OPIOIDS MOBILIZE CALCIUM FROM INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE STORES IN NG108-15 CELLS

Opioids elicit an increase in the intracellular free Ca2+ concentratio n ([Ca2+](j)) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in d ifferentiated cells or Ca2+ release from internal stores in undifferen tiated cells (Jin et al., 1992). In this report we describe fura-2-bas ed digital imaging studies that demonstrate that opioid-evoked Ca2+ re lease in these cells results from the activation of phospholipase C (P LC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (I P3)-sensitive store. D-Ala(2)-D-Leu(5)-enkephalin (DADLE) evoked conce ntration-dependent increases in [Ca2+](j) (EC(50) congruent to 4 nM). The response was blocked by naloxone (1 mu M) In single cells, sequent ial application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen(2), D-Pen(5)-enkephalin (DPDPE) > tran s-(+/-) dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacet amide (U50488) > D-ala(2), N-Me-Phe(4), Gly(5)-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent ( n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+](j) i ncrease of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 9 1% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE- evoked responses were not blocked by removal of extracellular Ca2+; in stead, they were abolished by treatment with 10 nM thapsigargin, an ag ent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 mu M concentration of U73122, an aminosteroid inhibitor of PLC, c ompletely blocked the DADLE-evoked [Ca2+](j) increase, while an inacti ve analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+](j) increases in NG108-1 5 cells, we pretreated cells with pertussis or cholera toxin; pertussi s toxin blocked the opioid-induced response while cholera toxin was wi thout effect, consistent with a G(i)- or G(o)-mediated effect. Activat ion of the opioid inhibitory pathway previously described for these ce lls appears to stimulate the phosphoinositide (PI) cascade as well. In cluding the PI cascade among the multiple second messenger systems mod ulated by opioids may be key to understanding the biochemical events t hat underlie acute and chronic opioid action.