Studies on Neuropeptide Receptors Using Genetic Engineering and Electrophysiological Techniques

利用基因工程和电生理技术研究神经肽受体

• 批准号: 06670062
• 负责人: YOSHIDA Shigeru
• 金额: $ 1.34万
• 依托单位: Fukui Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670062/
• 关键词: Neuropeptides; Vasoactive Intestinal Contractor (VIC); Endothelin; Xenopus Oocyte; Ca^<2+> Store; Ion Channels; Genetic Engineering; Electrophysiology; 受容体; 電気生理

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

为研究神经肽受体的特性，进行了以下实验：1)VIC受体在非洲爪哇卵母细胞中的功能表达。用从大鼠肠道提取的mRNA注射非洲爪哇卵母细胞，研究了内皮素(ET)家族血管活性肠缩窄物(VIC)受体的表达。2.在mRNA注射的卵母细胞中，VIC(1 nM-100 nm)的内向电流反应呈剂量依赖关系。3.对VIC的反应依赖于氯离子，并可被外加BAPTA/AM(10µM)抑制。4.反复应用VIC可使VIC反应减敏。对VIC有反应的卵母细胞在内皮素-1、-2或-3.5作用下不产生电流。结果表明，在注射…的非洲爪哇卵母细胞中，VIC的特异性受体在功能上表达更多的大鼠肠道mRNA，通过PTX敏感的G蛋白和Ca^&lt；2+&gt；-激活的氯离子通道发挥作用。B)从非洲爪哇卵母细胞内储存的钙释放机制1。方法：1.采用双电极电压钳技术测量全细胞电流，研究了改变胞外pH(PH_0)对非洲爪哇卵母细胞电生理特性的影响。PH_0从对照7.5升至碱性8.5~10.5时，产生膜超极化；pH_0降至5.5~6.5时，出现由快成分棘波和慢去极化组成的去极化。在电压钳条件下，碱性pH_0引起依赖于pH_0的外向电流，膜电导随之增加。结论是：(1)碱性pH_0引起的非洲爪哇卵母细胞外向电流依赖于K~(++)通道的激活，可能是通过环AMP途径；(2)碱性pH_0抑制细胞内钙离子的释放，(3)pH_0的酸性变化诱导细胞内钙离子释放，并开放激活的氯通道，引起内向电流反应；(4)外向电流和内向电流均起源于卵母细胞，而不是周围的卵泡细胞。提示卵母细胞周围碱性pH_0的生理意义在于通过超极化膜电位，抑制细胞内钙离子释放，使卵母细胞处于准备受精的状态。这也有助于受精后卵裂卵母细胞的发育。较少

项目成果

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Yoshida,S.,Yoshimura,M.& Taniyama,K.: "Expression of specific receptors for vasoactive intestinal contractor in Xenopus oocytes injected with mRNA from rat intestine." Clinical and Experimental Pharmacology and Physiology. 22. A53 (1995)

吉田，S.，吉村，M.

Yoshida,S.,Yoshimura,M.& Taniyama,K.: "Expression of specific receptors for vasoactive intestinal contractor in Xenopus oocytes injected with mRNA from rat intestine." Third Congress of Federation of Asian and Oceanian Physiological Societies. Abstract Vo

吉田，S.，吉村，M.

Yoshimura,M.,Yoshida,S.& Taniyama,K.: "Property of receptor for vasoactive intestinal contractor(VIC)expressed in Xenopus oocytes injected with mRNA from rat intestine." Life Sciences. (In Press). (1996)

吉村，M.，吉田，S.

S.Kan: "Specific receptor for vasoactive intestinal contractor in myenteric cholinergic neurones" European Journal of Pharmacology. 258. 139-143 (1994)

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