotides in the guinea-pig taenia coli: NAD acts indirectly on P1-purinoceptors; NADP acts like a P2-purinoceptor agonist.

The actions of the adenine dinucleotides beta-nicotinamide adenine dinucleotide (NAD) and beta-nicotinamide adenine dinucleotide phosphate (NADP) were examined on the carbachol-contracted taenia coli of the guinea-pig. Both were capable of inducing full relaxations in a concentration-dependent manner; NADP was 21.4 times more effective than NAD at EC50; the threshold for NADP was approximately 0.1 microM and for NAD approximately 1.0 microM. The P1-purinoceptor antagonist, 8-phenyltheophylline (10 microM), produced a large parallel rightward shift in the NAD concentration-response curve; in contrast it produced a small parallel leftward shift in the NADP concentration-response curve. Dipyridamole (0.2 microM), a purine nucleoside uptake inhbitor, markedly potentiated responses to NAD and slightly potentiated NADP. 8-Phenyltheophylline antagonized the dipyridamole potentiation of both NAD and NADP. By use of high performance liquid chromatography it was shown that the action of NAD involves a breakdown to adenosine. Apamin, a K+ channel blocker, which antagonizes P2-purinoceptor activation in the intestine, abolished responses to NADP but not to NAD. The alpha-and beta-adrenoceptor antagonists, phentolamine (1 microM) and propranolol (1 microM), did not affect responses to NAD or NADP. Tetrodotoxin, a neurotoxin, did not abolish responses to either NAD or NADP. It is concluded that NAD acts largely indirectly as a P1-purinoceptor agonist following its breakdown to adenosine by ectoenzymes, while NADP acts in a similar manner to a P2-purinoceptor agonist.

PACPX--a substituted xanthine--antagonizes both the A1 and A2 subclasses of the P1-purinoceptor: antagonism of the A2 subclass is competitive but antagonism of the A1 subclass is not.

1,3-Dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) was examined for its ability to antagonize adenosine acting on the A1 and A2 subclasses of the P1-purinoceptor. A1-purinoceptors were studied in the isolated, driven left atria of the guinea-pig, and A2-purinoceptors in the isolated, carbachol-contracted taenia coli of the guinea-pig. PACPX antagonized the actions of adenosine in both types of preparation and was a more potent antagonist than 8-phenyltheophylline. The antagonism at the A2-purinoceptor was competitive with a pA2 of 5.95. The antagonism at the A1-purinoceptor was not competitive, although antagonism at the A1-purinoceptor was greater than that at the A2-purinoceptor, based on a comparison of pD2 values. The manner of antagonism of PACPX on the A1-purinoceptors of the heart was different from that found for the A1-receptors in bovine brain, implying that there is a fundamental difference between these central and peripheral A1 subclasses of P1-purinoceptor.

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). 5. Thus, suramin at concentrations above 10 microM blocked actions mediated via P2x- and P2y-purinoceptors in the guinea-pig urinary bladder and taenia coli respectively. Potentiation of purinoceptor-mediated activity was seen only at a low concentration of suramin (1 microM) and only in the urinary bladder (P2x-purinoceptor). For its antagonistic activity suramin did not discriminate between P2X- and P2y-purinoceptors, but it was selective for P2-purinoceptor-mediated activity rather than that mediated via cholinoceptors, adrenoceptors or histamine receptors.

Evidence that the P1-purinoceptor in the guinea-pig taenia coli is an A2-subtype.

