CHARACTERIZATION OF ANF RECEPTORS IN CULTURED RENAL CORTICAL VASCULAR SMOOTH-MUSCLE CELLS

Because atrial natriuretic factor (ANF) has been demonstrated to decrease resistances in cortical renal vessels in vivo, we studied I-125-ANF binding and ANF-dependent guanosine 3',5'-cyclic monophosphate (cGMP) production in subcultured vascular smooth muscle cells (VSMC) prepared from the rabbit renal cortex. I-125-ANF specific binding at 4-degrees-C represented 70% of total binding and reached a plateau at 30-60 min. Equilibrium saturation binding curves suggested one group of high-affinity receptor sites (K(D) = 78 +/- 16 pM, B(max) = 45 +/- 11 fmol/mg) but were compatible with several groups exhibiting close binding parameters. ANF, [Ala7, Ala23]ANF (a linear analogue), and C-ANF-(4-23) (a ligand of C receptors) inhibited I-125-ANF binding at 37-degrees-C with nearly similar potencies. In contrast, at 4-degrees-C, complete or nearly complete inhibition of binding was obtained with ANF and linear ANF, the latter exhibiting the weakest potency, whereas C-ANF-(4-23) displaced only 35% of the tracer. ANF markedly stimulated cGMP accumulation, with a threshold concentration of 10 pM and a stimulation of 115 times basal value at 0.1-mu-M. Linear ANF was also stimulatory with a much weaker potency. Around 25% of I-125-ANF bound to cell surface was internalized at 37-degrees-C. Phenylarsine oxide partially inhibited internalization as well as the inhibitory potency of C-ANF-(4-23) on I-125-ANF binding. As shown by high-performance liquid chromatography extracellular I-125-ANF was rapdily degraded at 37-degrees-C into its I-125-COOH-terminal tripeptide and I-125-Tyr. These results indicate that renal cortical VSMC possess B receptors of high density linked to guanylate cyclase, C receptors, and ectoenzymes able to degrade ANF. Therefore this preparation seems particularly appropriate for studying the cellular mechanisms of the vasoreactive response of the renal cortical vessels to ANF.