Aldosterone enhances ligand-stimulated nitric oxide production in endothelial cells.
Chronic and acute actions of aldosterone have been shown recently to directly affect the cardiovascular system. However, it is unclear whether the acute effects of aldosterone on vasculature are constrictive or dilatory. Here, to clarify the nongenomic effects of aldosterone on endothelial function, we examined the effects of aldosterone on nitric oxide (NO) production in cultured endothelial cells (ECs) and on vascular tone. The intracellular NO production of bovine aortic ECs loaded with DAF-2 was determined using confocal microscopy. Accumulated NO in the culture medium was quantified by a microplate reader using membrane-impermeable DAF-2. Phosphorylation of endothelial NO synthase (eNOS) at Ser(1179) was assessed by Western blotting. Changes in intracellular Ca(2+) ([Ca(2+)](i)) were determined by confocal microscopy in ECs doubly loaded with fluo-4 and Fura Red. The effects of aldosterone, acetylcholine (ACh), and other signaling molecules on the tension of phenylephrine (PE)-contracted aortas of Sprague-Dawley rats were examined in an ex vivo organ bath chamber system. Short-term pre-exposure to aldosterone (1 x 10(-7) mol/L) enhanced ATP-induced NO production in ECs with increased phosphorylation of eNOS at Ser(1179). These effects were blocked by eplerenone, a mineralocorticoid receptor (MR) antagonist, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Notably, aldosterone alone did not affect ATP-induced [Ca(2+)](i) changes or the Ser(1179) phosphorylation. Similarly, aldosterone (1 x 10(-8) to 1 x 10(-7) mol/L) did not affect the tone of rat aortas pre-contracted by PE, but enhanced ACh-induced vasorelaxation, which was again reversed by eplerenone or LY29400. In contrast, sodium nitroprusside-induced vasorelaxation in endothelium-denuded aortas was not affected by aldosterone. Thus, aldosterone acutely enhances ligand-mediated endothelial NO production by eplerenone-sensitive mechanisms involving a PI3K that may synergize Ca(2+)-dependent eNOS phosphorylation at Ser(1179).
Mutoh
Akiko
A
Department of Nephrology and Endocrinology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
Isshiki
Masashi
M
Fujita
Toshiro
T
D000109:Acetylcholine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000450:Aldosterone / Q000494:pharmacology*; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008024:Ligands; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology; D014665:Vasodilator Agents / Q000494:pharmacology
D008024:Ligands; D014665:Vasodilator Agents; D009569:Nitric Oxide; D000450:Aldosterone; D000255:Adenosine Triphosphate; D052250:Nitric Oxide Synthase Type III; D000109:Acetylcholine; D002118:Calcium