Studies on Neuropeptide Receptors Using Genetic Engineering and Electrophysiological Techniques

为了研究神经肽受体的性质，进行了以下实验：A）维克受体在非洲爪蟾卵母细胞中的功能性表达1.研究了注射大鼠小肠组织中内皮素（ET）家族血管活性肠收缩因子（维克）受体的爪蟾卵母细胞的表达。电压钳法测量全细胞电流。2.维克效应的内向电流响应维克对VIC的反应依赖于Cl-，并且可被BAPTA/AM（10 μ M）或PTX抑制维克反应可被VIC的重复作用所抑制，而对维克有反应的卵母细胞在内皮素-1、-2或-3的刺激下不产生电流。 关于我们 大鼠肠道mRNA通过PTX敏感的G蛋白和Ca^<2+>激活的Cl-通道发挥作用。B）非洲爪蟾卵母细胞胞内储存的Ca^<2+>释放机制1.采用双电极电压钳法测量卵母细胞全细胞电流，研究了改变细胞外pH（pH_0）对卵母细胞电生理特性的影响. pH_0从对照7.5升高到碱性8.5-10.5时，膜发生超极化，而pH_0降低到5.5-6.5时，膜发生由尖峰状快成分和慢去极化组成的去极化。在电压钳条件下，碱性pH_0引起了一个依赖于pH_0的外向电流，并伴随着膜电导的增加.结论如下：（1）碱性pH_0引起的外向电流依赖于激活K^+通道，推测是通过cAMP途径，（2）碱性pH_0抑制胞内钙库的释放，（3）酸性pH_0的改变诱导胞内钙库的释放并打开钙激活的Cl-通道，导致内向电流反应;（4）外向电流和内向电流都起源于卵母细胞，但不起源于周围的卵泡细胞。提示卵母细胞外液pH值偏碱性的生理意义在于使卵母细胞膜电位超极化，抑制胞内Ca^2+释放，从而使卵母细胞处于受精前的状态。这也将有助于受精后分裂卵母细胞的发育。少

The follwoing experiments were carried out for studying the properties of neuropeptide receptors.A) Functional Expression of VIC Receptors in Xenopus Oocytes1. Expression of receptors for vasoactive intestinal contractor (VIC) of the endothelin (ET) family was studied in Xenopus oocytes injected with mRNA extracted from the rat intestine. Whole-cell currents were measured using the voltage-clamp method.2.Inward-current responses to VIC (1 nM-100nM) were evoked in a dose-dependent manner in mRNA-injected oocytes.3.The response to VIC was dependent on Cl-and was suppressed either by the external application of BAPTA/AM (10muM), a membrane-permeant intracellular Ca^<2+> chelator, or by PTX (pertussis toxin, 0.5mug/ml).4.The VIC response was desensitized with repetitive application of VIC.Oocytes which responded to VIC produced no currents when exposed to endothelin-1, -2 or -3.5.These results indicate that specific receptors for VIC,functionally expressed in Xenopus oocytes injected with … More rat intestinal mRNA,function via PTX-sensitive G-protein and Ca^<2+>-activated Cl-channels.B) Mechanisms of release of Ca^<2+> from intracellular stores in Xenopus oocytes1. Effects of changing the extracellular pH (pH_0) on the electrophysiological properties of Xenopus oocytes were investigated by measuring whole-cell currents using the two-electrode voltage-clamp method.2. Increasing pH_0 from control 7.5 to alkaline 8.5-10.5 produced a membrane hyperpolarization while lowering pH_0 to 5.5-6.5 generated a depolarization consisting of a spike-like fast component followed by a slow depolarization. Under voltage-clamp conditions, alkaline pH_0 elicited a pH_0-dependent outward current with a concomitant increase in the membrane conductance.3. Conclusions are : (1) the outward current elicited by alkaline pH_0 in Xenopus oocytes is dependent on the activation of K^+ channels presumably via the cyclic AMP pathway, (2) alkaline pH_0 inhibits release of Ca^<2+> from intracellular stores, (3) an acidic shift of pH_0 induces release of Ca^<2+> from intracellular stores and opens Ca^<2+>-activated Cl-channels, resulting in an inward current response, and (4) both outward and inward currents originate in the oocyte but not in the surrounding follicle cells.5. It is suggested that the physiological significance of the alkaline pH_0 of the fluid which surrounds oocytes is to keep the oocyte in a state prepared for fertilization by hyperpolarizing the membrane potential and inhibiting intracellular Ca^<2+> release. This would also assist development of cleaving oocytes after fertilization. Less