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Opioids mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells

W Jin, NM Lee, HH Loh and SA Thayer
Journal of Neuroscience 1 April 1994, 14 (4) 1920-1929; DOI: https://doi.org/10.1523/JNEUROSCI.14-04-01920.1994
W Jin
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
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NM Lee
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
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HH Loh
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
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SA Thayer
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
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Abstract

Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108–15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid- evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5- trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5- enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-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+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE- evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid- induced [Ca2+]i increases in NG108–15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.

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The Journal of Neuroscience: 14 (4)
Journal of Neuroscience
Vol. 14, Issue 4
1 Apr 1994
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Opioids mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells
W Jin, NM Lee, HH Loh, SA Thayer
Journal of Neuroscience 1 April 1994, 14 (4) 1920-1929; DOI: 10.1523/JNEUROSCI.14-04-01920.1994

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Opioids mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells
W Jin, NM Lee, HH Loh, SA Thayer
Journal of Neuroscience 1 April 1994, 14 (4) 1920-1929; DOI: 10.1523/JNEUROSCI.14-04-01920.1994
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