Characterization of neutral endopeptidase in vascular smooth muscle cells of rabbit renal cortex.
In addition to biological and clearance receptors for atrial natriuretic factor (ANF), cultured vascular smooth muscle cells (VSMC) from the rabbit renal cortex possess ectoenzymes degrading this hormone. We examined whether neutral endopeptidase (NEP) was implicated in this process. The presence of NEP in VSMC was demonstrated as follows. 1) NEP activity measured from the hydrolysis of a synthetic substrate by intact cells cultured in a medium containing 10% fetal calf serum was 1,609 +/- 65 pmol.min-1 x mg-1 [surface localization of the enzyme was confirmed by low activity (4% of total) in the cytosol; release of NEP activity in the medium was negligible]; 2) a monoclonal antibody directed against rabbit NEP specifically stained VSMC membranes; and 3) mRNA from VSMC hybridized a NEP cDNA probe with a single band as shown by Northern blot analysis. The role of NEP in ANF catabolism was demonstrated by incubating 125I-ANF or unlabeled ANF for increasing periods of time with VSMC in the presence of thiorphan (1-100 microM). Intact hormone estimated by trichloroacetic acid precipitation or radio-immunoassay, respectively, increased markedly compared with control in the presence of this specific inhibitor of NEP. NEP activity was stimulated (x1.6) in quiescent VSMC deprived from serum during 3 days. This effect was dose dependent and was not observed with creatine kinase activity measured as control. NEP expression at the cell surface estimated by sorting of immunostained cells was also increased in the absence of serum. Northern blot analysis showed increases in the mRNA band of NEP with increasing periods of serum deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)
Dussaule
J C
JC
Institut National de la Santé et de la Recherche Médicale 64, Hôpital Tenon, Paris, France.
Stefanski
A
A
Béa
M L
ML
Ronco
P
P
Ardaillou
R
R
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002417:Cattle / Q000097:blood / Q000196:embryology; D002462:Cell Membrane / Q000201:enzymology; D007672:Kidney Cortex / Q000098:blood supply; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D015260:Neprilysin / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012079:Renal Circulation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014018:Tissue Distribution
D012333:RNA, Messenger; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000001:Calcimycin; D009320:Atrial Natriuretic Factor; D015260:Neprilysin; D013755:Tetradecanoylphorbol Acetate