PROTEIN PHOSPHORYLATION AND EPITHELIAL ION TRANSPORT

长期的目标是阐明细胞凋亡的分子机制， 第二信使cAMP、cGMP和Ca调节电解质和水 在上皮中的运输。 Cl-或Na/K依赖性机制 渗透到细胞中是重要的， 和水穿过上皮细胞，并在各种 组织中 离子传输过程的故障，包括 阳离子耦合Cl进入机制，已牵连在一些 疾病，包括青光眼，囊性纤维化，腹泻和原发性 高血压 在上皮细胞中，净液体运输代表了一种平衡， 主动吸收和主动分泌之间的关系以及Na/Cl或Na/K/Cl 共转运机制是这两个过程的必要组成部分。 一个 细胞内Ca、cAMP含量增加，或者在某些情况下， cGMP调节大多数上皮细胞中的Na/Cl转运，导致细胞内Na/Cl转运减少。 吸收性上皮细胞中的液体吸收和液体吸收的增加 分泌上皮的分泌。 这些第二信使可能通过 特异性蛋白激酶。 该建议旨在将Ca-，cAMP-和 cGMP介导的Na/K/Cl共转运的变化与磷酸化 完整组织中的特异性蛋白质，并表征 运输相关磷蛋白。 模型系统是 冬比目鱼，其中Na/K/Cl共运输是重要的 监管机构。 这种简单的外延是相对均匀的， 具有良好表征的Na/K/Cl共转运机制， 由三个第二信使控制。 磷酸化的变化， 共转运将基于时间、剂量依赖性和 影响的可逆性。 特异性磷蛋白底物将是 通过一维和二维凝胶电泳鉴定， 放射自显影和运输将通过放射性同位素通量进行评估。 特异性底物和激酶将通过各种方法表征。 技术，包括亚细胞分级分离，光亲和标记， 肽图谱和磷酸氨基酸分析。 总体目标是 确定离子转运组分的分子性质; 第二信使特异性磷蛋白本身可能是转运蛋白 或其调制器。 这些研究将提供一个很好的模型， 在更复杂的上皮细胞中检查类似的转运机制， 哺乳动物回肠和哺乳动物Henle袢的粗升支 肾

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.