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

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.