The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [H-3]-cGMP to inside-out vesicles from human erythrocytes

An ATP-dependent transport system is responsible for the cellular extrusion of cGMP. The objective of the present study was to determine the effect of Mg2+, ATP and other nucleotides (2'-dATP, GTP and ADP), exogenous ATPase m odulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A(1) and o ligomycin A) on the cGMP transport. The uptake of [H-3]-cGMP (1 mu M) at 37 degrees C was studied in inside-out vesicles from human erythrocytes. Magn esium caused a maximal activation between 5 and 10 mM and the optimal ATP c oncentration was 1.25 mM with K-50-values of 0.3-0. 5 mM. Among other nucle otides tested, 2'-dATP (K-50 Of 0.7 mM) was nearly as effective as ATP, whe reas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate ( P-type ATPase inhibitor) showed to be competitive inhibitors with K-i value s of 0.15 mM and 10 mu m, respectively. NEM (a sulphydryl agent) reduced th e ATP-dependent uptake in a concentration-dependent manner with a K-i value of 10 mu M. Ouabain (Na+/K+ -ATPase inhibitor) had no effect. Bafilomycin A(1) (V-type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) wer e the most potent inhibitors with K-i values of 0.7 and 1.8 mu M, respectiv ely. The present study suggests that the cellular cGMP extrusion is energiz ed by an ATPase with a unique inhibitor profile, which clearly differentiat es it from the other major classes of membrane-bound ATPases.