Am J Physiol Regul Integr Comp Physiol. 2003 Jul.
Ascorbate inhibits iNOS expression and preserves vasoconstrictor responsiveness in skeletal muscle of septic mice.
Wu F, Wilson JX, Tyml K.
Dept. of Medical Biophysics, University of Western Ontario, London, ON, Canada.
[ PubMed ] [ Free full text ]

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.

Excerpts from the full text article:

In septic patients and animals, bacterial lipopolysaccharide (LPS) elicits the innate immune response and triggers the release of proinflammatory cytokines, including interleukin-1, interferon-gamma (IFN-gamma), and TNF-alpha. Induction of inducible nitric oxide synthase (iNOS) by LPS and proinflammatory cytokines contributes to the bactericidal effects of phagocytes (11, 16). LPS and cytokines also induce iNOS in vascular endothelial cells (ECs) and smooth muscle cells; this induction is, at least in part, responsible for vascular hyporeactivity, hypotension, and consequent multiple organ failure in sepsis (2, 33). In mice made septic by cecal ligation and puncture (CLP), vasoconstriction by norepinephrine (NE) in skeletal muscle arterioles is compromised (22). However, using the same model, arteriolar reactivity after CLP is maintained in iNOS-deficient mice. Moreover, these iNOS-deficient mice have improved survival (21). Although several drugs with relative selectivity for iNOS have been developed, their effectiveness in experimental sepsis therapy is limited because of their inhibition of all nitric oxide synthase (NOS) isoforms at relatively high doses (10, 25, 46).

Sepsis is also associated with elevated levels of reactive oxygen species (ROS; including superoxide, hydroxyl radicals, and hydrogen peroxide) (15, 27, 38, 47). ROS have been implicated in the potentiation of iNOS expression in a variety of cell types (3, 24, 44). Antioxidants (e.g., N-acetyl-L-cysteine, SOD/catalase) inhibit LPS- and cytokine-induced iNOS expression in ECs and other cell types (13, 32). Our recent in vitro results showed that ascorbate could blunt iNOS induction by LPS+IFN- in skeletal muscle ECs by reducing intracellular oxidative stress (45). However, endogenous ascorbate is depleted during sepsis (1, 4, 12). Although other antioxidants are also affected by sepsis, there is a strong correlation between survival and the plasma concentration of ascorbate (4, 15). Deficiency of endogenous ascorbate may worsen intracellular oxidative stress, thereby sensitizing cells to stimuli that induce iNOS. We hypothesize that administration of ascorbate can prevent sepsis induction of iNOS in vivo. The present study aimed to determine the effects of ascorbate administration on iNOS expression and vascular reactivity to NE in septic mice.

Materials and methods

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CLP. Sepsis was induced by CLP by using the procedures described by Armour et al. (1). Mice were anesthetized by 1% halothane (mixed with oxygen in the inspired gas). After laparotomy, the cecum was ligated distal to the ileocecal valve (bowel continuity preserved), punctured with an 18-gauge needle, and a small amount of cecal content was expressed through the puncture. Some mice were injected with a 0.1-ml bolus of sodium ascorbate (200 mg/kg body wt; dissolved in sterile 0.9% saline) through the tail vein at 30 min before the CLP procedure. The selected dose of ascorbate has been shown to prevent skeletal muscle microvascular dysfunction in CLP rats (1). CLP animals were resuscitated subcutaneously with a bolus of saline (50 ml/kg) containing buprenorphine (analgesic; 3 µg/ml). By 6 h after CLP, all of the mice had developed signs of severe sepsis, including lethargy, piloerection, and diarrhea. CLP caused 89% lethality at 24 h. Control mice were subjected only to subcutaneous bolus injection of saline containing buprenorphine, because sepsis was defined in the present study as the outcome of the laparotomy and CLP procedures.

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Results

Ascorbate inhibits iNOS expression. As shown in Fig. 1A, plasma NOx levels rose by 6 h after CLP, remained elevated at 12 h, and returned to baseline by 24 h. The time course of NOx production thus suggested that CLP increased NO synthesis at the early stage of sepsis. Figure 1B shows that ascorbate alone did not influence NOx production in non-CLP mice; however, ascorbate administration blocked the NOx elevation in the plasma of CLP mice at 6 h after CLP.

