Inhibition by guanosine cyclic monophosphate (cGMP) analogues of uptake of [3H]3',5'-cGMP without stimulation of ATPase activity in human erythrocyte inside-out vesicles

The cellular extrusion of guanosine 3 ' ,5 ' -cyclic monophosphate (3 ' ,5 ' -cGMP) is a unidirectional ATP-dependent process that is inhibited by probenecid, a non-selective transport inhibitor of organic anions. In the present study, various cGMP analogues were tested for their ability to inhibit 3 ' ,5 ' -cGMP efflux and stimulate the cGMP-selective ATPase in human erythrocytes. The difference in uptake of 1 muM [H-3]3 ' ,5 ' -cGMP to inside-out vesicles in the presence and absence of 1 mM ATP at 37 degrees was defined as active transport. Two ATP-dependent components were detected for unlabelled 3 ' ,5 ' -cGMP (0.01-100 muM) with respective K-i of 1.3 +/- 0.2 and 280 +/- 50 muM (mean +/- SEM, N = 3). The high-affinity transport was inhibited by the analogues with a typical pattern: Rp-monophosphorothioate guanosine 3 ' ,5 ' -cyclic monophosphate (Rp-cGMPS) > 3 ' ,5 ' -cGMP > 2 ' -O-monobutyryl guanosine 3 ' ,5 ' -cyclic monophosphate (O-mb-cCMP) approximate to N-2-monobutyryl guanosine 3 ' ,5 ' -cyclic monophosphate (N-mb-cGMP) greater than or equal to N-2,2 ' -O-dibutyryl guanosine 3 ' ,5 ' -cyclic monophosphate (Db-cGMP) approximate to 8 ' -bromo guanosine 3 ' ,5 ' -cyclic monophosphate (Br-cGMP) Guanosine 2 ' ,3 ' -cyclic monophosphate (2 ' ,3 ' -cGMP) > Sp-monophosphorothioate guanosine 3 ' ,5 ' -cyclic monophosphate (Sp-cGMPS). A concentration-dependent inhibition was found for the low-affinity transport, but no distinct order of potency was identified. Analysis according to Lineweaver-Burk of active [H-3]3 ' ,5 ' -cGMP transport (0.2-2 muM) gave a K-m value of 1.5 +/- 0.1 muM (mean +/- SEM, N = 3). The presence of 10 muM cGMP analogues did not change the ordinate intercept, but made the slopes steeper with a typical order: Rp-cGMPS > 3 ' ,5 ' -cGMP > N-mb-cGMP approximate to O-mb-cGMP approximate to db-cGMP approximate to 8-Br-cGMP > 2 ' ,3 ' -cGMP > Sp-cGMPS. Only 3 ' ,5 ' -cGMP and 2 ' ,3 ' -cGMP were able to activate the cGMP-specific ATPase, 640 +/- 200% and 430 +/- 160% (mean +/- SEM, N = 5) above basal levels, respectively. The present data show that the binding is less selective than ATPase activation of the cellular cGMP transport system. (C) 2001 Elsevier Science Inc. All rights reserved.