Biomedical subjects

S M Hourani

Publications and source records attributed to S M Hourani.

At least 37 records · Page 2Linked to original sources

PubMed1996

Differential ontogeny of adenosine receptors in the longitudinal muscle and muscularis mucosae of the rat isolated duodenum.

The ontogeny of P1 purinoceptors in the separated layers of the rat duodenum was investigated using functional assays. In the longitudinal muscle N6-cyclopentyladenosine (CPA) caused relaxations from day 20 that were inhibited by 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX) (10 nM) indicating an action via adenosine A1 receptors. 5'-N-ethylcarboxamidoadenosine (NECA) caused relaxations at day 15 that were inhibited by DPCPX (1 microM) while 2-p-(2-carboxyethl)phenylethylamino-5'-N-ethylcarboxamidoade nosine (CGS 21680) was almost inactive, indicating an action at adenosine A2B receptors. From day 20 NECA was inhibited by DPCPX (10 nM) but was not antagonised by DPCPX (1 microM) to the extent expected for an adenosine A1 receptor, suggesting activation of adenosine A1 and adenosine A2B receptors. In the muscularis mucosae, CPA and NECA caused contractions from day 10 inhibited by DPCPX (1 microM) while CGS 21680 was less potent, indicating activation of adenosine A2B receptors. These results show that adenosine A2B receptors are present early in the postnatal period, whereas adenosine A1 receptors develop after day 20.

Adenosine↗

PubMed1996

Differential distribution of adenosine A2 receptors in the epididymal and prostatic portions of the rat vas deferens.

In the rat vas deferens there are prejunctional A1 receptors mediating inhibition of transmitter release and post-junctional A1 and A2 receptors mediating enhancement and inhibition of contractions respectively. In this study the distribution of adenosine receptors in the prostatic and epididymal portions of the bisected rat vas deferens was investigated. The pre- and post-junctional A1 receptors were present on both portions of the bisected tissue. However, post-junctional A2 receptors appear to be present only in the prostatic region, showing that adenosine receptors are differentially distributed along the length of the rat vas deferens.

Adenosine↗

PubMed1996

P2T purinoceptors: ADP receptors on platelets.

ADP acts on platelets via the P2T purinoceptor to cause aggregation, but the way in which it does so is not fully understood. Most aggregating agents act via G protein-coupled receptors to stimulate phospholipase C (PLC) and so mobilize Ca2+ via inositol trisphosphate, whereas ADP clearly causes the mobilization of Ca2+ from internal stores but is only a weak activator of PLC. ADP also inhibits adenylate cyclase and it has been suggested that this effect is mediated by a different receptor, although evidence from antagonist studies argues against this. Studies of Ca2+ influx have shown that ADP is unique in causing a rapid influx of Ca2+, and patch-clamp studies have confirmed the activation by ADP of non-selective cation channels. This would imply the existence of two ADP receptors on platelets, a receptor-operated channel responsible for the rapid Ca2+ influx and a G protein-coupled receptor possibly linked to both inhibition of adenylate cyclase and mobilization of Ca2+. In this review the structure-activity relationships for aggregation, inhibition of adenylate cyclase and increases in cytoplasmic Ca2+ are summarized, and the relationship between these effects discussed.

Adenosine Diphosphate↗

PubMed1996

Selective enhancement by an adenosine A1 receptor agonist of agents inducing contraction of the rat vas deferens.

The adenosine analogue N6-cyclopentyladenosine (CPA), acting via postjunctional A1 receptors, has been shown to enhance contractions of the rat vas deferens induced by adenosine 5'-triphosphate (ATP), the sympathetic cotransmitter in this tissue. The aim of the present study was to examine the ability of CPA to enhance contractions induced by other contractile agents. CPA (0.01-0.3 microM) enhanced contractions induced by exogenous ATP (10 microM), 5-hydroxytryptamine (5-HT) (3 microM), tyramine (10 microM), 2-methyl-5-hydroxytryptamine (2-Me-5-HT) (10 microM) and KCl (35 mM) and this enhancement was blocked by an A1-selective concentration (3 nM) of 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX). CPA failed to enhance contractions induced by exogenous noradrenaline (NA) (1 microM or 10 microM), bradykinin (0.1 microM), phenylephrine (3 microM) or carbachol (10 microM). The contractions induced by ATP (10 microM), 5-HT (3 microM), 2-Me-5-HT (10 microM) and KCl (35 mM) were unaffected by tetrodotoxin (1 microM) as well as by desensitisation of the P2x-purinoceptors with the ATP analogue adenosine 5'-(alpha, beta-methylene) triphosphonate. The contractions induced by tyramine (10 microM) and 2-Me-5-HT (10 microM) were blocked by prazosin (100 nM) or by imipramine (1 microM). Ketanserin (10 nM) antagonised the response to 5-HT giving a dose-ratio of 12.9 corresponding to an apparent pA2 of 9.1. In conclusion, the A1-mediated effect was clearly selective for certain contractile agents and not due to a non-specific increase in contractility of the tissue. CPA enhanced contractions induced by both ATP and indirect sympathomimetics which release endogenous NA, and this enhancement of the two sympathetic cotransmitters may have a functional significance, and demonstrates the complexity of the neuromodulatory effects of adenosine in the rat vas deferens.

