title: Voltage-dependent inhibition of Ca2+ channels in GH3 cells by cloned mu- and delta-opioid receptors.
abstract: To study cloned opioid receptor binding and modulation of both adenylyl cyclase and ion channel activity, we stably expressed mu- and delta-opioid receptors in the rodent pituitary-derived GH3 cell line. GH3 cells express G proteins and voltage-activated Ca2+ channels (predominantly of the L-type). Activation of cloned rat mu-opioid receptors expressed in GH3 cells (termed GH3MOR cells) inhibits L-type Ca2+ channel activity. GH3MOR cells, further transfected with mouse delta receptor cDNA (termed GH3MORDOR cells), bound both [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) and [D-Pen2,D-Pen5]enkephalin (DPDPE). These opioid ligands inhibited adenylyl cyclase activity (IC50 = 174 and 0.53 nM, respectively). This action of DAMGO and DPDPE was attenuated selectively by mu- and delta-opioid receptor-specific antagonists. Activation of both opioid receptors also led to inhibition of Ca2+ channel activity, measured with Ba2+ as the charge carrier using the whole-cell patch-clamp technique. Both DAMGO (1 microM) and DPDPE (1 microM) reversibly inhibited Ba2+ currents (by 17.0 +/- 1.4% and 20.7 +/- 1.3%, respectively) in GH3MORDOR cells. The inhibitory action of DPDPE was dose dependent (IC50 = 1.6 nM) and was attenuated by pretreatment with pertussis toxin (200 ng/ml) or by the inclusion of guanosine-5'-O-(2-thio)diphosphate (2 mM) in the recording electrode. Ba2+ current inhibitions by both DAMGO and DPDPE were completely reversed by depolarizing (to > 50 mV) prepulses in GH3MORDOR cells. In summary, cloned mu- and delta-opioid receptors expressed in GH3 cells voltage-dependently couple through Gi/G(o) proteins to L-type Ca2+ channels.
authors.affiliation: Department of Psychiatry, University of California Los Angeles, School of Medicine 90024, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Piros
authors.forename: P L
authors.initials: PL
authors.lastname: Prather
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; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000502:physiology; D007700:Kinetics; D037342:Pertussis Toxin; D010902:Pituitary Gland / Q000166:cytology / Q000502:physiology; D051381:Rats; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases