Studies on Neuropeptide Receptors Using Genetic Engineering and Electrophysiological Techniques

为了研究神经肽受体的特性，进行了以下实验。A)爪蟾卵母细胞中VIC受体的功能表达1。研究人员在注射从大鼠肠道提取的 mRNA 的非洲爪蟾卵母细胞中研究了内皮素 (ET) 家族血管活性肠道收缩剂 (VIC) 受体的表达。使用电压钳法测量全细胞电流。2.在注射mRNA的卵母细胞中以剂量依赖性方式诱发对VIC（1nM-100nM）的内向电流反应。3.对VIC的反应依赖于Cl-并且通过外部应用BAPTA/AM（10μM）（一种膜渗透性细胞内Ca^2螯合剂）或PTX（百日咳）来抑制毒素，0.5mug/ml)。4.重复应用VIC使VIC反应脱敏。当暴露于内皮素-1、-2或-3.5时，对VIC作出反应的卵母细胞不产生电流。这些结果表明，注射了大鼠肠道mRNA的非洲爪蟾卵母细胞中功能性表达的VIC特异性受体，通过PTX敏感的G蛋白和Ca^<2>激活的Cl通道起作用。B)非洲爪蟾卵母细胞胞内储存的Ca ^ 2 释放机制1。采用二电极电压钳法测量全细胞电流，研究改变细胞外pH（pH_0）对非洲爪蟾卵母细胞电生理特性的影响。 2．将 pH_0 从对照 7.5 增加到碱性 8.5-10.5 会产生膜超极化，而将 pH_0 降低到 5.5-6.5 会产生由尖峰状快速成分和随后的缓慢去极化组成的去极化。在电压钳条件下，碱性pH_0会引发依赖于pH_0的外向电流，同时膜电导随之增加。3．结论是：(1)爪蟾卵母细胞中碱性pH_0引起的外向电流依赖于可能通过环AMP途径激活的K^通道，(2)碱性pH_0抑制细胞内储存的Ca^2释放，(3)pH_0的酸性转变诱导细胞内储存的Ca^2释放并打开Ca^2激活的Cl通道，导致内向电流响应，以及(4)外向电流和内向电流均起源于卵母细胞，而不起源于周围的卵泡细胞。5．这表明卵母细胞周围液体的碱性pH_0的生理意义是通过超极化膜电位和抑制细胞内Ca 2 释放来保持卵母细胞处于准备受精的状态。这也将有助于受精后卵母细胞分裂的发育。较少的

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