Adenosine↗

PubMed1996

Purinoceptors and platelet aggregation.

1. Adenosine 5'-diphosphate (ADP) is a physiologically important mediator, being released from damaged cells and from aggregating platelets. It acts on platelets to cause aggregation via a purinoceptor ('P2T-purinoceptor') at which adenosine 5'-triphosphate (ATP) is a competitive antagonist; however, the way in which it does so is not fully understood. 2. ADP activates a G12 protein, is a weak activator of phospholipase C but causes calcium mobilization from internal stores, and also inhibits adenylate cyclase. It seems likely that these effects are mediated by a single receptor but this is still unclear. 3. ADP also causes a rapid calcium influx which has the characteristics of a receptor-operated channel, and it has been suggested that this is due to a P2X1 receptor. This suggests the presence of at least two types of receptor responding to ADP, one G protein coupled and one a cation channel, and raises questions about the role of ATP in platelet function. 4. Adenosine acts via an A2a receptor to stimulate adenylate cyclase in platelets, and this nonselectively inhibits platelet activation. As ADP released from platelets is broken down to adenosine by ectonucleotidases on endothelial cells, this may provide an important mechanism for limiting inappropriate platelet aggregation in an intact blood vessel.

Adenosine Diphosphate↗

PubMed1996

Characterization of P1-purinoceptors on rat isolated duodenum longitudinal muscle and muscularis mucosae.

1. P1-purinoceptors mediating relaxation of the rat duodenum longitudinal muscle and contraction of the rat duodenum muscularis mucosae were characterized by the use of adenosine and its analogues, 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyl-adenosine (CPA), N6-(phenylisopropyl)adenosine (R-PIA), 2-chloroadenosine (2-CADO) and 2-p-((carboxyethyl)phenethylamino)-5'-carboxamidoadenosine (CGS21680), as well as the P1-purinoceptor antagonist 8-phenyltheophylline (8-PT) and the A1-selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). 2. In the rat duodenum longitudinal muscle, the order of potency of the adenosine agonists was CPA > NECA > adenosine > CGS21680. DPCPX antagonized responses to CPA and NECA at a concentration of 1 nM suggesting that they are acting at A1 receptors. A Schild plot versus CPA gave a slope near to unity (slope = 0.955) and a pA2 of 9.8 confirming that CPA was acting via A1 receptors. Schild analysis for DPCPX versus NECA, however, gave a slope of 0.674 suggesting that NECA was acting on both A1 and A2 receptors. CGS21680, a selective A2a agonist, was much less potent than adenosine suggesting that the A2 receptors are of the A2b subtype. 3. In the rat duodenum muscularis mucosae, the order of potency of the adenosine agonists was NECA > or = R-PIA = CPA > 2-CADO > adenosine, and DPCPX antagonized responses to CPA and NECA at a concentration of 1 microM. CGS21680, at a concentration of 10 microM, had no effect on this tissue. This suggests the presence of A2 receptors in this tissue and that they are of the A2b subtype. 4. These results are in agreement with previous studies in the whole duodenum showing the presence of A1 and A2b receptors causing relaxation, and this shows that the longitudinal muscle dominates the response of the whole tissue. In addition, a contractile A2b receptor has been revealed on the muscularis mucosae, the first time this subtype has been reported to elicit an excitatory response in a smooth muscle preparation.

Adenosine↗

PubMed1996

Responses of the longitudinal muscle and the muscularis mucosae of the rat duodenum to adenine and uracil nucleotides.

