delta-Opioids stimulate inositol 1,4,5-trisphosphate formation, and so mobilize Ca2+ from intracellular stores, in undifferentiated NG108-15 cells.
delta-Opioids mobilize Ca2+ from intracellular stores in undifferentiated NG108-15 cells, but the mechanism involved remains unclear. Therefore, we examined the effect of [D-Pen 2,5] enkephalin on inositol 1,4,5-trisphosphate formation in these cells. [D-Pen 2,5] enkephalin caused a dose-dependent (EC50= 3.1 nM) increase in inositol 1,4,5-trisphosphate formation (measured using a specific radioreceptor mass assay), which peaked (25.7+/-1.2 pmol/mg of protein with 1 microM, n=9) at 30 s and returned to basal levels (10.6+/-0.9 pmol/mg of protein, n=9) within 4-5 min. This response was fully naloxone (1 microM) reversible and pertussis toxin (100ng/ml for 24 h) sensitive. Preincubation with Ni2+ (2.5 mM) or nifedipine (1 microM) had no effect on the [D-Pen 2,5] enkephalin (1 microM)-induced inositol 1,4,5-triphosphate response, and K+ (80mM) was unable to stimulate inositol 1,4,5-trisphosphate formation, indicating Ca2+ influx-induced activation of phospholipase C is not involved. Preincubation with the protein kinase C inhibitor Ro 31-8220 (1 microM) enhanced, whereas acute expo sure to phorbol 12,13-dibutyrate (1 microM) abolished, the [D-Pen 2,5] enkephalin (0.1 microM)-induced inositol 1,4,5-triphosphate response, suggesting protein kinase C exerts an autoinhibitory feedback action. [D-Pen 2,5] Enkephalin also dose-dependently (EC50 =2.8 nM) increased the intracellular [Ca2+], which was maximal (24 nM increase with 1 microM, n=5) at 30 s. This close temporal and dose-response relationship strongly suggests that delta-opioid receptor-mediated increases in intracellular [Ca2+] results from inositol 1,4,5-trisphosphate-induced Ca2+ release from intracellular stores, in undifferentiated NG108-15 cells.
Smart
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Department of Anaesthesia, Leicester Royal Infirmary, England.
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D000700:Analgesics / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000494:pharmacology; D005910:Glioma; D015544:Inositol 1,4,5-Trisphosphate / Q000097:blood / Q000378:metabolism*; D009447:Neuroblastoma; D009532:Nickel / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D037342:Pertussis Toxin; D017465:Receptors, Opioid, delta / Q000502:physiology*; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000648:ultrastructure; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000700:Analgesics; D002121:Calcium Channel Blockers; D004745:Enkephalins; D017465:Receptors, Opioid, delta; D010566:Virulence Factors, Bordetella; D009532:Nickel; D015544:Inositol 1,4,5-Trisphosphate; D020881:Enkephalin, D-Penicillamine (2,5)-; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D009543:Nifedipine; D002118:Calcium