Autoregulatory Role of Endothelium-derived Nitric Oxide (NO) on Lipopolysaccharide-induced Vascular Inducible NO Synthase Expression and Function

Phuong A Vo,Bhavini Lad,James A.P Tomlinson,Stephanie Francis,Amrita Ahluwalia

doi:10.1074/jbc.m411317200

Phuong A Vo, Bhavini Lad + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m411317200

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Abstract

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.

Highlights

Bacterial sepsis is a systemic inflammatory state characterized by vascular smooth muscle dysfunction, leading to hypotension, inadequate tissue perfusion, and multiple organ failure [1]
Recent work using bone marrow-derived macrophages generated from endothelial NOS (eNOS) knock-out and wild-type mice indicate a reciprocal relationship between eNOS-derived Nitric oxide (NO) and NO produced by inducible nitric oxide synthase (iNOS), such that constitutive low level NO production is essential for maximal iNOS expression in response to LPS in these cells [15]
Using LPS, ex vivo, to mimic aspects of sepsis, we demonstrate that activation of constitutive eNOS activity is an essential prerequisite for efficient arterial expression and function of vascular iNOS

Summary

Autoregulation of Nitric Oxide Production in Sepsis

Concentrations of NO produced consequent to iNOS induction suppress constitutive NOS activity i.e. endothelial NOS (eNOS) and neuronal NOS activity [13]. Recent work using bone marrow-derived macrophages generated from eNOS knock-out and wild-type mice indicate a reciprocal relationship between eNOS-derived NO and NO produced by iNOS, such that constitutive low level NO production is essential for maximal iNOS expression in response to LPS in these cells [15]. Whether such a reciprocal interaction exists between vascular eNOS-derived NO and iNOS activity in terms of the vascular effects of NO in sepsis is unknown. In the present study, we investigated whether endothelial NO played a regulatory role on LPS-induced iNOS expression and function in rat isolated aortic rings, ex vivo, and in thoracic aortae from wild-type (WT) and eNOS knock-out (KO) mice, in vivo

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