Inhibition by guanosine cyclic monophosphate (cGMP) analogues of uptake of [(3)H]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 microM [(3)H]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 microM) with respective K(i) of 1.3 +/- 0.2 and 280 +/- 50 microM (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-cGMP) approximately N(2)-monobutyryl guanosine 3',5'-cyclic monophosphate (N-mb-cGMP) > or = N(2),2'-O-dibutyryl guanosine 3',5'-cyclic monophosphate (Db-cGMP) approximately 8'-bromo guanosine 3',5'-cyclic monophosphate (Br-cGMP) approximately 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 [(3)H]3',5'-cGMP transport (0.2--2 microM) gave a K(m) value of 1.5 +/- 0.1 microM (mean +/- SEM, N = 3). The presence of 10 microM 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 approximately O-mb-cGMP approximately db-cGMP approximately 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.

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 microM) at 37 degrees 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 microns, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 microM. 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 microM, 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.

Cyclic AMP stimulates the cyclic GMP egression pump in human erythrocytes: effects of probenecid, verapamil, progesterone, theophylline, IBMX, forskolin, and cyclic AMP on cyclic GMP uptake and association to inside-out vesicles.

The knowledge about the structure and function of the protein families responsible for cGMP synthesis and metabolic conversion has grown vastly the last years, whereas little is known about proteins that account for the cellular export of cGMP. In the present study, we have employed a model with inside-out vesicles prepared from human erythrocytes to characterize modulation and regulation of cellular cGMP extrusion. The active transport was saturable (Km of 2.4 +/- 0.2 microM, mean +/- SEM, n = 3) and coupled to ATP hydrolysis since no accumulation was detected in the presence of ATP-gamma-S and AMP-PNP. The observation that 100 microM of cAMP caused a minimal inhibition (14.4 +/- 0.3%) of active cGMP transport showed that the extrusion system for cGMP was not shared with cAMP, but a competitive interaction occurred for the ATP-independent association to the inside out vesicles. In contrast, the lowest, but physiological relevant cAMP concentrations (0.1-5 microM) stimulated the active cGMP transport with 30-35%, an observation that suggests cAMP as an allosteric regulator of the cGMP transporter. Several well-known modulators of other energy-requiring membrane transport systems caused a competitive and concentration-dependent inhibition, including verapamil (Ki = 13.0 +/- 2.4 microM), forskolin (Ki = 13.5 +/- 1.4 microM) and probenecid (Ki = 27.0 +/- 1.3 microM). Progesterone, which was the most potent inhibitor (Ki = 2.2 +/- 0.3 microM), interacted with the active cGMP transport in a noncompetitive manner. The highest concentration (100 microM) of IBMX and theophylline reduced the active cGMP uptake with 29.5 +/- 1.9% and 21.6 +/- 2.1%, respectively. None of these substances interfered with the association of cGMP to the vesicles in absence of ATP. The present results show that human erythrocytes possess a cell membrane cGMP transporter which is coupled to an ATPase. Its activity is regulated by cAMP in an apparent allosteric manner and inhibited by substances previously known to interact with other membrane transport systems.