title: Modulation of high affinity phenylalkylamine binding sites on cultured human embryonal vascular smooth muscle cells.
abstract: Two phenylalkylamine Ca2+ channel ligands, (+/-)-[3H]verapamil ((+/-)-[3H]V) (-)-[3H]desmethoxyverapamil ((-)-[3H]DV), were employed in whole cell binding assays to characterize the specific high affinity binding sites on Ca2+ channels, their cooperativity and modulations induced on cultured human embryonal vascular smooth muscle preparation (VSM) by: 1) Beta-adrenergic stimulation of the cell, 2) exposure to high K+ concentration, 3) exposure to high concentration of Mg2+ ions, 4) the presence of a benzothiazepine Ca2+ channel antagonist and modulator d-cis-diltiazem, and 5) guanylylimidodiphosphate. The total amounts of specific (+/-)-[3H]V and (-)-[3H]DV binding sites present on VSM cells increased significantly after beta-adrenergic receptor activation, following cell membrane depolarization induced by high concentrations of K+, in the presence of Ca2+ chelator Na3EDTA, and after incubation of VSM cells with a benzothiazine-type Ca2+ channel blocker d-cis-diltiazem. A marked reduction of (-)-[3H]DV binding was observed after permanent G-protein activation by a nonhydrolyzable analog of guanylylimidodiphosphate, after incubation of the cells with norepinephrine, and after incubation of VSM cells with millimolar concentration of Mg2+. The results suggest the existence of multiple modulations of specific (-)-[3H]DV binding sites on Ca2+ channel corresponding to the way of activation of the cell and also to the immediate "state" of the membrane bound Ca2+ channels present on VSM cells, the positive heterotropic interaction after beta-adrenergic stimulation, the homotropic positive allosteric interaction induced by d-cis-diltiazem and pure noncompetitive inhibition induced by guanylylimidodiphosphate. The presence of high concentrations of Mg2+ inhibited whereas the presence of Ca2+ chelator, of ethylenediamine-tetraacetic acid sodium salt, significantly increased the total number of specific high affinity (-)-[3H]DV binding sites on VSM cells.
authors.affiliation: Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava.
authors.forename: J
authors.initials: J
authors.lastname: Drímal
mesh_terms: D001665:Binding Sites; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004110:Diltiazem / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D014700:Verapamil / Q000031:analogs & derivatives / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D006165:Guanylyl Imidodiphosphate; C043184:4-desmethoxyverapamil; D014700:Verapamil; D019204:GTP-Binding Proteins; D004110:Diltiazem; D008274:Magnesium; D007545:Isoproterenol; D011188:Potassium