甲硫—脑啡肽对大鼠DRG分离神经元ATP—激活内向电流的抑制

本研究探讨了甲硫-脑啡肽(met-Enk)对ATP-激活电流(IATP)的调制作用.实验在大鼠新鲜分离背根神经节(DRG)神经元上进行.应用全细胞膜片钳技术所记录的IATP为内向电流.在被检测的DRG神经元中,90.0%(45/50)的细胞对ATP有反应.在45个对ATP敏感的细胞中对大部分细胞(29/45)施加met-Enk(10-9～10-5mol/L)也引起一内向电流;少部分细胞(9/45)为外向电流;其余的细胞(7/45)未引起可检测的膜反应.预加met-Enk后IATP明显地被抑制,此种抑制作用为剂量依赖性的.在预加10-9、10-8、10-7、10-6、10-5mol/Lmet-Enk后,IATP的抑制分别为13.2±5.4%(n=5)、39.2±8.6%(n=8)、54.1±8.6%(n=8)、43.3±7.9%(n=7);43.1±7.9%(n=7)(mean±SKM).阿片肽拮抗剂纳洛酮能翻转此种抑制效应.IATP的量-效关系表明,预加met-Enk后曲线明显压低,在浓度为10-3mol/L时IATP下降约25%,而Kd值几乎不变.应用二次钳压技术胞内透析H-9(PKA抑制剂)能取消此种抑制作用.上述结果提示met-Enk对IATP的抑制效应为非竞争性抑制作用,可能是由于阿片受体激活后,经相应的胞内信号转导途径使ATP受体磷酸化所致.

Inhibition of ATP-activated current by met-Enk in isolated DRG neurons of the rat

The present study aimed to explore modulation of ATP-activated currents (I(ATP)) by met-Enk in rat DRG neurons. I(ATP) was recorded using the whole-cell patch clamp technique. The majority of the neurons examined responded to ATP (90.0 percent;, 45/50) with inward currents. In the 45 ATP sensitive neurons three kinds of responses to application of met-Enk were distinguished: (l) inward currents (29/45), (2) outward currents (9/45), and (3) no effect (7/45). Pretreatment with met-Enk (10(-9)~10(-5) mol/L) suppressed I(ATP) (10(-4) mol/L) in 29 neurons responding to met-Enk with inward currents. The inhibition by met-Enk of I(ATP) could be blocked by naloxone (10(-7) mol/L) in a concentration-dependent manner. Met-Enk of 10(-9), 10(-8), 10(-7), 10(-6) and 10(-5) mol/L suppressed I(ATP) by l3.2+/-5.4 percent; (n=5); 39.2+/-8.6 percent; (n=8), 54.l+/-8.6 percent; (n=8),43.3+/-7.9 percent; (n=7) and 43.l+/-7.9 percent; (n=7) (mean+/-MSE), respectively. A comparison of concentration - response relations of ATP with and without preapplication of met-Enk indicated that after pretreatment with met-Enk (10(-7) mol/L) the curve shifted downward markedly with a decrease of 25 percent; of the maximum value of I(ATP) and unchanged K(d) value. The suppression of I(ATP) by met-Enk was reversed as evidenced by intracellular dialysis of H-9 by using the repatch technique. Taken together, it is suggested that the inhibition by met-Enk of I(ATP) is caused by activation of opiate receptor, which eventually results in phosphorylation of ATP receptor, mediated by modulation of G protein coupling and intracellular signal transduction.