Genistein may improve vascular function, but the mechanism of this effect is unclear. We tested the hypothesis that genistein directly regulates vascular function through stimulation of endothelial nitric oxide synthesis. Genistein activated endothelial nitric oxide synthase (eNOS) in intact bovine aortic endothelial cells and human umbilical vein endothelial cells over an incubation period of 10 min. The maximal eNOS activity was at 1 M genistein. Consistent with this activation pattern, 1 M genistein maximally stimulated the phosphorylation of eNOS at serine 1179 at 10 min of incubation. The rapid activation of eNOS by genistein was not dependent on RNA transcription or new protein synthesis and was not blocked by a specific estrogen receptor antagonist. In addition, inhibition of MAPK or phosphatidylinositol 3-OH kinase/Akt kinase had no affect on eNOS activation by genistein. Furthermore, the genistein effect on eNOS was also independent of tyrosine kinase inhibition. However, inhibition of cAMP-dependent kinase [protein kinase A (PKA)] by H89 completely abolished the genistein-stimulated eNOS activation and phosphorylation, suggesting that genistein acted through a PKA-dependent pathway. These findings demonstrated that genistein had direct nongenomic effects on eNOS activity in vascular endothelial cells, leading to eNOS activation and nitric oxide synthesis. These effects were mediated by PKA and were unrelated to an estrogenic effect. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for soy phytoestrogens. (Endocrinology 145: 5532-5539, 2004)

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The glucagon-like peptide-1 (GLP-1) receptor is expressed on alpha-cells, though its functional significance is unknown. The endogenous beta-cell GLP-1 receptor is coupled to adenylyl cyclase, cell depolarization, activation of voltage-dependent Ca2+ channels (VDCC) and extracellular Ca2+ influx (Lu et al., 1993 b). In contrast, the signaling pathways of the GLP-1 receptor in alpha-cells are poorly understood. To determine the signaling mechanisms of the alpha-cell GLP-1 receptor, we established a stable pancreatic islet alpha-cell line expressing the recombinant rat GLP-1 receptor (INR1-SF2), using INRl-G9 cells. These INRl-G9 cells do not express endogenous GLP-1 receptor. In INR1-SF2 cells, GLP-1 bound to the recombinant receptor (Kd = 0.9 nM) and increased cAMP (ED50 = 0.6 nM). GLP-1 increased the free cytosolic Ca2+ ([Ca2+]i) (ED50 = 50 nM) by release from intracellular stores, but did not affect INR1-SF2 cell phosphoinositol turnover. Despite expressing VDCC, the INR1-SF2 cells were not depolarized by GLP-1, even in the presence of glucose. This contrasts with the depolarizing action of GLP-1 in beta-cells in the presence of glucose (Lu et al., 1993 b). This study establishes that a single GLP-1 receptor species can mediate the effects of GLP-1 through multiple signaling pathways, including the adenylyl cyclase system and intracellular Ca2+ release, in an alpha-cell type. Furthermore, since GLP-1 is unable to cause cellular depolarization or activate VDCC in INR1-SF2 cells, these data suggest that glucose-induced membrane depolarization may be crucial for GLP-1 to further activate VDCC and potentiate glucose-stimulated insulin release in beta-cells. Finally this study describes a cell line that can be used as a model system for evaluation of GLP-1 signaling in alpha-cells.

We have synthesized an analog of dehydroepiandrosterone (DHEA, 1) containing both a benzophenone (BP) and a biotin (Bt) group (DHEA-BP-Bt, 8). Compound 8 was prepared by functionalization on C-17 of 1. Biocytin was reacted with 4-benzoylbenzoic acid and the product was condensed with 1 containing a diamine-hexane linker. We detected specific protein bands of approximately 55, 80, and 150 kDa by SDS-PAGE analysis of vascular endothelial cell plasma membranes which had been photoirradiated in the presence of 8. KeywordsDHEA; photoaffinity; biotin; benzophenone; chemical probe There is extensive epidemiological evidence that plasma concentrations of the adrenal steroid dehydroepiandrosterone (5-androsten-3β-ol-17-one, DHEA, 1) are related to vascular function. [1][2][3][4][5] Hormonal steroids activate well characterized intracellular receptors, but may also activate cellular signaling by binding to receptors expressed at the plasma membrane. 6 However, there is no known receptor for 1 and the molecular mechanisms underlying its putative vascular benefits are unknown. 7 We and others have shown that 1 binds to specific, high affinity, plasma membrane sites on vascular endothelial and other cells, to activate cellular signaling in a G-protein-dependent manner. [8][9][10] Interaction of 1 with this plasma membrane receptor has been proposed as a potential mechanism of its vascular effects. 8 Characterization of the putative receptor involved in this pathway is crucial to advance these studies.Many investigators have successfully used the high affinity of avidin for biotin in protocols to affinity purify hormonal receptors by initially biotinylating the cognate ligand. Thus, Supplementary data associated with this article can be found, in the online version, at doi:Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. While the use of biotinylated ligands for receptor isolation has been a great advance in this field, the solubilization of plasma membrane proteins, required for receptor purification, can significantly decrease the affinity of the receptor for the biotinylated ligand and impair the ability to isolate low abundance receptors by avidin chromatography. Modification of ligands to include photoirradiation-activated cross-linking groups can facilitate extensive solubilization without loss of ligand receptor interaction after cross-linking. 20,21 We therefore took advantage of photoaffinity cross-linking and avidin-biotin affinity techniques, to design and synthesize a novel photoactivatable biotinylated analog of 1, in order to isolate the high affinity plasma membra...

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