Parviz Meghji

8-Phenyltheophylline: A potent P1-purinoceptor antagonist

8-Phenyltheophylline was more potent than theophylline in antagonizing the inhibitory effects of adenosine in guinea-pig driven left atrium, rabbit basilar artery and electrically stimulated guinea-pig ileum preparations. In guinea-pig atrium and ileum, the antagonism of adenosine responses by the methylxanthines is of a competitive nature, but in rabbit basilar artery it does not appear to be so. In all three preparations, the effects of theophylline and 8-phenyltheophylline could be reversed by washing. It is concluded that 8-phenyltheophylline is a more potent P1-purinoceptor antagonist than theophylline.

Distribution of P1- and P2- purinoceptors in the guinea-pig and frog heart

1 The effects of adenyl compounds were examined on the guinea‐pig and frog heart in terms of the P1/P2‐purinoceptor hypothesis. 2 The effects of two slowly degradable adenosine 5′‐triphosphate (ATP) analogues; β, γ‐methylene adenosine 5′‐triphosphate (APPCP) and α, β‐methylene adenosine 5′‐triphosphate (APCPP) were also examined. 3 Adenosine, adenosine 5′‐monophosphate (AMP), adenosine 5′‐diphosphate (ADP), ATP and APPCP produced inhibitory effects in guinea‐pig atria. These inhibitory effects were antagonized competitively by theophylline and potentiated by dipyridamole. APCPP did not produce a similar inhibitory response. 4 Guinea‐pig ventricles were insensitive to adenyl compounds. 5 ATP and ADP produced initial excitatory effects in frog atria which were followed by inhibitory effects. Adenosine and AMP produced inhibitory effects alone whereas APCPP produced excitatory effects only. The inhibitory effects were antagonized competitively by theophylline and potentiated by dipyridamole. 6 ATP, ADP, APPCP and APCPP evoked excitatory responses in frog ventricles. These responses were not affected by theophylline or dipyridamole. Adenosine and AMP were inactive on frog ventricles. 7 It is concluded that only P1‐receptors are present in guinea‐pig atria; that both P1‐ and P2‐receptors are present in frog atria; and that only P2‐receptors are present in frog ventricles. No evidence was found for the presence of either P1 or P2‐purinoceptors in guinea‐pig ventricles.

Studies on the stereoselectivity of the P2‐purinoceptor

1 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. 2 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. 3 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. 4 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. 5 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.

The effect of adenyl compounds on the rat heart.

1 The effects of adenyl compounds were examined on the rat atrium and ventricle. 2 Adenosine, adenosine 5′‐monophosphate, adenosine 5′‐diphosphate, adenosine 5′‐triphosphate (ATP) and β, γ‐methylene ATP (APPCP) produced negative inotropic effects on the rat atrium. These inhibitory effects were antagonized by 8‐phenyltheophylline (8‐PT), a P1‐purinoceptor antagonist, and potentiated by erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (EHNA), an adenosine deaminase inhibitor, but were not affected by dipyridamole, which blocks adenosine uptake. 3 α, β‐Methylene ATP (APCPP), which is resistant to degradation, did not produce a similar inhibitory response in the rat atrium. 4 Adenosine did not affect the basal developed force of the rat ventricle nor did it affect the β‐adrenoceptor‐mediated positive inotropic effect. 5 Very high concentrations of ATP (0.1–3 mM) produced negative inotropic effects in the rat ventricle. APPCP (0.3 mM) also produced an inhibitory response, which was significantly smaller than that produced by ATP (0.3 mM). APCPP elicited excitation rather than the expected inhibitory response. 6 The inhibitory effect of ATP in the rat ventricle was not blocked by indomethacin, 8‐PT or atropine. 7 It impossible that the action of ATP in the rat ventricle is mediated via P2‐purinoceptors and that the lack of inhibitory action of APCPP on the rat ventricle is due to the difference in structural conformation between ATP and APCPP. 8 It seems likely that inhibitory P1‐purinoceptors are present in the rat atrium and that the inhibitory responses produced by ATP in the rat ventricle may be mediated via P2‐purinoceptors.

Inhibition of extracellular ATP degradation in endothelial cells.