1. Previous studies have shown that the rat duodenum contains P1 and P2Y purinoceptors via which it relaxes to adenosine and adenosine 5'-triphosphate (ATP) respectively. It has also been shown to contract to uridine 5'-triphosphate (UTP) and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), and based on their differential inhibition by the P2 antagonist suramin it has been suggested that they act via two separate receptors. In addition, the rat duodenum has been shown to dephosphorylate ATP rapidly via ectonucleotidases and adenosine deaminase. In this study the responses of two preparations from the rat duodenum, the longitudinal muscle and the muscularis mucosae, were investigated using a series of nucleotides and suramin. 2. 2-Methylthioadenosine 5'-triphosphate (2-MeSATP), ATP, ATP-gamma-S and adenosine 5'-alpha,beta-methylene-triphosphonate (AMPCPP) each relaxed the longitudinal muscle, with an agonist potency order of 2-MeSATP > ATP = ATP-gamma-S > AMPCPP, while UTP and uridine 5'-diphosphate (UDP) were not observed to elicit relaxation. This indicates the presence of a relaxant P2Y-purinoceptor on the longitudinal muscle. The longitudinal muscle did not contract to any of the agonists at concentrations of 300 microM, apart from ATP-gamma-S which caused very weak contractions. 3. ATP-gamma-S, adenosine 5'-methylenediphosphonate (AMPCP), AMPCPP, ATP, UTP, adenosine 5'-diphosphate (ADP), UDP and 2-MeSATP each contracted the muscularis mucosae with an agonist potency order of ATP-gamma-S > or = AMPCP > or = AMPCPP = ATP = UTP = ADP = UDP >> 2-MeSATP, although maximal responses were not obtained at concentrations of 300 microM. The muscularis mucosae did not relax to any of the agonists at concentrations of 300 microM. 4. Suramin (1 mM) inhibited relaxations induced by ATP on the longitudinal muscle, shifting the relaxation concentration-response curve to the right. This further supports the presence of a P2Y-purinoceptor on this muscle layer. Suramin (1 mM) inhibited contractions induced by AMPCPP, but not those induced by ATP, UTP or ATP-gamma-S, in the muscularis mucosae. Desensitization of the muscularis mucosae was seen with AMPCPP, but not with UTP or ATP-gamma-S, and no cross-desensitization between AMPCPP and UTP or ATP-gamma-S was observed. This suggests there are two receptors which mediate contraction on the rat duodenum muscularis mucosae, one suramin-sensitive and the other suramin-insensitive. 5. ATP was rapidly degraded by the muscularis mucosae to ADP, adenosine 5'-monophosphate (AMP) and inosine, with no adenosine being detected. A similar rate of degradation was seen for UTP with UDP, uridine 5'-monophosphate (UMP) and uridine being formed and for 2-MeSATP with 2-methylthioadenosine 5'-diphosphate (2-MeSADP), 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) and 2-methylthioadenosine being formed. AMPCPP and ATP-gamma-S were both degraded more slowly, AMPCPP being degraded to AMPCP, and ATP-gamma-S to ADP, AMP and inosine. Suramin (1 mM), did not significantly affect the rate and pattern of degradation of these nucleotides, apart from AMPCPP which was degraded slightly more slowly in the presence of suramin. 6. These results show that there is a P2Y-purinoceptor which mediates relaxation in the rat duodenum longitudinal muscle. They also show that there is a contraction-mediating suramin-sensitive receptor on the rat duodenum muscularis mucosae which is desensitized by AMPCPP, and thus is probably of the P2X subtype. In addition, there is a contraction-mediating suramin-insensitive receptor on the rat duodenum muscularis mucosae which is not desensitized by UTP or ATP-gamma-S, and at which ATP and UTP show equal potency, and is thus probably of the P2U subtype. In addition, the rat duodenum muscularis mucosae contains ectonucleotidases and adenosine deaminase, which rapidly degrade nucleotides, although the inhibition by suramin of this deg

Adenosine Triphosphate↗

PubMed1996

Activation of multiple sites by adenosine analogues in the rat isolated aorta.

