CHARACTERIZATION OF NEUTRAL ENDOPEPTIDASE IN VASCULAR SMOOTH-MUSCLE CELLS OF RABBIT RENAL-CORTEX

In addition to biological an clearance receptors for atrial natriuretic factor (ANF), culture vascular smooth muscle cells (VSMC) from the rabbit renal cortex possess ectoenzymes degrading this hormone. We examined whether neutral endopeptidase (NEP) was implicated in this process. The presence of NEP in VSMC was demonstrated as follows. 1) NEP activity measured from the hydrolysis of a synthetic substrate by intact cells cultured in a medium containing 10% fetal calf serum was 1,609 +/- 65 pmol . min-1 . mg-1 [surface localization of the enzyme was confirmed by low activity (4% of total) in the cytosol; release of NEP activity in the medium was negligible]; 2) a monoclonal antibody directed against rabbit NEP specifically stained VSMC membranes; and 3) mRNA from VSMC hybridized a NEP cDNA probe with a single band as shown by Northern blot analysis. The role of NEP in ANF catabolism was demonstrated by incubating I-125 ANF or unlabeled ANF for increasing periods of time with VSMC in the presence of thiorphan (1-100 muM). Intact hormone estimated by trichloroacetic acid precipitation or radioimmunoassay, respectively, increased markedly compared with control in the presence of this specific inhibitor of NEP. NEP activity was stimulated (x1.6) in quiescent VSMC deprived from serum during 3 days. This effect was dose dependent and was not observed with creatine kinase activity measured as control. NEP expression at the cell surface estimated by sorting of immunostained cells was also increased in the absence of serum. Northern blot analysis showed increases in the mRNA band of NEP with increasing periods of serum deprivation. The effect of serum deprivation was potentiated by dexamethasone (x1.23 at 100 nM) and by 8-bromoguanosine 3',5'-cyclic monophosphate (cGMP) (x1.62 at 10 muM), whereas it was abolished by a combined treatment with phorbol 12-myristate 13-acetate, an activator of protein kinase C, and A23187 calcium ionophore. These results demonstrate the role of NEP in the catabolism of ANF by VSMC and the inhibitory influence on the expression of this enzyme of unknown serum factors contrasting with the stimulatory effect of glucocorticoids and cGMP.