title: Alpha adrenergic receptor subtypes involved in prostaglandin synthesis are coupled to Ca++ channels through a pertussis toxin-sensitive guanine nucleotide-binding protein.
abstract: Previously, we have shown that alpha-2C and alpha-1A adrenergic receptors (AR) stimulate prostacyclin (PGI2) synthesis through a pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in vascular smooth muscle cells (VSMC). The purpose of this study was to assess the role of Ca++ in PGI2 production elicited by alpha-AR activation and to investigate the modulation of the Ca++ channel by G proteins coupled to these alpha-AR in VSMC. PGI2 was measured as immunoreactive 6-keto-PGF1 alpha by radioimmunoassay and cytosolic calcium ([Ca++]i) by spectrofluorometry using fura-2. Norepinephrine, methoxamine and UK-14304 enhanced 6-keto-PGF1 alpha production and [Ca++]i, which was inhibited by depletion of extracellular Ca++ and by Ca++ channel antagonists (verapamil, nifedipine and PN 200-110). Moreover, the Ca++ channel activator Bay K 8644 increased 6-keto-PGF1 alpha production in a nifedipine-sensitive manner, indicating the involvement of dihydropyridine-sensitive Ca++ channels in VSMC. Pertussis toxin inhibited AR agonist-induced 6-keto-PGF1 alpha production and the increase in [Ca++]i. Alpha AR agonists increase Ca++ influx in the presence of guanosine 5'-0-(2- thiodiphosphate) (GTP-gamma-S), and this effect was blocked in the presence of guanine 5'-O-(2-thiodiphosphate) (GDP-beta-S) and antiserum against Gi alpha 1-2 protein in reversibly permeabilized cells with beta-escin. VSMC of rabbit aortae contain a G protein(s) that was recognized by Gi alpha 1-2 but not Gi alpha 3 or G0 antibodies at 1:200 dilution. The calmodulin inhibitor W-7 blocked AR agonist and Bay K 8644-stimulated 6-keto-PGF1 alpha production. The phospholipase A2 inhibitors 7,7-dimethyleicosadienoic acid and oleoyloxyethyl phosphocholine but not phospholipase C inhibitor U-73122 reduced 6-keto-PGF1 alpha production in VSMC. These data suggest that a pertussis toxin-sensitive G protein, probably Gi alpha 1-2, coupled to alpha AR regulates Ca++ influx, which, in turn, by interacting with calmodulin, increases phospholipase A2 activity to release arachidonic acid for PGI2 synthesis in VSMC of rabbit aortae.
authors.affiliation: Department of Pharmacology, College of Medicine, University of Tennessee, Memphis.
authors.forename: C
authors.initials: C
authors.lastname: Nebigil
authors.forename: K U
authors.initials: KU
authors.lastname: Malik
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000502:physiology*; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009281:Naphthalenes; D037342:Pertussis Toxin; D010741:Phospholipases A / Q000502:physiology; D054467:Phospholipases A2; D011083:Polycyclic Compounds / Q000494:pharmacology; D011453:Prostaglandins / Q000096:biosynthesis*; D011817:Rabbits; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
chemical_list: D015220:Calcium Channels; D009281:Naphthalenes; D011083:Polycyclic Compounds; D011453:Prostaglandins; D011942:Receptors, Adrenergic, alpha; D013449:Sulfonamides; D010566:Virulence Factors, Bordetella; C017967:W 7; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D037342:Pertussis Toxin; D010741:Phospholipases A; D054467:Phospholipases A2; D019204:GTP-Binding Proteins; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium