Increased Nitrovasodilator Sensitivity in Endothelial Nitric Oxide Synthase Knockout Mice

Abstract

Abstract-Endogenously produced nitric oxide (NO) modulates nitrovasodilator-induced relaxation. We investigated the underlying mechanism in wild-type (WT) mice and endothelial NO synthase knockout (eNOS Ϫ/Ϫ ) mice to determine whether a chronic lack of endothelial NO alters the soluble guanylyl cyclase (sGC) pathway. In aortic segments from eNOS Ϫ/Ϫ mice, the vasodilator sensitivity to sodium nitroprusside (SNP) was significantly greater than that in WT mice. There was no difference in sensitivity to the G-kinase I activator 8-para-chlorophenylthio-cGMP or to cromakalim. N -Nitro-L-arginine had no effect on the SNP-induced relaxation in eNOS Ϫ/Ϫ but increased the sensitivity in WT mice so it was no longer different than that of eNOS Ϫ/Ϫ . Basal cGMP levels in aortic rings were significantly lower in eNOS Ϫ/Ϫ mice than in WT mice. SNP (300 nmol/L) induced a significantly greater cGMP accumulation in eNOSmice than in WT mice. The maximal SNP-induced (10 mol/L) increase in cGMP was similar in both strains. SNP-stimulated sGC activity was significantly greater in eNOS Ϫ/Ϫ mice than in WT mice. Incubation of aortic segments from WT mice with N -nitro-L-arginine increased sGC activity, an effect prevented by coincubation with SNP (10 mol/L). The aortic expressions of the sGC ␣1 and ␤1 subunits in WT and eNOS Ϫ/Ϫ mice were identical as determined with Western blot analysis. These data suggest that chronic exposure to endothelium-derived NO, as well as acute exposure to nitrovasodilator-derived NO, desensitizes sGC to activation by NO but does not alter sGC expression. Both the acute cessation of endothelial NO formation in WT mice and the chronic deficiency of NO in eNOS Ϫ/Ϫ mice restore the NO sensitivity of sGC and enhance vascular smooth muscle relaxation in response to nitrovasodilator agents. Key Words: nitric oxide Ⅲ mice Ⅲ genes Ⅲ vasodilator agents V ascular relaxation responses elicited by nitrovasodilator agents and by endothelium-derived nitric oxide (NO) are mainly mediated via the activation of soluble guanylyl cyclase (sGC) and a subsequent increase in intracellular cGMP levels. 1 Interactions between endogenous and exogenous NO have been reported to modulate vasodilatory responsiveness. 2-5 Different mechanisms may underlie this phenomenon, such as the downregulation of sGC, sGC desensitization, or inhibition of the cGMP-dependent signal transduction cascade. In the present study, we investigated the mechanism by which endothelium-derived NO affects the nitrovasodilator-induced relaxation in wild-type (WT) mice and in endothelial NO synthase knockout mice (eNOS Ϫ/Ϫ ). Methods Animals and Tissue PreparationsWT c57 black b6 mice were purchased from Charles River, and eNOS Ϫ/Ϫ mice and age-matched control animals were obtained from the Department of Physiology at Heinrich Heine Universität Düssel-dorf. 6 Mice were housed under conditions that conformed with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication No. 85-23).Mice were killed by cervical dislocation. T...

Defective Insulin and Acetylcholine Induction of Endothelial Cell–Nitric Oxide Synthase Through Insulin Receptor Substrate/Akt Signaling Pathway in Aorta of Obese Rats

“…However, after endothelium denudation, the potency of GTN was potentiated in both groups (pEC 50 : 7.75 Ϯ 0.01 and 7.82 Ϯ 0.01, control vs. obese, respectively; data not shown). It has been reported that the endothelium-independent relaxations induced by NO donors are potentiated in eNOS knockout mice (34) or not affected after endothelium denudation in rats (35). Our results demonstrate a significant decrease in the potency of GTN in endotheliumintact aorta of obese rats.…”

Increased eNOS accounts for changes in connexin expression in renal arterioles during diabetes

“…A similar decrease in NO-induced vasorelaxation has been reported in aorta of streptozotocin-induced diabetic rats due to decrease in guanylate cyclase and protein kinase G-protein (Zanetti et al, 2005). Interestingly, the activity of guanylate cyclase was increased in eNOS knockout mice (Brandes et al, 2000). Further experimentation is therefore necessary to clarify the involvement of guanylate cyclase and ROS in the mechanism by which elevated eNOS protein can alter Cx expression.…”

Preserved coronary arteriolar dilatation in patients with type 2 diabetes mellitus: Implications for reactive oxygen species

“…Brandes et al reported that both acute cessation of endothelial NO production in wild type mice and chronic deficiency of NO in eNOS knockout mice increase the NO sensitivity of vascular smooth muscle cells in response to nitrovasodilator agents [6]. They proposed that an enhanced sensitivity of soluble guanylate cyclase (sGC) to NO may compensate for the reduced NO availability [6].…”

“…They proposed that an enhanced sensitivity of soluble guanylate cyclase (sGC) to NO may compensate for the reduced NO availability [6]. In this context, it has been also found that administration of exogenous NO decreased sGC activity acutely and decreased its protein expression over time [43].…”

“…It seems plausible that the lack of NO may lead to enhanced sGC activity in vascular smooth muscle cells [6]. Although it has been proposed that oxidative and/or nitrosative stress can lead to inactivation of sGC in long-term [36], other studies have demonstrated that acute exposure to H 2 O 2 can lead to activation of sGC, contributing to the relaxation of the bovine pulmonary artery [7,55].…”