CA2-CYCLASE MODULATION BY CLONED MU-OPIOID RECEPTORS IN GH(3) CELLS( CHANNEL AND ADENYLYL)

Members of the three classes of opioid receptors (mu, delta, and kappa ) have been cloned and characterized in unexcitable cell lines using b iochemical 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 in to clonal pituitary GH(3) cells, GH(3) cells expressing mu-opioid rece ptors (GH(3)MOR cells) bound the receptor-specific ligands [D-Ala(2),M e-Phe(4),Gly-ol(5)]-enkephalin (DAMGO) and morphine with high affinity (K-i = 1.0 and 7.2 nM, respectively), and these ligands also potently inhibited adenylyl cyclase activity (IC50 = 21.9 and 55.2 nM, respect ively). Functional coupling of mu-opioid receptors to voltage-activate d Ca2+ channels was compared with that of endogenous somatostatin (SRI F) receptors in GH(3)MOR cells, using the patch-clamp technique, with Ba2+ as the charge carrier. DAMGO (1 mu M) and SRIF (1 mu M) inhibited Ba2+ currents by 23.8 +/- 1.0% and 22.9 +/- 2.5%, respectively. DAMGO (0.1 nM to 10 mu M) dose-dependently inhibited Ba2+ currents, with an IC50 of 105 nM. The mu-opioid receptor agonist morphine (1 mu M) inhi bited currents by 13.5 +/- 1.1% and the delta-opioid receptor-selectiv e ligand [D-Pen(2,5)]-enkephalin (1 mu M) caused only 3.5 +/- 2.1% inh ibition. The inhibitory actions of DAMGO, morphine, and [D-Pen(2,5)]-e nkephalin 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 GH(3)MOR ce lls.