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Biomedical subjects

N J Cusack

Publications and source records attributed to N J Cusack.

At least 37 records · Page 2Linked to original sources
PubMed1985

Characterization of ectonucleotidases on vascular smooth-muscle cells.

We compared the properties of the ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5'-nucleotidase, EC 3.1.3.5) in intact pig aortic smooth-muscle cells in culture with the properties that we previously investigated for ectonucleotidases of aortic endothelial cells [Cusack, Pearson & Gordon (1983) Biochem. J. 214, 975-981]. In experiments with nucleotide phosphorothioate diastereoisomers, stereoselective catabolism of adenosine 5'-[beta-thio]triphosphate, but not of adenosine 5'-[alpha-thio]triphosphate, by the triphosphatase and stereoselective catabolism of adenosine 5'-[alpha-thio]diphosphate by the diphosphatase were found, as occurs in endothelial cells. In contrast with endothelial ecto-5'-nucleotidase, the smooth-muscle-cell enzyme catabolized adenosine 5'-monophosphorothioate (AMPS) to adenosine: the affinity of the enzyme for AMPS was greater than for AMP, and Vmax for AMPS was about one-sixth that for AMP. In both cell types AMPS was an apparently competitive inhibitor of AMP catabolism by 5'-nucleotidase. The relative rates of catabolism of nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety were similar to those in endothelial cells. No ectopyrophosphatase activity was detected in smooth-muscle cells, in contrast with endothelial cells, where modest activity is present.

5'-Nucleotidase↗
PubMed1985

Specificity of P2-purinoceptor that mediates endothelium-dependent relaxation of the pig aorta.

ATP (EC50 5 microM) induced endothelium-dependent relaxation of the isolated aorta of the newborn pig, but the other naturally occurring nucleotides CTP, GTP, ITP and UTP were more than 100 times less potent. 2-Methylthio-ATP (EC50 0.1 microM) was 50 times more potent than ATP, but the unnatural enantiomers L-ATP and 2-methylthio-L-ATP were virtually inactive. beta,gamma-Imido-ATP and beta,gamma-methylene-ATP, both of which are resistant to degradation by ectonucleotidases on cultured pig endothelial cells, were much less potent than ATP. ADP beta S, which is also resistant to degradation, was equipotent with ATP at low concentrations but achieved a maximal relaxation of only 50% that of ATP. The Rp and Sp diastereoisomers of ATP beta S were both equipotent with ATP at low concentrations and both achieved approximately 60% of the maximal relaxation of ATP. The Rp and Sp diastereoisomers of ADP alpha S were both less potent than ATP and achieved only approximately 25% of the maximal relaxation of ATP. These results demonstrate that the P2-purinoceptor mediating endothelium-dependent relaxation of the pig aorta exhibits a high degree of specificity for the adenine base, is stereospecific for the D-ribofuranosyl moiety, requires a phosphate chain of 2 or 3 units but is not stereoselective toward this phosphate chain. These structural requirements have some features in common with the P2-purinoceptors on smooth muscle and on platelets, and are quite different from those of the ectonucleotidases present on pig endothelial cells in culture.

Adenosine Triphosphate↗
PubMed1985

L-AMP-PCP, an ATP receptor agonist in guinea-pig bladder, is inactive on taenia coli.

L-Adenyl 5'-(beta, gamma-methylene)-diphosphonate (L-AMP-PCP), a potent ATP receptor agonist in the guinea-pig bladder, was tested on the guinea-pig taenia coli. L-AMP-PCP, unlike ATP, did not relax the taenia coli, and it neither enhanced nor inhibited the action of ATP. Unlike ATP, L-AMP-PCP was not degraded by ectonucleotidases on the taenia coli. The lack of pharmacological effect of L-AMP-PCP on the taenia coli supports the suggestion that the ATP receptors here differ from those in the guinea-pig bladder.

5'-Nucleotidase↗
PubMed1985

Studies on the stereoselectivity of the P2-purinoceptor on the guinea-pig vas deferens.

