PROTEIN PHOSPHORYLATION AND EPITHELIAL ION TRANSPORT

我们的长期目标是阐明 第二信使cAMP、cGMP和Ca调节电解质和水 在上皮细胞中的运输。氯离子的钠或钠/钾依赖机制 进入细胞的渗透在盐的媒介运输中都是重要的 和水穿过上皮细胞，并在各种不同的体积调节 纸巾。离子传输过程出现故障，包括 阳离子偶联氯进入机制，已被牵连在许多 疾病，包括青光眼、囊性纤维化、腹泻和 高血压。在上皮细胞中，液体的净运输代表着平衡。 主动吸收和主动分泌与钠/氯或钠/钾/氯的关系 共传输机制是这两个过程的必要组成部分。一个 细胞内钙、cAMP含量的增加，在某些情况下， CGMP，调节大多数上皮细胞的钠/氯转运，导致 吸收上皮细胞的液体吸收和液体增加 分泌上皮细胞的分泌物。这些第二信使很可能通过 特定的蛋白激酶。这项提议旨在将钙离子、cAMP和 CGM介导的钠/钾/氯共转运与磷酸化的变化 在完整组织中的特定蛋白质，并表征 运输相关的磷蛋白。模型系统是 冬季比目鱼，在那里钠/钾/氯共同运输在 渗透调节。这个简单的上皮组织相对均匀，而且 拥有一种特性良好的钠/钾/氯共转运机制，可以 受到所有三个第二信使的监管。磷酸化和磷酸化的变化 共传输将基于时间、剂量依赖和 效果的可逆性。特定的磷蛋白底物将是 通过一维和二维凝胶电泳法鉴定 放射自显影和传输将通过放射性同位素通量进行评估。 特定的底物和激酶将由各种不同的 技术，包括亚细胞分离，光亲和标记， 多肽图谱和磷酸氨基酸分析。总体目标是 确定离子传输组分的分子性质； 第二信使特异的磷酸蛋白本身可能是转运蛋白 或其调制器。这些研究将为 研究更复杂的上皮细胞中类似的转运机制，如 哺乳动物的回肠和哺乳动物的粗大的亨勒氏环升支 肾脏。

The long-term goal is to elucidate the molecular mechanisms by which the second messengers cAMP, cGMP and Ca regulate electrolyte and water transport in epithelia. Sodium- or Na/K-dependent mechanisms of Cl permeation into cells are important both in the vectorial transport of salt and water across epithelia and in volume regulation in a variety of tissues. Malfunctioning of ion transport processes, including cation-coupled Cl entry mechanisms, has been implicated in a number of diseases, including glaucoma, cystic fibrosis, diarrhea and essential hypertension. In epithelia, net fluid transport represents a balance between active absorption and active secretion and Na/Cl or Na/K/Cl cotransport mechanisms are necessary components of both processes. An increase in the intracellular content of either Ca, cAMP or, in some cases, cGMP, regulates Na/Cl transport in most epithelia, leading to a decrease in fluid absorption in absorptive epithelia and to an increase in fluid secretion in secretory epithelia. These second messengers probably act via specific protein kinases. This proposal aims to correlate Ca-, cAMP- and cGM-mediated changes in Na/K/Cl cotransport with phosphorylation of specific proteins in intact tissues and to characterize the transport-related phosphoproteins. The model system is the intestine of the winter flounder, where Na/K/Cl cotransport is important in osmoregulation. This simple epi thelium is relatively homogeneous, and possesses a well-characterized Na/K/Cl cotransport mechanism that can be regulated by all three second messengers. Changes in phosphorylation and cotransport wil be correlated on the basis of time, dose dependency and reversibility of effects. Specific phosphoprotein substrates will be identified by one- and two-dimensional gel electrophoresis followed by autoradiography, and transport will be assessed by radioisotope fluxes. Specific substrates and kinases will be characterized by various techniques, including subcellular fractionation, photoaffinity labelling, peptide mapping, and phosphoaminoacid analysis. The overall objective is to identify the molecular nature of ion transport components; second-messenger-specific phosphoproteins could be the transporter per se or modulaters thereof. These studies will provide an excellent model for examining similar transport mechanisms in more complex epithelia such as mammalian ileum and the thick ascending limb of Henle's loop in mammalian kidney.