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

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-Pen^2,5]-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.