Export of guanosine 3′,5′-cyclic monophosphate (cGMP) from human erythrocytes characterized by inside-out membrane vesicles

Abstract

The present study was undertaken to characterize the export of cGMP from human erythrocytes at 37°C. Inside-out membrane vesicles were exposed to cGMP and [³H]-cGMP in the presence and absence of 2mmol l⁻¹ ATP. In the absence of ATP, an equilibrium was reached within 15 min for the lowest tested concentration (0.65 μmol l⁻¹), and the amount of cGMP in the vesicles was linearly correlated to the cGMP concentrations in the incubate. These observations suggest that the ATP-independent process represents passive diffusion or non-saturated binding to membrane components. In the presence of ATP, cGMP accumulated linearly during the test period (up to 120 min) and the transport into the inside-out vesicles was dependent on both low- and high-K_m transport. The kinetic parameters for the low-K_m process were determined after 5 and 120 min, the K_m values being 4.6 (SD 1.9) and 4.7 (SD 1.1) μmol 1⁻-¹ (n = 3), respectively. The corresponding V_max values were 400 (SD 50) and 440 (SD 70) fmol mg⁻¹ min⁻¹. The high-K_m process was characterized by K_m = 170 (SD 50) pmol 1⁻-¹ and V_max=1610 (SD 280) fmol mg⁻¹ min⁻¹ (n = 5). The present data demonstrate an ATP-requiring saturable transport system for cGMP in human erythrocytes.

Key Words:

• ATP cGMP
• Erythrocytes
• inside-out vesicles
• transport