Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells.
Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I-ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 +/- 60 nM and Vmax of 35 +/- 14 pmol of ANP degraded/10 min/10(5) cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0-8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when 125I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.
Frost
S J
SJ
Biomedical Operations and Research Branch, NASA Johnson Space Center, Houston, Texas 77058.
Whitson
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011480:Protease Inhibitors; D009320:Atrial Natriuretic Factor; D010450:Endopeptidases