Activity and localization of neutral metalloendopeptidase (EC 3. 4. 24. 11) (NEP), the degradative enzyme for atrial natriuretic peptide (ANP), in rat kidney

Atrial natriuretic peptide (ANP) is degraded by neutral metalloendopeptidase (EC 3. 4. 24. 11) (NEP), and the kidney is the major site of ANP clearance. The regional distribution of NEP in rat kidney was investigated by an enzymatic method and by in vitro autoradiography. The activity of NEP, measured with an enzymatic fluorimetric method employing N-dansy l-D-alanyl-glycyl-L-4-nitrophenylalany 1-glycine as a synthetic substrate, was 18 times higher in the outer stripe and 8 times higher in the inner cortex than in the outer cortex. Low concentrations of NEP were found in the outer cortex, in the inner stripe and in the inner medulla. NEP activity in rat kidney was inhibited by specific NEP inhibitors (phosphoramidon, thiorphan, SCH39370, SCH47896 and SCH 48446) at micromolar concentrations. SCH 47896 is a phenolic derivative of SCH39370 which can be radioiodinated with 125I. SCH48446 is a di-iodo analog of SCH47896. Thus, [125I] SCH47896 retains the full enzymatic inhibitory activity and full biological potency to bind to the active site of NEP. Autoradiographs using [125I] SCH47896 demonstrated maximal binding to regions of the outer stripe of the outer medulla and to the inner cortex, which was consistent with binding to the deep proximal tubules. These bindings were displaced in a dose-dependent manner by NEP inhibitors. Enalaprilat did not displace [125I] SCH47896 binding. EDTA inhibited these bindings by 90%. The present result suggests that degradation of ANP by NEP occurs mainly in the deep proximal tubules, and that the proximal convoluted tubule in the outer cortex is not a major site of location of NEP. These properties enable [125I] SCH47896 to be used as a radioligand for in vitro autoradiography. Using these methods, investigation of the regulation of NEP under different physiological and pathophysiological conditions is possible.