Inhibition by guanosine cyclic monophosphate (cGMP) analogues of uptake of[H-3]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 pro benecid, a non-selective transport inhibitor of organic anions. In the pres ent study, various cGMP analogues were tested for their ability to inhibit 3 ' ,5 ' -cGMP efflux and stimulate the cGMP-selective ATPase in human eryt hrocytes. The difference in uptake of 1 muM [H-3]3 ' ,5 ' -cGMP to inside-o ut vesicles in the presence and absence of 1 mM ATP at 37 degrees was defin ed as active transport. Two ATP-dependent components were detected for unla belled 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 inhi bited by the analogues with a typical pattern: Rp-monophosphorothioate guan osine 3 ' ,5 ' -cyclic monophosphate (Rp-cGMPS) > 3 ' ,5 ' -cGMP > 2 ' -O-m onobutyryl guanosine 3 ' ,5 ' -cyclic monophosphate (O-mb-cCMP) approximate to N-2-monobutyryl guanosine 3 ' ,5 ' -cyclic monophosphate (N-mb-cGMP) gr eater than or equal to N-2,2 ' -O-dibutyryl guanosine 3 ' ,5 ' -cyclic mono phosphate (Db-cGMP) approximate to 8 ' -bromo guanosine 3 ' ,5 ' -cyclic mo nophosphate (Br-cGMP) Guanosine 2 ' ,3 ' -cyclic monophosphate (2 ' ,3 ' -c GMP) > Sp-monophosphorothioate guanosine 3 ' ,5 ' -cyclic monophosphate (Sp -cGMPS). A concentration-dependent inhibition was found for the low-affinit y transport, but no distinct order of potency was identified. Analysis acco rding 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 o f 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 abl e to activate the cGMP-specific ATPase, 640 +/- 200% and 430 +/- 160% (mean +/- SEM, N = 5) above basal levels, respectively. The present data show th at the binding is less selective than ATPase activation of the cellular cGM P transport system. (C) 2001 Elsevier Science Inc. All rights reserved.