REGULATION OF SODIUM/INOSITOL COTRANSPORTER

过去十年的研究表明，肌醇在人体内起着重要作用 在保护细胞免受高渗应激中的作用。肾髓质是 只有哺乳动物的组织在正常情况下会有很大的张力变化。 这些变化是产生浓缩或浓缩的机制的内在原因。 稀释尿液。这种压力是由于高度集中的 迅速出现的细胞内钾，以平衡细胞外 音调。然而，经过一段时间，延髓中的细胞降低了 它们的钾浓度与等渗细胞中的钾浓度相同 其他器官通过积累小的有机溶质，如肌醇， 不要像高浓度的钾那样扰乱细胞的功能。 在一些异常状态下，如慢性高钠血症，大脑也会 以非干扰性渗透分子的形式积聚肌醇。在高渗培养中 来自许多组织的培养细胞，包括肾、脑、内皮和 眼睛，积聚肌醇。积累是增加的结果 钠/肌醇共转运体将肌醇带入体内的活性 细胞对抗巨大(超过500倍)的浓度梯度。 我们已经克隆了共转运蛋白的cDNA，并证明了 高张力增加了其基因的转录。我们还发现了 其他步骤监管的证据。我们建议研究这一机制。 从转录水平开始参与调控。 增强剂是一种DNA序列元件，它可以提高 对特定刺激作出反应的特定基因的转录 由与增强子结合的蛋白质介导。我们将确定 基于其刺激报告基因转录的增强子 作为对高张力的反应。与之相互作用的蛋白质(S) 增强子将被克隆，针对该蛋白质的抗体将被提高。我们 将研究高张力如何调节增强子结合蛋白 MRNA表达水平，与增强子的结合/解离， 磷酸化，以及与其他蛋白质的相互作用。我们已经确定了 转录后调控细胞活性的机制 辅助传送器。我们将确定所需的信使核糖核酸序列 用于通过紧张度调节RNA周转。细胞质蛋白(S) 与这个序列相互作用并改变RNA的稳定性将是 已确认身份。探索翻译后的第三个层面的监管， 我们将确定参与抑制的磷酸化位点。 蛋白激酶C和蛋白激酶A转运获得的信息 从这些研究中可以洞察细胞是如何感觉到高渗的 压力以及信号是如何传递的。

Research in the last decade has revealed that inositol plays an important role in protecting cells from hypertonic stress. The renal medulla is the only tissue in mammals that normally undergoes large changes in tonicity. The changes are intrinsic to the mechanism for producing concentrated or dilute urine. The stress is the result of the high concentration of intracellular potassium that rapidly occurs to balance extracellular tonicity. Over a period of time, however, the cells in the medulla lower their concentration of potassium to the same level as in isotonic cells in other organs by accumulating small organic solutes, such as inositol, that do not perturb cell function the way high concentrations of potassium do. In some abnormal states, such as chronic hypernatremia, brain also accumulates inositol as a non-perturbing osmolyte. In hypertonic culture medium cells from many tissues including kidney, brain, endothelium, and eye, accumulate inositol. The accumulation is the result of increased activity of the sodium/inositol cotransporter that brings insitol into cells against enormous (over 500-fold) concentration gradients. We have cloned the cDNA for the cotransporter and demonstrated that hypertonicity increases transcription of its gene. We have also found evidence for regulation at other steps. We propose to study the mechanisms involved in the regulation starting at the level of transcription. Enhancers are DNA sequence elements that increase the rate of transcription of specific genes in response to specific stimuli typically mediated by proteins that bind to the enhancer. We will identify the enhancer based on its ability to stimulate transcription of reporter genes in response to hypertonicity. The protein(s) that interacts with the enhancer will be cloned and antibodies to the protein will be raised. We will examine how hypertonicity regulates the enhancer binding protein at the level of mRNA expression, binding/dissociation to the enhancer, phosphorylation, and interaction with other proteins. We have identified a post-transcriptional mechanism of regulation of the activity of the cotransporter. We will identify the sequence of the mRNA that is required for the regulation of RNA turnover by tonicity. The cytoplasmic protein(s) that interacts with this sequence and changes the RNA stability will be identified. To explore the third, post-translational, level of regulation, we will determine phosphorylation sites involved in the inhibition of transport by protein kinase C and protein kinase A. Information obtained from these studies should provide insight into how cells sense hypertonic stress and how the signal is transduced.