VOLTAGE-DEPENDENT INHIBITION OF CA2-OPIOID AND DELTA-OPIOID RECEPTORS( CHANNELS IN GH(3) CELLS BY CLONED MU)

To study cloned opioid receptor binding and modulation of both adenyly l cyclase and ion channel activity, we stably expressed mu- and delta- opioid receptors in the rodent pituitary-derived GH(3) cell line. GH(3 ) cells express G proteins and voltage-activated Ca2+ channels (predom inantly of the L-type). Activation of cloned rat mu-opioid receptors e xpressed in GH(3) cells (termed GH(3)MOR cells) inhibits L-type Ca2+ c hannel activity. GH(3)MOR cells, further transfected with mouse delta receptor cDNA (termed GH(3)MORDOR cells), bound both [D-Ala(2),N-MePhe (4),Gly-ol(5)]enkephalin (DAMGO) and [D-Pen(2),D-Pen(5)]enkephalin (DP DPE). These opioid ligands inhibited adenylyl cyclase activity (IC50 = 174 and 0.53 nM, respectively). This action of DAMGO and DPDPE was at tenuated selectively by mu- and delta-opioid receptor-specific antagon ists. Activation of both opioid receptors also led to inhibition of Ca 2+ channel activity, measured with Ba2+ as the charge carrier using th e whole-cell patch-clamp technique. Both DAMGO (1 mu M) and DPDPE (1 m u M) reversibly inhibited Ba2+ currents (by 17.0 +/- 1.4% and 20.7 +/- 1.3%, respectively) in GH(3)MORDOR cells. The inhibitory action of DP DPE was dose dependent (IC50 = 1.6 nM) and was attenuated by pretreatm ent with pertussis toxin (200 ng/ml) or by the inclusion of guanosine- 5'-O-(2-thio)diphosphate (2 mM) in the recording electrode. Ba2+ curre nt inhibitions by both DAMGO and DPDPE were completely reversed by dep olarizing (to >50 mV) prepulses in GH(3)MORDOR cells. In summary, clon ed mu- and delta-opioid receptors expressed in GH(3) cells voltage-dep endently couple through G(i)/G(o) proteins to L-type Ca2+ channels.