Nucleotide-evoked relaxation of rat vas deferens: possible mechanisms.

ATP causes relaxation of the K(+)-contracted rat vas deferens. Possible sites of action were investigated. ATP and adenosine relaxed the vas deferens precontracted with 80 mM K(+); EC(50) values and maximal relaxations averaged, respectively, 760 microM and 56% for ATP and 74 microM and 30% for adenosine. The adenosine P1 receptor antagonist 8-(para-sulfophenyl)theophylline (8-SPT) reduced relaxations caused by adenosine and low concentrations of ATP, as did the Rp-diastereomer of adenosine 3',5'-cyclic phosphorothioate (Rp-cAMPS), an inhibitor of protein kinase A. The phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) augmented responses to adenosine and low concentrations of ATP. alpha,beta-Methylene ADP, an inhibitor of 5'-nucleotidase, reduced relaxations caused by ATP to a similar extent as did 8-SPT. In the presence of an almost saturating concentration of adenosine, ATP caused further relaxation. Conversely, in the presence of ATP, adenosine had little effect. Like ATP, UTP and other nucleoside triphosphates relaxed the vas deferens. The P2 receptor antagonists reactive blue 2, acid blue 25 and 4,4'-diisothiocyanotostilbene-2,2'-disulphonate (DIDS) attenuated the relaxation caused by ATP; suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), Evans blue, trypan blue, reactive red 2 and brilliant blue G had no effect. Three non-selective inhibitors of protein kinases, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), staurosporine and (8R*,9S*,11S*)-(-)-9-hydroxy-9-carboxy-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (K-252b), markedly reduced the relaxation caused by ATP. The results indicate that adenosine, derived from enzymatic dephosphorylation, contributes to the relaxant effect of ATP, presumably by activation of a smooth muscle adenosine receptor linked to the accumulation of cAMP and activation of protein kinase A. Yet, the main part of the response to ATP is mediated by a site distinct from the adenosine receptor. The pharmacological properties of this site differ from known P2 receptor subtypes. Possibly, the nucleotide-evoked relaxation is due to a phosphoryl transfer catalyzed by an ecto-protein kinase.

Nucleotide-evoked relaxation of rat vas deferens--a possible role for endogenous ATP released upon alpha(1)-adrenoceptor stimulation.

The possibility was tested that endogenous ATP released upon alpha(1)-adrenoceptor activation causes relaxation of the rat vas deferens smooth muscle. ATP, 2-methylthio ATP and adenosine relaxed the vas deferens precontracted with 80 mM K(+). The metabolically stable P2 receptor agonists alpha,beta-methylene ATP (alpha,beta-MeATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) had little or no effect. The adenosine P1 receptor antagonist 8-(para-sulfophenyl)theophylline did not significantly affect the response to ATP. The P2 receptor antagonist reactive blue 2 markedly reduced the relaxation (by up to 73%); suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and acid blue 129 caused no change. ATP, but not alpha,beta-MeATP, also attenuated contractions elicited by noradrenaline at resting tension; reactive blue 2 blocked the inhibitory effect of ATP. Reactive blue 2, by itself, enhanced the response to noradrenaline (by up to 36%); suramin, PPADS and acid blue 129 caused no change. In the presence of the ATP-degrading enzymes apyrase and nucleotide pyrophosphatase, the facilitatory effect of reactive blue 2 was lost. Apyrase, by itself, enhanced the response to noradrenaline (by 13%). The results indicate that endogenous ATP, released from rat vas deferens smooth muscle upon alpha(1)-adrenoceptor stimulation, causes relaxation. The site of action of ATP is not a typical smooth muscle P2Y receptor.

P2 receptor-mediated inhibition of dopamine release in rat neostriatum.

