Molecular Mechanisms of HCO3- Secretion by the Pancreatic Duct

描述(申请人提供)：胰腺导管的液体和电解质运输在分泌腺中是独一无二的。该管道不吸收钠，并分泌近140毫米HCO3-。导管功能对胰腺健康至关重要，这在慢性胰腺炎和胰腺炎中是明显的。虽然cftr的Cl功能是导管分泌所必需的，但最近的研究表明，cftr通过调节参与胆道液和HCO3-分泌的几个Cl-和HCO3-转运蛋白，特别是SLC26转运蛋白家族(SLC26Ts)的成员，对上皮功能起着全球性的调节作用。事实上，我们已经证明了：a)SLC26T的电活性和异构体特异性化学计量，b)SLC26T在导管HCO3分泌中的作用，c)SLCT和CFTR的相互调节，d)CFTRR结构域和SLC26TS STAS结构域之间的相互作用，e)WNKS激酶对SLC26T的调节。这些发现导致了一种关于导管液体和HCO3分泌的新假说，其中大部分HCO3分泌是由不同的SLC26T介导的，这些SLC26T受CFTR调控。反过来，SLC26T在静息状态下抑制CFTR活性，在刺激状态下增强CFTR活性。CFTR通过控制胰管的膜电位来确定胰液中HCO3-的最终浓度。我们将在WT和slc26a6-/-小鼠的模型系统和自然胰管中验证这一假说，并在四个特定目的中验证HCO3分泌在胰腺炎中的作用：1.确定STAS-R结构域的共性Ser/Thr/Tyr磷酸化位点在STAS-R结构域相互作用中的作用以及CFTR和slc26a3/6的相互调节。2.研究WNKS对slc26a3/6的调节以及体内外CFTR和slc26a3/6的相互调节。3.研究WNKS上游和WNKS下游的激酶及其信号通路在体内外调节slc26a3/6中的作用。4.检测sLC6a6和HCO3-分泌在胰腺炎中的作用。HCO3-分泌受损的slc26a6/-小鼠将被用来确定HCO3-分泌在急性胰腺炎的诱发和发展中的作用。这项拟议的工作将促进我们对胰管液体和HCO3-分泌的了解，并产生与胰腺炎和囊性纤维化相关的信息。 与公共健康相关：胰腺分泌消化酶和含有大量HCO3的液体--将这些酶清洗到肠道。HCO3-是防止酶过早激活所必需的，这样它们就不会消化胰腺本身。当HCO3分泌异常时，胰腺会自行消化，就像囊性纤维化和胰腺炎一样。这项工作旨在了解胰腺是如何分泌HCO3的，以及为什么HCO3分泌在胰腺炎和囊性纤维化中是异常的。

DESCRIPTION (provided by applicant): Fluid and electrolyte transport by the pancreatic duct is unique among secretory glands. The duct does not absorb Na and secrets nearly 140 mM HCO3-. Ductal function is critical for pancreatic health, as evident in CF and pancreatitis. Although the Cl function of CFTR is required for ductal secretion, recent findings show that CFTR is a global regulator of epithelial function by regulating several Cl- and HCO3- transporters that participate in duntal fluid and HCO3- secretion, in particular members of the SLC26 transporters family (SLC26Ts). Indeed, we have shown that a) the electrogenicity and isoform specific stoichiometry of the SLC26Ts, b) the function of the SLC26Ts in ductal HCO3- secretion, c) the mutual regulation of the SLCTs and CFTR, d) the interaction between the CFTR R domain and SLC26Ts STAS domain, e) the regulation of the SLC26Ts by the WNKs kinases. These findings led to a new hypothesis of ductal fluid and HCO3- secretion in which the bulk of HCO3- secretion is mediated by different SLC26Ts that are regulated by CFTR. In turn, the SLC26Ts supress CFTR activity in the resting state and enhance CFTR activity at the stimulated state. CFTR determines the final HCO3- concentration in the pancreatic juice by controlling the membrane potential of the duct. We will test this hypothesis in model systems and native pancreatic ducts of WT and slc26a6-/- mice and the role of HCO3- secretion in pancreatitis in four specific aims: 1. Determine the role of coserved Ser/Thr/Tyr phosphortlation sites suggested by the NMR structure od STAS-R domains in the STAS-R domain interaction and mutual regulation of CFTR and slc26a3/6. 2. Study regulation of slc26a3/6 by the WNKs kinases and in the reciprocal regulation of CFTR and slc26a3/6 in vitro and in vivo. 3. study the role of kinases and signaling pathways upstream of the WNKs and kinases downstrean of the WNKs in the regulation of slc26a3/6 in vivtro and in vivo. 4. Examine the role of slc6a6 and HCO3- secretion in pancreatitis. The slc26a6-/- mice in which HCO3- secretion is compromized, will be used to determine the role of HCO3- secretion in induction and progression of acute pancreatitis. The proposed work should advance our understanding of pancreatic duct fluid and HCO3- secretion and result in information relevant to pancreatitis and Cystic Fibrosis. Public Health Relevance: The pancreas secretes digestive enzymes and fluid that contains a lot of HCO3- that wash the enzymes to the intestine. The HCO3- is needed to prevent premature activation of the enzymes so that they will not digest the pancreas itself. When HCO3- secretion is aberrant the pancreas digests itself as occur in the diseases Cystic Fibrosis and pancreatitis. This work is aimed to understand how the pancreas secretes HCO3- and why HCO3- secretion is aberrant in pancreatitis and Cystic Fibrosis.