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that the proteolytic degradation of the endogenous nNOS in
brain homogenate, as measured by Western blotting, is pre-
vented by the peptidic calpain inhibitor (Fig. 7). Calpains are
ubiquitous Ca2ϩ-dependent proteases that selectively cleave
a wide variety of substrates. Furthermore, the earlier report
that leupeptin, pepstatin A, phenylmethylsulfonyl fluoride,
and soybean trypsin inhibitor do not prevent the loss of
nNOS activity in rat brain homogenates is consistent with a
key role for calpain versus other proteolytic activities in
triggering the inactivation of the nNOS isoforms (Mittal and
Hadhav, 1994). The finding that calpain plays a key role in
the degradation of nNOS is in accord with the report that
calpain helps to limit the activity of iNOS in RAW 264.7 cells
(Walker et al., 1996).
⅐
Because NO is a short-lived free radical, regulation of
⅐
signaling occurs largely at the level of NO synthesis. Both
the nNOS␣ and nNOS isoforms are rapidly degraded by a
Ca2ϩ-dependent protease; therefore, it is possible to envision
⅐
a regulatory effect of protein degradation on NO production.
Activation of nNOS by Ca2ϩ would be followed by Ca2ϩ
-
dependent activation of calpain, which in turn would contrib-
⅐
ute to control of the level of NO biosynthesis through nNOS
catabolism. Proteolytic digestion of the enzyme, like its ca-
talysis-dependent inactivation, provides a mechanism for
⅐
preventing the elevated concentrations of NO that are toxic
to the cell.
The degradation of nNOS by calpain may play a significant
role in Duchenne muscular dystrophy. It has been reported
that nNOS is absent from the skeletal muscle sarcolemma of
Duchenne muscular dystrophy patients (Brenman et al.,
1995; Chang et al., 1996). nNOS is also deficient in the
skeletal muscle of young mdx mice, an animal model for
Duchenne muscular dystrophy (Chang et al., 1996). Further-
more, calpain is overexpressed and activated in the skeletal
muscle sarcolemma of deceased mdx mice (Spencer et al.,
1995; Spencer and Tidball, 1996). Our results suggest that
elevated levels of active calpain may accelerate the degrada-
tion of nNOS and thus contribute to the deficiency of nNOS
in the skeletal muscle sarcolemma of Duchenne muscular
dystrophy patients and mdx mice.
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11467.
In conclusion, the nNOS␣ and nNOS isoforms are bio-
chemically closely related but are not identical. Of particular
interest is the demonstration that nNOS is rapidly degraded
by calpain, a Ca2ϩ-dependent protease. This degradation
may be involved in regulation of the cellular concentration of
⅐
NO and may contribute to the deficiency of nNOS in some
muscular disorders.
Acknowledgments
Russell DH and Snyder SH (1969) Amine synthesis in regenerating rat liver: ex-
tremely rapid turnover of ornithine decarboxylase. Mol Pharmacol 5:253–262.
Sato K, Saito Y, and Kawashima S (1995) Identification and characterization of
membrane-bound calpains in clathrin-coated vesicles from bovine brain. Eur
J Biochem 230:25–31.
Schmidt HH, Pollock JS, Nakane M, Gorsky LD, Forstermann U, and Murad F
(1991) Purification of a soluble isoform of guanylyl cyclase-activating-factor syn-
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Sheta EA, McMillan K, and Masters BSS (1994) Evidence for a bidomain structure
of constitutive cerebellar nitric oxide synthase. J Biol Chem 269:15147–15153.
Silvagno F, Xia H, and Bredt DS (1996) Neuronal nitric-oxide synthase-, an alter-
natively spliced isoform expressed in differentiated skeletal muscle. J Biol Chem
271:11204–11208.
Spencer MJ, Croall DE, and Tidball JG (1995) Calpains are activated in necrotic
fibers from mdx dystrophic mice. J Biol Chem 270:10909–10914.
Spencer MJ and Tidball JG (1996) Calpain translocation during muscle fiber necro-
sis and regeneration in dystrophin-deficient mice. Exp Cell Res 226:264–272.
We thank David S. Bredt and Houhui Xia (University of Califor-
nia, San Francisco, CA) for the nNOS cDNA, Emanuel E. Strehler
(Mayo Clinic, Rochester, MN) for the human CaM cDNA, and M.
Almira Correia (University of California, San Francisco, CA) for the
rat brain and muscle tissue.
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