Molecular Mechanisms of HCO3- Secretion by the Pancreatic Duct

描述（由申请人提供）：通过胰管输送液体和电解质在分泌腺中是独一无二的。导管不吸收Na，并分泌近140 mM的HCO3-。导管功能对胰腺健康至关重要，如CF和胰腺炎。虽然CFTR的Cl功能是导管分泌所必需的，但最近的研究结果表明，CFTR通过调节参与管液和HCO3分泌的几种Cl-和HCO3-转运蛋白，特别是SLC26转运蛋白家族（SLC26Ts）的成员，是上皮功能的全局调节剂。事实上，我们已经证明了a) SLC26Ts的电性和同工异构体特异性化学测量，b) SLC26Ts在导管HCO3-分泌中的功能，c) SLC26Ts和CFTR的相互调节，d) CFTR R结构域和SLC26Ts STAS结构域之间的相互作用，e) WNKs激酶对SLC26Ts的调节。这些发现导致了导管液体和HCO3-分泌的新假设，其中大部分HCO3-分泌是由CFTR调节的不同slc26t介导的。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最终的HCO3-浓度。我们将在WT和slc26a6-/-小鼠的模型系统和天然胰管中验证这一假设，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NMR结构提示的保守Ser/Thr/Tyr磷酸化位点在CFTR和slc26a3/6的STAS-R结构域相互作用和相互调控中的作用。2. 在体外和体内研究WNKs激酶对slc26a3/6的调控以及CFTR和slc26a3/6的相互调控。3. 研究WNKs上游激酶和下游激酶在体内外调控slc26a3/6中的作用和信号通路。4. 探讨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导和进展中的作用。这项工作将促进我们对胰管液和HCO3-分泌的理解，并获得与胰腺炎和囊性纤维化相关的信息。

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.