Characterization of membrane-associated peptidase activities expressed by endothelial cells of the ovine median eminence.
The capillary endothelial cells of the median eminence represent a potential site for the degradation/modification of both circulating and hypothalamic peptides passing through the hypophysial portal system toward the pituitary. This study examines endothelial cell peptidase expression in vitro by monitoring the metabolism of gonadotropin-releasing hormone (GnRH) by cultured endothelial cells from sheep median eminence. Cleavage of GnRH by median eminence endothelial cell membranes generated GnRH1-5 as the primary stable product, which was then degraded to GnRH1-3 and free amino acids. Degradation of GnRH was completely inhibited by TPCK, ZnCl2 and N-ethylmaleimide, and partially inhibited by EDTA and by a specific inhibitor of the metalloendopeptidase EC 3.4.24.15, CFP-AAY-pAB. Interestingly, an increase in GnRH1-9 production was seen with the latter inhibitors, suggesting a two-step mechanism of GnRH degradation involving a primary cleavage at the Pro9-Gly10-NH2 bond, inhibitable by TPCK, ZnCl2, and NEM, followed by cleavage by EC 3.4.24.15 to generate GnRH1-5. Phosphoramidon and angiotensin converting enzyme inhibitors (as well as other non-specific inhibitors) were without effect, indicating that endopeptidase EC 3.4.24.11 and angiotensin converting enzyme are not involved. Neither bovine aortic endothelial cell nor AtT-20 cell membranes exhibited this pattern of peptidase activity. Degradation of GnRH by intact median eminence endothelial cells in culture was also observed, suggesting an extracellular orientation for these enzymes; the potential role of such peptidases in the fine regulation of both pituitary function and local blood flow is currently under investigation.
Lew
R A
RA
Peptide Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia.
Tetaz
T
T
Smith
A I
AI
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008473:Median Eminence / Q000098:blood supply / Q000166:cytology / Q000201:enzymology*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000096:biosynthesis*; D012039:Regional Blood Flow / Q000502:physiology; D012756:Sheep
D004791:Enzyme Inhibitors; D007987:Gonadotropin-Releasing Hormone; D010447:Peptide Hydrolases