We next determined the effect of ascorbate on the protein expression of all three NOS isoforms, and on the enzymatic activity of both cNOS and iNOS, within the EDL muscles of CLP mice. A 6-h CLP insult caused a clear iNOS induction in the skeletal muscle at both protein (Fig. 2, A and B) and activity (Fig. 2C) levels. Ascorbate administration completely blocked the iNOS induction but did not influence the cNOS (including eNOS and nNOS) protein (Fig. 2, A and B) and activity (Fig. 2C) expression pattern in the skeletal muscles of CLP mice.

We further determined the effect of ascorbate on the mRNA levels of all three NOS isoforms in ECs freshly harvested from EDL skeletal muscles of CLP mice. iNOS mRNA was induced in the skeletal muscle ECs at 3 h after CLP; this induction was completely blocked by prior ascorbate administration (Fig. 3, A and B). Neither CLP nor ascorbate administration plus CLP altered the eNOS mRNA transcription pattern (Fig. 3, A and B). nNOS mRNA was not detectable in these ECs.

The results above indicate that ascorbate selectively inhibited the CLP-stimulated iNOS expression while at the same time maintaining the normal cNOS protein and activity levels. Furthermore, the RT-PCR analysis reveals that iNOS mRNA was induced in the microvascular ECs from the septic skeletal muscle and that ascorbate inhibitory effect occurred at the transcription level.

Ascorbate prevents CLP-stimulated oxidative stress. Ascorbate is a potent antioxidant that scavenges ROS, including superoxide anion and hydroxyl radicals (8, 30). We determined the effect of ascorbate on oxidant levels in the septic skeletal muscle. As shown in Fig. 4A, ascorbate administration significantly elevated the ascorbate concentration in the skeletal muscles of control and CLP mice. Moreover, ascorbate prevented the increase of oxidant levels in the skeletal muscle of CLP mice (Fig. 4B). These results indicate that ascorbate acts as an antioxidant in skeletal muscle exposed to septic insult.

Ascorbate preserves microvascular reactivity to catecholamines. CLP decreased the baseline arterial pressure (Fig. 5). Ascorbate administration partially prevented this decrease (Fig. 5). Our next experiments determined the effect of ascorbate on the vascular response to NE within the EDL muscle. As shown in Fig. 6, CLP impaired both arteriolar constriction (Fig. 6A) and arterial pressure (Fig. 6B) responses to NE. Ascorbate administration completely prevented these impairments. Ascorbate administration did not affect the changes in vascular diameter and arterial pressure caused by NE in non-CLP mice (data not shown). Together, these results indicate that ascorbate administration partially restored the baseline arterial pressure and completely preserved the microvascular constriction and arterial pressure responses to NE in CLP mice.

Discussion

Oxidative stress occurs in skeletal muscle early after the onset of systemic microbial infection (27, 38). Using an oxidant-sensitive fluorescence probe, DHR, to determine the total oxidant levels in the EDL muscle, we showed that sepsis causes an increase of ROS generation at 6 h after CLP (Fig. 4B). However, the increased oxidative stress could also reflect a decreased supply of antioxidants. Indeed, it has been reported that endogenous ascorbate is depleted during sepsis (1, 4, 12). The present results also indicate that ascorbate concentration in the skeletal muscle tended to decrease at 6 h after CLP (Fig. 4A). This is likely because cellular mechanisms of ascorbate production are impaired in sepsis (23). Contrastively, ascorbate administration to the septic mice increased ascorbate concentration and abolished the CLP-stimulated oxidative stress within the skeletal muscle.

