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Estrogen effect on endothelial nitric oxide (no) production Rahimian, Roshanak


Pre-menopausal women have a much lower incidence of coronary heart disease than men, and the difference appears to be related to the estrogen circulating in women. Estrogen has two protective cardiovascular effects: one is on the blood lipid profile, and the other is a direct effect on the blood vessel wall and the generation of nitric oxide (NO). In this connection, the modulatory effects of chronic subcutaneous or oral estrogen, LY117018 and raloxifene, selective estrogen receptor modulators (SERMs), on the release of NO was studied in the rings of rat aorta. Treatment of ovariectomized rats with estrogen and LY117018 enhanced cholinergic, endothelium-dependent vasodilation of the aorta, and secondly, the inhibition of NO synthase (NOS) caused a greater enhancement of adrenergic vasoconstriction in estrogen, LY117018 and raloxifenetreated animals than those in male, ovariectomized progesterone plus estrogen-treated animals (P < 0.05). These effects occurred without changes in the sensitivity of smooth muscle cells to either NO donors, or to an adrenergic agonist. We, therefore, proposed that estrogen and SERMs exert their vasomotor effects primarily through enhancing endothelial-dependent vasodilation by increasing basal and stimulated release of NO. In the next phase, the modulatory effects of chronic estrogenic treatment on the responses to cyclopiazonic acid (CPA), an sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor, was studied in rings of rat aorta. In phenylephrine (PE, 2xl0⁻⁶ M) pre-contracted rings with intact endothelium, CPA (10⁻⁷ to 3xl0⁻⁵ M) produced endothelium-dependent relaxations in a concentration dependent manner. The CPA dilation as a percentage loss of PE tone was greater in aortic rings from female and estrogen -treated rats compared to those from male or ovariectomized rats (P < 0.05). These relaxation responses of CPA were converted to contractions by pre-treatment with an inhibitor of NOS. There were no differences in CPA-induced contractions of aortas excised from either estrogen treated or untreated ovariectomized rats. These results demonstrate that CPA causes a greater endothelium-dependent dilation in estrogentreated ovariectomized and control female rats. Depletion of endoplasmic reticulum (ER) Ca²⁺ by CPA discharges Ca²⁺ from intracellular stores in endothelial cells which in turns triggers influx of Ca²⁺ from the extracellular space via receptor operated channels (ROCs)/ or store operated channels (SOCs) of the plasma membrane, and subsequently stimulates NOS. Although the passive Ca²⁺ leak, ROCs/ or SOCs are voltage independent, membrane potential (Em) plays an important role in regulating Ca²⁺ entry. The next set of experiment was designed to investigate the role of Em in the regulation of Ca²⁺ entry triggered by agonist/ or SERCA inhibitors. [Ca²⁺]i was measured by fura-2/AM fluorescence imaging microscopy in freshly isolated rabbit aortic endothelial cells. No changes in [Ca²⁺]i in response to PE (5 μM) was observed indicating PE (a selective agonist of α₁ receptor), contraction may be used as a test system for basal NO release. Acetylcholine (Ach; 10 μM) and CPA (10 μM) increased [Ca²⁺]i. The maintained [Ca²⁺]i increase upon agonist or Ca²⁺ pump blocker application was blocked by tetraethylamonium (TEA; 3 mM), a K⁺ channel blocker, indicating involvement of K⁺ channel activity. ROCs were found to be responsible for the [Ca²⁺]i increase, since SK&F96365 (50 μM), a ROC blocker greatly reduced the maintained [Ca²⁺]i increase caused by Ach and CPA. When Ach and CPA were added together the induced Ca²⁺ plateau was less sensitive to TEA but could be abolished by a combination of TEA and the CI⁻ channel inhibitor NPPB (50 μM). These data suggested that maintenance of a polarized membrane potential by activity of K⁺ and Cl⁻ channels is a requisite for Ca²⁺ influx through ROCs/ or SOCs and, therefore, for the synthesis/release of NO. The possibility that enhanced Ca²⁺ stimulation of endothelial NOS contributes to estrogenic effects has not been previously investigated. The last phase of experiment was therefore designed to determine whether estrogen enhances NO release, at least in part, by raising [Ca²⁺]i. [Ca²⁺]i was measured by fura-2/AM fluorescence imaging microscopy in freshly isolated valvular endothelial cells taken from female and male rats. The basal level of [Ca²⁺ ]i was significantly elevated in female valvular endothelial cells when compared to males (P < 0.05). Inhibition of SERCA with CPA (10 μM) caused a greater increase in the [Ca²⁺]i in female than male endothelial cells. Removal of extracellular Ca²⁺ returned the [Ca²⁺]i to the basal level in both female and male endothelium. The rate of [Ca²⁺]i decline was significantly (P < 0.05) slower in female endothelial cells compared to males. There were no differences in the unstimulated rate of Mn²⁺ quenching between two groups. In conclusion, these results indicate a novel mechanism for the protective action of estrogen in the blood vessels. It shows that a difference in Ca²⁺ homeostasis leading to greater basal [Ca²⁺]i in female than male rats may be responsible for enhanced CPA endothelium-dependent vasodilation and NO secretion in female and estrogen-treated ovariectomized female rats, when compared to male or ovariectomized rats.

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