title: Ca2+ channel and adenylyl cyclase modulation by cloned mu-opioid receptors in GH3 cells.
abstract: Members of the three classes of opioid receptors (mu, delta, and kappa) have been cloned and characterized in unexcitable cell lines using biochemical techniques. However, an important function of these cloned receptors, their coupling to voltage-activated Ca2+ channels, remains untested. We stably transfected cloned rat mu-opioid receptor cDNAs into clonal pituitary GH3 cells. GH3 cells expressing mu-opioid receptors (GH3MOR cells) bound the receptor-specific ligands [D-Ala2,Me-Phe4,Gly-ol5]-enkephalin (DAMGO) and morphine with high affinity (Ki = 1.0 and 7.2 nM, respectively), and these ligands also potently inhibited adenylyl cyclase activity (IC50 = 21.9 and 55.2 nM, respectively). Functional coupling of mu-opioid receptors to voltage-activated Ca2+ channels was compared with that of endogenous somatostatin (SRIF) receptors in GH3MOR cells, using the patch-clamp technique, with Ba2+ as the charge carrier. DAMGO (1 microM) and SRIF (1 microM) inhibited Ba2+ currents by 23.8 +/- 1.0% and 22.9 +/- 2.5%, respectively. DAMGO (0.1 nM to 10 microM) dose-dependently inhibited Ba2+ currents, with an IC50 of 105 nM. The mu-opioid receptor agonist morphine (1 microM) inhibited currents by 13.5 +/- 1.1% and the delta-opioid receptor-selective ligand [D-Pen2,5]-enkephalin (1 microM) caused only 3.5 +/- 2.1% inhibition. The inhibitory actions of DAMGO, morphine, and [D-Pen2,5]-enkephalin were reversed by naloxone. Ba2+ current inhibitions by DAMGO and SRIF were attenuated by pertussis toxin pretreatment. Nimodipine reduced the amplitude of Ba2+ current inhibition by DAMGO, suggesting that mu-opioid receptors couple to L-type Ca2+ channels in GH3MOR cells.
authors.affiliation: Department of Psychiatry and Biobehavioral Sciences, University of California, School of Medicine, Los Angeles 90024, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Piros
authors.forename: P L
authors.initials: PL
authors.lastname: Prather
authors.forename: H H
authors.initials: HH
authors.lastname: Loh
authors.forename: P Y
authors.initials: PY
authors.lastname: Law
authors.forename: C J
authors.initials: CJ
authors.lastname: Evans
authors.forename: T G
authors.initials: TG
authors.lastname: Hales
mesh_terms: D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001464:Barium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000378:metabolism / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D017136:Ion Transport; D008564:Membrane Potentials; D009020:Morphine / Q000378:metabolism / Q000494:pharmacology; D009270:Naloxone / Q000494:pharmacology; D037342:Pertussis Toxin; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017450:Receptors, Opioid, mu / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013004:Somatostatin / Q000494:pharmacology; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D015220:Calcium Channels; D004745:Enkephalins; D017450:Receptors, Opioid, mu; D010566:Virulence Factors, Bordetella; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D001464:Barium; D009270:Naloxone; D013004:Somatostatin; D009020:Morphine; D020881:Enkephalin, D-Penicillamine (2,5)-; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases