δ‐ and μ‐opioid receptor mobilization of intracellular calcium in SH‐SY5Y human neuroblastoma cells

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Abstract
1 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. 2 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. 3 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. 4 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. 5 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. 6 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. 7 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. 8 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. 9 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. 10 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.