The effects of 5'-N-ethylcarboxamidoadenosine (NECA), L-NECA, 2-chloroadenosine, N6-phenylisopropyladenosine (L-PIA and D-PIA), cyclohexyladenosine (CHA), and adenosine were examined on the guinea-pig taenia coli. All the analogues except L-NECA caused relaxations; the order of potency for the series was: NECA greater than 2-chloroadenosine greater than L-PIA greater than CHA greater than D-PIA greater than adenosine. L-PIA was twice as potent as D-PIA in inducing relaxations of the guinea-pig taenia coli. Adenosine and its analogues that induce relaxation all caused a slow membrane hyperpolarization; differences in the rates of hyperpolarization and latencies were apparent, although not statistically significant. The duration of the response to adenosine was significantly less than that for any adenosine analogue. Ion studies, using the sucrose gap, revealed that responses to the analogues were attenuated in elevated extracellular potassium or reduced extracellular chloride. 8-Phenyltheophylline, a potent P1-purinoceptor antagonist, caused a rightward shift of all the adenosine and analogue concentration-response curves. Dipyridamole, an adenosine uptake inhibitor, potentiated the relaxations to adenosine but had no significant effect on the relaxations induced by the analogues. It is concluded that NECA, 2-chloroadenosine, L-PIA, CHA, D-PIA and adenosine mediate their relaxant effects via an extracellular P1-purinoceptor which displays characteristics of the A2-subtype as determined by the rank order of agonist potency. Electrophysiological analysis of the responses to each of the analogues did not reveal any marked differences in the modes of action even between NECA and L-PIA (preferential A2- and A1-receptor agonists, respectively).

Pre- and postjunctional actions of purine and xanthine compounds in the guinea-pig caecum circular muscle.

1. The sucrose-gap technique was used to study pre- and postjunctional actions of P1-purinoceptor and P2-purinoceptor agonists and a range of xanthine derivatives in the guinea-pig caecum circular muscle. 2. Adenosine, 2-chloroadenosine (2-ClAd), ATP and alpha,beta-methylene ATP all caused concentration-dependent hyperpolarization of the smooth muscle membrane with a rank order of potency of 2-ClAd greater than alpha,beta-methylene ATP greater than adenosine. 3. The xanthine derivatives caffeine, theophylline, 8-phenyltheophylline and 1,3-dipropyl-8-(2-amino-4-chlorophenyl) xanthine (PACPX) at submicromolar concentrations evoked depolarization of the smooth muscle membrane. At higher concentrations, all these compounds and enprofylline caused concentration-dependent hyperpolarization. 4. All the purine compounds tested caused a reduction in the amplitude of the non-adrenergic, non-cholinergic inhibitory junction potential (i.j.p.). For the P1-purinoceptor agonists adenosine and 2-ClAd this was almost entirely a prejunctional effect. For the P2-purinoceptor agonists this was mostly a postjunctional effect because both ATP and alpha,beta-methylene ATP caused significantly greater increases in the conductance of the smooth muscle membrane than did adenosine or 2-ClAd. 5. All the xanthine compounds tested (up to 100 microM), except enprofylline, were capable of increasing the amplitude of the i.j.p. At millimolar concentrations both caffeine and theophylline could reduce the i.j.p. amplitude. 6. It is concluded that there are inhibitory prejunctional P1-purinoceptors on the i.j.p.-producing neurones in the guinea-pig caecum circular muscle and that, of the xanthine derivatives tested, none of them would be suitable to use as a P1-purinoceptor antagonist in this preparation because of their own direct effects.

Effects of divalent cations and La3+ on contractility and ecto-ATPase activity in the guinea-pig urinary bladder.

1. Several cations (Ba2+, Cd2+, Co2+, Cu2+, Mn2+, Ni2+, Zn2+ and La3+, all as chloride salts, 1-1000 microM) were tested in the guinea-pig urinary bladder for their ability to: (i) modify contractile responses to electrical field stimulation (EFS), ATP, alpha,beta-methylene ATP (alpha,beta-meATP), carbachol (CCh), and KCl; (ii) affect ecto-ATPase activity. 2. Ba2+ (10-1000 microM) concentration-dependently potentiated contractile responses evoked by EFS (4-16 Hz), ATP (100 microM), alpha,beta-meATP (1 microM), CCh (0.5 microM), and KCl (30 mM). Ni2+ at concentrations of 1-100 microM also potentiated contractility of the urinary bladder, but at concentrations tested its effect was not concentration-dependent. Cu2+ at a concentration of 10 microM and Cd2+ at a concentration of 1 microM potentiated responses to all stimuli, except KCl. Ni2+ at a concentration of 1000 microM and Cd2+ at a concentration of 100 microM inhibited contractions evoked by all stimuli, and at a concentration of 1000 microM Cd2+ abolished any contractions. Responses to ATP and alpha,beta-meATP were selectively inhibited by Cu2+, Zn2+ or La3+, each at a concentration of 1 mM. 3. Cu2+, Ni2+, Zn2+ and La3+ (100-1000 microM) concentration-dependently inhibited ecto-ATPase activity in the urinary bladder smooth muscle preparations, while Ba2+ and Mn2+ were without effect, and Cd2+ and Co2+ caused significant inhibition only at a concentration of 1000 microM. 4. There was no correlation between the extent of ecto-ATPase inhibition and the effect on contractile activity of any of the cations. 5. In conclusion, the ability of some divalent cations to inhibit ecto-ATPase activity and to potentiate or inhibit contractile responses in the guinea-pig urinary bladder appear to be independent effects.