ATP, 2-chloro-ATP, 2-methylthio-ATP and their unnatural L-enantiomers, Rp and Sp diastereoisomers of the ATP phosphorothioate analogues, ATP alpha S and ATP beta S, were tested on the guinea-pig vas deferens. The 2-substituted analogues of ATP were no more effective than ATP in causing contraction of the vas deferens. However, stereoselectivity was observed with each pair of enantiomers of ATP, 2-chloro-ATP and 2-methylthio-ATP. No stereoselectivity was observed for the phosphorothioate analogues. Rp- and Sp-ATP beta S were more effective than ATP at eliciting contractions of the vas deferens. These results show that unlike the P2-purinoceptor mediating excitatory responses in the guinea-pig bladder, the P2-purinoceptor mediating contraction in the guinea-pig vas deferens displays stereoselectivity.

Adenosine Triphosphate↗
PubMed1984

Effects of phosphorothioate analogues of ATP, ADP and AMP on guinea-pig taenia coli and urinary bladder.

Phosphorothioate analogues of ATP, ADP and AMP were tested on the guinea-pig taenia coli and urinary bladder. The Rp diastereoisomers of the phosphorothioate analogues, ATP-alpha-S and ADP-alpha-S were respectively 7 and 3 times more effective than the Sp diastereoisomers in causing relaxation of the taenia coli. No stereoselectivity was observed for the diastereoisomers of ATP-beta-S. In guinea-pig bladder, no stereoselectivity was observed for any of the phosphorothioate analogues. These results show that P2-purinoceptors mediating inhibitory responses in the guinea-pig taenia coli show marked stereoselectivity, while P2-purinoceptors mediating excitatory responses in the guinea-pig bladder show little stereoselectivity.

Adenosine Diphosphate↗
PubMed1984

Some pharmacological and biochemical interactions of the enantiomers of adenylyl 5'-(beta, gamma-methylene)-diphosphonate with the guinea-pig urinary bladder.

Adenosine 5'-triphosphate (ATP) and adenylyl 5'-(beta, gamma-methylene)-diphosphonate (AMP-PCP) both contracted the guinea-pig urinary bladder, but the response to AMP-PCP was much greater. We synthesized the enantiomer of AMP-PCP, L-adenylyl 5'-(beta, gamma-methylene)-diphosphonate (L-AMP-PCP), and tested it on the guinea-pig bladder. L-AMP-PCP contracted the guinea-pig bladder, and was more potent than AMP-PCP and much more potent than ATP. The potential breakdown product of L-AMP-PCP, L-adenosine, unlike adenosine (the breakdown product of AMP-PCP), did not inhibit contractions of the guinea-pig bladder. ATP and its enantiomer L-adenosine 5'-triphosphate (L-ATP) were rapidly degraded by the muscle, and AMP-PCP was also degraded, but more slowly. L-AMP-PCP, however, was completely resistant to degradation. L-AMP-PCP would appear to be a useful ATP analogue, as it is potent and resistant to degradation, and its potential breakdown product, L-adenosine, is inactive.

Adenosine↗
PubMed1983

Stereoselectivity of ectonucleotidases on vascular endothelial cells.

We have investigated the stereoselectivity of ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5'-nucleotidase, EC 3.1.3.5) on pig aortic endothelial cells using two classes of nucleotide analogue. In experiments with nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety, the rate of catabolism of 100 microM-L-ATP was one-fifth that of D-ATP, the rate of catabolism of 100 microM-L-ADP was one-fifteenth that of D-ADP and there was no detectable catabolism of 100 microM-L-AMP. Each of the L-enantiomers inhibited, apparently competitively, the catabolism of the corresponding D-enantiomer; Ki values were approx. 0.6 mM, 1.0 mM and 3.9 mM for L-ATP, L-ADP and L-AMP respectively. Experiments with adenosine 5'-[beta, gamma-imido]triphosphate and with D- and L-enantiomers of adenosine 5'-[beta, gamma-methylene]triphosphate revealed modest ectopyrophosphatase activity, undetectable in experiments with natural nucleotides, which was also stereoselective. Use of phosphorothioate nucleotide analogues demonstrated that ATP catabolism was virtually stereospecific with respect to the geometry of the thiol group substituted on the beta-phosphate: the Rp isomer was degraded, whereas there was little or no breakdown of the Sp isomer. ADP catabolism was also stereospecific with respect to the geometry of the thiol group substituted on the alpha-phosphate: the Sp isomer but not the Rp isomer was degraded. The geometry of thiol-group substitution on the alpha-phosphate had no effect on ATP catabolism to ADP. There was no detectable catabolism of analogues with thiol-group substitution on the terminal phosphate. Each of the phosphorothioate analogues that was catabolized broke down at a rate similar to that of the natural nucleotide from which it was derived. These results demonstrate that the ectonucleotidases on pig aortic endothelial cells exhibit a high degree of stereoselectivity, characteristic for each enzyme, both with respect to the ribofuranosyl moiety and to the phosphate side chain.