Axon terminal nucleotide P2 receptors mediating an inhibition of transmitter release have, so far, been detected in various sympathetically innervated tissues,(8,27) and on central noradrenergic,(14,26) glutamatergic(15) and serotonergic neurons. (28) We have now investigated the effect of ATP and related nucleotides on the release of endogenous dopamine from slices of rat neostriatum using fast cyclic voltammetry. Mutual interactions between the two neurotransmitters have been observed previously: ATP and related nucleotides induce a release of dopamine in PC12 pheochromocytoma cells, a frequently used model for sympathetic neurons;(10,22) they also increase the dopamine concentration in rat brain measured by in vivo microdialysis(16,32) and stimulate the uptake of dopamine by rat striatal synaptosomes.(3) Dopamine, in contrast, facilitates activation of ligand-gated cation channels (i. e. P2X(2) receptors) by ATP.(11,20) Here, we show that ATP and two of its analogues decrease the electrically evoked release of endogenous dopamine in rat neostriatum. The inhibitory effect of ATP is blocked by the P2 receptor antagonists suramin, reactive blue 2 and cibacron blue 3GA. Suramin, in addition, partly prevents the attenuation of dopamine release evoked by a single stimulus that follows a brief train of high-frequency pulses.These findings suggest the existence of release-inhibiting P2 receptors on dopaminergic nerve terminals and indicate that dopaminergic transmission in rat neostriatum might be modulated by an endogenous P2 receptor ligand, presumably ATP.

A contraction-mediating receptor for UTP, presumably P2Y2, in rat vas deferens.

The possible existence of a contraction-mediating P2-receptor for uracil nucleotides was investigated in the rat vas deferens. In order to minimize breakdown of nucleotides, Evans blue was used as an inhibitor of ectonucleotidases. UTP was degraded by rat vas deferens tissue, and the degradation was inhibited by Evans blue (100 microM). In the absence of other drugs, UTP and UDP elicited marginal contractions. Evans blue (100 microM) greatly enhanced contractions elicited by the uracil nucleotides. When the medium contained alpha,beta-MeATP (100 microM) in addition to Evans blue in order to desensitize contraction-mediating P2X1-receptors, responses to UTP and UDP were not changed; in contrast, responses to alpha,beta-MeATP were virtually abolished and contractions elicited by ATP and ADP were greatly reduced; EC50 values were 122 microM for UTP and 58 microM for ATP under these conditions. The P2-receptor antagonist suramin attenuated contractions elicited by UTP (320 microM) and alpha,beta-MeATP (32 microM) in the presence of Evans blue; pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS) also reduced responses to alpha,beta-MeATP but, at up to 100 microM, did not alter contractions elicited by UTP. Incubation of vasa deferentia in nominally calcium-free medium almost abolished the response to alpha,beta-MeATP (32 microM), while a major part of the contraction elicited by UTP (320 microM) was preserved. In the presence of Evans blue and alpha,beta-MeATP, prior addition of UDP (3200 microM) or ATP (320 microM), without washout, markedly reduced the response to UTP (320 microM); UTP and ATP also reduced the response to UDP; in contrast, prior addition of UTP or UDP did not alter the contraction to ATP. The results demonstrate the existence of a contraction-mediating, uracil nucleotide-sensitive P2Y-receptor in rat vas deferens, distinct from the P2X1-receptor. Pharmacological analysis indicates that it is P2Y2.

Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens.

In order to assess the consequences of a concomitant blockade of P2X-receptors and ecto-nucleotidases, effects of 13 P2-receptor antagonists were investigated on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP) and ATP and on the removal of ATP from the incubation medium by vas deferens tissue. Increasing concentrations of all antagonists reduced and finally abolished contractions elicited by alpha,beta-MeATP (3 microM), with IC50-values ranging from 1.1 to 100 microM. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), 6-azophenyl-4-amino-5-hydroxy-naphthalene-1,3-disulphonate (NH02), 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A also progressively reduced and finally abolished contractions elicited by ATP (1 mM). 8,8'-[Carbonylbis(imino-3, 1-phenylenecarbonyl-imino)]-bis-(1,3,5-naphthalenetrisulphonate ) (NF023), suramin, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), trypan blue and reactive blue 19, in contrast, caused only partial blockade, by 34-43% maximally; reactive blue 2 and reactive red 2 had no effect; and 6,6'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-4-amino-5-hydroxy-naphtha lene-1,3-disulphonate (NH01) and Evans blue even enhanced the response to ATP. For antagonists causing full or partial inhibition, the IC50-values against ATP were close to those against alpha,beta-MeATP. All antagonists attenuated the removal of ATP, with IC25%-values ranging from 0.8 microM to >320 microM. The results confirm the frequent combination, in one antagonist molecule, of P2-receptor blockade and blockade of ecto-nucleotidases. This dual action underlies the effect of such compounds on contractions of the vas deferens elicited by ATP which, for certain substances (e.g., suramin, reactive blue 2), can be explained by a simple model in which the antagonist simultaneously blocks the degradation of ATP and a single contraction-mediating receptor (P2X1). Several observations, however, do not conform with this model, and the existence of multiple contraction-mediating receptors for ATP or multiple, pharmacologically distinct ecto-nucleotidases has to be considered.

