Studies on Neuropeptide Receptors Using Genetic Engineering and Electrophysiological Techniques

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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