Resistance to Endotoxic Shock in Endothelial Nitric-oxide Synthase (eNOS) Knock-out Mice

Abstract

The expression of inducible nitric-oxide synthase (iNOS) and subsequent "high-output" nitric oxide (NO) production underlies the systemic hypotension, inadequate tissue perfusion, and organ failure associated with septic shock. Therefore, modulators of iNOS expression and activity, both endogenous and exogenous, are important in determining the magnitude and time course of this condition. We have shown previously that NO from the constitutive endothelial NOS (eNOS) is necessary to obtain maximal iNOS expression and activity following exposure of murine macrophages to lipopolysaccharide (LPS). Thus, eNOS represents an important regulator of iNOS expression in vitro. Herein, we validate this hypothesis in vivo using a murine model of sepsis. A temporal reduction in iNOS expression and activity was observed in LPS-treated eNOS knock-out (KO) mice as compared with wild-type animals; this was reflected in a more stable hemodynamic profile in eNOS KO mice during endotoxaemia. Furthermore, in human umbilical vein endothelial cells, LPS leads to the activation of eNOS through phosphoinositide 3-kinase-and Akt/protein kinase B-dependent enzyme phosphorylation. These data indicate that the pathogenesis of sepsis is characterized by an initial eNOS activation, with the resultant NO acting as a co-stimulus for the expression of iNOS, and therefore highlight a novel pro-inflammatory role for eNOS.The systemic hypotension, organ failure, and morbidity that occur during bacterial sepsis are associated with the expression of inducible nitric-oxide synthase (iNOS) 1 and excessive production of nitric oxide (NO). This is evidenced by the increased levels of nitrite (NO 2 Ϫ ) and nitrate (NO 3 Ϫ ; stable metabolites of NO) measured in the plasma of septic patients (1, 2), inflammation-induced iNOS expression (3, 4), and by the ability of selective iNOS inhibitors to restore blood pressure in experimental models of sepsis and reverse hypotension in human endotoxaemia (5-10).Inducible NOS is effectively absent under physiological conditions but is expressed in many cell types in response to pro-inflammatory cytokines and lipopolysaccharides (LPS); in accord, induction of this NOS isoform is necessary for "high output" NO production and cytostatic and cytotoxic effects that facilitate host defense (11). While iNOS expression is essential to combat bacterial infection, the sustained overproduction of NO is deleterious to the host, as exemplified by the cardiovascular dysfunction during sepsis. Since iNOS is regulated primarily at a transcriptional level (12-14), a better understanding of the mechanisms involved in iNOS mRNA and protein expression should lead to improved treatment for endotoxaemia and other inflammatory cardiovascular disorders.In contrast to iNOS, a potential role for endothelial NOS (eNOS) in the pathogenesis of sepsis is unsubstantiated. Initial studies suggested that eNOS knock-out (KO) animals respond to endotoxin in an identical manner to wild-type (WT) littermates (15), and more recently, mice overexpressing eNO...

Induction of IRAK-M Is Associated with Lipopolysaccharide Tolerance in a Human Endotoxemia Model

“…Interestingly, del Fresno et al (52) have shown previously that IRAK-M expression can be induced in vitro in isolated human monocytes by the NO donor GNSO in a TNF-dependent manner. Also, endothelial-derived NO synthase appears crucial in the reactions observed in mice injected with LPS, and indeed NO may enhance TNF-␣ production by human mononuclear cells (53,54). Thus, inflammatory reactions dependent on NO may be accelerated through endothelial-derived NO production.…”

Prostaglandin F₂_α Regulates the Nitric Oxide Generating System in Bovine Luteal Endothelial Cells

“…Indeed, an in vivo experiment showed that PGF stimulates eNOS mRNA and protein expressions in the luteal tissue of the cow [10], sheep [47] and rabbit [23]. Recently, iNOS expression has been found to be reduced in eNOS knockout mice [48], suggesting that there is an important interaction between eNOS and iNOS expression. Since the results of the present study shown a direct effect of PGF on iNOS, but not on eNOS expression, a basal level of eNOS expression may be necessary for increasing iNOS expression in response to PGF.…”

eNOS phosphorylation: A pivotal molecular switch in vasodilation and cardioprotection?

“…In addition to the potential cardioprotective effects triggered by eNOS phosphorylation, recent studies also indicated a detrimental or pro-inflammatory role of eNOS phosphorylation [32,33]. It has been shown that Akt-mediated eNOS phosphorylation played a causal role in the pathogenesis of endotoxic shock [33] as well as anaphylactic shock [32].…”

“…It has been shown that Akt-mediated eNOS phosphorylation played a causal role in the pathogenesis of endotoxic shock [33] as well as anaphylactic shock [32]. Under these pathological situations, genetic deletion of eNOS was proved to be protective against the lethal hypotensive reaction caused by eNOS-derived or -triggered overproduction of NO [32,33].…”