Nucleotide-evoked relaxation of human coronary artery.

Endothelium-dependent dilation of coronary blood vessels in response to ATP and related nucleotides has been demonstrated in various animal species. The aim of the present study was to investigate a possible relaxant effect of ATP, the adenine nucleotides 2-methylthio ATP (MeSATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS), and the pyrimidine nucleotide UTP in isolated human coronary artery. In endothelium-intact rings of human coronary artery precontracted with K+ (20-40 mM), the nucleotides caused relaxation. Average maximal percentage relaxations and average EC50 values (concentrations causing half-maximal relaxation) were 89% and 47.1 microM for ATP, 28% and 0.3 microM for MeSATP, 35% and 0.6 microM for ADPbetaS, and 49% and 1.6 microM for UTP. For each of the four agonists, the potency to elicit relaxation varied greatly between individual rings, so that equi-relaxing concentrations spanned several orders of magnitude. Moreover, the sensitivities to ATP and UTP, when tested in the same ring, were not correlated. Mechanical removal of the endothelium as well as NG-nitro-L-arginine methyl ester (L-NAME; 30 microM), an inhibitor of nitric oxide synthase, abolished the relaxation caused by MeSATP, ADPbetaS and UTP and greatly attenuated the response to lower concentrations of ATP (3.2-320 microM), but high concentrations of ATP (320 and 1000 microM) caused relaxation also in endothelium-denuded preparations and in the presence of L-NAME. High concentrations of ADPbetaS (32 and 100 microM) and UTP (320 and 1000 microM) caused contraction of endothelium-denuded preparations. Thus, extracellular nucleotides cause endothelium-dependent, primarily nitric oxide-mediated relaxation of human coronary artery. ATP in addition causes endothelium-independent relaxation. The receptors activated by the nucleotides appear to be unevenly distributed on the coronary endothelium.

On the suitability of adenosine 3'-phosphate 5'-phosphosulphate as a selective P2Y receptor antagonist in intact tissues.

Agonist and antagonist effects of the putative P2Y1 receptor antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC50 3.3 microM). The response was attenuated by the P2 receptor antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and reactive red 2 with apparent Kd values (0.27 and 0.29 microM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of alpha,beta-methylene ATP (alpha,beta-MeATP) in the taenia coli. Incubation with PAPS (10-100 microM) attenuated the P2Y receptor-mediated relaxation caused by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS); PAPS (100 microM) also attenuated the relaxation caused by alpha,beta-MeATP, as well as the alpha1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC50 24.7 microM), which was reduced by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; 1 microM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 microM) attenuated the P2Y receptor-mediated relaxation caused by ADPbetaS; PAPS (100 microM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 microM) attenuated the P2X receptor-mediated contraction elicited by alpha,beta-MeATP but did not alter the alpha1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

On the suitability of adenosine 3'-phosphate 5'-phosphosulphate as a selective P2Y receptor antagonist in intact tissues.

Agonist and antagonist effects of the putative P2Y1 receptor antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC50 3.3 microM). The response was attenuated by the P2 receptor antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and reactive red 2 with apparent Kd values (0.27 and 0.29 microM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of alpha,beta-methylene ATP (alpha,beta-MeATP) in the taenia coli. Incubation with PAPS (10-100 microM) attenuated the P2Y receptor-mediated relaxation caused by 5'-O-(2-thiodiphosphate) (ADPbetaS); PAPS (100 microM) also attenuated the relaxation caused by alpha,beta-MeATP, as well as the alpha1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC50 24.7 microM), which was reduced by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; 1 microM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 microM) attenuated the P2Y receptor-mediated relaxation caused by ADPbetaS; PAPS (100 microM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 microM) attenuated the P2X receptor-mediated contraction elicited by alpha,beta-MeATP but did not alter the alpha1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

Depression of C fiber-evoked activity by intrathecally administered reactive red 2 in rat thalamic neurons.