1. The presence of A2 receptors mediating relaxation in the rat isolated aorta has been previously demonstrated. However, agonist dependency of the degree of rightward shift elicited by 8-sulphophenyltheophylline (8-SPT) led to the suggestion that the population of receptors in this tissue is not a homogeneous one. In this study we have re-examined the effects of 8-SPT in the absence and presence of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and investigated antagonism of responses by the potent A2a receptor ligands PD 115,199 (N-[2-dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 dipropyl-1H-purin-8-yl)) benzene sulphonamidexanthine), ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol), and CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine). We have also investigated the antagonist effects of BWA1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine) which has been shown to have affinity at rat A3 receptors. 2. Adenosine, R-PIA (N6-R-phenylisopropyl adenosine), CPA (N6-cyclopentyladenosine) and NECA (5'-N-ethylcarboxamidoadenosine) all elicited relaxant responses in the phenylephrine pre-contracted rat isolated aorta with the following potency order (p[A50] values in parentheses): NECA (7.07 +/- 0.11) > R-PIA (5.65 +/- 0.10) > CPA (5.05 +/- 0.12) > adenosine (4.44 +/- 0.12). 3. 8-SPT (10-100 microM) caused parallel rightward shifts of the E/[A] curves to NECA (pKB = 5.23 +/- 0.16). A smaller rightward shift of E/[A] curves to CPA was observed (pA2 = 4.85 +/- 0.17). However, no significant shifts of E/[A] curves to either adenosine or R-PIA were observed. 4. In the absence of endothelium E/[A] curves to NECA and CPA were right-shifted compared to controls. However, removal of the endothelium did not produce a substantial shift of adenosine E/[A] curves, and E/[A] curves to R-PIA were unaffected by removal of the endothelium. 5. In the presence of L-NAME (100 microM) E/[A] curves to NECA and CPA were right-shifted. However, no further shift of the CPA E/[A] curve was obtained when 8-SPT (50 microM) was administered concomitantly. The locations of curves to R-PIA and adenosine were unaffected by L-NAME (100 microM). 6. In the presence of PD 115,199 (0.1 microM) a parallel rightward shift of NECA E/[A] curves was observed (pA2 = 7.50 +/- 0.19). PD 115,199 (0.1 and 1 microM) gave smaller rightward shifts of E/[A] curves to R-PIA and CPA, but E/[A] curves to adenosine were not significantly shifted in the presence of PD 115,199 (0.1 or 1 microM). 7. The presence of ZM 241385 (3 nM-0.3 microM) caused parallel rightwad shifts of NECA E/[A] curves (pKB = 8.73 +/- 0.11). No significant shifts of E/[A] curves to adenosine, CPA or R-PIA were observed in the presence of 0.1 microM ZM 241385. 8. CGS 21680 (1 microM) elicited a relaxant response equivalent to approximately 40% of the NECA maximum response. In the presence of this concentration of CGS 21680, E/[A] curves to NECA were right-shifted in excess of 2-log units, whereas E/[A] curves to R-PIA were not significantly shifted. 9. BWA1433 (100 microM) caused a small but significant right-shift of the E/[A] curve to R-PIA yielding a pA2 estimate of 4.1 IB-MECA (N6-(3-iodo-benzyl)adenosine-5(1)-N-methyl uronamide) elicited relaxant responses which were resistant to blockade by 8-SPT (p[A]50 = 5.26 +/- 0.13). 10. The results suggest that whereas relaxations to NECA (10 nM-1 microM) are mediated via adenosine A2a receptors, which are located at least in part on the endothelium, R-PIA and CPA may activate A2b receptors on the endothelium and an additional, as yet undefined site, which is likely to be located on the smooth muscle and which is not susceptible to blockade by 8-SPT, PD 115,199 or ZM 241385. This site is unlikely to be an A3 receptor since the very small shift obtained in the presence of BWA1433 (100 microM), and the low potency of IB-MECA is not consistent with the affin

Adenosine↗

PubMed1996

Differential development of adenosine A1 and A2b receptors in the rat duodenum.

1. The development of the adenosine A1 and A2b receptors inducing relaxation of the rat duodenum was studied by use of a combination of functional and radioligand binding assays on rats aged between 5 and 30 days and compared with results previously found in adult rat duodenum. 2. 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) bound with high affinity to a single site in duodenum preparations from rats aged 20, 25 and 30 days. At 10 and 15 days there was no detectable specific binding of [3H]-DPCPX. 3. The affinity (KD) of the binding site for [3H]-DPCPX was similar in membrane preparations from 20, 25 and 30 day old animals (1.58-2.27 nM), but the density (Bmax) of binding sites was found to increase up to 25 days where peak levels (72.0 +/- 9.5 fmol mg-1 protein) were observed and then decline at 30 days (45.5 +/- 2.9 fmol mg-1 protein) to levels commensurate with those previously determined in the adult rat duodenum. 4. In duodenum from 10 day old rats no responses to N6-cyclopentyladenosine (CPA, 1 nM-10 microM) were observed, at 15 days the duodenum responded to the highest concentration of CPA (3 microM) only, and at 20-30 days concentration-related responses were observed, with the potency of CPA increasing with an increase in age. DPCPX (10 nM) abolished the responses to CPA except at the highest concentration of CPA (3 microM) where the response was markedly attenuated, suggesting the presence of an A1, receptor. 5. In rat duodenum from animals of all ages (5-30 days), concentration-related responses to 5'-N-ethylcarboxamidoadenosine (NECA) were observed. The potency of NECA remained constant with an increase in age, whereas the maximum relaxation response increased from 20% at 5 days to 110% at 25 and 30 days. In the presence of 1 microM DPCPX a right-ward shift in the concentration-response curve to NECA was observed at all ages. In the presence of 10 nM DPCPX, the response to NECA was unaffected in the duodenum from animals aged 10 and 15 days. However, in duodenum from animals aged 20-30 days the concentration-response curve to NECA was shifted to the right suggesting that there is an A1 component to the action of NECA at these ages. Schild analysis of the effects of increasing concentrations of DPCPX versus NECA on the duodenum from 25 day old animals generated a slope of 0.62 suggesting that NECA acts at A1 and A2b receptors as in the adult. 6. The A2b-selective analogue, 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) (10 nM-10 microM) was without effect on the carbachol-contracted duodenum from 15 day old rats and the duodenum from 25 day old rats responded to the highest concentration of CGS 21680 only, suggesting that the A2 receptors here, as in the adult, are not of the A2a subtype. The adenosine antagonist, 8-phenyltheophylline (8-PT) (10 microM), abolished the inhibitory effects of NECA (100 nM-100 microM) on 10, 15 and 25 day old rat duodenum indicating that the responses to NECA were not mediated via an adenosine A3 receptor. 7. These results show that adenosine A1 receptors in rat duodenum are present and functionally viable from day 20 onwards and that the density of A1 receptors varies with age, increasing up to day 25 and then declining at day 30 to a density commensurate with that found in the adult. The responses to CPA, mediated via the A1 receptor, increase with age in a similar fashion. In contrast however, the response to NECA was evident from day 5, the earliest age studied, and from days 5-15 NECA acted via the A2b receptor subtype. However, from day 20 onwards NECA acted at a mixed population of A1 and A2b receptors. These results demonstrate the differential development of the A1 and the A2b receptors in the rat duodenum.

