Localization and characterization of neutral metalloendopeptidase (EC 3.4.24.11), the degradative enzyme for atrial natriuretic peptide, in rat kidney using a radioiodinated neutral metalloendopeptidase inhibitor.
Atrial natriuretic peptide (ANP) is rapidly degraded by neutral metalloendopeptidase (EC 3.4.24.11, 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 radioinhibitor 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 125I. NEP catalytic activity in the rat kidney was inhibited by SCH47896 and its di-iodo analog SCH48446. Specific binding of [125I]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, [125I]SCH47896 retains full enzymatic inhibitory activity and full binding to the active site of the NEP. Autoradiographs using [125I]SCH47896 demonstrated maximal binding to deep proximal renal tubules. This binding was displaced in a dose-dependent manner by NEP inhibitors. Renal NEP was inhibited by SCH39370. Inhibition of ANP degradation by NEP in the kidney by the new NEP or atriopeptidase inhibitors may explain their natriuretic and diuretic effect in the absence of changes in plasma ANP levels. These studies will allow investigation of the regulation of NEP and the role inhibition of tissue NEP plays in the actions of the new atriopeptidase inhibitors. Furthermore, this method of radioinhibitor binding is applicable to any enzyme, provided a suitable radioligand can be constructed.
Kanazawa
M
M
Department of Medicine, University of Melbourne, Austin Hospital, Heidelberg, Victoria, Australia.
Casley
D J
DJ
Sybertz
E J
EJ
Haslanger
M F
MF
Johnston
C I
CI
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D001345:Autoradiography; D001667:Binding, Competitive; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004347:Drug Interactions; D006020:Glycopeptides / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism* / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D015244:Thiorphan / Q000494:pharmacology
D004151:Dipeptides; D006020:Glycopeptides; D007457:Iodine Radioisotopes; D010636:Phenols; C060631:Sch 39370; C075066:SCH 47896; D010649:Phenylalanine; D009320:Atrial Natriuretic Factor; D015244:Thiorphan; D015260:Neprilysin; C008890:phosphoramidon