Adenine nucleotide analogues, including gamma-phosphate-substituted analogues, are metabolised extracellularly in innervated frog sartorius muscle

Abstract

The metabolism of adenine nucleotides and of their analogues by ecto-enzymes in the innervated frog sartorius muscle was investigated with HPLC. The breakdown of beta, gamma-methylene-ATP was also evaluated by studying the ability of the adenosine uptake inhibitor, dipyridamole, and of the adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), to modify the effect of beta, gamma-methylene-ATP on nerve-evoked twitches. ATP-gamma-S at low (10 microM) but not at high (> or = 100 microM) concentrations was quickly metabolised into a compound with a higher negative charge. L-ATP, homo-ATP and 2-methylthio-ATP were metabolised into compounds with a lower negative charge. Beta-gamma-Imido-ATP and gamma-anilino-ATP were only metabolised slightly. As determined by HPLC, beta, gamma-methylene-ATP was not metabolised. In contrast, this ATP analogue inhibited nerve-evoked twitch responses, an effect which was potentiated by dipyridamole and antagonised by DPCPX. Alpha, beta-Methylene-ATP was dephosphorylated into alpha, beta-methylene-ADP, which was virtually resistant to metabolism in the absence of ATP. In the presence of ATP, alpha, beta-methylene-ADP was transiently phosphorylated into alpha, beta-methylene-ATP. Formation of ATP from ADP was observed even in the absence of an exogenous phosphate donor, and was prevented by the adenylate kinase inhibitor, P1P5-di-(adenosine-5')pentaphosphate (AP5A). AP5A caused only partial inhibition of AMP formation from ADP. The results suggest that some ATP analogues with substitutions in the gamma-phosphate, such as ATP-gamma-S and beta, gamma-methylene-ATP, are metabolised in the innervated frog sartorius muscle. The ADP analogue, alpha, beta-methylene-ADP, might be a substrate for an ecto-nucleoside diphosphate kinase. ADP, besides being dephosphorylated, is also a substrate for an ecto-adenylate kinase in innervated frog sartorius muscle.