Rapid receptor-mediated catabolism of 125I-atrial natriuretic factor by vascular endothelial cells

Abstract

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.

Characterization of atrial natriuretic peptide degradation by cell‐surface peptidase activity on endothelial cells

“…1B). The peaks at 15 min and 17 min corresponded to lZ5I-tyrZ8 and arg27-1251-tyr28 respectively, based on previously published data using CIS HPLC columns with similar gradients [6,17,181. Since this degradation occurred at 4"C, proteolytic activity on the surface of CPA47 cells is suggested.…”

Differential antimitogenic effectiveness of atrial natriuretic peptides in primary versus subcultured rat aortic smooth muscle cells: Relationship to expression of ANF‐C receptors

“…Rate of degradation of ANF is similar in primary and subcultured aortic smooth muscle cells The ability of vascular smooth muscle cell cultures to degrade ANF is well established (Johnson et al, 1989). Therefore, the differential effect of ANF in primary vs. subcultured cells could be related to a difference in the degradation of ANF in culture.…”

“…Indeed, a peptidase present in vascular smooth muscle cells that specifically removes the COOH-terminal tripeptide of ANF has been recently demonstrated (Johnson et al, 1989). It is well documented that the endopeptidases 24.11 (enkephalinases) catalyze the cleavage of the Serlz3 Phelz4 bond of rANF,,.,,, (Stephenson and Kenny, 1987), rendering the peptide inactive, at least relative to biological effects thought to be mediated via cGMP as the second messenger.…”

Biochemistry of natriuretic peptides

“…It is possible that other degradative enzymes may fulfil this role. Three enzyme candidates, with similar specificity in cleaving the carboxy-terminal end of ANP, have been found in bovine vascular smooth muscle and endothelial cells [154]. rat brain [155], and neuroblastoma cells, [156] but their importance in ANP metabolism has yet to be determined.…”