Adenosine↗

PubMed1996

The ontogenetic profiles of the pre- and postjunctional adenosine receptors in the rat vas deferens.

1. The ontogenetic profiles of the prejunctional A1 and postjunctional A1 and A2 receptors on the rat vas deferens were investigated, using a combination of functional and radioligand binding assays to follow the A1 receptors and functional assays alone to follow the development of the A2 receptors. 2. The prejunctional A1 receptor, assessed by the inhibitory action of N6-cyclopentyladenosine (CPA) (3 nM-3 microM) on nerve-mediated contractions, was present from day 15 onwards, day 15 being the earliest age at which nerve-mediated contractions could be detected. The potency of CPA was constant across the ages studied, with pD2 values ranging from 6.4-7.1, not significantly different from that previously observed in adult rat vas deferens. 3. The postjunctional A2 receptors, assessed by the inhibitory action of 5'-N-ethylcarboxamidoadenosine (NECA) (10 nM-30 microM) on KCl-induced contractions were present from day 10 onwards, day 10 being the earliest age at which responses to KCl could be observed. The potency of NECA remained constant with an increase in age, with potency values, expressed as pEC25 values, ranging from 6.5-7.0. 4. The postjunctional A1 receptor displayed a different development profile from that of the prejunctional A1 and postjunctional A2 receptors. Postjunctional A1 receptors were identified by the enhancement of KCl-induced contractions by CPA (10 nM-0.3 microM). At 10 and 15 days, CPA failed to enhance KCl-induced contractions. From day 20 to day 40, this enhancement increased with an increase in age and the level of enhancement achieved statistical significance from day 30. 5. Radioligand binding studies using 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) revealed binding sites characteristic of A1 receptors on the vas deferens from rats aged 20 days onwards. The density (Bmax) of A1 receptors expressed relative to protein content was greatest at day 20 (153 +/- 33 fmol mg-1 protein) and declined at day 30 (43.9 +/- 3.7 fmol mg-1 protein) to a level commensurate with that previously determined in adult rat vas deferens (43.3 +/- 12 fmol mg-1 protein). However, when expressed relative to tissue wet weight little variation in receptor density was observed between these ages (Bmax 0.13 +/- 0.02 fmol mg-1 wet weight at 20 days; 0.17 +/- 0.01 fmol mg-1 wet weight at 30 days). The binding affinity (KD) remained constant with an increase in age and was similar to the KD value previously generated for adult rat vas deferens (approximately 1 nM). At ages 10 and 15 days no reproducible binding could be detected. 6. These results show the differential development of the adenosine receptors on the rat vas deferens with postjunctional A1 receptors demonstrating delayed development, while prejunctional A1 and postjunctional A2 receptors were present from the earliest ages studied. In addition, comparison of binding studies and functional studies suggests that the binding studies detect only the A1 receptors present on the smooth muscle and not those present on the nerve terminals.

Adenosine↗

PubMed1995

Effects of suramin on contractions of the guinea-pig vas deferens induced by analogues of adenosine 5'-triphosphate.

