The H295R human adrenocortical cell line contains functional atrial natriuretic peptide-receptors that inhibit aldosterone biosynthesis

The inhibitory effect of atrial natriuretic peptide (ANP) on angiotensin Ii (AII)-stimulated aldosterone secretion has been previously studied in rat and bovine adrenal zona glomerulosa cells in primary culture. However the understanding of the mode of action of ANP at the molecular level has been hampered by limitations of those primary cell culture systems and by the lack of cell lines from human adrenal cortex. Here we demonstrate the presence of fully functional ANP receptors in the recently characterized AII-responsive adrenocortical carcinoma cell line H295R. Specific saturable binding of I-125-rANP to H295R cell membrane preparations revealed a single class of high affinity binding sites with a density of 20 fmol/mg of protein. The pharmacological profile of this ANP receptor was documented by competitive binding of I-125-rANP with naturally occurring natriuretic peptides. rANP was the most potent with a K-d of 42 pM. pBNP32 was less potent with a K-d of 174 pM. I-125-rANP binding was not competed by pCNP (NPRB-specific ligand) nor by C-ANF (NPRC-specific ligand). Photoaffinity labeling of membrane preparations with I-125-BPA-ANP revealed a single specific protein of molecular weight around 130 kDa. This protein was further identified by immunodetection with a specific antibody directed to the human ANP-specific receptor NPRA. Natriuretic peptides stimulated cGMP production by the receptor-coupled guanylate cyclase with the same specificity. Aldosterone production by AII-stimulated H295R cells was dose-dependently inhibited by rANP with an ED, of 1.5 nM. In addition, we used this model to test two chimeric analogs of ANP and BNP. pBNP1 and pBNP3 were, respectively, 4- and 2-fold more potent than rANP in competing for I-125-rANP binding with K-d of 10 and 20 pM. pBNP1 was 24-fold more potent in inhibiting AII-stimulated aldosterone production with ED(50) of 63 pM. pBNP1 is therefore the most potent natriuretic peptide analog tested. In summary, the human H295R cell line contains NPRA receptors positively coupled to the particulate guanylate cyclase and that antagonize angiotensin II stimulation of aldosterone secretion.