Dual excitatory and inhibitory effects of opioids on intracellular calcium in neuroblastoma x glioma hybrid NG108-15 cells.
The intracellular free calcium concentration ([Ca2+]i) was measured in single NG108-15 cells using indo-1-based microfluorimetry. In cells differentiated for 6-14 days in serum-free, forskolin (5 microM)-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 microM, also produced rapid transient increases in [Ca2+]i (EC50 = 10 nM). The [Ca2+]i increases elicited by DADLE did not correlate with the inhibitory effects of the peptide. DADLE-induced [Ca2+]i 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 delta-opioid receptor. The response was completely blocked by removal of extracellular Ca2+ or application of 1 microM nitrendipine, indicating that the increase in [Ca2+]i results from Ca2+ influx via dihydropyridine-sensitive, voltage-gated Ca2+ channels. Substitution of N-methyl-D-glucamine for extracellular Na+ or application of 1 microM 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 delta-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.
Jin
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Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
Lee
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NM
Loh
H H
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Thayer
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D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D004745:Enkephalins / Q000494:pharmacology; D005910:Glioma; D006822:Hybrid Cells; D015640:Ion Channel Gating / Q000187:drug effects; D009294:Narcotics / Q000494:pharmacology*; D009447:Neuroblastoma; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology; D017465:Receptors, Opioid, delta / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D004745:Enkephalins; D009294:Narcotics; D011759:Pyrrolidines; D017465:Receptors, Opioid, delta; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D015240:Phorbol 12,13-Dibutyrate; D016308:Enkephalin, Leucine-2-Alanine; D003994:Bucladesine; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D020881:Enkephalin, D-Penicillamine (2,5)-; D002118:Calcium