The Na+/H+ exchanger isoform 3 (NHE3) constitutes a major Na+ transport process, which moves a large quantity of salt and water from the mucosal side toward the serosal side. Our long-term goal is to understand the molecular mechanisms that control NHE3 activity in the intestine. Because NHE3 is often targeted in many diarrheal diseases and the mechanisms underlying inhibition of NHE3 have often been studied. However, the mechanisms and biological factors activating NHE3 are less well understood. This is an important aspect as a better understanding of the mechanisms activating NHE3 should aid the development of improved therapeutic modalities to counteract diarrhea caused by inhibition of Na+/H+ exchange. The goals of this application are to delineate the mechanisms of NHE3 activation by glucocorticoids and lysophosphatidic acid (LPA). In the previous grant, we investigated the regulation of NHE3 by glucocorticoids, which is a major hormone activating NHE3 under physiologic and pathophysiologic conditions. Our studies revealed that the serum and glucocorticoid-inducible kinase 1 (SGK1) plays a central role in regulation of NHE3. Moreover, transcriptional regulation of NHE3 gene expression by glucocorticoids is not sufficient to result in an increase in NHE3 activity. Glucocorticoids activate SGK1, which phosphorylates NHE3 and facilitates trafficking of NHE3 protein to the surface membrane. A goal of this application is to further delineate the roles of SGK1 and the NHE regulatory factor 2 (NHERF2) in vivo using transgenic animals. We will also test a specific hypothesis that SGK2 plays a role in activation of NHE3 in the intestine. In addition to glucocorticoids, we recently found that LPA is a potent activator of NHE3 in intestinal epithelial cells. We hypothesize that LPA activates NHE3 via LPA5 receptor, which is highly expressed in the intestine. We will define the mechanism of LPA- mediated activation of NHE3 and determine the physiologic effect of LPA on NHE3. In Aim 1, we will investigate regulation of NHE3 by dexamethasone in vivo and determine the role of SGK2. In Aim 2 and Aim 3, we propose to determine the mechanism of LPA-mediated activation of NHE3 and determine its effect in an experimental model of diarrhea. PUBLIC HEALTH RELEVANCE: The Na+/H+ exchanger 3 (NHE3) is the major Na+ absorptive process in the intestine and colon and is frequently inhibited in many diarrheal diseases. The goals of this application are to determine the effects of glucocorticoids and lysophosphatidic acids and to delineate the mechanisms resulting in the activation of NHE3. The outcome of the proposed studies should enhance our understanding of the regulation of NHE3 and may aid in the development of improved therapeutic modalities to neutralize diarrhea caused by Na+ malabsorption.