To investigate the possible role of spinal purinoceptors in nociception, the potent P2-purinoceptor antagonist reactive red 2 was studied in rats under urethane anesthesia in which nociceptive activity was elicited by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intrathecal (i.t.) application of reactive red 2 (6-200 micrograms) caused a dose-dependent reduction of the evoked activity in thalamic neurons. The estimated ED50 was 30 micrograms, and the maximum depression of nociceptive activity amounted to about 70% of the control activity at a dose of 100 micrograms. Morphine, administered i.t. at a maximally effective dose (80 micrograms), inhibited the evoked nociceptive activity by only up to 55% of the control activity. An i.t. co-injection of reactive red 2 (100 micrograms) and morphine (80 micrograms) caused a maximum reduction of the evoked thalamic activity by up to 85% of the control activity, thus, exceeding significantly the effect elicited by either drug alone. Similarly, i.t. co-injection of almost equipotent dosages of reactive red 2 (30 micrograms) and morphine (30 micrograms) caused a maximum reduction of the evoked activity by up to 72% of the control activity, which again exceeded significantly the effect of either drug alone. The results suggest that in rats reactive red 2 exerts antinociception by blockade of P2-purinoceptors in the spinal cord and, hence, support the idea that ATP may play an important role in spinal transmission of nociceptive signals. An activation of the spinal opioid system does not seem to contribute to the effect of reactive red 2 but might act additive or even synergistically with its antinociceptive action.

P2-receptor antagonists: IV. Blockade of P2-receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2.

Effects of reactive blue 2 and twelve structurally related compounds were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP; mediated by P2X-receptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y-receptors), and the degradation of ATP by rat vas deferens tissue. All compounds, except acid blue 41 and acid blue 129 (at up to 100 microM), shifted the concentration-response curve of alpha,beta-MeATP in the rat vas deferens to the right. Most increased, but uniblue A greatly decreased, the maximum of the curve. In the case of cibacron blue 3GA and reactive blue 19, of which three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from unity. The apparent Kd values of the effective substances ranged between 0.7 and 111 microM. Most compounds increased the contraction of the rat vas deferens elicited by high K+. In the guinea-pig taenia coli, all compounds, except uniblue A and reactive blue 19 (at up to 100 microM), shifted the concentration-response curve of ADPbetaS to the right in a parallel manner. In the case of acid blue 129 and acid blue 80, of which three concentrations were tested, the slope of the Arunlakshana-Schild regression did not differ from unity. The apparent Kd values of the effective substances were between 0.7 and 69 microM. Most compounds also reduced the relaxation of the guinea-pig taenia coli elicited by noradrenaline. The removal of ATP from the medium by vas deferens tissue was decreased only by reactive blue 2, cibacron blue 3GA, uniblue A and reactive blue 19, with IC25% values between 17 and 62 microM. The structure-activity relationships for P2X- and P2Y-receptor blockade in this series are strikingly dissimilar. In reactive blue 2 and its isomers, for example, both the 1-amino-anthraquinone-2-sulphonate core and the 'side-chain' of the molecule are involved in P2X-receptor binding; P2Y-receptor affinity, in contrast, resides largely or totally in the anthraquinone core. The most promising antagonists are uniblue A which is P2X- versus P2Y-selective and acid blue 129 which is P2Y- versus P2X-selective, both with few, if any, non-P2-receptor effects at concentrations blocking the respective P2-subtype.

Characterization by antagonists of P2-receptors mediating endothelium-dependent relaxation in the rat aorta.

