This proposal will advance understanding of mechanisms of stimulation of the intestinal Na absorptive cell brush border (BB) Na/H exchanger NHE3 and the intestinal neutral NaCl absorptive process that NHE3 is part of. The studies are relevant to normal digestive physiology and to the pathobiology of diarrheal diseases. The overall goal is to understand the mechanisms by which NHE3 is acutely stimulated by neurohumoral agonists and growth factors. We have shown that several such agonists acutely stimulate NHE3 by altering NHE3 trafficking and increasing the amount of NHE3 on the BB. The mechanism by which this stimulation occurs is not understood. These studies will determine how stimulation occurs, examining mechanisms such as rapid changes in turnover number, endocytosis, half-life and size of the BB. Stimulation in some models requires NHERF2. The mechanism by which NHERF2 allows rapid stimulation of NHE3 will be examined. NHE3 binds to ezrin. It will be tested whether ezrin serves as an intermediate in acute regulation of NHE3 or in rearrangement in the BB, which accompanies some NHE3 stimulation. NHE3 is present in small intestinal BB in three pools. These include detergent soluble, detergent resistant-cytoskeleton and detergent resistant-lipids rafts. In addition, NHE3 is present in large BB complexes with appearance of larger complexes when NHE3 is acutely stimulated. As further evidence of NHE3 presence in multiple BB pools, in the renal proximal tubule cell line OK, NHE3 is distributed in microvilli (MV) in the central region of the apical surface rather than being present in all MV. We will test the hypothesis that NHE3 in the multiple BB pools has the different activities and takes part in different aspects of acute stimulation. In order to examine NHE3 in these several BB pools, we will make use of NHE3 expressed in cell culture models of intestinal absorptive cells (Caco-2) and renal proximal tubules (OK cells) and compare results with ileal Na absorptive cells. We will measure NaCl absorption and NHE3 activity using both isotopes ad fluorometery. This will be correlated with biochemical measurements in the same tissue or cells, including density gradient centrifugation to separate the NHE3 pools. NHE3 and its regulatory molecules will be molecularly identified and expressed in Caco-2 and OK cells to understand NHE3 regulation. Cell biologic approaches, especially confocal microscopy will examine localization of BB NHE3 relative to its regulatory molecules as altered with acute stimulation. We also will test effects of stimulation of NHE3 on its BB mobility by use of fluorescence recovery after photobleaching (FRAP).
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