Description:
In this study we have investigated δ and μ opioid receptor‐mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line, SH‐SY5Y.The Ca2+‐sensitive dye, fura‐2, was used to measure [Ca2+]i in confluent monolayers of SH‐SY5Y cells. Neither the δ‐opioid agonist, DPDPE ([D‐Pen2,5]‐enkephalin) nor the μ‐opioid agonist, DAMGO (Tyr‐D‐Ala‐Gly‐N‐Me‐Phe‐Gly‐ol enkephalin) elevated [Ca2+]i when applied alone. However, when either DPDPE or DAMGO was applied in the presence of the cholinoceptor agonist, carbachol (100 nM‐1 mM) they evoked an elevation of [Ca2+]i above that caused by carbachol alone.In the presence of 1 μm or 100 μm carbachol, DPDPE elevated [Ca2+]i with an EC50 of 10 nM. The elevation of [Ca2+]i was independent of the concentration of carbachol. The EC50 for DAMGO elevating [Ca2+]i in the presence of 1 μm and 100 μm carbachol was 270 nM and 145 nM respectively.The δ‐receptor antagonist, naltrindole (30 nM), blocked the elevations of [Ca2+]i by DPDPE (100 nM) without affecting those caused by DAMGO while the μ‐receptor antagonist, CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Pen‐Thr‐NH2) (100 nM‐1 μm) blocked the elevations of [Ca2+]i caused by DAMGO (1 μm) without affecting those caused by DPDPE.Block of carbachol activation of muscarinic receptors with atropine (10 μm) abolished the elevation of [Ca2+]i by the opioids. The nicotinic receptor antagonist, mecamylamine (10 μm), did not affect the elevations of [Ca2+]i caused by opioids in the presence of carbachol.Muscarinic receptor activation, not a rise in [Ca2+]i, was required to reveal the opioid response. The Ca2+ channel activator, maitotoxin (3 ng ml−1), also elevated [Ca2+]i but subsequent application of opioid in the presence of maitotoxin caused no further changes in [Ca2+]i.The elevations of [Ca2+]i by DPDPE and DAMGO were abolished by pretreatment of the cells with pertussis toxin (200 ng ml−1, 16 h). This treatment did not significantly affect the response of the cells to carbachol.The opioids appeared to elevate [Ca2+]i by mobilizing Ca2+ from intracellular stores. Both DPDPE and DAMGO continued to elevate [Ca2+]i when applied in nominally Ca2+ ‐free external buffer or when applied in a buffer containing a cocktail of Ca2+ entry inhibitors. Thapsigargin (100 nM), an agent which discharges intracellular Ca2+ stores, also blocked the opioid elevations of [Ca2+]i.δ and μ Opioids did not appear to mobilize intracellular Ca2+ by modulating the activity of protein kinases. The application of H‐89 (10 μm), an inhibitor of protein kinase A, H‐7 (100 μm), an inhibitor of protein kinase C, protein kinase A and cyclic GMP‐dependent protein kinase, or Bis I, an inhibitor of protein kinase C, did not alter the opioid mobilization of [Ca2+]i.Thus, in SH‐SY5Y cells, opioids can mobilize Ca2+ from intracellular stores but they require ongoing muscarinic receptor activation. Opioids do not elevate [Ca2+]i when applied alone.