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J Biol Chem, Vol. 275, Issue 4, 2376-2380, January 28, 2000
From the Department of Pharmacology, University of Michigan
Medical School, Ann Arbor, Michigan 48109-0632
Guanabenz, a metabolism-based irreversible
inactivator of neuronal nitric-oxide synthase (nNOS) in
vitro, causes the loss of immunodetectable nNOS in
vivo. This process is selective in that the slowly reversible
inhibitor NG-nitro-L-arginine did
not decrease the levels of nNOS in vivo. To better
understand the mechanism for the loss of nNOS protein in
vivo, we have investigated the effects of guanabenz and
NG-nitro-L-arginine in HEK 293 cells stably transfected with the enzyme. We show here that guanabenz,
but not NG-nitro-L-arginine, caused
the inactivation and loss of nNOS protein in the HEK 293 cells. In
studies with cycloheximide or in pulse-chase experiments with
[35S]methionine, we demonstrate that the loss of nNOS was
due in large part to enhanced proteolysis of the protein with the
half-life decreasing by one-half from 20 to 10 h. Other
metabolism-based irreversible inactivators to nNOS,
NG-methyl-L-arginine, and
N5-(1-iminoethyl)-L-ornithine, but
not the reversible inhibitor 7-nitroindazole (7-NI), caused a similar
decrease in the half-life of nNOS. Proteasomal inhibitors, lactacystin,
Cbz-leucine-leucine-leucinal, and
N-acetyl-leucine-leucine-norleucinal, but not the lysosomal protease inhibitor leupeptin, were found to effectively inhibit the
proteolytic degradation of nNOS. Thus we have shown for the first time
that the irreversible inactivators of nNOS, perhaps through covalent
alteration of the enzyme, enhance the proteolytic turnover of the
enzyme by a mechanism involving the proteasome.
Guanabenz-mediated Inactivation and Enhanced Proteolytic
Degradation of Neuronal Nitric-oxide Synthase*
,
*
This work was supported by National Institutes of Health
Grant ES08365 (to Y. O.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Postdoctoral Fellow of the University of Michigan and Parke-Davis
Partnership in Chemical and Biological Sciences.
§
Trainee under Pharmacological Sciences Training Program GM07767
from the National Institutes of Health.
¶
Recipient of the Burroughs Wellcome Fund New Investigator
Award in Toxicology. To whom correspondence should be addressed: Dept.
of Pharmacology, University of Michigan Medical School, Medical Science
Research Bldg. III, Ann Arbor, MI 48109-0632. Tel.: (734) 936-5797;
Fax: (734) 763-4450; E-mail: osawa@umich.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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