Characterization of angiotensin IV-degrading enzymes and receptors on rat mesangial cells.
Because mesangial cells (MC) are a target and a degradation site for angiotensin II (ANG II), we characterized the degrading enzymes and receptors of ANG IV, a metabolite of ANG II, on these cells. ANG IV was metabolized into its NH2-terminal deleted peptides, ANG II-(4-8), ANG II-(5-8), and ANG II-(6-8) by rat MC. Total protection of ANG IV was obtained only when PC-18, a specific aminopeptidase N (APN) inhibitor, and JFH-27A, a mixed inhibitor of dipeptidylaminopeptidase (DAP) and neutral endopeptidase (NEP), were simultaneously added. In contrast, thiorphan, an NEP inhibitor, was inactive. These results demonstrate the exclusive role of APN and DAP in ANG IV degradation. 125I-labeled ANG IV binding was studied in the presence of PC-18 and JFH-27A to suppress ligand degradation. Under these conditions, ANG IV-specific receptors could be demonstrated with a KD of 1.8 nM and a density of 55 fmol/mg. In contrast with MC, no evidence for ANG IV receptors could be obtained in freshly isolated glomeruli. ANG IV stimulated cytosolic calcium concentration in MC, whereas its NH2-terminal deleted metabolites were inactive. Therefore, ANG IV must be protected from degradation by APN and DAP in studies on the nonimmediate biological effects of this peptide.
Institut National de la Santé et de la Recherche Médicale (INSERM) 64, Hôpital Tenon, 75020 Paris, France.
D
D
Chansel
S
S
Czekalski
S
S
Vandermeersch
E
E
Ruffet
M C
MC
Fournié-Zaluski
R
R
Ardaillou
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011945:Receptors, Angiotensin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D010446:Peptide Fragments; D011945:Receptors, Angiotensin; D011994:Recombinant Proteins; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D007703:Peptidyl-Dipeptidase A; D002118:Calcium