Adenine nucleotide metabolism and nucleoside transport in human erythrocytes under ATP depletion conditions

Abstract

The adenine nucleotides of human red cells were labeled by incubation of the cells with [ 3H]adenosine. Then, the cells were incubated in Tris-saline with various supplements that cause the loss of cellular ATP, and the degradation products were quantitated as a function of time of incubation at 37°C. Incubation of the cells with 2.5 or 5 mM iodoacetate, iodoacetamide or 1 mM HCHO in combination with 5 mM KF and 50 mM deoxyglucose, 50 mM d-glucose or 10 mM inosine was most efficient in depleting the cells of ATP (100% in 0.5–1 h) without causing cell lysis. In iodoacetate- and iodoacetamide-treated cells practically all catabolism of ATP occurred via ADP → AMP → IMP → inosine → hypoxanthine with hypoxanthine accumulating in the medium. In HCHO-treated cells and in cells incubated in Tris-saline or in Tris-saline with deoxyglucose with and without KF, a substantial proportion of ATP (up to 50%) was catabolized via ADP → AMP → adenosine → inosine → hypoxanthine . Under all conditions, AMP deamination and IMP and AMP hydrolysis were rate-limiting reactions. IMP degradation was more rapid in iodoacetamide- and HCHO-treated than in iodoacetate-treated red cells. It was also more rapid in fresh than in outdated red cells, and it was inhibited by P i. Treatment with iodoacetamide and HCHO under ATP-depletion conditions resulted in a 60–80% inhibition of uridine transport by the cells. Treatment with iodoacetate or deoxyglucose plus KF had only minor effects on nucleoside transport; thus, cells treated in this manner might be useful for studying the transport of adenosine and deoxyadenosine under conditions were their phosphorylation is prevented.