The activation of P1- and P2-purinoceptors in the guinea-pig left atrium by diadenosine polyphosphates.

1. The effects of P1, P2-di(adenosine) pyrophosphate (AP2A), P1, P3-di(adenosine) triphosphate (AP3A), P1,P4-di(adenosine) tetraphosphate (AP4A), P1,P5-di(adenosine) pentaphosphate (AP5A), ATP, alpha, beta-methylene ADP and 2-chloroadenosine (2-ClAd) were examined in the guinea-pig driven left atrium. 2. All these purine compounds except alpha, beta-methylene ADP produced a negative inotropic response with a rank order of potency of: 2-ClAd > > AP2A > or = ATP > or = AP4A = AP3A = AP5A. The EC50 value for 2-ClAd was approximately 1 microM, while those for the remaining compounds were in the range 10 microM-100 microM, alpha, beta-Methylene ADP (10-300 microM), a selective P2Y-purinoceptor agonist, produced a small positive inotropism. 3. The P1-purinoceptor antagonist, 8-para-sulphophenyltheophylline (8-pSPT, 20 microM) caused a right-ward shift in the concentration-response curves for 2-ClAd, ATP and AP2A, but converted the responses of AP3A, AP4A, and AP5A into positive inotropisms. 4. The non-selective P2-purinoceptor antagonist, suramin (300 microM), had no significant effect on the concentration-response curves for 2-ClAd, ATP or AP2A, but significantly antagonized inhibitory responses to AP3A, AP4A and AP5A, and excitatory responses to alpha, beta-methylene ADP. 5. In the presence of 8-pSPT (20 microM), suramin (300 microM) abolished the positive inotropic responses evoked by the dinucleotides. 6. ATP was degraded far more rapidly than any of the dinucleotides, and AP3A was the least stable of the diadenosine compounds. The relative order of stability was AP2A > AP4A = AP5A > AP3A > > ATP. Suramin (300 microM) reduced the rate of degradation of ATP and AP3A by approximately 30%. Suramin had no significant effect on the degradation of AP2A, AP4A or AP5A. 7. It is concluded that the diadenosine polyphosphates cause negative inotropic responses via P1-purinoceptors and a hitherto undefined suramin-sensitive P2-purinoceptor, and that they appear to have positive inotropic effects mediated via another suramin-sensitive P2-purinoceptor.

Effects of cyclopiazonic acid on contractility and ecto-ATPase activity in guinea-pig urinary bladder and vas deferens.

1. Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic ATPase, was tested on guinea-pig urinary bladder and vas deferens for its ability: (1) to modify contractile responses to electrical field stimulation (EFS), exogenous ATP, alpha,beta-methylene ATP (alpha,beta-MeATP), carbachol, noradrenaline (NA), histamine, and KCl; (2) to affect ecto-ATPase activity; (3) to modify the release of ATP evoked by EFS. 2. In the urinary bladder, CPA (10 microM) potentiated contractile responses to EFS, exogenous ATP (100 microM), alpha,beta-meATP (1 microM), carbachol (0.5 microM), histamine (30 microM) and KCl (30 mM). In the vas deferens, CPA (10 microM) potentiated responses to EFS, ATP, alpha,beta-meATP, NA (100 microM) and KCl. CPA at a concentration of 1 microM had no effect on ATP-induced relaxation of carbachol-precontracted guinea-pig taenia coli, and at a concentration of 10 microM it markedly increased spontaneous contractile activity of taenia. 3. Ecto-ATPase was estimated to have Vmax and Km values of 0.98 nmol Pi 30 min-1 mg-1 wet tissue and 881 microM ATP in the urinary bladder, and 0.75 nmol Pi 30 min-1 mg-1 wet tissue and 914 microM ATP in the vas deferens, respectively. CPA at a concentration of 10 microM significantly inhibited ecto-ATPase activity by 18% in the urinary bladder and by 24% in the vas deferens. 4. In the guinea-pig vas deferens, CPA significantly potentiated ATP release evoked by EFS from 2.2 +/- 0.8 (6) pmol ATP min-1 g-1 wet tissue to 35.2 +/- 4.8 (6) pmol ATP min-1 g-1 wet tissue (P < 0.01). 5. In conclusion, the potentiation of contractile responses of the guinea-pig urinary bladder and vas deferens by CPA has a non-specific character. CPA inhibited ecto-ATPase activity and increased ATP release, but these effects do not appear to contribute to the potentiation of Pu-purinoceptor-mediated responses since the contractile actions of all the agonists studied were potentiated to the same extent.