ROS, superoxide in particular, may act as initiating factors in the pathogenesis of sepsis (15, 47). When SOD, the superoxide anion scavenger, was given alone as a pretreatment, the blood pressure was maintained and survival was improved in septic rats (39) and mice (7). However, SOD had no effect on the blood pressure or survival when given to rabbits 1 h after infection with live Escherichia coli (6) or when given to rats after the onset of sepsis (40). It seems that SOD administration is effective only when given before the onset of sepsis (15). ROS could have mediated the systemic inflammatory response in sepsis, which ultimately leads to the expression of vascular iNOS (17). In the present studies, administration of ascorbate before sepsis decreased ROS levels and consequently attenuated iNOS expression. However, prior ascorbate administration only partially restored the decrease of baseline arterial pressure caused by CLP. The lack of complete restoration of baseline blood pressure by ascorbate could be due to possible iNOS-independent mechanisms of blood pressure regulation in sepsis or due to vasodilative ability of ascorbate itself. For instance, ascorbate has been reported to directly modulate the receptor- and voltage-operated Ca2+ channels, leading to vasodilation (9). In addition, a recent report suggests that acidosis rather than excessive NO may cause the systemic hypotension during sepsis through reducing myocardial contractility (34).

The level of iNOS activity in septic skeletal muscle was much lower than that of cNOS (i.e., eNOS+nNOS; Fig. 2). Our data thus suggest that the iNOS expression in this tissue may be compartmentalized in the relatively small volume of vascular ECs and/or smooth muscle cells. To assess the possibility of induction of iNOS expression in ECs, we isolated ECs from the EDL skeletal muscles of CLP mice and conducted RT-PCR analysis of iNOS mRNA in these cells. The results demonstrated that CLP can induce iNOS mRNA transcription in the ECs of skeletal muscles and that this induction can be completely blocked by prior ascorbate administration (Fig. 3). Other investigators have reported that LPS induces iNOS expression in vivo in ECs of several tissues (26, 36). Our laboratory has shown that cultured ECs of rat EDL muscle origin express substantial iNOS on exposure to inflammatory stimulation (44). We have further demonstrated that iNOS expression in these cells is reduced by intracellular ascorbate in vitro (45). ECs play a pivotal role in the development of vascular dysfunction in sepsis (42). The present study demonstrated that ascorbate improvement of vascular reactivity could be due to protecting ECs from iNOS induction, which otherwise produces excessive NO.

It has been extensively documented that iNOS-derived NO contributes to the decreased systemic vascular resistance, impaired vascular responsiveness to catacholamines, and consequent hypotension present in sepsis (2, 21, 22, 33). However, the use of selective iNOS inhibitors for sepsis therapy is limited because they may also inhibit cNOS activity. For example, aminoguanidine was shown to prevent arterial pressure changes induced by LPS in rats (43). Further studies showed, however, that aminoguanidine enhanced LPS-induced intestinal vascular leakage due to inhibition of vascular cNOS activity (25). The present study showed that ascorbate injection selectively inhibited induction of iNOS by a septic insult. Furthermore, this was associated with complete prevention of vascular hyporeactivity and arterial pressure hyporesponsiveness to NE in CLP mice.

Ascorbate has been used to treat inflammatory symptoms of influenza and cold (14, 19). Injection of ascorbate also prevents skeletal muscle microvascular dysfunction in rats subjected to CLP (1). Our present data suggest that the anti-inflammatory property of ascorbate includes inhibition of iNOS expression. Additionally, ascorbate may preserve the activity of cNOS. Specifically, ascorbate maintains tetrahydrobiopterin, a critical cofactor for eNOS, in the reduced state (20). This effect of ascorbate is important in sepsis in maintaining the physiological functions of NO, such as preventing platelet and leukocyte adherence to endothelium (37). Another added benefit of therapy with ascorbate may be the protection of catecholamines from oxidation by superoxide (29). The inactivation of catecholamines by superoxide and NO has also been implicated in the pathogenesis of vascular hyporesponsiveness in sepsis (29). Because hospital infection constitutes a major cause of sepsis (10), acute administration of high-dose ascorbate along with antibiotic and supportive treatments before surgery, hemodialysis, or transplantation may reduce the morbidity and mortality associated with these interventions. Thus ascorbate could be used as an adjunct treatment in sepsis therapy.

In summary, the present data show for the first time that a bolus intravenous injection of ascorbate inhibits CLP-stimulated iNOS expression in mouse EDL muscle at both protein and activity levels. This effect of ascorbate is associated with reduced oxidative stress within the skeletal muscle. Our data suggest that ascorbate inhibition of iNOS expression contributes to the restored microvascular constriction and arterial pressure responses to NE.