Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells ( The inhibitory effect of estrogen on the development of atherosclerosis has been suggested by abundant human epidemiological and animal experimental data (1-9). The incidence of atherosclerotic diseases is lower in premenopausal women than in men, steeply rises in postmenopausal women, and is reduced to premenopausal levels in postmenopausal women who receive estrogen therapy (10 -12). Until recently, the atheroprotective effects of estrogen were attributed principally to the effects on serum lipid concentrations. However, estrogeninduced alterations in serum lipids account for only approximately one-third of the observed clinical benefits of estrogen (12)(13)(14). Recent evidence suggests that the direct actions of estrogen on blood vessels contribute to the cardioprotective effects of estrogen (13, 15). There are many kinds of direct effects of estrogen on blood vessels, such as estrogen-induced increases of vasodilatation and inhibition of the response of blood vessels to injury and the development of atherosclerosis. However, the molecular mechanism underlying the estrogeninduced vasodilatation has not yet been determined. Several studies suggest that a key mediator of this vasodilator response could be the endothelium-derived relaxing factor nitric oxide (NO), and that brief treatment with estrogen increases basal NO release in endothelial cells without elevation of eNOS mRNA or protein (16). Estrogen activates endothelial nitric oxide synthase (eNOS) without altering expression of the eNOS gene in vascular endothelium (17)(18)(19)(20). However, the details of the mechanism of the estrogen-induced eNOS activation are not yet well understood.The serine/threonine kinase termed Akt or protein kinase B (PKB) 1 is an important regulator of various cellular processes, including glucose metabolism and cell survival (21, 22). Activation of receptor tyrosine kinases and G-protein-coupled receptors, and stimulation of cells by mechanical force, can lead to the phosphorylation and activation of . Akt was identified as a downstream component of survival signaling through phosphatidylinositol 3-kinase (PI3K) (26 -30). Akt may be regulated by both phosphorylation and the direct binding of PI3K lipid products to the Akt pleckstrin homology domain. Akt can then phosphorylate substrates such as glycogen synthase kinase-3, 6-phosphofructo-2-kinase, and BAD. More recently, it was found that eNOS is also an Akt substrate and is activated by Akt-dependent phosphorylation to release NO in endothelial cells (31-34).The actions of estrogen can be mediated by the classical nuclear receptors, ER␣ and ER␤ (35,36) or through other putative membrane receptors. By definition, rapid effects of estrogen that involve nongenomic mechanisms are independent of transcriptional activation by the nuclea...

17␤-Estradiol activates endothelial nitric oxide synthase (eNOS),The cardioprotective effects of estrogen are diverse, including both rapid non-genomic and delayed genomic effects on the blood vessel wall (reviewed in Ref. 1). Specific, rapid vascular effects, such as moderation of vasomotor tone, have been linked to an estrogen-stimulated increase in bioavailable nitric oxide (NO) 1 (2-4). 17␤-estradiol (E2) treatment of human endothelial cells (EC) induces rapid release of NO by estrogen receptor (ER)-dependent activation of endothelial nitric oxide synthase (eNOS) (5). Many factors regulate eNOS enzyme activity, including fatty acid modification, subcellular localization, and binding to numerous proteins and cofactors, including calmodulin, caveolin-1, the 90-kDa heat shock protein (HSP90), and tetrahydrobiopterin (see Ref. 6 for review). eNOS is a Ca 2ϩ / calmodulin-dependent enzyme, the activity of which is also regulated by phosphorylation. Specific phosphorylation of eNOS by the serine/threonine kinase Akt renders the enzyme more active at much lower Ca 2ϩ concentrations (7,8). We demonstrated previously that the ER-dependent activation of eNOS occurs at resting Ca 2ϩ concentrations and requires activation of the phosphatidylinositol-3-OH kinase (PI3-kinase)/ Akt pathway (9). The regulatory subunit of PI3-kinase, P85, acts to stabilize and inhibit the catalytic activity of PI3-kinase. Recently, ER was shown to specifically bind to P85 in vitro (10). The E2-induced