The receptors through which 2-methylthio ATP (MeSATP), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), UTP and ATP elicit endothelium-dependent relaxation of noradrenaline-precontracted rings of the rat aorta were characterized by means of a series of antagonists. The acetylcholine-induced relaxation and the degradation of MeSATP, UTP and ATP were also studied. The potency of the nucleotides at producing relaxation decreased in the order MeSATP (EC50 0.24 microM) > ADP beta S (0.43 microM) > UTP (1.09 microM) > ATP (3.53 microM). MeSATP, ADP beta S and UTP did not cause relaxation when the endothelium had been destroyed; high concentrations of ATP still caused some relaxation. The relaxation by MeSATP, ADP beta S and UTP became very small after treatment of the rings with NG-nitro-L-arginine methyl ester; the relaxation by ATP was less affected. Pre-exposure to MeSATP (100 microM) abolished or almost abolished the relaxation normally elicited by MeSATP and ADP beta S, did not change that elicited by UTP and slightly enhanced the relaxation elicited by ATP. Of nine compounds examined as antagonists, six attenuated selectively the effect of some or all of the nucleotides (as compared to acetylcholine): suramin, reactive blue 2, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), reactive red 2 and 5,5'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-7-amino -6-hydroxy-naphthalene-1,4-disulphonate (NH05). Decreases of maximal relaxations and slopes different from unity in Schild plots often indicated non-competitive kinetics of the antagonism. For each of the six 'selective' antagonist, the apparent Kd values against MeSATP and against ADP beta S were similar: none of the six differentiated between MeSATP and ADP beta S. Also, for each of four 'selective' antagonists, the apparent Kd values against UTP and against ATP were similar: none of the four differentiated between these two nucleotides (two antagonists did not act against UTP and ATP in the 'selective' concentration range). On the other hand, for five of the six 'selective' antagonists (the exception being NH05), the apparent Kd values against MeSATP and ADP beta S were considerably lower than those against UTP and ATP. At the highest concentrations tested against agonist-evoked relaxations, the antagonists did not alter the removal from the incubation medium, by pieces of rat aorta, of MeSATP, UTP and ATP. It is concluded that nucleotides cause endothelium-dependent relaxation of the rat aorta through two sites: a P2Y-receptor and a P2U-receptor. The receptors may be pharmacologically similar to a bovine endothelial P2Y (P2Y1) and a cloned rat P2U (P2Y2) receptor, respectively. ATP acts mainly through the P2U-receptor. Suramin, reactive blue 2, iso-PPADS, PPADS and reactive red 2 are more potent at the P2Y- than the P2U-receptor. NH05 does not discriminate between the two receptors but is the most potent P2U antagonist so far described.

Vasoconstrictor and vasodilator effects of guanine nucleotides in the rat aorta.

Although GTP, like ATP and UTP, is stored in platelet dense granules, little is known about its vascular effects. The present study was carried out in order to characterize the effects of GTP and related compounds in the rat aorta. Contractions were examined in aortic rings at resting tension. In rings with intact endothelium, GTP, GDP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) caused small contractions. In endothelium-denuded rings, the contractions were unchanged or increased and persisted after desensitization of P2X-receptors by alpha,beta-methylene ATP. Relaxations were examined in aortic rings precontracted with noradrenaline. In rings with intact endothelium, GTP (EC50 131 microM), GDP (no maximal effect obtained), GTP gamma S (EC50 6.8 microM) and guanosine (EC50 822 microM) caused prominent relaxation, whereas GDP beta S caused further contraction. In endothelium-denuded rings, the relaxant effect of GTP was greatly reduced, that of GDP and guanosine was unchanged, and that of GTP gamma S was abolished. Relaxations by GTP and GTP gamma S in endothelium-intact rings were studied in more detail. The relaxation by GTP was slightly and the relaxation by GTP gamma S greatly reduced after treatment with NG-nitro-L-arginine methyl ester. Pre-exposure to a high concentration of the P2Y-receptor agonist 2-methylthio ATP (MeSATP) did not attenuate the effects of GTP and GTP gamma S. Four compounds previously identified as antagonists at the P2Y- and P2U-receptors of rat aortic endothelium--suramin, reactive blue 2, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS) and 5,5'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-7-amino-6- hydroxynaphthalene-1,4-disulphonate (NH05)--were tested against GTP and GTP gamma S. Suramin, reactive blue 2 and iso-PPADS were much less potent against GTP and GTP gamma S than previously found against (the P2Y effect of) MeSATP. Suramin, iso-PPADS and NH05 were about as potent against GTP and GTP gamma S as previously found against (the P2U effect of) UTP and in particular ATP. It is concluded that guanine nucleotides can cause both contraction and relaxation of the rat aorta. The high concentrations of GTP and GDP required, and in the case of contraction the small size of the response, make a physiological role of the vascular effects of these nucleotides unlikely. GTP and GTP gamma S elicit endothelium-dependent relaxation through P2U-receptors. GTP in addition relaxes the aorta through smooth muscle receptors, possibly by way of its degradation product guanosine. The stable analog GTP gamma S is a relatively potent and selective agonist for the endothelial P2U-receptor.

P2-purinoceptor antagonists: I. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by small aromatic isothiocyanato-sulphonates.

