Ascorbate inhibits iNOS expression and preserves vasoconstrictor responsiveness in skeletal muscle of septic mice

Abstract

Inducible nitric oxide synthase (iNOS) expression in blood vessels contributes to the vascular hyporeactivity characteristic of sepsis. Our previous work demonstrated in vitro that ascorbate inhibits iNOS expression in lipopolysaccharide- and interferon-gamma-stimulated skeletal muscle endothelial cells (ECs) through an antioxidant mechanism. The present study evaluated in vivo the hypothesis that administration of ascorbate decreases oxidative stress, prevents endothelial iNOS expression, and improves vascular reactivity in septic skeletal muscle. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Plasma nitrite and nitrate (NOx) levels were elevated by 6 h after CLP. Prior ascorbate bolus injection (200 mg/kg body wt iv) blocked the elevation of plasma NOx and abolished the expression of iNOS protein and activity in the septic skeletal muscle. We also demonstrated that iNOS mRNA determined by RT-PCR was induced in the microvascular ECs of the muscle at 3 h after CLP. This induction was attenuated by prior ascorbate administration. Ascorbate inhibition of iNOS expression was associated with decreased oxidant levels in the septic muscle. Moreover, ascorbate administration restored partially the baseline arterial pressure and preserved completely the microvascular constriction and arterial pressure responses to norepinephrine in CLP mice. These results suggest that early administration of ascorbate may be a valuable adjunct treatment of sepsis.

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“…Neither CLP nor ascorbate injection alters the eNOS mRNA levels measured in endothelial cells at 3 h post-CLP, which is a time when the iNOS mRNA levels in these cells are elevated (63). However, the eNOS activity in skeletal muscle is decreased by CLP and parenteral ascorbate (200 mg/kg) blocks this loss of activity (68) (Fig.…”

“…of ascorbate (76-200 mg/ kg) attenuates the loss of arteriolar and blood pressure responsiveness to the vasoconstrictors norepinephrine and angiotensin II in CLP rats and mice (2,40,63,64). An important mechanism for the protection of arteriolar reactivity by ascorbate is inhibition of iNOS expression in endothelial cells.…”

“…Part of the evidence for this mechanism is that injection of ascorbate (200 mg/kg) prevents CLP from elevating the concentration of NO metabolites in plasma (64). Further, ascorbate injection blocks the increase by CLP of iNOS mRNA expression in microvascular endothelial cells at 3 h after CLP, apparently due to inhibition by ascorbate of NADPH oxidase activity (63,65). Parenteral ascorbate also blocks the increases in total NOS, iNOS, and neuronal NOS (nNOS) activities in skeletal muscle of CLP mice (68) (Fig.…”

“…Bolus injection i.v. of ascorbate blocks hypotension from developing in endotoxemic rats (53) Ascorbate also prevents hypotension for 24 h in CLP rats (2,58), attenuates the fall in baseline blood pressure for 6 h in CLP mice (63), and fails to protect baseline blood pressure in FIP mice (59). The reasons for this variation between the different septic peritonitis models may be that, compared to salinevehicle injected CLP rats (75% survival at 24 h), sepsis Ascorbate also blocked the increases in iNOS and nNOS activities, and the decrease in eNOS activity (B).…”

“…High levels of ascorbate are effective for inhibiting the induction of iNOS protein and elevation of iNOS activity by septic insult in cultures of microvascular endothelial cells (24,62,66). In CLP animals, too, ascorbate (200 mg/kg) injection blocks CLPinduced increases in iNOS mRNA in freshly harvested microvascular endothelial cells, iNOS protein and activity in skeletal muscles, and NO metabolites in plasma (63,64). This may explain, at least in part, the observation that ascorbate treats septic endothelial barrier dysfunction in cell culture and animal models of sepsis.…”