The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [3H]-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 modulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A1 and oligomycin A) on the cGMP transport. The uptake of 3H]-cGMP (1 mu M) at 37 C was studied in inside-out vesicles from human erythrocytes. Magnesium caused a maximal activation between 5 and 10 mM and the optimal ATP concentration was 1.25 mM with K50-values of 0.3-0.5 mM. Among other nucleotides tested, 2'-dATP (K50 of 0.7 mM) was nearly as effective as ATP, whereas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate (P-type ATPase inhibitor) showed to be competitive inhibitors with Ki values of 0.15 mM and 10 mu m, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 mu M. Ouabain (Na+/K+-ATPase inhibitor) had no effect. Bafilomycin A1 (V- type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) were the most potent inhibitors with Ki values of 0.7 and 1.8 mu M, respectively. The present study suggests that the cellular cGMP extrusion is energized by an ATPase with a unique inhibitor profile, which clearly differentiates it from the other major classes of membrane-bound ATPases.