Rapid receptor-mediated catabolism of 125I-atrial natriuretic factor by vascular endothelial cells.
The binding, internalization and degradation of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) by cultured bovine aortic endothelial cells (BAECs) were studied at 37 degrees C. 125I-hANF was rapidly cleared from the extracellular medium (t1/2 approximately 10 min), whereas preincubation of the cells in the presence of 20 mM-NH4Cl or 0.2 mM-chloroquine resulted in a significant inhibition of this process. The BAECs rapidly produce three major degradation products of 125I-hANF, namely [125I]iodotyrosine 126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg125-[125I]iodotyrosine126(125I-FRY), which were detected in the extracellular medium. NH4Cl and chloroquine acted to inhibit the generation of 125I-Y and 125I-RY, but not that of 125I-FRY. Furthermore, excess unlabelled hANF (300 nM) completely blocked the rapid production of 125I-Y and 125I-RY in the first 5 min, but only partially (49%) inhibited the generation of 125I-FRY. Thus, in contrast with our previous findings with cultured smooth-muscle cells [Johnson, Arik & Foster (1989) J. Biol. Chem. 264, 11637-11642], BAECs bind, internalize and rapidly degrade 125I-hANF, resulting in the release of 125I-Y and 125I-RY into the extracellular medium. Similarly to smooth-muscle cells, the BAECs generate 125I-FRY from 125I-hANF via an extracellular proteolytic event. The rapidity of the receptor-mediated process and its sensitivity to NH4Cl and chloroquine suggest that the 125I-hANF is proteolytically processed in the endosomes of BAECs and that its receptors cycle between the cell surface and intracellular stores.
Johnson
G R
GR
Section of Cardiovascular Biochemistry, Schering-Plough Research, Bloomfield, NJ 07003.
Arik
L
L
Pitts
B J
BJ
Foster
C J
CJ
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007470:Monoiodotyrosine / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*
D004151:Dipeptides; D007457:Iodine Radioisotopes; D009842:Oligopeptides; D011956:Receptors, Cell Surface; C064796:arginyliodotyrosine; C064797:phenylalanyl-arginyl-iodotyrosine; D000643:Ammonium Chloride; D009320:Atrial Natriuretic Factor; D002738:Chloroquine; D017461:Receptors, Atrial Natriuretic Factor; D007470:Monoiodotyrosine