Opioid receptor-mediated inhibition of ω-conotoxin GVIA-sensitive calcium channel currents in rat intracardiac neurons

Modulation of depolarization-activated ionic conductances by opioid receptor agonists was investigated in isolated parasympathetic neurons from neonatal rat intracardiac ganglia by using the whole cell perforated patch clamp technique. Met-enkephalin (10 mu M) altered the action potential waveform, reducing the maximum amplitude and slowing the rate of rise and repolarization but the afterhyperpolarization was not appreciably altered. Under voltage clamp, 10 mu M Met-enkephalin selectively and reversibly inhibited the peak amplitude of high-voltage-activated Ca2+ channel currents elicited at 0 mV by similar to 52% and increased three- to fourfold the time to peak. Met-enkephalin had no effect on the voltage dependence of steady-state inactivation but shifted the voltage dependence of activation to more positive membrane potentials whereby stronger depolarization was required to open Ca2+ channels. Half-maximal inhibition of Ba2+ current (I-Ba) amplitude was obtained with 270 nM Met-enkephalin or Leu-enkephalin. The opioid receptor subtype selective agonists, DAMGO and DADLE, but not DPDPE, inhibited I-Ba and were antagonized by the opioid receptor antagonists, naloxone and naltrindole with IC(50)s of 84 nM and 1 mu M, respectively. The kappa-opioid receptor agonists, bremazocine and dynorphin A, did not affect Ca2+ channel current amplitude or kinetics. Taken together, these data suggest that enkephalin-induced inhibition of Ca2+ channels in rat intracardiac neurons is mediated primarily by the mu-opioid receptor type. Addition of Met enkephalin after exposure to 300 nM omega-conotoxin GVIA, which blocked similar to 75% of the total Ca2+ channel current, failed to cause a further decrease of the residual current. Met-enkephalin inhibited the omega-conotoxin GVIA-sensitive but not the omega-conotoxin-insensitive I-Ba in rat intracardiac neurons. Dialysis of the cell with a GTP-free intracellular solution or preincubation of the neurons in Pertussis toxin (PTX) abolished the attenuation of I-Ba by Met-enkephalin, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. The activation of mu-opioid receptors and subsequent inhibition of N-type Ca2+ channels in the soma and terminals of postganglionic intracardiac neurons, is likely to inhibit the release of ACh and thereby regulate vagal transmission to the mammalian heart.