Smart D, Lambert D G
Department of Anaesthesia, Leicester Royal Infirmary, England.
J Neurochem. 1996 Apr;66(4):1462-7. doi: 10.1046/j.1471-4159.1996.66041462.x.
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.
δ-阿片类物质可促使未分化的NG108-15细胞内的钙离子从细胞内储存库中释放出来，但其中涉及的机制仍不清楚。因此，我们研究了[D-青霉胺2,5]脑啡肽对这些细胞中肌醇1,4,5-三磷酸生成的影响。[D-青霉胺2,5]脑啡肽导致肌醇1,4,5-三磷酸生成呈剂量依赖性增加（EC50 = 3.1 nM）（使用特异性放射受体质量分析法测定），在30秒时达到峰值（1 μM时为25.7±1.2 pmol/mg蛋白质，n = 9），并在4-5分钟内恢复到基础水平（10.6±0.9 pmol/mg蛋白质，n = 9）。该反应完全可被纳洛酮（1 μM）逆转且对百日咳毒素（100 ng/ml，作用24小时）敏感。用Ni2+（2.5 mM）或硝苯地平（1 μM）预孵育对[D-青霉胺2,5]脑啡肽（1 μM）诱导的肌醇1,4,5-三磷酸反应无影响，且K+（80 mM）无法刺激肌醇1,4,5-三磷酸的生成，表明不涉及钙离子内流诱导的磷脂酶C激活。用蛋白激酶C抑制剂Ro 31-8220（1 μM）预孵育可增强[D-青霉胺2,5]脑啡肽（0.1 μM）诱导的肌醇1,4,5-三磷酸反应，而急性暴露于佛波醇12,13-二丁酸酯（1 μM）则消除该反应，提示蛋白激酶C发挥自抑制反馈作用。[D-青霉胺2,5]脑啡肽还呈剂量依赖性增加细胞内钙离子浓度（EC50 = 2.8 nM），在30秒时达到最大值（1 μM时增加24 nM，n = 5）。这种紧密的时间和剂量反应关系强烈表明，在未分化的NG108-15细胞中，δ-阿片受体介导的细胞内钙离子浓度增加是由肌醇1,4,5-三磷酸诱导的细胞内储存库钙离子释放所致。