The plasma membrane ATPase on the human umbilical vein endothelial cell line (ECV304) was demonstrated to be an ecto-enzyme. Hydrolysis of ATP was measured by monitoring the appearance of inorganic phosphorus. Hydrolysis of extracellular ATP was insensitive to oligomycin, vanadate, ouabain and N-ethylmaleimide, compounds that inhibit the intracellular ion pumping ATPases. Beta-Glycerophosphate (1-10 mM) or p-nitrophenyl phosphate (1-10 mM) did not inhibit hydrolysis of ATP, ruling out the involvement of non-specific phosphatases. Enzyme activity in buffer that had previously been incubated with cells was < 7%, showing that the enzyme activity measured did not result from release of intracellular enzymes. Consistent with this, the cell preparations used were estimated to be > 95% intact as judged by release of cytosolic enzyme lactate dehydrogenase. The enzyme activity was Ca2-/Mg2- dependent. Gramicidin S (20 microM), suramin (100-300 microM), chlorpromazine (250 microM), trifluoperazine (50-250 microM), and thioridazine (100 microM) inhibited the hydrolysis of ATP (3 mM) by 45-80%. The percentage inhibition produced by these substances was not altered in the presence of a concentration of alpha, beta-methylene ADP (10 microM) which inhibited hydrolysis of AMP (3 mM) by 90%, suggesting that these compounds inhibit ecto-ATPase and/or ecto-ADPase. Measurements of absolute amounts of ATP released from various tissues, including the heart, have been hindered because ATP is rapidly and sequentially hydrolysed to adenosine. Identification of compounds that inhibit ATP degradation would prove to be useful to overcome this problem and would lead to the development of invaluable pharmacological tools in many other aspects of purine research.

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 > 2‐chloroadenosine > 2‐azidoadenosine > 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 > 2‐chloro‐l‐adenosine > 2‐azido‐l‐adenosine > 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.

The effect of adenyl compounds on the heart of the dogfish, Scyliorhinus canicula

The effects of adenyl compounds were examined on dogfish atria and ventricles. Adenosine, ATP, beta, gamma-methylene ATP ( APPCP ) and 2-chloroadenosine produced negative inotropic and chronotropic effects on the dogfish atrium, which were antagonized by 8-phenyltheophylline, a P1- purinoceptor antagonist. alpha-beta-Methylene ATP ( APCPP ), which is resistant to degradation, did not produce a similar inhibitory response in the dogfish atrium. Atropine did not affect the responses to adenosine, indicating that adenosine did not produce its effects indirectly by the release of acetylcholine. The effects of adenosine and ATP were not potentiated by dipyridamole, which blocks adenosine uptake; and 2-chloroadenosine, which is reported to be resistant to uptake and deamination, was equipotent with adenosine; this suggests the absence of an adenosine uptake system. Dogfish ventricles were insensitive to adenyl compounds. Adrenaline, noradrenaline and acetylcholine produced positive inotropic effects on the ventricle. It is concluded that inhibitory P1- purinoceptors are present in the dogfish atrium. However, adenyl compounds had no direct action on the contractility of the dogfish ventricle.

Actions of some autonomic agents on the heart of the trout (Salmo gairdneri) with emphasis on the effects of adenyl compounds

The effects of some autonomic agents including adrenaline, noradrenaline, dopamine, 5-hydroxytryptamine (5-HT), histamine, acetylcholine and adenyl nucleotides and nucleosides were examined on atria and ventricles of the trout (Salmo gairdneri). In atria, adrenaline, noradrenaline and dopamine produced positive chronotropic and negative or positive inotropic effects. Acetylcholine and 5-HT produced negative inotropic effects; 5-HT also produced small positive chronotropic effects. Adenosine and ATP produced negative inotropic and positive chronotropic effects; these effects were not antagonized by 8-phenyltheophylline nor were they potentiated by dipyridamole. alpha, beta-Methylene ATP, which is resistant to degradation, was inactive. These results suggest that, unlike all other vertebrate hearts studied, ATP and adenosine do not act via P1- or P2-purinoceptors. In the ventricle, adrenaline and noradrenaline produced positive inotropic effects. Acetylcholine and adenyl compounds had no direct action on contractility.

An unusual excitatory action of adenosine on the ventricular muscle of the south african clawed toad (Xenopus laevis)

The effects of adenyl compounds and some of their analogues were examined on atrial and ventricular muscle of Xenopus laevis. In contrast to all heart muscle preparations studied previously, adenosine produced excitation of the ventricle of Xenopus. 2-Chloroadenosine, ATP, beta, gamma-methylene ATP (APPCP) and alpha, beta-methylene ATP (APCPP) also elicited excitation of Xenopus ventricles; the order of potency being 2-chloroadenosine greater than ATP greater than or equal to adenosine greater than APPCP = APCPP. The excitatory effects of 2-chloroadenosine, adenosine, ATP and APPCP were antagonised by 8-phenylthe-ophylline (8-PT). A small excitatoty response to ATP persisted in the presence of 8-PT. The effects of adenosine were not potentiated by dipyridamole. Propranolol antagonised the excitatory responses to adrenaline, phenylephrine, 5-hydroxytryptamine (5-HT) and dopamine. High concentrations of histamine were needed to produce even small excitatory responses. Neither propranolol nor phentolamine affected the responses to adenosine and ATP. Adenosine, ATP and APPCP elicited inhibition of Xenopus atria which was antagonised by 8-PT but was not potentiated by dipyridamole. APCPP, which is resistant to degradation, did not produce a similar inhibitory response. It is concluded that excitation, mediated largely by excitatory P1- and possibly by some P2-purinoceptors, occurs in the Xenopus ventricle and that there are inhibitory P1-purinoceptors in the Xenopus atrium.