1. Adenosine 5'-triphosphate (ATP) and some of its analogues contract the guinea-pig vas deferens, acting via receptors which have been classified as P2X-purinoceptors. We have recently shown, however, that the effects of ATP are enhanced, rather than inhibited, by the non-selective P2 antagonist, suramin, and that this enhancement could not easily be explained in terms of inhibition by suramin of the breakdown of ATP. We therefore investigated the effects of suramin on contractions induced by ATP analogues, to define the structure-activity relationships of the suramin-resistant response. 2. In the absence of suramin, the order of potency for ATP analogues was adenosine 5'-(alpha,beta-methylene)triphosphonate (AMPCPP) = P1,P5-diadenosine pentaphosphate (Ap5A) = adenosine 5'-tetraphosphate (Ap4) > adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) = adenylyl 5'-(beta,gamma-methylene) diphosphonate (AMPPCP) > P1,P5-diadenosine tetraphosphate (Ap4A) > adenosine 5'-O-(2- thiodiphosphate) (ADP beta S) > 2-methylthioadenosine 5'-triphosphate (MeSATP) > or = ATP > adenosine 5'-diphosphate (ADP). This is generally in agreement with previously reported structure-activity relationships in this tissue. 3. In the presence of suramin (1 mM), responses to Ap5A, Ap4A, AMPPCP, ADP beta S and ADP were abolished or greatly reduced, and contractions induced by AMPCPP, Ap4 and ATP gamma S were inhibited. Contractions induced by MeSATP however, like those induced by ATP itself, were not reduced, but at concentrations above 100 microM were enhanced. In the presence of suramin (1 mM) the order of potency of analogues was therefore AMPCPP = Ap4> ATP = MeSATP> ATP gamma S, with all other analogues tested being essentially inactive at concentrations up to 500 microM.4. Contractile responses of the vas deferens to transmural nerve stimulation (1-50 Hz) in the presence of the alpha-adrenoceptor antagonist, phentolamine (10 microM), were abolished by suramin (1 mM). This is in agreement with previous reports that suramin inhibits the excitatory junction potential, a response thought to be mediated by P2 purinoceptors. It is however hard to reconcile the evidence implicating ATP as the non-adrenergic transmitter responsible for this response with the failure of suramin to inhibit the contractions induced by ATP itself while abolishing nerve-mediated contractions.5. In conclusion, these results confirm our previous findings of a suramin-resistant component to the ATP-induced contraction in the guinea-pig vas deferens, and show that the structure-activity relationships of this response are not identical to those of any known P2-purinoceptor subclass. Although the inhibition by suramin of the breakdown of ATP may contribute to the suramin-resistance of some of the ATP analogues, it does not appear to provide the full explanation.

Adenosine Triphosphate↗

PubMed1994

Effects of extracellular divalent cations on responses of human blood platelets to adenosine 5'-diphosphate.

The effects of extracellular divalent cations on the responses of human platelets to adenosine 5'-diphosphate (ADP) and on its inhibition by the competitive antagonist adenosine 5'-triphosphate (ATP) were investigated. Two responses were studied, shape change and the inhibition of prostaglandin E1 (PGE1)-stimulated adenylate cyclase, and experiments were carried out in the presence of divalent cations (Ca2+ and Mg2+, 1 mM) or in their absence. For each response there was a small leftward shift of the concentration-response curve to ADP in the absence of divalent cations compared to that in their presence, and this leftward shift disappeared when the results were plotted in terms of ADP3- rather than total ADP concentration. The shape change results were, however, complicated by a reduction in the maximal response to ADP in the absence of divalent cations. For each response there was also a marked increase in the pA2 value of ATP in the absence of divalent cations compared to that in their presence, and this difference disappeared if the results were calculated in terms of ATP4- instead of total ATP. These results suggest that the human platelet ADP receptor, in common with other receptors for adenine nucleotides, recognises predominantly the uncomplexed forms of ADP and ATP as ligands.

Adenosine Diphosphate↗

PubMed1994

Effects of suramin on increases in cytosolic calcium and on inhibition of adenylate cyclase induced by adenosine 5'-diphosphate in human platelets.

The effects of the P2-purinoceptor antagonist, suramin, on ADP-induced increases in human platelet cytosolic calcium concentration ([Ca2+]i) and inhibition of prostaglandin E1 (PGE1)-stimulated adenylate cyclase activity were investigated. Suramin (50-200 microM) acted as an antagonist of ADP-induced increases in [Ca2+]i, causing parallel, rightward shifts of the log concentration-response curve to ADP with no apparent depression of the maximal response. However, the slope of the Schild plot was 2.3 +/- 0.3, similar to that obtained in previous studies on aggregation, indicating that the antagonism was not simply competitive. The apparent pA2 for suramin, taken from the Schild plot, was 4.63, similar to that for suramin's inhibition of aggregation, which suggests that these two effects are closely related. Suramin was not specific for the ADP receptor, however, as it was also able to inhibit, non-competitively, increases in [Ca2+]i induced by 5-hydroxytryptamine. Suramin (50-400 microM) also inhibited the effect of ADP on PGE1-stimulated accumulation of cyclic AMP, causing parallel shifts of the log concentration-response curve to ADP, with a Schild plot slope of 1.00 +/- 0.10, suggesting competitive antagonism, and a pA2 value of 5.09. Suramin (400 microM) did not reduce the inhibition of cyclic AMP accumulation by adrenaline, although it was able to inhibit the accumulation of cyclic AMP caused by PGE1, again showing that suramin has some non-specific effects. These data suggest that suramin is an antagonist at the platelet ADP receptor mediating increases in [Ca2+]i and inhibition of adenylate cyclase, but that it also shows non-specific effects and can depress platelet responses to other agonists. In addition, the similar pA2 value of suramin for the two effects of ADP does not support suggestion that they are mediated by two different receptors on human platelets.

