Lipinsky D, Gershengorn M C, Oron Y
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel.
Pflugers Arch. 1993 Oct;425(1-2):140-9. doi: 10.1007/BF00374514.
To dissect the cellular events responsible for the prolonged latency of the response to thyrotropin-releasing hormone (TRH) in Xenopus oocytes we interfered with different steps of the signal transduction pathway. Preincubation of oocytes with cis-vaccenic acid (a membrane-fluidizing agent) shortened the latency, suggesting a contribution of membranal processes. TRH-induced depletion of cellular calcium stores prolonged latency (up to threefold), which returned to control levels upon repletion of the stores. Injection of D-2,3-diphosphoglycerate (PGA), which inhibits inositol (1,4,5)-trisphosphate (InsP3) dephosphorylation, alone evoked a small, prolonged depolarizing current and significantly shortened the latency of the response to TRH. Injection of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inactivates guanine nucleotide-binding regulatory proteins, decreased the amplitude of the response and increased latency. Injection of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) immediately before the challenge with TRH did not shorten the latency of the response. Decreasing the effective receptor density with chlordiazepoxide, an antagonist of the TRH receptor, resulted in an extension of latency, whereas the expression of a large number of TRH receptors by injection of RNA transcribed from cloned receptor DNA (10-100 ng/oocyte) shortened the latency to below 2 s. Our results suggest that the latency of the response to TRH reflects the activation of a late step in the signal transduction sequence, most likely the release of calcium by InsP3. We propose that this process is kinetically controlled by an early rate-limiting event, involving the activation of a guanine nucleotide-binding protein by the TRH receptor.
为了剖析非洲爪蟾卵母细胞中促甲状腺激素释放激素（TRH）反应延迟延长所涉及的细胞事件，我们干扰了信号转导途径的不同步骤。用顺式vaccenic酸（一种膜流化剂）预孵育卵母细胞可缩短延迟时间，提示膜相关过程的作用。TRH诱导的细胞钙库耗竭会延长延迟时间（延长至三倍），而钙库补充后延迟时间又恢复到对照水平。注射抑制肌醇（1,4,5）-三磷酸（InsP3）去磷酸化的D-2,3-二磷酸甘油酸（PGA），单独即可诱发一个小的、持续时间长的去极化电流，并显著缩短对TRH反应的延迟时间。注射使鸟嘌呤核苷酸结合调节蛋白失活的鸟苷5'-O-（2-硫代二磷酸）（GDPβS），会降低反应幅度并增加延迟时间。在TRH刺激前立即注射鸟苷5-O-（3-硫代三磷酸）（GTPγS），并未缩短反应的延迟时间。用TRH受体拮抗剂氯氮卓降低有效受体密度，会导致延迟时间延长，而通过注射从克隆受体DNA转录的RNA（10 - 100 ng/卵母细胞）来表达大量TRH受体，则可将延迟时间缩短至2秒以下。我们的结果表明，对TRH反应的延迟反映了信号转导序列中晚期步骤的激活，最有可能是由InsP3释放钙。我们提出，这个过程在动力学上受一个早期限速事件控制，该事件涉及TRH受体对鸟嘌呤核苷酸结合蛋白的激活。
