Adenine nucleotide analogues, including γ-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 β,γ-methylene-ATP was also evaluated by studying the ability of the adenosine uptake inhibitor, dypiridamole, and of the adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), to modify the effect of β,γ-methylene-ATP on nerve-evoked twitches. ATP-γ-S at low (10 μM) but not at high (⩾ 100 μM) 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. β-γ-Imido-ATP and γ-anilino-ATP were only metabolised slightly. As determined by HPLC, β,γ-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. α,β-Methylene-ATP was dephosphorylated into α,β-methylene-ADP, which was virtually resistant to metabolism in the absence of ATP. In the presence of ATP, α,β-methylene-ADP was transiently phosphorylated into α,β-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, P 1P 5-di-(adenosine-5′)pentaphosphate (AP 5A). (AP 5A) caused only partial inhibition of AMP formation from ADP. The results suggest that some ATP analogues with substitutions in the γ-phosphate, such as ATP-γ-S and β,γ-methylene-ATP are metabolised in the innervated frog sartorius muscle. The ADP analogue, α,β-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.