Adenosine Diphosphate↗

PubMed1994

Receptors for ADP on human blood platelets.

It is well established that ADP causes aggregation of human blood platelets, and indeed it was the first aggregating agent to be studied, but the ways in which platelets respond to ADP are still relatively obscure. Although it is apparent that increases in intracellular Ca2+ concentrations are of major importance in activating platelets, it is not clearly understood how ADP causes these increases and what other signal transduction mechanisms it uses. It is not even clear whether ADP causes its effects by interacting with only one receptor, or whether multiple receptors for ADP exist on platelets. In this review, Susanna Hourani and David Hall examine some of the conflicting evidence in this field, and draw some tentative conclusions about the number and nature of receptors for ADP on human platelets.

Adenosine Diphosphate↗

PubMed1994

Characterization of adenosine receptors in the rat isolated aorta.

1. Adenosine and its analogues relaxed the isolated rat aorta by an endothelium-dependent mechanism with an order of potency of 5'-N-ethylcarboxamidoadenosine (NECA) > 2-(p-(2-carboxy-ethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > adenosine = N6-(2-(4-amino-phenyl)ethyl)adenosine (APNEA) = N6-cyclopentyladenosine (CPA) > 5'-methylthioadenosine (MTA), although the maximal response achieved by CGS 21680 was less than that achieved by NECA. 2. Both 8-sulphophenyltheophylline (8-SPT) and MTA antagonized responses to the adenosine analogues, but there were some anomolous features of this antagonism and NECA was inhibited more powerfully than the other agonists. This suggests that as well as A2a receptors mediating relaxation, the rat aorta may relax to adenosine analogues by other mechanisms.

Adenosine↗

PubMed1994

Post-junctional excitatory adenosine A1 receptors in the rat vas deferens.

1. At concentrations between 1 nM and 1 microM, the A1-selective agonists N6-cyclopentyladenosine (CPA) and (R)-N6-phenylisopropyladenosine (R-PIA) each enhanced contractions of the rat vas deferens induced by ATP (10 microM), and this enhancement was blocked by an A1-selective concentration (1 nM) of the antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). 2. No such enhancement was observed with the non-selective agonists adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) at concentrations between 1 nM and 100 microM, which instead inhibited the contractions. 3. These results show that in addition to the previously demonstrated inhibitory A1 and A2 adenosine receptors, the rat vas deferens also possesses post-junctional excitatory A1 adenosine receptors.

Adenosine↗

PubMed1994

Differential effects of suramin on P2-purinoceptors mediating contraction of the guinea-pig vas deferens and urinary bladder.

