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

Mark Connor*, Graeme Henderson

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

59 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)333-340
Number of pages8
JournalBritish Journal of Pharmacology
Volume117
Issue number2
Publication statusPublished - 1996
Externally publishedYes

Keywords

  • Calcium mobilization
  • Intracellular calcium
  • Muscarinic receptors
  • Opioids
  • SH-SY5Y

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