Role of Neuronal Nitric-Oxide Synthase in Estrogen-Induced Relaxation in Rat Resistance Arteries

Abstract

Estrogen has antihypertensive and vasorelaxing properties, partly via activation of endothelial nitric-oxide synthase (eNOS). Recently, neuronal nitric-oxide synthase (nNOS) has been detected in vascular cells, although the significance of this is unclear. Estrogen was found to stimulate nNOS in certain cultured cells. We hypothesized that estrogen regulates vascular tone partly via endothelium-derived nNOS. Human umbilical vein endothelial cells were used to test whether acute (5 min) stimulation with 17␤-estradiol (E2) at 1 or 10 nM affected nNOS activity. Small mesenteric arteries from Sprague-Dawley rats were examined for relaxation to E2 (0.001-10 M) in the absence or presence of selective nNOS inhibitor [N-propyl-Larginine (L-NPA); 2 M] or pan-NOS inhibitor [N-nitro-L-arginine methyl ester (L-NAME); 100 M] using a wire myograph. Immunostaining was used to visualize nNOS in rat mesenteric artery cross-sections. Western blotting measured total and phospho-nNOS in endothelial cell lysates and thoracic aorta homogenates. E2 rapidly increased (p Ͻ 0.001) activating phosphorylation of nNOS and nitric oxide (NO) production (as measured by 4-amino-5-methylamino-2,7-difluorofluorescein fluorescence) in endothelial cells. Likewise, E2 caused dosedependent relaxation of arteries from female rats, which was blunted by both L-NPA and L-NAME (p Ͻ 0.001). In contrast, E2 response was modest in male animals and unaffected by NOS inhibition. It is noteworthy that there was a greater baseline presence of phospho-nNOS in male relative to female aortas. Although eNOS is believed to be the main source of NO in the vascular endothelium, we confirmed nNOS expression in endothelial cells. Endothelial nNOS mediated E2 relaxation in isolated arteries from female animals. Altogether, these data suggest vascular nNOS as a novel mechanism in E2 signaling.

Estradiol attenuates high glucose-induced endothelial nitrotyrosine: role for neuronal nitric oxide synthase

“…Several groups have used L-NPA at micromolar concentrations in different systems to demonstrate selective nNOS inhibition (3,11). A recently published study from our group has shown that similar concentrations of L-NPA can indeed inhibit nNOS in the vasculature without affecting eNOS-dependent vascular functions (33). Ongoing work in our laboratory also suggests similar effects of 2 M L-NPA compared with small interfering RNA-mediated downregulation of nNOS protein in HUVECs (data not shown).…”

“…Classically, eNOS has been considered the main source of NO in the vasculature, although there is increasing evidence that nNOS in the endothelium plays an important function (4,9,10,17). Recently, we have demonstrated the presence of endothelial nNOS in rat mesenteric arteries where this enzyme contributes to a vasorelaxant role (33). The beneficial vascular effects of estrogen are believed to be largely mediated through increased endothelial NO synthesis (1,2,22).…”

The Contribution of Endothelial Marker Proteins in the Determination of Vascular Angiogenic Potential, in Normal Physiological Conditions and in Neoplasia

“…Physiologically, the two forms of nitrogen oxide synthase can be distinguished as the constitutive form (cNOS) and the inductive form (iNOS) [37]. The constitutive form, present in nervous tissue (nNOS) and in endothelial cells (eNOS), comprises up to 95% of the total synthase activity [25]. Interestingly, the nNOS isoform was discovered only a few years ago in endothelial cells.…”

The Changes of Aortic Stiffness During Normal Menstrual Cycle

“…In addition, increased oestrogen may cause peripheral artery vasodilation and reduce the TPR, which may cause lower AI (Hayashi et al 1995). Oestrogen has been demonstrated to increase production of both nitric oxide (NO) and prostacyclin by upregulating endothelial nitric oxide synthase (eNOS) activity and prostacyclin synthase via a receptormediated system in which it promotes vasodilation (Hayashi et al 1995;Massion & Balligand 2003;Lekontseva et al 2011). Further study has reported that the physiological changes observed during the luteal phase of menstrual cycle does cause a marked decrease in TPR and a significant decrease in mean arterial pressure in the mid luteal phase corresponding to oestrogen level (Hassan et al 1990).…”