Inhibitory effects of opioids on voltage-dependent Ca2+ channels and catecholamine secretion in cultured porcine adrenal chromaffin cells

The inhibitory effects of opioids on voltage-dependent calcium channels (VDCCs) were investigated in cultured porcine adrenal chromaffin cells using whole-cell patch clamp technique. The effects of the opioid on [Ca2+], increase and catecholamine secretion induced by high K+ were also examined in single cells by fura-2 micro fluorimetry and amperometry. A depolarizing pulse to 0 mV (test pulse) from a holding potential of -80 mV evoked an inward barium current (I-Ba), which was reversibly inhibited by methionine-enkephalin. This inhibitory effect of methionine-enkephalin was abolished by naloxone. Selective agonists of opioid receptor subtypes (DAMGO: mu, DPDPE: 8, U50488: kappa) dose-dependently inhibited I-Ba. In inhibitory potency, the order was DAMGO>U50488>DPDPE. These agonists applied sequentially produced a reversible I-Ba inhibition in the same cells. The inhibitory effect of DAMGO on I-Ba almost disappeared in the presence of omega-conotoxin GVIA but not omega-agatoxin IVA plus nifedipine. Application of a conditioning prepulse to + 100 mV prior to the test pulse partly retrieved the I-Ba inhibition by DAMGO, suggesting the involvement of voltage-sensitive components in opioid-induced VDCC inhibition. Intracellular application of GDPbetaS or GTP-gammaS as well as pretreatment with pertussis toxin significantly reduced the extent of I-Ba inhibition induced by DAMGO. DAMGO reversibly inhibited the [Ca2+], increase and catecholamine release induced by high K+. RT-PCR revealed the expression of mu-, delta- and K-Opioid receptor mRNAs in cultured adrenal chromaffin cells. These results suggest that porcine adrenal chromaffin cells possess mu-, delta- and kappa-opioid receptors and activation of Opioid receptors mainly inhibits N-type VDCCs via pertussis toxin-sensitive G-proteins. (C) 2002 Elsevier Science B.V. All rights reserved.