5'-Nucleotidase↗
PubMed1983

Studies on the stereoselectivity of the P2-purinoceptor.

ATP, 2-chloro-ATP, 2-methylthio-ATP, and their unnatural L-enantiomers, were synthesized and their effects tested on the guinea-pig taenia coli and urinary bladder, and the stimulated frog ventricle. The potent P2-purinoceptor agonists, 2-chloro-ATP and 2-methylthio-ATP were, respectively, 30 and 200 times more effective than ATP in relaxing the guinea-pig taenia, but approximately as effective as ATP in contracting the guinea-pig bladder and augmenting the force of contraction of the frog ventricle. A high degree of stereoselectivity was observed for relaxations of the guinea-pig taenia coli produced by the P2-purinoceptoragonists, and 2-methylthio-ATP was over 700 times more effective than its L-enantiomer. In contrast, stereoselectivity for contraction of the guinea-pig bladder was observed only at low concentrations with each pair of enantiomers, and a similar low stereoselectivity was displayed by the frog ventricle. These results show that P2-purinoceptors mediating inhibitory responses in the guinea-pig taenia coli can show a high degree of stereoselectivity, while P2-purinoceptors mediating excitatory responses in the guinea-pig bladder and in the frog ventricle show little stereoselectivity. The partial stereoselectivity of the P2-purinoceptor in smooth muscle contrasts with the absolute stereospecificity of P1-purinoceptors for adenosine on smooth muscle and autonomic nerve terminals and the absolute stereospecificity of the receptor for ADP on the human platelet.

Adenosine Triphosphate↗
PubMed1982

Evidence for stereospecificity of the P1-purinoceptor.

1 The effects of adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloroadenosine, 2-azidoadenosine, and their L-enantiomers were examined on driven left atria, trachea and transmurally stimulated ileum of the guinea-pig. 2 In each tissue the order of potency of the D-enantiomers for producing inhibitory effects was NECA greater than 2-chloroadenosine greater than 2-azidoadenosine greater than adenosine. 3 The log concentration-response curve of each agonist was shifted to the right in the presence of the P1-purinoceptor antagonist, theophylline. 4 Dipyridamole, which blocks adenosine uptake, potentiated the effects of adenosine but not those of the D-enantiomers of adenosine analogues. 5 The greater potency of the adenosine analogues therefore, is at least partly due to their resistance to tissue uptake and subsequent enzymatic destruction. 6 The L-enantiomers of adenosine and its analogues did not produce inhibitory responses in the driven left atria or transmurally stimulated ileum. At high concentrations relaxations of the tracheal muscle were obtained, with the potency series L-NECA greater than 2-chloro-L-adenosine greater than 2-azido-L-adenosine greater than L-adenosine. 7 It is concluded that the postsynaptic P1-purinoceptors in the guinea-pig atria and trachea and the presynaptic P1-purinoceptors on cholinergic nerve terminals in guinea-pig ileum are stereospecific for the D-enantiomers of adenosine and its analogues.

2-Chloroadenosine↗
PubMed1982

Differential inhibition by adenosine or by prostaglandin E1 of human platelet aggregation induced by adenosine 5'-O-(1-thiodiphosphate) and adenosine 5'-O-(2-thiodiphosphate).

Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and inhibits stimulated adenylate cyclase. Adenosine 5'-O-(1-thiodiphosphate) (ADP-alpha-S) and adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) act at the ADP receptor and achieve the same maximal rate of human platelet aggregation as each other. Adenosine and prostaglandin E1, which noncompetitively inhibit ADP-induced aggregation by stimulating adenylate cyclase, inhibit aggregation induced by ADP-x-S more than aggregation induced by ADP-beta-S. ADP-x-S, unlike ADP-beta-S and ADP itself, does not inhibit stimulated adenylate cyclase. This suggests that the inhibition of stimulated adenylate cyclase by ADP, although not a cause of aggregation, may be of physiological importance in reducing the effects of endogenous agents such as adenosine and prostaglandins (for example, prostacyclin) to which the platelet may be exposed.

Adenosine Diphosphate↗
PubMed1982

Specific but noncompetitive inhibition by 2-alkylthio analogues of adenosine 5'-monophosphate and adenosine 5'-triphosphate of human platelet aggregation induced by adenosine 5'-diphosphate.

Some 2-alkylthio derivatives of adenosine 5'-monophosphate (AMP), adenosine 5'-monophosphorothioate (AMPS) and adenosine 5'-triphosphate (ATP) were examined as inhibitors of human platelet aggregation. 2-Methylthio-AMP, 2-ethylthio-AMP, 2-(pentan-l-yl)thio-AMP, 2-ethylthio-AMPS, 2-methylthio-ATP and 2-ethylthio-ATP (100 microM) each inhibited aggregation induced by adenosine 5'-diphosphate (ADP) but not by 11 alpha, 9 alpha-epoxymethano prostaglandin H2, a stable endoperoxide analogue. Log dose-response curves to ADP in the absence and presence of each inhibitor were not parallel and the inhibition could not be overcome by high concentrations of ADP. The ATP analogues achieved greater inhibition of aggregation induced by ADP (5 microM) than did the AMP analogues. The order of potency of the AMP analogues was 2-ethylthio-AMPS greater than 2-ethylthio-AMP greater than 2-(pentan-l-yl)thio-AMP greater than 2-methylthio-AMP, and 2-methylthio-ATP was more potent than 2-ethylthio-ATP. These 2-alkylthio substituted analogues of AMP and ATP are specific but noncompetitive inhibitors of ADP-induced human platelet aggregation.

Adenosine Diphosphate↗
PubMed1982

Adenosine 5-diphosphate antagonists and human platelets: no evidence that aggregation and inhibition of stimulated adenylate cyclase are mediated by different receptors.

1 Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and noncompetitively inhibits stimulated human platelet adenylate cyclase; it has been suggested that these two effects are mediated by separate ADP receptors on the platelet surface. 2 Adenosine 5'-triphosphate and seven adenine nucleotide analogues were tested as inhibitors of both effects of ADP on human platelets, and were found to be competitive. 3 pA2 values were calculated for each antagonist for inhibition of both effects of ADP, and a good correlation (correlation coefficient 0.87; p less than 0.01) was found between the pA2 values for inhibition of ADP-induced aggregation and the pA2 values for inhibition of the effect of ADP on stimulated adenylate cyclase. 4 Such a correlation does not support the suggestion that ADP-induced aggregation and the inhibition by ADP of stimulated adenylate cyclase are mediated by two separate receptors.

Adenosine Diphosphate↗
PubMed1982

Competitive inhibition by adenosine 5'-triphosphate of the actions on human platelets of 2-chloroadenosine 5'-diphosphate, 2-azidoadenosine 5'-diphosphate and 2-methylthioadenosine 5'-diphosphate.

1 Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and noncompetitively inhibits stimulated human platelet adenylate cyclase; these two effects are mediated by the same ADP receptor, at which adenosine 5'-triphosphate (ATP) is a competitive antagonist. 2 Two ADP analogues, 2-azidoadenosine 5'-diphosphate (2-azido-ADP) and 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP) have been reported to be more potent as inhibitors of adenylate cyclase than they are as aggregating agents, but no evidence has been presented that these actions are mediated solely by the ADP receptor. 3 We therefore tested the ability of ATP to inhibit the actions of these compounds and of another ADP analogue, 2-chloroadenosine 5'-diphosphate (2-chloro-ADP). 4 2-Chloro-ADP, 2-azido-ADP and 2-methylthio-ADP each induced aggregation and inhibited stimulated adenylate cyclase. Both of these actions were competitively inhibited by ATP (50 microM) with pA2 values similar to those previously found for inhibition by ATP of these effects of ADP. 5 The reported greater potency of 2-azido-ADP and of 2-methylthio-ADP as inhibitors of adenylate cyclase than as aggregating agents is therefore due only to their greater efficacy for this effect, not to some extra actions elsewhere.

