Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C2C12muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

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The effect of the nitric oxide donor sodium nitroprusside on glucose uptake in human primary skeletal muscle cells

Nitric Oxide ◽

10.1016/j.niox.2009.06.002 ◽

2009 ◽

Vol 21 (2) ◽

pp. 126-131 ◽

Cited By ~ 16

Author(s):

Darren C. Henstridge ◽

Brian G. Drew ◽

Melissa F. Formosa ◽

Alaina K. Natoli ◽

David Cameron-Smith ◽

...

Keyword(s):

Nitric Oxide ◽

Skeletal Muscle ◽

Sodium Nitroprusside ◽

Glucose Uptake ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Nitric Oxide Donor

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Stretch-induced nitric oxide modulates mechanical properties of skeletal muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.00018.2004 ◽

2004 ◽

Vol 287 (2) ◽

pp. C292-C299 ◽

Cited By ~ 47

Author(s):

Jingying Sarah Zhang ◽

William E. Kraus ◽

George A. Truskey

Keyword(s):

Nitric Oxide ◽

Mechanical Properties ◽

Skeletal Muscle ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Calpain Inhibitor ◽

No Donor ◽

Static Stretch ◽

Force Microscopy ◽

Protein Levels

In this study, we examined the hypothesis that stretch-induced (nitric oxide) NO modulates the mechanical properties of skeletal muscles by increasing accumulation of protein levels of talin and vinculin and by inhibiting calpain-induced proteolysis, thereby stabilizing the focal contacts and the cytoskeleton. Differentiating C2C12 myotubes were subjected to a single 10% step stretch for 0–4 days. The apparent elastic modulus of the cells, Eapp, was subsequently determined by atomic force microscopy. Static stretch led to significant increases ( P < 0.01) in Eapp beginning at 2 days. These increases were correlated with increases in NO activity and neuronal NO synthase (nNOS) protein expression. Expression of talin was upregulated throughout, whereas expression of vinculin was significantly increased only on days 3 and 4. Addition of the NO donor l-arginine onto stretched cells further enhanced Eapp, NOS activity, and nNOS expression, whereas the presence of the NO inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) reversed the effects of mechanical stimulation and of l-arginine. Overall, viscous dissipation, as determined by the value of hysteresis, was not significantly altered. For assessment of the role of vinculin and talin stability, cells treated with l-NAME showed a significant decrease in Eapp, whereas addition of a calpain inhibitor abolished the effect. Thus our results show that NO inhibition of calpain-initiated cleavage of cytoskeleton proteins was correlated with the changes in Eapp. Together, our data suggest that NO modulates the mechanical behavior of skeletal muscle cells through the combined action of increased talin and vinculin levels and a decrease in calpain-mediated talin proteolysis.

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Regulation of inducible nitric oxide synthase expression in L6 rat skeletal muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.1.c35 ◽

1997 ◽

Vol 272 (1) ◽

pp. C35-C40 ◽

Cited By ~ 30

Author(s):

S. Okuda ◽

F. Kanda ◽

Y. Kawahara ◽

K. Chihara

Keyword(s):

Nitric Oxide ◽

Skeletal Muscle ◽

Protein Kinase ◽

Nitric Oxide Synthase ◽

Tyrosine Kinase ◽

Muscle Cells ◽

Inos Expression ◽

Skeletal Muscle Cells ◽

Rat Skeletal Muscle ◽

Oxide Synthase

Cytokine-stimulated expression of inducible type of nitric oxide synthase (iNOS) seems to be regulated by various signal pathways in a cell-specific manner. In this study, we examined how it was regulated in L6 rat skeletal muscle cells. In L6 cells, the combination of interleukin-1 beta and interferon-gamma induced a marked accumulation of nitrite, a stable metabolite of nitric oxide. In parallel with this reaction, iNOS mRNA expression was achieved at a maximum between 3 and 6 h, and iNOS protein was detectable at 6 h and peaked at 24 h after stimulation. Tyrosine kinase inhibitors, herbimycin A, and genistein suppressed cytokine-induced iNOS expression and nitrite production. Forskolin, an adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) activator, and phorbol 12-myristate 13-acetate, a protein kinase C (PKC)-activating phorbol ester, enhanced these cytokine-induced reactions. These results indicate that iNOS expression by cytokines is mediated via a protein tyrosine kinase-dependent pathway and is positively modulated by both PKA- and PKC-dependent pathways in this cell type.

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