Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase

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Abstract
We used cultured rat lung fibroblasts to evaluate the role of particulate and soluble guanylate cyclase in the atrial natriuretic factor (ANF)-induced stimulation of cyclic GMP. ANF receptors were identified by binding of 125I-ANF to confluent cells at 37.degree.C. Specific ANF binding was rapid and saturable with increasing concentrations of ANF. The equilibrium dissociation constant (KD) was 0.66 .+-. 0.077 nM and the Bmax. was 216 .+-. 33 fmol bound/106 cells, which corresponds to 130 000 .+-. 20 000 sites/cell. The molecular characteristics of ANF binding sites were examined by affinity cross-linking of 125I-ANF to intact cells with disuccinimidyl suberate. ANF specifically labelled two sites with molecular sizes of 66 and 130 kDa, which we have identified in other cultured cells. ANF and sodium nitroprusside produced a time- and concentration-dependent increase in intracellular cyclic GMP. An increase in cyclic GMP by ANF was detected at 1 nM, and at 100 nM an approx. 100-fold increase in cyclic GMP was observed. Nitroprusside stimulated cyclic GMP at 10 nM and at 1 mM a 500-600-fold increase in cyclic GMP occurred. The simultaneous addition of 100 nM-ANF and 10 .mu.M-nitroprusside to cells resulted in cyclic GMP levels that were additive. ANF increaed the activity of particulate guanylate cyclase by about 10-fold, but had no effect on soluble guanylate cyclase. In contrast, nitroprusside did not alter the activity of particulate guanylate cyclase, but increased the activity of soluble guanylate cyclase by 17-fold. These results demontrate that rat lung fibroblasts contain ANF receptors and suggest that the ANF-induced stimulation of cyclic GMP is mediated entirely by particulate guanylate cyclase.