Selective antagonism by PPADS at P2X-purinoceptors in rabbit isolated blood vessels.

1. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a P2-purinoceptor antagonist, was investigated for its ability to antagonize: (1) P2X-purinoceptor-mediated contractions of the rabbit central ear artery and saphenous artery evoked by either alpha,beta-methylene ATP (alpha,beta-MeATP) or electrical field stimulation (EFS); (2) P2Y-purinoceptor-mediated relaxations of the rabbit mesenteric artery; (3) endothelium-dependent and endothelium-independent, P2Y-purinoceptor-mediated relaxations of the rabbit aorta. 2. alpha,beta-MeATP (0.1-100 microM) caused concentration-dependent contractions of the rabbit ear and saphenous arteries. The negative log[alpha,beta-MeATP] that produced a contraction equivalent to the EC25 for noradrenaline (ear artery) or histamine (saphenous artery) in the absence of PPADS was 6.60 +/- 0.18 (9) and 6.18 +/- 0.17 (9) in the ear artery and saphenous artery, respectively. These effects of exogenous alpha,beta-MeATP were concentration-dependently inhibited by PPADS (1-30 microM). In the ear artery, the negative log[alpha,beta-MeATP] producing a contractile response equivalent to the EC25 of noradrenaline, in the presence of PPADS at 1, 3 and 10 microM was 6.16 +/- 0.18 (8), 5.90 +/- 0.18 (8) and 4.72 +/- 0.36 (8), respectively (P < 0.01). In the saphenous artery, the negative log[alpha,beta-MeATP] values equivalent to the EC25 for histamine in the presence of PPADS at concentrations of 1, 3, 10 and 30 microM were 5.90 +/- 0.19 (8), 5.73 +/- 0.16 (8), 4.99 +/- 0.14 (8) and 4.51 +/- 0.13 (8), respectively (P < 0.01). 3. PPADS at a concentration of 1 microM had no effect on contractions of the ear artery evoked by EFS (4-64 Hz; 1 microM phentolamine present). At higher concentrations (3-30 MicroM) it caused concentration dependent inhibition of neurogenic contractions. In the saphenous artery, PPADS (1-30 MicroM) concentration-dependently inhibited contractions evoked by EFS at frequencies of 4, 8 and 16 Hz. Contractions evoked by EFS at frequencies of 32 and 64 Hz were significantly inhibited by PPADS only at concentrations of 10 and 30 MicroM.4. PPADS (30 MicroM) had no effect on relaxations to 2-methylthio ATP (3 nM-3 MicroM) in rabbit mesenteric artery and to ATP (1 MicroM-I mM) in rabbit aorta (with endothelium intact or removed). In addition,PPADS (30 MicroM) had no significant influence on the contractile potency of noradrenaline and histamine in rabbit ear and saphenous artery, respectively.5. In conclusion, these results support the evidence that PPADS is a selective antagonist of P2X-purinoceptor-mediated responses.

Inhibitory action of PPADS on relaxant responses to adenine nucleotides or electrical field stimulation in guinea-pig taenia coli and rat duodenum.