Adenosine Diphosphate↗
PubMed1981

Partial agonist behaviour of adenosine 5'-O-(2-thiodiphosphate) on human platelets.

1 The effects of an adenosine diphosphate (ADP) analogue, adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S), in which a terminal phosphate oxygen has been replaced by sulphur, were studied on human platelets. 2 ADP-beta-S induced platelet aggregation and inhibited prostaglandin E1 (PGE1)-stimulated adenylate cyclase but in both cases was less potent than ADP and did not achieve the same maximal effects. 3 Both actions of ADP could be inhibited by the simultaneous addition of ADP-beta-S (50 microM). 4 Aggregation induced by 11 alpha, 9 alpha-epoxymethano prostaglandin H2 (a stable endoperoxide analogue) was not inhibited by simultaneous addition of ADP-beta-S (50 microM). 5 The behaviour of ADP-beta-S towards human platelets was consistent with it being a partial agonist.

Adenosine Diphosphate↗
PubMed1981

Effects of RP and SP diastereoisomers of adenosine 5'-O-(1-thiodiphosphate) on human platelets.

1 RP and SP diastereoisomers of adenosine 5'-O-(1-thiodiphosphate) ((R)-ADP-alpha-S and (S)-ADP-alpha-S), an adenosine 5'-diphosphate (ADP) analogue, were tested on intact human platelets. 2 Each diastereoisomer induced aggregation, (S)-ADP-alpha-S being 5 times more potent than (R)-ADP-alpha-S but they achieved only 75% of the maximal effect of ADP. 3 Aggregation induced by each diastereoisomer was competitively inhibited by ATP (50 microM). 4 Simultaneous addition of each diastereoisomer inhibited aggregation induced by ADP but not by 11 alpha, 9 alpha-epoxymethano prostaglandin H2, a stable endoperoxide analogue. Both diastereoisomers are therefore partial agonists at the ADP receptor mediating aggregation. 5 Unlike ADP, neither diastereoisomer inhibited prostaglandin E1 (PGE1)-stimulated adenylate cyclase, but each competitively inhibited the effect of aDP, with (S)-ADP-alpha-S again being 5 times more potent than (R)-ADP-alpha-S. 6 These are the first reported examples of ADP analogues to induce platelet aggregation without inhibiting PGE1-stimulated adenylate cyclase.

Adenosine Diphosphate↗
PubMed1981

5'-N-ethylcarboxamidoadenosine: a potent inhibitor of human platelet aggregation.

1 5'-N-ethylcarboxamidoadenosine (NECA) is an adenosine analogue which is 22,900 times more potent than adenosine as a vasodilator. Adenosine and some of its analogues are also inhibitors of human platelet aggregation. NECA was tested for its effects on human platelets. 2 NECA (1 microM) inhibited human platelet aggregation induced by adenosine 5'-diphosphate (ADP), adrenaline, 5-hydroxytryptamine (5-HT) and thrombin more powerfully than adenosine. NECA was 5 to 10 times more potent than adenosine at inhibiting ADP- and adrenaline-induced aggregation. 3 NECA, like adenosine, caused dose-dependent increases in levels of platelet adenosine 3',5'-cyclic monophosphate (cyclic AMP), which were competitively inhibited by theophylline, an adenosine antagonist. 4 These effects of NECA, like those of adenosine, were completely stereospecific as the L-enantiomer of NECA was inactive. 5 NECA did not interfere with the inhibition by ADP of prostaglandin E1 (PGE1)-stimulated adenylate cyclase. 6 NECA is the most potent analogue of adenosine tested so far on human platelets, and is the first example of a 5' modification to retain affinity for the platelet adenosine receptor.

Adenosine↗