Dual excitatory and inhibitory effects of opioids on intracellular calcium in neuroblastoma x glioma hybrid NG108-15 cells

W. Jin, N. M. Lee, Horace H Loh, Stanley A Thayer

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The intracellular free calcium concentration ([Ca2+];) was measured in single NG108-15 cells using indo-1-based microfluorimetry. In cells differentiated for 6-14 days in serum-free, forskolin (5 μM)-supplemented medium, application of micromolar concentrations of [D-Ala2, D-Leu5]-enkephalin (DADLE) inhibited Ca2+ influx mediated by voltage-gated Ca2+ channels. DADLE, at concentrations ranging from 1 nM to 1 μM, also produced rapid transient increases in [Ca2+]i (EC50 = 10 nM). The [Ca2+], increases elicited by DADLE did not correlate with the inhibitory effects of the peptide. DADLE-induced [Ca2+], increases were blocked by naloxone. In single cells, sequential application of selective opioid agonists (30 nM) evoked responses of the rank order DADLE = [D-Pen2,D-Pen5]-enkephalin > (trans)-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide > [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, consistent with activation of a δ-opioid receptor. The response was completely blocked by removal of extracellular Ca2+ or application of 1 μM nitrendipine, indicating that the increase in [Ca2+], results from Ca2+ influx via dihydropyridine-sensitive, voltage-gated Ca2+ channels. Substitution of N-methyl-o-glucamine for extracellular Na+ or application of 1 μM tetrodotoxin greatly reduced, and in some cases blocked, the DADLE-induced [Ca2+]i increase, consistent with amplification of the response by voltage-gated Na+ channels. The [Ca2+]i increase was mimicked by both dibutyryl-cAMP and phorbol 12,13-dibutyrate. These findings are consistent with a δ-opioid-induced depolarization, possibly mediated by a second messenger, that subsequently recruits voltage-sensitive Ca2+ channels. In contrast to differentiated cells, undifferentiated cells responded to DADLE with a modest [Ca2+]i increase that was not sensitive to nitrendipine. In these cells, activation of the same second messenger system may elevate [Ca2+]i by mobilization from intracellular stores rather than influx. In addition to previously described inhibitory coupling to adenylyl cyclase and Ca2+ channels in NG108-15 cells, these results suggest that a novel, excitatory, effector system may also couple to opioid receptors.

Original languageEnglish (US)
Pages (from-to)1083-1089
Number of pages7
JournalMolecular Pharmacology
Issue number6
StatePublished - Dec 1992


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