1. The effect of the P2-purinoceptor antagonist, suramin, was investigated on contractions of the guinea-pig vas deferens and urinary bladder induced by adenosine 5'-triphosphate (ATP) and by the other naturally occurring nucleoside triphosphates. 2. ATP, guanosine 5'-triphosphate (GTP), cytidine 5'-triphosphate (CTP), inosine 5'-triphosphate (ITP) and uridine 5'-triphosphate (UTP) (0.1-500 microM) each contracted both the guinea-pig bladder and the guinea-pig vas deferens. In the vas deferens the order of potency of the nucleotides was ATP >> CTP > GTP > or = UTP = ITP, and in the bladder it was ATP >> CTP = GTP > UTP = ITP, although maximal responses to these agonists were not achieved in either tissue. 3. Suramin (30 microM-1 mM) dose-dependently inhibited ATP-induced contractions of the bladder in an apparently non-competitive manner, causing a reduction in the slope of the concentration-response curve to ATP. In contrast, suramin (5 microM-1 mM) had little inhibitory effect on ATP-induced contractions of the vas deferens, and indeed at concentrations of 100 microM and above markedly potentiated high concentrations of ATP (100-500 microM). The contractions induced by CTP, GTP, UTP and ITP (1-500 microM) were, however, abolished by suramin (1 mM) in each tissue. 4. Desensitization of the P2X purinoceptors in the guinea-pig vas deferens with adenosine 5'-alpha,beta-methylenetriphosphonate (AMPCPP) (300 microM) abolished contractions induced by ATP (1 microM-1 mM) in the absence of suramin. However, the contractions induced in the presence of suramin were unaffected by prior desensitization, indicating that they were not mediated by P2X-purinoceptors.5. ATP (100 MicroM) was dephosphorylated by both isolated tissue preparations under the conditions of these experiments, breakdown products being detectable after 2 min, with the major breakdown product in the bladder being inosine whereas that in the vas deferens was adenosine. Approximately 35% of the ATP remained intact after incubation for 30 min with the bladder, and approximately 45% remained after incubation for 30 min with the vas deferens. In each tissue this degradation was inhibited by suramin (1 mM), so that after incubation of ATP (100 MicroM) in the presence of suramin for 30 min,approximately 50% remained in the case of the bladder and approximately 65% remained in the vas deferens. However, inhibition of the production of the inhibitory agonist, adenosine by suramin did not appear to be responsible for the potentiation observed in the vas deferens, as the PI-purinoceptor antagonist 8-sulphophenyltheophylline (100 MicroM) did not reduce this potentiation.6. Chelation of divalent cations did not appear to account for the enhancement by suramin of ATP-induced contractions of the vas deferens, as the enhancement was still observed when Mg2+ was omitted from the buffer or when its concentration (normally 1.2 mM) was increased ten fold to 12 mM,or when the concentration of Ca2+ (normally 2.5 mM) was reduced to 0.83 mM. Even in the absence of Mg2+ and with the Ca2+ concentration reduced to 0.83 mM, no inhibition by suramin (1 mM) of ATP-induced contractions was observed.7. The most likely explanation for the potentiation by suramin of the ATP-induced contractions of the vas deferens is the co-existence of inhibitory P2Y-purinoceptors. However, no consistent relaxations to ATP (1-100 MicroM) or to the more potent P2Y-purinoceptor agonist 2-methylthioadenosine 5'-triphosphate(2-MeSATP) (0.01-100 MicroM) could be detected in the vas deferens precontracted with KCl (35 mM), even after desensitization of P2x-purinoceptors with AMPCPP (300 MicroM). Similarly, ATP (1-100 MicroM) or 2-MeSATP (0.01-1100 MicroM) added before KCI (35 mM), carbachol (10 JM) or noradrenaline (10 MicroM) did not reduce subsequent contractions to these agents.8. The differential effect of suramin on the contractions induced by ATP in the bladder and the vas deferens was unexpected, and shows that the receptor populations by which ATP acts in these tissues may not be identical. The failure of suramin to inhibit responses to ATP in the vas deferens suggests that this tissue, in addition to possessing P2x-purinoceptors may also possess a suramin-insensitive contractile ATP receptor revealed in the presence of suramin.

Adenosine Triphosphate↗

PubMed1994

Contractile effects of uridine 5'-triphosphate in the rat duodenum.

1. Previous studies have shown that the rat duodenum relaxes to adenosine and adenosine 5'-triphosphate (ATP) via P1 and P2Y purinoceptors respectively, but in preliminary studies uridine 5'-triphosphate (UTP) was found to contract this tissue. The non-selective P2 antagonist suramin and a number of nucleotides were therefore used to investigate this response further. 2. ATP, UTP, adenosine 5'-diphosphate (ADP), adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), guanosine 5'-triphosphate (GTP) and uridine 5'-diphosphate (UDP) each relaxed the duodenum, with an agonist potency order of ATP = ADP > ATP-gamma-S >> GTP >> UTP = UDP, consistent with the presence of a P2Y purinoceptor mediating relaxation. 3. ATP-gamma-S, UTP and UDP each contracted the duodenum with an agonist potency order of ATP-gamma-S > UTP > UDP, although maximal responses to these agonists were not obtained at a concentration of 267 microM (ATP-gamma-S) and 300 microM (UTP and UDP). No contractions were observed with any of the other agonists at concentrations up to 300 microM. 4. Indomethacin (25 microM) did not inhibit the contractions induced by UTP, indicating that they were not mediated via production of prostaglandins. 5. Suramin (100 microM and 1 mM) inhibited relaxations induced by ATP, shifting the concentration-response curve to the right, with the maximal response to ATP being decreased by the higher concentration of suramin (1 mM). Suramin (1 mM) inhibited relaxations induced by ATP-gamma-S, shifting the concentration-response curve to the right, and completely abolished contractions induced by ATP-gamma-S. In contrast, suramin (100 JAM and 1 mM) had no effect on contractions induced by UTP.Contractions induced by UTP were, however, less sustained in the presence of suramin, which also affected the basal tone of some tissues when precontracted with carbachol (0.1 microM). In the presence of suramin (I mM), no contractions to ATP were observed.6. These results confirm that in the rat duodenum there is a P2Y purinoceptor that mediates relaxation in response to a number of purine nucleotides, and at which the pyrimidine nucleotides UTP and UDP are almost inactive. There are also receptors at which UTP and ATP-y-S act to cause contraction.Suramin discriminates between the contractile effects of these two agonists, which may indicate the presence of a suramin-insensitive pyrimidinoceptor as well as a suramin-sensitive receptor for ATP-y-S.An alternative explanation is that the differential effects of suramin are via its actions as an antagonist in addition to its action as an ectonucleotidase inhibitor.

Adenine Nucleotides↗