Metabolism of 125I-atrial natriuretic factor by vascular smooth muscle cells. Evidence for a peptidase that specifically removes the COOH-terminal tripeptide.
The addition of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) to cultured bovine aortic smooth muscle cells at 37 degrees C resulted in a rapid clearance from the medium (t1/2 approximately 7.5 min). Within 5 min, [125I]iodotyrosine126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg-[125]iodotyrosine126 (125I-FRY) appeared in the medium. The identities of these degradation products were confirmed by 1) retention time on high performance liquid chromatography (HPLC) relative to standards, 2) products generated by digestion with aminopeptidase M, and 3) the absence of the Met110. Preincubation of the cells with ammonium chloride or chloroquine resulted in a significant increase in the intracellular accumulation of radiolabel, indicative of endocytosis and rapid delivery of 125I-hANF to an acidic intracellular compartment (endosome and/or lysosome). Neither ammonium chloride, chloroquine, nor excess unlabeled hANF blocked the rapid appearance in the medium of 125I-RY or 125I-FRY. Bestatin inhibited the generation of 125I-RY, with a concomitant increase in 125I-FRY, suggesting that the 125I-RY is produced by aminopeptidase action on 125I-FRY. The endopeptidase 24.11 (enkephalinase) inhibitor, SCH 39370, did not inhibit the formation of 125I-FRY. These results provide evidence of a peptidase capable of specifically removing the COOH-terminal tripeptide from 125I-hANF. The COOH-terminal tripeptide, Phe124-Arg-Tyr126, was also isolated from cell digests of hANF by HPLC and its identity confirmed by amino acid analysis. Since it is generally believed that the COOH-terminal tripeptide is critical to many of atrial natriuretic factor-(99-126)'s bioactivities, this enzyme may be involved in the inactivation of atrial natriuretic factor-(99-126) in target tissues.
Johnson
G R
GR
Section of Cardiovascular Biochemistry, Schering-Plough Research, Bloomfield, New Jersey 07003.
Arik
L
L
Foster
C J
CJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors; D006801:Humans; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*
D000596:Amino Acids; D004791:Enzyme Inhibitors; D007457:Iodine Radioisotopes; D009320:Atrial Natriuretic Factor