Ca²⁺ channel and adenylyl cyclase modulation by cloned μ-opioid receptors in GH₃ cells

Members of the three classes of opioid receptors (μ, δ, and κ) 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 μ-opioid receptor cDNAs into clonal pituitary GH₃ cells. GH₃ cells expressing μ-opioid receptors (GH₃MOR cells) bound the receptor-specific ligands [D-Ala²,Me-Phe⁴,Gly-ol⁵]-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, respectively). Functional coupling of μ-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 μM) and SRIF (1 μM) inhibited Ba²⁺ currents by 23.8 ± 1.0% and 22.9 ± 2.5%, respectively. DAMGO (0.1 nM to 10 μM) dose-dependently inhibited Ba²⁺ currents, with an IC₅₀ of 105 nM. The μ-opioid receptor agonist morphine (1 μM) inhibited currents by 13.5 ± 1.1% and the δ-opioid receptor-selective ligand [D-Pen^2,5]-enkephalin (1 μM) caused only 3.5 ± 2.1% inhibition. The inhibitory actions of DAMGO, morphine, and [D-Pen^2,5]-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 μ-opioid receptors couple to L-type Ca²⁺ channels in GH₃MOR cells.