Effects of eight small aromatic isothiocyanato-sulphonates, of the aliphatic 2-isothiocyanatoethene-1-sulphonate (IES), and of the parent amines were studied on contractions of the rat vas deferens elicited by alpha, beta-methylene ATP (alpha, beta-MeATP; mediated by P2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S; mediated by P2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. The aromatic isothiocyanato-sulphonates all reduced contractions of the rat vas deferens elicited by alpha, beta-methylene ATP. The antagonism was non-competitive, with depression of the maximum of the concentration-response curve of alpha, beta-MeATP and incomplete reversibility. The IC50 values were between 11 and 54 microM. In the guinea pig taenia coli, the aromatic compounds shifted the concentration-response curve of ADP beta S to the right in a surmountable manner (one exception), and where three concentrations were tested, the Arunlakshana-Schild regression was linear and its slope did not differ from 1. The apparent Kd values were between 10 and 214 microM. The removal of ATP from the medium by vas deferens tissue was decreased by the aromatic isothiocyanates with IC25% values between 25 and 464 microM. IES and the parent amines were inactive or almost inactive (parent amines not tested on ATP breakdown). The results indicate that the isothiocyanato residue as well as the aromatic core are essential for P2-purinoceptor blockade. At the P2X-purinoceptor, potency increases with the size of the molecules but is independent of the position of the isothiocyanato and sulphonate substituents. No simple structure-activity relationship for the P2Y-purinoceptor and the ATP-degrading ecto-nucleotidases can be derived beyond the apparent lack of a major influence of the position of the substituents. 2-Isothiocyanatonaphthalene-1-sulphonate (beta-INS) seems to be interesting because of relatively high P2X-selectivity versus both the P2Y-purinoceptor and ecto-nucleotidases.

P2-purinoceptor antagonists: II. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to Evans blue and trypan blue.

Effects of Evans blue and four derivatives as well as of trypan blue and four derivatives, mostly smaller fragments but two compounds with an additional ethylene bridge in the center of the molecule, were studied on contractions of the rat vas deferens elicited by alpha, beta-methylene ATP (alpha, beta-MeATP; mediated by P2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S; mediated by P2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. All compounds shifted the concentration-response curve of alpha, beta-MeATP in the rat vas deferens to the right, and most compounds increased the maximum of the curve. Each member of the Evans blue series was similar in potency to the corresponding member of the trypan blue series. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent Kd values were between 0.8 and 385 microM. In the guinea-pig taenia coli, only the members of the trypan blue group were relatively potent, shifting the concentration-response curve of ADP beta S to the right in a surmountable manner. In 2 of 3 cases where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent Kd values in the trypan blue group were between 5.2 and 324 microM. The removal of ATP from the medium by vas deferens tissue was decreased mainly by the members of the Evans blue group, with IC25% values between 13 and 158 (in 1 case > 1000) microM. The results indicate that the position of the sulphonate residues at the terminal naphthalene rings of these compounds hardly influences P2X purinoceptor affinity but greatly influences P2Y affinity and ecto-nucleotidase blockade. Among active compounds, apparent purinoceptor affinity and ecto-nucleotidase blockade increase with the size of the molecules up to Evans blue and trypan blue themselves; introduction of a central ethylene bridge does not result in a further gain in potency. NH01, the desmethyl derivative of Evans blue, seems to be interesting because it is the compound with the highest P2X- versus P2Y-selectivity presently available.

P2-purinoceptor antagonists: III. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to suramin.

Effects of suramin and five analogs or fragments of suramin were studied on contractions of the rat vas deferens elicited by alpha, beta-methylene ATP (alpha, beta-MeATP; mediated by P2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S; mediated by P2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. One compound, NF023, differed from suramin by removal of two p-methylbenzamido groups, whereas another, BSt101, differed from NF023 by additional removal of the three sulphonate residues from one of the terminal naphthalene rings. The compounds all shifted the concentration-response curve of alpha, beta-MeATP in the rat vas deferens to the right and simultaneously increased the maximum of the curve. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent Kd values were between 1 and 3672 microM. In the guinea-pig taenia coli, the compounds shifted the concentration-response curve of ADP beta S to the right in a parallel manner, but in the one case where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent Kd values were between 10 and 786 microM. The removal of ATP from the medium by vas deferens tissue was decreased only by suramin, NF023 and BSt101, with IC25% values between 170 and 590 microM.

Opposite modulation of noradrenaline and ATP release in guinea-pig vas deferens through prejunctional beta-adrenoceptors: evidence for the beta 2 subtype.

Activation of prejunctional beta-adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of beta-adrenoceptor involved. In [3H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the beta 2-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 microM). Isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin- and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained concentration elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the beta 1-selective antagonist 1-[2-((3-carbamoyl- 4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM). The results confirm the opposite beta-adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional beta-adrenoceptor is of the beta 2-subtype.

Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli.

The guinea-pig taenia coli possesses two relaxation-mediating receptors for nucleotides: a prototypic P2Y-purinoceptor, which is activated by adenosine 5'-O-(2-thio-diphosphate) (ADP beta S), and a separate receptor for alpha, beta-methylene ATP (alpha,beta-MeATP). Effects of several as yet incompletely characterized P2-purinoceptor antagonists at these receptors were examined. The concentration-relaxation curve of ADP beta S was shifted to the right by reactive blue 2, suramin, 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulp honic acid (XAMR0721; at 1000 microM only), pyridoxalphosphate-6-azophenyl-2',5'-disulphonic acid (iso-PPADS), pyridoxal 5-phosphate, trypan blue and Evans blue (at 320 microM only). Schild plots for the antagonism of reactive blue 2, suramin, iso-PPADS and pyridoxal 5-phosphate against ADP beta S had slopes < 1. The concentration-relaxation curve of alpha,beta-MeATP was shifted to the right by reactive blue 2, suramin, XAMR0721, iso-PPADS, pyridoxal 5-phosphate and trypan blue but not by Evans blue (320 microM). Schild plots for the antagonism of suramin, XAMR0721 and iso-PPADS against alpha,beta-MeATP had slopes > 1. Only XAMR0721 differed clearly in potency against the two nucleotides: it was considerably more potent against alpha,beta-MeATP than against ADP beta S. 2-Methylthio ATP (MeSATP; 1 microM) and ATP (100 microM) were degraded by pieces of taenia coli. All antagonists except trypan blue attenuated the degradation of either or one of the two nucleotides. The selective effect of XAMR0721 against alpha,beta-MeATP confirms the existence of two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli. Comparison of the apparent affinities of the antagonists for the two taenia coli receptors with affinities for the P2X-purinoceptor of the rat vas deferens shows that reactive blue 2, suramin, iso-PPADS, pyridoxal 5-phosphate and trypan blue have little selectivity for any of the three receptors. XAMR0721, which has been shown to possess relatively high affinity for the P2Y-purinoceptor in turkey erythrocytes, was very weak at the P2Y-receptor of the taenia, thus supporting the existence of pharmacologic P2Y-receptor subtypes. Evans blue, with little effect in the taenia coli but a marked effect in the rat vas defrens, is the most selective P2X- (versus P2Y-) purinoceptor antagonists presently known, although its effect on the degradation of nucleotides must be kept in mind.

ATP release and its prejunctional modulation.

We studied some properties of the release of noradrenaline and ATP in isolated sympathetically innervated tissues. Release was elicited by electric stimulation and assessed as overflow of tritiated compounds (after labelling with [3H]noradrenaline) and enzymically measured ATP, respectively. Evans blue, which inhibits ectonucleotidases, greatly increased the evoked overflow of ATP, indicating that a major part of the ATP was metabolized after release. Much of the ATP was postjunctional in origin. The neural fraction was isolated when postjunctional release was suppressed by prazosin (alpha 1-adrenoceptor antagonist) and suramin (P2 purinoceptor antagonist). Comparison of neural ATP and [3H]-noradrenaline release showed that prostaglandin E2 reduced the release of both co-transmitters to a similar extent. Activation of prejunctional alpha 2-adrenoceptors, however, preferentially reduced the release of [3H]noradrenaline, and activation of prejunctional A1 purinoceptors reduced preferentially the release of ATP. Nucleotides such as ATP depressed the release of [3H]noradrenaline through two receptors: the well-known prejunctional A1 receptors and a separate group of prejunctional P2 purinoceptors. P2 antagonists increased the release of [3H]-noradrenaline. Overall, the results indicate differential storage, release and modulation of release of the two sympathetic co-transmitters. They also indicate that postganglionic sympathetic axons possess receptors for both co-transmitters: alpha 2 and P2 autoreceptors.

