REGULATION OF NA+/K+ TRANSPORT BY THYROID HORMONE

该项目将继续致力于获得 对细胞内Na、K的作用机制的认识 运输受甲状腺激素的调节。尤其是， 申请者将继续研究最初提出的假设 这一奖项的申请表明著名的刺激者 活性Na，K的转运是通过甲状腺激素而不是通过 可归因于Na，K泵的初级诱导(Na，K- ATPase)，正如先前假设的那样，是由于 限速性被动细胞内钠和钙离子内流增加 K的外流。在当前项目期内，申请人有 成功地为这一假设获得了确凿的证据， 包括对连续细胞系(ARL 15)的第一次描述 大鼠肝细胞)，其对甲状腺激素的反应是 Na、K主动转运增加及进一步论证 这种刺激确实伴随着相应的 被动钠内流和被动钾流出均增加，AS 假设的。这种方便的甲状腺激素的可用性 响应性细胞培养系统，尤其是 方便的甲状腺激素反应细胞培养系统，并在 第二个大鼠肝细胞系(克隆9)的特殊之处 申请者最近也被证明是类似的 对甲状腺激素的反应，现在使实验成为可能 旨在阐明甲状腺的具体机制 激素调节钠和钾的运输。 为所要求的项目延期两年提出的研究 期间将在上述克隆9号细胞中进行 文化体系。T_3对活性的影响假说 Na、K转运归因于被动运动的增强 Na和K的泄漏，而不是对Na，K-的初级诱导 ATPase，将通过研究时间进程和 T_3对活性影响的量效关系 Na，K转运，被动Na和K通量，细胞内Na和 K浓度和Na，K-ATPase水平。另外， 特异膜转运特性的研究 T_3引起细胞内游离钠升高的途径(S) 进入和钾外流将使用特定的抑制剂 以及对媒介成分的操纵。最后， 细胞内游离钙水平变化在脑缺血再灌注损伤中的可能作用 甲状腺激素的调节作用将被调查 使用钙敏感染料的荧光光谱。

The project will continue to be directed toward obtaining an understanding of the mechanism by which cellular Na+and K+ transport is regulated by thyroid hormone. In particular, the applicant will pursue the hypothesis proposed in the initial application for this Award that the well-known stimulation of active Na, K transport by thyroid hormone, rather than being attributable to a primary induction of the Na, K pump (Na, K- ATPase) as has previously been postulated, is instead due to an increase in the rate-limiting passive cellular influx of Na+ and efflux of K+. During the current project period the applicant has succeeded in obtaining substantial evidence for this hypothesis, including the first description of a continuous cell line (ARL 15 rat liver cells) which responds to thyroid hormone with an increase in active Na, K transport, and the further demonstration that this stimulation is indeed accompanied by corresponding increases in both passive Na+ influx and passive K+ efflux, as postulated. The availability of this convenient thyroid-hormone responsive cell culture system, and in particular of this convenient thyroid-hormone responsive cell culture system, and in particular of a second line of rat liver cells (Clone 9) which the applicant has more recently demonstrated to be similarly responsive to thyroid hormone, now make possible experiments designed to elucidate the specific mechanisms by which thyroid hormone regulates Na+ and K+ transport. The studies proposed for the requested 2-year project extension period will be carried out in the above mentioned Clone 9 cell culture system. The hypothesis that the effect of T3+ on active Na, K transport is attributable to an enhancement of the passive leak of Na and K, rather than to a primary induction of the Na, K- ATPase, will be directly tested by studying the time-course and concentration-response relationship of the effects of T3 on active Na, K transport, passive Na+ and K+ fluxes, intracellular Na+ and K+ concentrations, and Na, K-ATPase levels. Additionally, studies to characterize the specific membrane transport pathway(s) responsible for the T3-induced increase in passive Na+ entry and K+ efflux will be carried out using specific inhibitors and manipulations of the constituents of the medium. Finally, the possible role of changes in intracellular free calcium levels in mediating the effects of thyroid hormone will be investigated using fluorescence spectroscopy with calcium-sensitive dyes.