Characterization, localization and quantification of neutral metalloendopeptidase, the degradative enzyme for atrial natriuretic peptide

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Atrial natriuretic peptide (ANP) is rapidly degraded by neutral metalloendopeptidase (NEP), with the kidney being a major site of ANP clearance. NEP has been anatomically localized in the rat kidney by in vitro autoradiography and the active site studied by a radioinhibitory binding assay (RIBA) using a newly developed radioinhibitor as a radioligand. SCH47896 is a phenolic derivative of SCH39370, a potent specific inhibitor of NEP, which can be radioiodinated with ^<125>I.NEP catalytic activity in the rat kidney was inhibited by SCH47896 and its di-iodo analog SCH48446. Specific binding of [^<125>I]SCH47896 to renal plasma membranes fitted a single-site model with Kd = 43.3 nM and maximal binding site density = 13.8 pmol/mg protein. Thus, [^<125>I]SCH47896 retains full enzymatic inhibitory activity and full binding to the active site of the NEP.Autoradiographs using [^<125>I]SCH47896 demonstrated maximal binding to deep proximal renal tubules. This binding was displaced in a dose-dependent manner by NEP inhibitors. The present studies suggest 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 [^<125>I]SCH47896 to be used as a radioligand for RIBA and in vitro autoradiography. Using these methods, investigation of the regulation of NEP under different physiological and pathophysiological conditions is possible. Furthermore, this method of radioinhibitor binding is applicable to any enzyme, provided a suitable radioligand can be constructed.