BACKGROUND & AIMS-Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na + /H + exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na + /H + exchanger activity and Na + -dependent fluid absorption in the intestine.

The mammalian kidney plays an essential role in the control of systemic water, ion, and acid-base balance. Na ϩ /H ϩ exchanger type 3 (NHE3) plays a pivotal role in salt and fluid reabsorption in the proximal tubule (1), accounting for ϳ50% of NaCl and 70% of NaHCO 3 reabsorbed from the glomerular filtrate (2). In fact, mice deficient in NHE3 expression are relatively hypotensive, even when NHE3 in the intestine is rescued by transgenic expression (3).The renin-angiotensin system is critically involved in regulation of body blood pressure and fluid balance. The kidney secretes renin when blood pressure is low that stimulates the production of ANG 3 I, which is subsequently converted into ANG II by angiotensin-converting enzyme. The presence of ANG II receptor on the membrane of renal proximal tubules was first reported in the 1980s (4). Through the binding with its cognate receptor(s), ANG II is importantly involved in fluid reabsorption in the proximal tubules. Substantial earlier evidence from Cogan and co-workers (5-8) has demonstrated that ANG II is a potent agonist of H ϩ secretion and HCO 3 Ϫ absorption in rat proximal tubules through mechanisms dependent on decrease of cAMP, increase of [Ca 2ϩ ] i , and activation of protein kinase C (PKC). It was later shown that ANG II, at low concentrations, stimulates NHE3 activity in renal proximal tubule cells (9 -11). Studies have implicated the roles of PKC and c-Src in ANG II-mediated activation of NHE3 (9, 12). In addition, ANG II stimulates NHE3 activity through increased exocytotic insertion of NHE3 in a phosphatidylinositol 3-kinase (PI3K)-dependent manner (13). Nevertheless, the molecular mechanisms underlying NHE3 activation by ANG II remain incompletely delineated.IRBIT was initially identified as an IP 3 receptor-binding protein and was shown to be a competitive inhibitor of Ca 2ϩ release by IP 3 receptor (14,15). We have recently identified IRBIT as a novel NHE3-interacting protein by yeast two-hybrid screening of a kidney library (16). Our study demonstrated that IRBIT binds the C-terminal domain of NHE3 and activates NHE3 activity in response to thapsigargin or ionomycin-induced rise of [Ca 2ϩ ] i in PS120 fibroblast cells (16). IRBIT mRNA is ubiquitously present in all tissues, but the highest expression was reported in the brain, reproductive tissues, and kidney (14). These findings prompted us to hypothesize that IRBIT might play an important role in NHE3 regulation in the kidney. In this work, we investigated the role of IRBIT in the regulation of NHE3 by ANG II. Our findings show that IRBIT is critically involved in the activation of NHE3 by ANG II, and this regulation is Ca 2ϩ -CaMKII-dependent.

Calcium (Ca 2؉ ) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca 2؉ regulates the activity of Na ؉ /H ؉ exchanger 3 (NHE3). However, the effect

A major of Na+ absorptive process in the proximal part of intestine and kidney is electroneutral exchange of Na+ and H+ by Na+/H+ exchanger type 3 (NHE3). During the past decade, significant advance has been achieved in the mechanisms of NHE3 regulation. A bulk of the current knowledge on Na+/H+ exchanger regulation is based on heterologous expression of mammalian Na+/H+ exchangers in Na+/H+ exchanger deficient fibroblasts, renal epithelial, and intestinal epithelial cells. Based on the reductionist's approach, an understanding of NHE3 regulation has been greatly advanced. More recently, confirmations of in vitro studies have been made using animals deficient in one or more proteins but in some cases unexpected findings have emerged. The purpose of this paper is to provide a brief overview of recent progress in the regulation and functions of NHE3 present in the luminal membrane of the intestinal tract.

Na(+) absorption is a vital process present in all living organisms. We have reported previously that lysophosphatidic acid (LPA) acutely stimulates Na(+) and fluid absorption in human intestinal epithelial cells and mouse intestine by stimulation of Na(+)/H(+) exchanger 3 (NHE3) via LPA(5) receptor. In the current study, we investigated the mechanism of NHE3 activation by LPA(5) in Caco-2bbe cells. LPA(5)-dependent activation of NHE3 was blocked by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 and U0126, but not by phosphatidylinositol 3-kinase inhibitor LY294002 or phospholipase C-β inhibitor U73122. We found that LPA(5) transactivated the epidermal growth factor receptor (EGFR) and that inhibition of EGFR blocked LPA(5)-dependent activation of NHE3, suggesting an obligatory role of EGFR in the NHE3 regulation. Confocal immunofluorescence and surface biotinylation analyses showed that LPA(5) was located mostly in the apical membrane. EGFR, on the other hand, showed higher expression in the basolateral membrane. However, inhibition of apical EGFR, but not basolateral EGFR, abrogated LPA-induced regulation of MEK and NHE3, indicating that LPA(5) selectively activates apical EGFR. Furthermore, transactivation of EGFR independently activated the MEK-ERK pathway and proline-rich tyrosine kinase 2 (Pyk2). Similarly to MEK inhibition, knockdown of Pyk2 blocked activation of NHE3 by LPA. Furthermore, we showed that RhoA and Rho-associated kinase (ROCK) are involved in activation of Pyk2. Interestingly, LPA(5) did not directly activate RhoA but was required for transactivation of EGFR. Together, these results unveil a pivotal role of apical EGFR in NHE3 regulation by LPA and show that the RhoA-ROCK-Pyk2 and MEK-ERK pathways converge onto NHE3.