delta- and mu-opioid receptor mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells

Br J Pharmacol. 1996 Jan;117(2):333-40. doi: 10.1111/j.1476-5381.1996.tb15195.x.


1. In this study we have investigated delta and mu opioid receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line, SH-SY5Y. 2. The Ca(2+)-sensitive dye, fura-2, was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither the delta-opioid agonist, DPDPE ([D-Pen2,5]-enkephalin) nor the mu-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 microM or 100 microM 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 microM and 100 microM carbachol was 270 nM and 145 nM respectively. 4. The delta-receptor antagonist, naltrindole (30 nM), blocked the elevations of [Ca2+]i by DPDPE (100 nM) without affecting those caused by DAMGO while the mu-receptor antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2) (100 nM-1 microM) blocked the elevations of [Ca2+]i caused by DAMGO (1 microM) without affecting those caused by DPDPE. 5. Block of carbachol activation of muscarinic receptors with atropine (10 microM) abolished the elevation of [Ca2+]i by the opioids. The nicotinic receptor antagonist, mecamylamine (10 microM), 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 Ca(2+)-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. delta and mu Opioids did not appear to mobilize intracellular Ca2+ by modulating the activity of protein kinases. The application of H-89 (10 microM), an inhibitor of protein kinase A, H-7 (100 microM), 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brain Neoplasms / metabolism*
  • Calcium / metabolism*
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Humans
  • Inositol 1,4,5-Trisphosphate / metabolism
  • Muscarinic Agonists / pharmacology
  • Neuroblastoma / metabolism*
  • Opioid Peptides / antagonists & inhibitors
  • Opioid Peptides / pharmacology
  • Pertussis Toxin
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • Receptors, Opioid, delta / agonists
  • Receptors, Opioid, delta / antagonists & inhibitors
  • Receptors, Opioid, delta / metabolism*
  • Receptors, Opioid, mu / agonists
  • Receptors, Opioid, mu / antagonists & inhibitors
  • Receptors, Opioid, mu / metabolism*
  • Tumor Cells, Cultured
  • Virulence Factors, Bordetella / pharmacology


  • Muscarinic Agonists
  • Opioid Peptides
  • Receptors, Opioid, delta
  • Receptors, Opioid, mu
  • Virulence Factors, Bordetella
  • Inositol 1,4,5-Trisphosphate
  • Pertussis Toxin
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C
  • Calcium