1. The effect of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on the relaxant response to adenine nucleotides was examined in the carbachol-contracted guinea-pig taenia coli and rat duodenum, two tissues possessing P2y-purinoceptors. In addition, in the taenia coli PPADS was investigated for its effect on relaxations evoked by adenosine, noradrenaline and electrical field stimulation. In order to assess the selectivity of PPADS between P2-purinoceptor blockade and ectonucleotidase activity, its influence on ATP degradation was studied in guinea-pig taenia coli. 2. The resulting rank order of potency for the adenine nucleotides in guinea-pig taenia coli was: 2-methylthio ATP >> ATP > alpha,beta-methylene ATP with the respective pD2-values 7.96 +/- 0.08 (n = 23), 6.27 +/- 0.12 (n = 21) and 5.88 +/- 0.04 (n = 24). 3. In guinea-pig taenia coli, PPADS (10-100 microM) caused a consistent dextral shift of the concentration-response curve (CRC) of 2-methylthio ATP and ATP resulting in a biphasic Schild plot. A substantial shift was only observed at 100 microM PPADS, the respective pA2-values at this particular concentration were 5.26 +/- 0.16 (n = 5) and 5.15 +/- 0.13 (n = 6). Lower concentrations of PPADS (3-30 microM) antagonized the relaxant effects to alpha,beta-methylene ATP in a surmountable manner. An extensive shift of the CRC was produced only by 30 microM PPADS (pA2 = 5.97 +/- 0.08, n = 6), and the Schild plot was again biphasic. 4. The relaxant responses to electrical field stimulation (80 V, 0.3 ms, 5 s, 0.5-16 Hz) in guinea-pigtaenia coli were concentration-dependently inhibited by PPADS (10-100 microM).5. In guinea-pig taenia coli, the potency of ATP in inducing relaxation appeared to be independent of its rate of degradation by ecto-nucleotidases, since the Km-value (366 microM) obtained in the enzyme assay was much higher than the functional EC50-value (0.45 microM) of ATP. PPADS (3-100 microM) was only weakly active in inhibiting ecto-nucleotidase activity leaving a residual activity of 81.8 +/- 5.1% at 100 microM.Enzyme inhibition by PPADS was concentration-independent and non-competitive.6. In rat duodenum, the rank order of potency was: 2-methylthio ATP >ATP> >alpha,beta-methylene ATP,the respective pD2-values being 6.98 +/- 0.04 (n = 76), 6.26 +/- 0.02 (n = 6) and 4.83 +/- 0.02 (n = 6). Among these agonists, 2-methylthio ATP displayed the lowest apparent efficacy.7. The CRC of 2-methylthio ATP in rat duodenum was shifted to the right by PPADS (10-100 microM) ina concentration-dependent manner, and Schild analysis gave a pA2-value of 5.09 +/- 0.06 (slope = 1.02,n=14).8 PPADS was without any effect on the carbachol-induced contraction in guinea-pig taenia coli or rat duodenum and on the relaxation to noradrenaline or adenosine in guinea-pig taenia coli.9 In conclusion, the antagonistic properties of PPADS at the taenia coli and rat duodenum P2y-purinoceptors were different from those recently described at the P2x-subtype: inhibition of P2y-purinoceptor-mediated responses was observed at higher concentrations (3-100 microM vs. 1-10 (30) microM).Furthermore, we conclude that in addition to the classical P2y-subtype, which is largely PPADS-resistant,the guinea-pig taenia coli may be endowed with a distinct relaxation-mediating P2-purinoceptor subtype which is sensitive to PPADS.

PPADS selectively antagonizes P2X-purinoceptor-mediated responses in the rabbit urinary bladder.

1. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), an inhibitor of P2X-purinoceptor-mediated responses in rabbit vas deferens, was investigated for its ability to antagonize contractions evoked by alpha,beta-methylene ATP (alpha,beta-MeATP), carbachol and electrical field stimulation in the rabbit urinary bladder detrusor muscle. 2. PPADS. (1-30 microM) caused concentration-dependent inhibition of contractions to the stable P2X-purinoceptor agonist, alpha,beta-MeATP, decreasing the maximum response to alpha,beta-MeATP (30 microM) at concentrations of 3-30 microM. The pD2 value for alpha,beta-MeATP in the absence of PPADS was 6.52 +/- 0.10 (8). In the presence of PPADS at concentrations of 1, 3, 10 and 30 microM the negative log concentrations of alpha,beta-MeATP that cause the same contractile response as the pD2 value were significantly different from control, being respectively 6.17 +/- 0.09 (8), 5.64 +/- 0.12 (7), 5.15 +/- 0.23 (7) and 4.78 +/- 0.22 (5). 3. PPADS (1-30 microM) caused concentration-dependent inhibition of contractions to stimulation of intramural purinergic nerves (1-32 Hz). There was a greater inhibition at lower frequencies (1-8 Hz) than at higher frequencies (16-32 Hz). PPADS, 30 microM, did not produce significantly greater antagonism than 10 microM. 4. PPADS (30 microM) had no significant influence on the contractile potency of carbachol: the pD2 values of carbachol in the absence and presence of PPADS were not significantly different being 6.42 +/- 0.16 (5) and 6.33 +/- 0.18 (5), respectively. However, PPADS caused a small, but significant, suppression of the maximal response of carbachol, reducing it by approximately 9%. 5. Radioligand binding studies carried out on rabbit bladder membranes with [3H]-alpha,beta-methylene ATP([3H]-alpha,beta-MeATP) showed that PPADS concentration-dependently inhibited the binding of [3H]-alpha,beta-MeATP to P2X-purinoceptors, while the binding of [3H]-quinuclidinyl benzilate to muscarinic cholinoceptors was not affected.6. Thus, PPADS (1-30 microM) antagonized responses mediated via P2X-purinoceptors in the rabbit urinary bladder. It was selective for P2-purinoceptor-mediated contractions rather than those mediated via muscarinic receptors. Binding studies demonstrated that the antagonistic effect of PPADS is via a direct interaction with P2x-purinoceptors.

Heme oxygenase-2 and nitric oxide synthase in guinea-pig intracardiac neurones.

There is increasing evidence that carbon monoxide (CO), like nitric oxide (NO), may be a neuronal messenger molecule. This study investigated the expression of heme oxygenase-2 (HO-2), the enzyme responsible for the synthesis of CO, by intracardiac neurones. Many, if not all newborn guinea-pig intracardiac neurones in culture were HO-2-immunoreactive. Furthermore, double labelling showed that a relatively small subpopulation of these neurones also expressed NO synthase/nicotinamide dinucleotide phosphate (NADPH)-diaphorase (NOS/NADPH-d) activity. These findings suggest that intracardiac neurones can synthesize CO and that CO may be fundamental to their function. Comparison of the proportions of intracardiac neurones that contain HO-2 with those that express NOS/NADPH-d activity also indicates that CO may be more important than NO in the intrinsic neuronal control of the heart.

Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurones.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.

Neuropeptide families: evolutionary perspectives.

Examination of neuropeptide families can provide information about phyletic relationships and evolutionary processes. In this article the oxytocin/vasopressin family, growth hormone releasing factor (GRF) superfamily and the substance P/tachykinin family have been considered in detail because they have been isolated from an extraordinarily diverse array of species from several vertebrate classes and invertebrate phyla. More important is that the nucleotide sequence of mRNA or cDNA encoding many of these peptides has been determined, which has allowed evolutionary distances to be estimated based on the DNA mutation rate. The origin of a given family lies in a primordial gene that arose many millions of years ago, and through time, exon duplication and insertion, gene duplication, point mutation and exon loss, the family developed into the forms that are now recognised. For example, in birds, GRF and pituitary adenylate cyclase activating peptide (PACAP) are encoded by the same gene, which probably arose as a result of exon duplication and tandem insertion of the ancestral GRF gene. In mammals GRF is the sole product on one gene, and PACAP is the product of a gene that also produces PACAP-related peptide (PRP), which is homologous to GRF. Thus it appears that between birds and mammals the GRF/PACAP gene duplicated: exon loss gave rise to the mammalian GRF gene, while mutation led to the formation of the mammalian PRP/PACAP gene. The neuropeptide Y superfamily is considered briefly, as is cionin, which is an invertebrate peptide that is closely related to the mammalian gastrin/cholecystokinin family.

