Neuronal Nitric Oxide Synthase Isoforms α and μ Are Closely Related Calpain-Sensitive Proteins

Laine, Romuald; Montellano, Paul R. Ortiz de

doi:10.1124/mol.54.2.305

Cited by 54 publications

(36 citation statements)

References 37 publications

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“…-dependent protease calpain could also be involved in nNOS degradation, as previously described [20]. We found 20% decreased expression of calpain-3 and -1/2 in muscle from fa/fa rats (p <0.05) (Fig.…”

Section: +supporting

confidence: 65%

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Mezghenna¹,

Leroy²,

Azay-Milhau³

et al. 2013

Diabetologia

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Aims/hypothesis Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect. Methods Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays. Results nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulininduced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes. Conclusions/interpretation Recovery of nNOS in insulinresistant muscles should be considered a potential new approach to address insulin resistance.

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Section: +supporting

confidence: 65%

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Mezghenna¹,

Leroy²,

Azay-Milhau³

et al. 2013

Diabetologia

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“…Based on the effects of ALLN, the protease responsible for the enhanced degradation of inducible NOS was identified as calpain. Calpain also appears to be responsible for the proteolytic degradation of nNOS in neurotoxin-treated cortical cells (38) as well as in in vitro systems (39). Our studies with the use of lactacystin strongly suggest that the proteasome is the major protease responsible for degradation of nNOS in our cellular system.…”

Section: Discussionmentioning

confidence: 61%

Guanabenz-mediated Inactivation and Enhanced Proteolytic Degradation of Neuronal Nitric-oxide Synthase

Noguchi¹,

Jianmongkol

Bender³

et al. 2000

Journal of Biological Chemistry

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Nitric-oxide synthases (NOS)1 are cytochrome P450-like hemoprotein enzymes that catalyze the conversion of L-arginine to citrulline and nitric oxide (1-4). Nitric oxide is a signaling molecule that is involved in a variety of physiological processes, including neurotransmission, vasorelaxation, platelet aggregation, and penile erection as well as in a variety of pathological conditions including septic shock, reperfusion injury, arthritis, atherosclerosis, diabetes, and graft rejection (5-8). It has been noted (9) that because nitric oxide is not stored, released, or inactivated after synaptic release by conventional regulatory mechanisms the biosynthetic regulation of the enzyme is of great importance. For the neuronal isoform the Ca 2ϩ -mediated activation is of prime importance. However, the factors that regulate proteolytic degradation of the enzyme have not been investigated. One approach that has been successfully utilized for the study of the proteolytic turnover of other P450 cytochromes is the use of suicide inactivators (10 -13). We wished to utilize this approach for the study of NOS turnover.Suicide inactivators are chemically inert molecules that mimic the natural substrate of the enzyme and become metabolized to a highly reactive intermediate that can covalently alter important active site entities, resulting in inactivation of the enzyme (14). In effect, the compound causes the enzyme to catalyze its own demise. Because the compound must not only have affinity for the active site but also must be metabolized in a manner to generate a reactive intermediate that is in close proximity to an important active site entity, these agents have the potential to be highly specific in vivo. In addition, they react with the form of the enzyme that is engaged in catalysis and thus are useful mechanistic probes into the nature of the bioactivation reaction. In some cases, these agents covalently alter P450 cytochromes in a manner that enhances their proteolysis and turnover (10 -13, 15-17).Guanabenz, a clinically used antihypertensive agent with a guanidino moiety, was recently shown, with the use of brain and penile cytosol, to be a metabolism-based inactivator of neuronal nitric-oxide synthase (nNOS) (18). Moreover, the treatment of rats with guanabenz was found to cause not only a decrease in activity, but also a concomitant loss of immunodetectable nNOS protein. To further understand the molecular mechanisms responsible for the loss of nNOS protein in vivo, we chose to model the effects of guanabenz with the use of HEK 293 cells stably transfected with nNOS. In the current study, we have shown that guanabenz causes the enhanced proteolytic turnover of nNOS. Suicide inactivators of nNOS, such as N G -methyl-L-arginine or N 5 -(1-iminoethyl)-L-ornithine, also caused the enhanced proteolytic degradation of the enzyme, whereas the slowly reversible inhibitor N G -nitro-L-arginine or the reversible inhibitor 7-NI did not. Studies with protease inhibitors indicate that the proteasome is responsible, in part, for r...

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“…The preferential induction of mRNA transcripts coding for nNOS after ischemic PC ( Figure 5) further supports this notion. Although nNOS and nNOS␣ have been reported to possess similar enzymatic activities in vitro, 24,34 their behavior in vivo under various conditions is completely unknown and warrants future investigation.…”

Section: Role Of Nnos In Late Pcmentioning

confidence: 99%

“…This phenomenon may reflect the fact that the half-life of nNOS␣ is only 24% of that of nNOS. 34 Alternatively, it may result from a regulatory mechanism at the translational level that favors the expression of the nNOS protein. In this regard, it is noteworthy that nNOS is one of those genes whose tissue-and developmental stage-specific expression are extensively regulated by translational mechanisms.…”

Section: Role Of Nnos In Late Pcmentioning

confidence: 99%

Cardioprotection During the Final Stage of the Late Phase of Ischemic Preconditioning Is Mediated by Neuronal NO Synthase in Concert With Cyclooxygenase-2

Wang

Kodani

Wang

et al. 2004

Circulation Research

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Abstract-The infarct-sparing effect of the late phase of ischemic preconditioning (late PC) lasts for 72 hours. Upregulation of both cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) has been shown to be essential to the protection in the initial stage of late PC (24 hours after PC); however, the mechanisms underlying the protection in the final stage of late PC (48 to 72 hours after PC) are unknown. Conscious rabbits were preconditioned with six cycles of 4-minute coronary occlusion/4-minute reperfusion. At 72 hours after PC, powerful protection against infarction was associated with increased myocardial levels of COX-2 mRNA, protein, and cardioprotective prostaglandins (PGI 2 and PGE 2 ). The COX-2-selective inhibitor NS-398 completely blocked the protection. Surprisingly, iNOS expression was not increased at 72 hours; instead, upregulation of neuronal NO synthase (nNOS) was evident at both the mRNA (ϩ266Ϯ20%, PϽ0.005) and the protein levels (ϩ195Ϯ66%, PϽ0.005), which was accompanied by an increase in myocardial nitrite/nitrate (ϩ20Ϯ4%, PϽ0. 05). The nNOS-selective inhibitors N-propyl-L-arginine or S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea completely blocked the protection of late PC at 72 hours, whereas the iNOSselective inhibitor S-methylisothiourea had no effect. In line with these findings, the disappearance of protection at 120 hours after PC was associated with the return of nNOS mRNA, protein, and activity to control levels. Although expression of COX-2 protein was still elevated at 120 hours, only a marginal increase in PGI 2 and PGE 2 levels was detected. In contrast to 72 hour after PC, nNOS was not upregulated at 24 hour after PC. We conclude that (1)

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Neuronal Nitric Oxide Synthase Isoforms α and μ Are Closely Related Calpain-Sensitive Proteins

Cited by 54 publications

References 37 publications

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Guanabenz-mediated Inactivation and Enhanced Proteolytic Degradation of Neuronal Nitric-oxide Synthase

Cardioprotection During the Final Stage of the Late Phase of Ischemic Preconditioning Is Mediated by Neuronal NO Synthase in Concert With Cyclooxygenase-2

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