association correlated with increases in PI3-kinase activity in EC. However, the specific mechanism for E2 activation of PI3-kinase is not known.Evidence is emerging that membrane forms of steroid hormone receptors exist and participate in signaling pathways (11)(12)(13)(14). The activity of E2 at the cell membrane has been shown in EC, neurons, and breast cancer cell lines. We previously determined that rapid E2 activation of eNOS and MAP kinase occurs through a membrane-associated ER (9, 12). The EC line EAhy.926 used in these experiments exhibits rapid E2-induced signaling but is unable to stimulate ER-dependent gene transactivation. Additionally, EAhy.926 cells do not express the traditional 66-kDa ER␣ or ER␤ but express a 46-kDa protein immunoreactive with C-terminal ER antibodies. Recently, a protein of similar size reactive with E2 and anti-ER antibodies was found to be associated with the plasma membrane in MCF-7 cells (13,14). Additionally, a 46-kDa putative ER, reactive with anti-ER antibodies, was found in wild-type and in the initial ER␣ knockout mice. This form of the receptor was thought to be responsible for E2 enhancement of basal NO production in the initial ER␣ knockout mice, because this E2 effect was lost in the complete ER␣ knockout mouse (15). In human ECs expressing both the 66-and the 46-kDa receptor, both rapid signaling to MAP kinase and gene transactivation of estrogen-responsive element-luciferase reporter was stimulated with E2 treatment (12). As previously indicated, the specific mechanism of membrane-associated ER ...

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Regulation of the mitogen-activated protein kinase (MAPK) family by gonadotropin-releasing hormone (GnRH) in the gonadotrope cell line L␤T2 was investigated. Treatment with gonadotropin-releasing hormone agonist (GnRHa) activates extracellular signal-regulated kinase (ERK) and c-Jun NH 2 -terminal kinase (JNK). Activation of ERK by GnRHa occurred within 5 min, and declined thereafter, whereas activation of JNK by GnRHa occurred with a different time frame, i.e. it was detectable at 5 min, reached a plateau at 30 min, and declined thereafter. GnRHa-induced ERK activation was dependent on protein kinase C or extracellular and intracellular Ca 2؉ , whereas GnRHa-induced JNK activation was not dependent on protein kinase C or on extracellular or intracellular Ca 2؉ . To determine whether a mitogen-activated protein kinase family cascade regulates rat luteinizing hormone ␤ (LH␤) promoter activity, we transfected the rat LH␤ (؊156 to ؉7)-luciferase construct into L␤T2 cells. GnRH activated the rat LH␤ promoter activity in a time-dependent manner. Neither treatment with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD98059, nor cotransfection with a catalytically inactive form of a mitogenactivated protein kinase construct inhibited the induction of the rat LH␤ promoter by GnRH. Furthermore, cotransfection with a dominant negative Ets had no effect on the response of the rat LH␤ promoter to GnRH. On the other hand, cotransfection with either dominant negative JNK or dominant negative c-Jun significantly inhibited the induction of the rat LH␤ promoter by GnRH. In addition, GnRH did not induce either the rat LH␤ promoter activity in L␤T2 cells transfected stably with dominant negative c-Jun. These results suggest that GnRHa differentially activates ERK and JNK, and a JNK cascade is necessary to elicit the rat LH␤ promoter activity in a c-Jun-dependent mechanism in L␤T2 cells. GnRH,1 a hypothalamic decapeptide, serves as a key regulator of the reproductive system. GnRH acts on anterior pituitary gonadotropes to stimulate the synthesis and release of the pituitary gonadotropins LH and FSH. The gonadotropins are subunit hormones, each containing noncovalently linked ␣-and ␤-subunits (1, 2). Within a species, the ␣-subunits are identical, while the ␤-subunits differ and confer the physiological specificity of the heterodimeric hormone. Each ␤-subunit as well as the common ␣-subunit is encoded by different genes on separate chromosomes. When GnRH binds to its seven-transmembrane receptor (3), it induces interaction of the receptor with the heterotrimeric G q protein, which leads to activation of phospholipase C and formation of inositol 1,4,5-triphosphate and diacylglycerol, leading to elevation of intracellular Ca 2ϩ