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 17β-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 μM) in the absence or presence of selective nNOS inhibitor [N-propyl-l-arginine (l-NPA); 2 μM] or pan-NOS inhibitor [Nω-nitro-l-arginine methyl ester (l-NAME); 100 μM] 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 < 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 dose-dependent relaxation of arteries from female rats, which was blunted by both l-NPA and l-NAME (p < 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.
Footnotes
This study was supported by a grant from the Heart and Stroke Foundation of Alberta, Northwest Territories and Nunavut (to S.T.D.). O.L. is supported by an Alberta Heritage Foundation for Medical Research Doctoral Scholarship. S.C. is supported by a Fellowship from the Canadian Institutes for Health Research. C.C. was awarded an Alberta Heritage Foundation for Medical Research summer studentship. S.D. is funded by Alberta Innovates Health Solutions as an Alberta Heritage Foundation for Medical Research Scientist and is a Canada Research Chair, Tier 1 in Women's Cardiovascular Health.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.183798.
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ABBREVIATIONS:
- eNOS
- endothelial nitric-oxide synthase
- CCRC
- cumulative concentration-response curve
- DAF-FM
- 4-amino-5-methylamino-2,7-difluorofluorescein
- E2
- 17β-estradiol
- ER
- estrogen receptor
- HUVEC
- human umbilical vein endothelial cell
- l-NPA
- N-propyl-l-arginine
- l-NAME
- Nω-nitro-l-arginine methyl ester
- MCh
- methacholine
- NA
- noradrenaline
- NO
- nitric oxide
- nNOS
- neuronal nitric-oxide synthase
- VSMC
- vascular smooth muscle cell
- vWF
- von Willebrand factor
- ANOVA
- analysis of variance
- RMANOVA
- repeated-measures ANOVA
- ICI 182780
- 7α,17β-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol
- DAPI
- 4,6-diamidino-2-phenylindole.
- Received May 10, 2011.
- Accepted July 29, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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