Cyclic GMP down-regulates atrial natriuretic peptide receptors on cultured vascular endothelial cells.

Down-regulation of atrial natriuretic peptide (ANP) receptors was investigated using a cultured bovine pulmonary artery endothelial (CPAE) cell line. Endothelial cells have been shown to possess two subtypes of ANP receptors, a guanylate cyclase-coupled receptor (B-receptor) and a clearance receptor (C-receptor). The treatment with APIII, rat ANP (103-126), at concentrations of 10(-8) to 10(-6) M for 24 h, resulted in a significantly (p less than 0.01) greater decrease in maximum 125I-APIII binding to CPAE cells than the identical concentration of API, rat ANP (103-123). APIII at concentrations of 10(-8) to 10(-6) M stimulated cyclic GMP (cGMP) production 3.3-17.5-fold greater than similar concentrations of API. From these findings, we hypothesized that cGMP produced following ANP binding to the B-receptor participates in ANP receptor regulation. M&B 22948, a selective inhibitor of cGMP-specific phosphodiesterase, significantly (p less than 0.01) potentiated the effect of both API and APIII on 125I-APIII binding, while M&B 22948 itself had no significant effect on 125I-APIII binding. Treatment of the cells with 1 mM 8-bromo-cGMP also significantly (p less than 0.01) decreased 125I-APIII binding to the cells, and a potentiation of this effect was observed by M&B 22948. Scatchard analysis of binding data from 8-bromo-cGMP-treated cells showed a significant decrease in Bmax (1.79 +/- 0.15 to 1.20 +/- 0.07 fmol/mg protein, p less than 0.05) without a significant change in Kd. Affinity cross-linking of 125I-APIII to 8-bromo-cGMP-treated cells showed a decrease in the labeling of 60- and 70-kDa bands corresponding to the C-receptor. In addition, the APIII-stimulated cGMP response remained unchanged in the 8-bromo-cGMP-treated cells, indicating that the B-receptor was not down-regulated. We conclude that cGMP regulates ANP-binding sites on the endothelial cell and that the evidence indicates that the C-receptor may preferentially be down-regulated by cGMP in CPAE cells.