mu-Opioid receptor stimulation of inositol (1,4,5)trisphosphate formation via a pertussis toxin-sensitive G protein.
The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both mu- and delta-opioid receptors), we demonstrated that fentanyl, a mu-preferring opioid, caused a dose-dependent (EC50 = 16 nM) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1-2 min. This response was of similar magnitude (25.4 +/- 0.8 pmol/mg of protein for 0.1 microM fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 mM carbachol (18.3 +/- 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole- (a delta antagonist), reversible. Further studies using [D-Ala2, MePhe4, Gly(ol)5]enkephalin and [D-Pen2,5]enkephalin confirmed that the response was specific for the mu receptor. Incubation with Ni2+ (2.5 mM) or in Ca(2+)-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 microM fentanyl, 26.9 +/- 1.5 pmol/mg of protein; pertussis-treated plus 0.1 microM fentanyl, 5.1 +/- 1.3 pmol/mg of protein). In summary, we have demonstrated a mu-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca(2+)-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca2+, could play a part in the cellular mechanisms of opioid action.
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University Department of Anaesthesia, Leicester Royal Infirmary, England.
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D002118:Calcium / Q000502:physiology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000494:pharmacology; D005283:Fentanyl / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis*; D009020:Morphine / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D037342:Pertussis Toxin; D017450:Receptors, Opioid, mu / Q000502:physiology*; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D004745:Enkephalins; D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D010566:Virulence Factors, Bordetella; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009020:Morphine; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium; D005283:Fentanyl