Voltage-dependent inhibition of Ca²⁺ channels in GH₃ cells by cloned μ- and δ-opioid receptors

To study cloned opioid receptor binding and modulation of both adenylyl cyclase and ion channel activity, we stably expressed μ- and δ-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 μ-opioid receptors expressed in GH₃ cells (termed GH₃MOR cells) inhibits L-type Ca²⁺ channel activity. GH₃MOR cells, further transfected with mouse δ 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 μ- and δ-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 μM) and DPDPE (1 μM) reversibly inhibited Ba²⁺ currents (by 17.0 ± 1.4% and 20.7 ± 1.3%, respectively) in GH₃MORDOR cells. The inhibitory action of DPDPE was close 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 μ- and δ- opioid receptors expressed in GH₃ cells voltage-dependently couple through G(i)G(o) proteins to L-type Ca²⁺ channels.