title: Role of putative membrane receptors in the effects of estradiol on human vascular cell growth.
abstract: The present study was designed to determine whether some of the effects of estrogen on human vascular cell growth are exerted through membrane-binding sites, using native as well as novel protein-bound, membrane non-permeant estrogenic complexes. We measured changes in DNA synthesis and creatine kinase-specific activity (CK), after treatment with estradiol-17beta (E(2)), estradiol-17beta-6-(O)-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (E(2)-BSA), 6-carboxymethyl genistein (CG) or 6- carboxymethyl genistein bound to the high molecular protein keyhole limpet hemocyanin (CG-KLH), and 7-(O)-carboxymethyl daidzein (CD) or 7-(O)-carboxymethyl daidzein linked to keyhole limpet hemocyanin (CD-KLH). High concentrations of either E(2) or E(2)-BSA inhibited DNA synthesis in vascular smooth muscle cells (VSMC) (-39% +/- 28% v -32% +/- 15%). Estradiol as well as CG and CD increased DNA synthesis dose dependently in endothelial ECV-304 cells. The CG and CD, as well as CG-KLH and CD-KLH, stimulated DNA synthesis dose dependently in VSMC (66% +/- 2%, 100% +/- 12%, 66% +/- 6%, and 41% +/- 8% at 300 nmol/L, respectively). In contrast all forms of protein-bound hormones were unable to affect DNA synthesis in ECV-304 cells or CK in either cell type. In VSMC, both free and bound hormones increased mitogen-activated protein-kinase (MAPK)-kinase activity, which was blocked by UO126, an inhibitor of MAPK-kinase. Furthermore, the effects of E(2), E(2)-BSA, or CG-KLH on DNA synthesis were inhibited by UO126. Using the E(2)-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for E(2) in VSMC and ECV 304 cells. Hence, the effects of E(2) on DNA synthesis in human VSMC, but not in endothelial cells, are apparently exerted by membrane-binding sites for E(2) and do not require intracellular entry of E(2) through the classic nuclear receptor route.
authors.affiliation: Institute of Endocrinology, Metabolism, and Hypertension, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel.
authors.forename: Dalia
authors.initials: D
authors.lastname: Somjen
authors.forename: Fortune
authors.initials: F
authors.lastname: Kohen
authors.forename: Batya
authors.initials: B
authors.lastname: Gayer
authors.forename: Orly
authors.initials: O
authors.lastname: Sharon
authors.forename: Merav
authors.initials: M
authors.lastname: Baz
authors.forename: Rona
authors.initials: R
authors.lastname: Limor
authors.forename: Tikva
authors.initials: T
authors.lastname: Kulik
authors.forename: Esther
authors.initials: E
authors.lastname: Knoll
authors.forename: Naftali
authors.initials: N
authors.lastname: Stern
mesh_terms: D002070:Butadienes / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000008:administration & dosage*; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D009570:Nitriles / Q000037:antagonists & inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D011960:Receptors, Estrogen / Q000187:drug effects / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D002070:Butadienes; D004791:Enzyme Inhibitors; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D009570:Nitriles; D012333:RNA, Messenger; D011960:Receptors, Estrogen; C113580:U 0126; D004958:Estradiol; D004247:DNA; D020928:Mitogen-Activated Protein Kinases; D003402:Creatine Kinase