Studies on Neuropeptide Receptors Using Genetic Engineering and Electrophysiological Techniques

为了研究神经肽受体的性质，我们进行了以下实验。A)爪蟾卵母细胞中VIC受体的功能表达研究了内皮素（ET）家族血管活性肠收缩受体（VIC）在注射大鼠肠道mRNA的爪蟾卵母细胞中的表达。采用电压钳法测量全细胞电流。注射mrna的卵母细胞对VIC （1 nM-100nM）的内电流反应呈剂量依赖性。对VIC的反应依赖于cl，并且可以通过外用BAPTA/AM (10muM)（一种膜渗透细胞内Ca^<2+>螯合剂）或PTX（百日咳毒素，0.5mug/ml）来抑制。反复应用VIC可使VIC反应脱敏。当暴露于内皮素-1、-2或-3.5时，对VIC有反应的卵母细胞不产生电流。这些结果表明，在注射了更多大鼠肠道mRNA的爪蟾卵母细胞中，VIC特异性受体通过ptx敏感的g蛋白和Ca^<2+>激活的cl -通道发挥功能。B)爪蟾卵细胞内Ca^<2+>释放机制[j]。采用双电极电压钳法测量全细胞电流，研究了改变细胞外pH值（pH_0）对爪蟾卵母细胞电生理特性的影响。将pH_0从对照7.5增加到碱性8.5-10.5产生超极化，而将pH_0降低到5.5-6.5产生由尖峰样快速组分和缓慢去极化组成的去极化。在电压箝位条件下，碱性pH_0引发了一个依赖于pH_0的向外电流，同时膜电导率也随之增加。结论是：(1)碱性pH_0在爪虫卵母细胞中引发的向外电流依赖于K^+通道的激活，可能是通过循环AMP途径；(2)碱性pH_0抑制细胞内储存的Ca^<2+>的释放；(3)pH_0的酸性转移诱导细胞内储存的Ca^<2+>的释放，打开Ca^<2+>激活的cl -通道，导致向内电流响应。(4)向外和向内的电流都起源于卵母细胞，而不是周围的卵泡细胞。提示卵母细胞周围液碱性pH_0的生理意义是通过超极化膜电位和抑制细胞内Ca^<2+>的释放，使卵母细胞处于准备受精的状态。这也有助于受精后卵细胞分裂的发育。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