Opposite modulation of cotransmitter release in guinea-pig vas deferens: increase of noradrenaline and decrease of ATP release by activation of prejunctional beta-adrenoceptors.

Effects of isoprenaline on contraction, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) as well as on contractions elicited by exogenous noradrenaline and ATP were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In [3H]-noradrenaline-pretreated tissues, electrical stimulation elicited an overflow of tritium and ATP and a biphasic contraction. Isoprenaline (1-100 nM) reduced the contraction, mainly phase I, and enhanced the evoked overflow of tritium: evoked overflow of ATP was not changed significantly. No, or almost no, contraction remained in [3H]-noradrenaline-pretreated tissues exposed to both prazosin (0.3 microM) and suramin (300 microM), and the evoked overflow of ATP was reduced by about 82%. Under these conditions, isoprenaline (1-100 nM) again enhanced the evoked overflow of tritium, but it now decreased the evoked overflow of ATP. Propranolol (1 microM), when added on top of prazosin and suramin, prevented the effects of isoprenaline (1-100 nM). In some tissues not pretreated with [3H]-noradrenaline, purinergic and adrenergic components of the neurogenic contraction (again to 210 pulses, 7 Hz) were isolated by exposure to prazosin (0.3 microM) and suramin (300 microM), respectively. Isoprenaline (1-100 nM) decreased the isolated purinergic component but did not change significantly the isolated adrenergic component. Contractions elicited by ATP (1000 microM) were not changed and contractions elicited by noradrenaline (100 microM) were slightly increased by isoprenaline (1-100 nM). Isoprenaline (100 nM) did not change the degradation of ATP (100 microM) by pieces of the vas deferens. It is concluded that, in the guinea-pig vas deferens, activation of prejunctional beta-adrenoceptors modulates the neural release of noradrenaline and ATP in opposite directions: release of noradrenaline is enhanced, whereas release of ATP is decreased.

Reactive red 2: a P2y-selective purinoceptor antagonist and an inhibitor of ecto-nucleotidase.

Effects of reactive red 2 and its parent compound acid red 33 were studied in rat vas deferens and guinea-pig taenia coli. In rat vas deferens, reactive red 2(1 to 10 microM) shifted the concentration-response curve for the P2X-purinoceptor-mediated contraction effect of alpha, beta-methylene ATP slightly to the right and progressively decreased the maximum (apparent antagonist Kd value 0.42 microM). Acid red 33 (1000 microM) shifted the curve to the right without changing the maximum (apparent Kd 386 microM). The concentration-contraction curve of noradrenaline was not altered by reactive red 2. In the carbachol-precontracted guinea-pig taenia coli, reactive red 2 (0.1 to 10 microM) shifted the concentration-response curve for the P2Y-purinoceptor-mediated relaxation effect of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) progressively to the right; only at the highest concentration of antagonist (10 microM) was the maximum slightly depressed; a pA2 value of 7.55 (Kd 0.028 microM) was derived from the shift. Acid red 33 (1000 microM) shifted the concentration-relaxation curve of ADP beta S to the right without changing the maximum (apparent Kd 171 microM). Reactive red 2 (1 to 10 microM) also shifted the concentration-response curve for the relaxation effect of alpha, beta-methylene ATP, which is mediated by an unclassified P2-purinoceptor, progressively to the right but simultaneously decreased the maximum (apparent Kd 1.6 microM). The concentration-relaxation curve of 2-chloroadenosine was not altered by reactive red 2. Pieces of vas deferens and taenia coli degraded 76 and 66% of added ATP (10 microM) within 30 min, respectively. Reactive red 2(0.1 to 100 microM) progressively reduced this degradation by up to 95%, with IC50 values of 3.9 +/- 0.6 and 3.9 +/- 2.3 microM, respectively. Acid red 33 (1000 microM) reduced the degradation by 30 and 20%, respectively. The results indicate that reactive red 2 is a relatively potent antagonist at both P2X-purinoceptors in rat vas deferens and P2Y-purinoceptors in guinea-pig taenia coli, with a 15 fold selectively for the P2Y-purinoceptor. It inhibits ecto-nucleotidase in both tissues. The dichloro-triazine residue that distinguishes the compound from acid red 33 greatly enhances the potency at both receptor subtypes as well as at the nucleotidase. As regards P2-purinoceptor subtypes, the results confirm the existence of two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli.