REGULATION OF NA+/K+ TRANSPORT BY THYROID HORMONE

该项目将继续致力于获得 了解细胞Na+和K+ 运输受甲状腺激素调节。 特别是 申请人将继续进行最初提出的假设， 申请这个奖项，众所周知的刺激 主动钠，钾运输甲状腺激素，而不是被 由于Na，K泵（Na，K- ATP酶），如先前所假设的，而是由于 Na+的限速性被动细胞内流增加， K+外流。 在目前的项目期间，申请人已 成功地获得了这一假设的实质性证据， 包括首次描述连续细胞系（ARL 15 大鼠肝细胞），其对甲状腺激素的反应是 增加Na、K的主动运输，进一步证明 这种刺激确实伴随着相应的 被动Na+内流和被动K+外流均增加， 假设的。 这种方便的甲状腺激素的可用性 反应性细胞培养系统，特别是这种 方便的甲状腺激素应答细胞培养系统， 特别是第二系大鼠肝细胞（克隆9）， 申请人最近已经证明， 对甲状腺激素有反应，现在可以进行实验 旨在阐明甲状腺功能亢进的具体机制， 激素调节Na+和K+转运。 为请求的两年项目延期而提议的研究 将在上述克隆9细胞中进行 培养体系 假设T3+对活性的影响 Na、K的转运是由于被动转运的增强， Na和K的泄漏，而不是Na，K-的主要诱导， ATP酶，将通过研究时间过程直接测试， T3对活性的影响的浓度-反应关系 Na+、K+转运、被动Na+和K+通量、细胞内Na+和 K+浓度和Na，K-ATP酶水平。 此外，本发明还 表征特定膜转运的研究 负责T3诱导的被动Na+增加的途径 进入和K+流出将使用特定的抑制剂进行 和对培养基成分的操纵。 最后 细胞内游离钙水平的变化可能在 将研究介导甲状腺激素的作用 使用荧光光谱法和钙敏感染料。

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.