Analysis of pancreatic polypeptide cDNA from the house musk shrew, Suncus murinus, suggests a phylogenetically closer relationship with humans than for other small laboratory animal species.

Pancreatic polypeptide was isolated and sequenced from endocrine cells of the pancreas from an insectivore, the house musk shrew, Suncus murinus. The primary sequence was APLEPAYPGD(10)NATPEQMAQY(20)AAELRKYINM(30)VTRPRYamide. This is the first polypeptide hormone to be characterised from this species and is typical of the primary sequences of pancreatic polypeptide of other animals, being a C-terminal-amidated peptide with 36 residues. Comparison with several vertebrate sequences shows that it has more in common with the human form than do the forms from common laboratory animals such as rabbits, rats, mice and guinea-pigs.

Neuropeptide families and their receptors: evolutionary perspectives.

Examination of families of neuropeptides and their receptors can provide information about phyletic relationships and evolutionary processes. Within an individual a given signal molecule may serve many diverse functions, mediated via subtypes of the receptor which may be coupled to their transduction mechanisms in different ways. The rate of evolution of a peptide may reflect or be reflected in the rate of evolution of its receptor. For example, in the neuropeptide Y (NPY) family, pancreatic polypeptide (PP) shows significant structural diversity, while NPY is highly conserved. Molecular forms of a given subtype of NPY receptor that is selectively activated by NPY (Y1 or Y2 or Y5) are also highly conserved, but the subtype that is primarily activated by PP (Y4), shows remarkable diversity. Also, between receptor subtypes there can be remarkable diversity. This is evident in several neuropeptide families, where a neuropeptide sequence is highly conserved across a wide range of species but where the receptor homology of subtypes with species tends to be much lower than homology between species. For example, human and rat vasopressin are identical, but the human V(1)- or V(2)-vasopressin receptors are approximately 80% homologous with rat V(1)- or V(2)-receptors, but within humans or rats the V(1)-receptor is less than 50% homologous with the V(2)-receptor. Furthermore, duplication of an ancestral gene is thought to have led to the co-presence in eutherian mammals of oxytocin and vasopressin, which have maintained a close structural similarity, yet in many species the oxytocin receptor is only 30 to 50% homologous with vasopressin receptors. Thus it appears that there has been greater evolutionary pressure to conserve the signal molecule, than to conserve the structure of the receptor. Evaluation of the evolution of neuropeptides and their receptors may be useful in determining phyletic relationships. Traditional classification places the guinea pig as a hystricomorph rodent within the same order (Rodentia) as the muriform or myomorph rat and mouse. However, molecular analyses of polypeptides have led to the suggestion that guinea pigs belong to a distinct order. Analysis of several neuropeptide sequences and the Y4 receptor supports this view. In general terms for both neuropeptides and receptors, sequence homology reflects phylogeny and taxonomy as based on morphological features. Within the oxytocin/vasopressin family in which peptides and receptors have been characterised in invertebrate representatives as well as fish and amphibia in addition to mammals, the molecular diversity correlates well with evolutionary diversity.

Evidence that ATP is a neurotransmitter in the frog heart.

In the presence of atropine, electrical field stimulation of frog atria produced a positive inotropic response. This response had two components. The initial phase was blocked by selective desensitization of P2-purinoceptors with alpha, beta-methylene ATP. The second phase, which was not always clearly separated from the initial phase, was blocked by the beta-adrenoceptor antagonist propranolol. The present results suggest that ATP may be a cotransmitter released with adrenaline from nerve terminals of the vagosympathetic trunk supplying the frog atria.

Isolated human bladder: evidence for an adenine dinucleotide acting on P2X-purinoceptors and for purinergic transmission.

In isolated strips of human urinary bladder detrusor muscle, ATP, alpha, beta-methylene ATP and P1,P6-diadenosine hexaphosphate caused concentration-dependent contractions. ATP was less potent than the two synthetic purine compounds and gave smaller maximum responses. Responses to ATP, P1,P6-diadenosine hexaphosphate and noncholinergic nerve stimulation were blocked following desensitization of P2X-purinoceptors by alpha,beta-methylene ATP. Thus, adenine dinucleotides can act on P2X-purinoceptors and there is an element of purinergic neuromuscular transmission in the human urinary bladder.