In rat CCDs, Na,K-ATPase activity measured as ouabain-sensitive currents was upregulated by exogenous aldosterone under a Na+-rich diet.13 This effect was eliminated by inhibition of ENaC-mediated Na+ access with coinfused amiloride,13 suggesting cross-talk between ENaC and Na,K-ATPase. Na,K-ATPase, which may allow principal cells to maintain intracellular Na+ concentrations within thin limits. The fine-tuning of Na+ balance is critical for the homeostasis of body fluid compartments. A variety of disorders and diseases, Antitumor agent-2 such as hypertension and edema, result at least partly from disturbances of Na+ homeostasis.1 The final regulation of renal Na+ reabsorption takes place in aldosterone-responsive distal tubules and collecting ducts.2 The bulk of Na+ transport in the collecting duct (CD) occurs in principal cells, where Na+ enters the cell the epithelial sodium channel (ENaC) and is extruded into the interstitial compartment Na,K-ATPase.3 Thus, tight control of vectorial Na+ transport must be exerted on CD principal cells to achieve whole-body Na+ homeostasis. According to dietary Na+ intake and aldosterone levels, CD principal cells are exposed to large physiologic variations of Na+ transport.2,3 Meanwhile, intracellular Na+ concentration must be maintained within narrow ranges, which is essential for vital cellular functions, such as control of osmolality, ionic strength, and membrane potential. Therefore, apical Na+ access Antitumor agent-2 and basolateral Na+ extrusion must be rapidly and tightly coordinated in order to match variations of Na+ transport while minimizing fluctuations of intracellular Na+ concentration. The mechanisms mediating this coordination remain largely unknown. Control of exocytosis/endocytosis is a common mechanism for modulating the abundance and function of membrane proteins. For example, increasing the activity of the AMP-activated protein kinase (AMPK), as a result of increased ATP consumption, modulated Na,K-ATPase endocytosis in cultured renal epithelial MDCK cells.4 Among several actions, activation of p38 kinase, a member of the MAP kinase family, regulates the endocytosis of a variety of cell surface proteins.5 We reported previously that aldosterone treatment which stimulates active transcellular Na+ reabsorption reduced p38 kinase activation, but not that of ERK1/2, in renal CD principal cells.6 Activation of p38 kinase is essential for EGFR Antitumor agent-2 endocytosis and lysosomal degradation.7C9 Interestingly, p38 kinase controls the phosphorylation and ubiquitinylation of aquaporin-2 (AQP2), triggering its endocytosis and degradation in renal CD principal cells. 10 We hypothesized that CD principal cells exhibit tight coordination of apical and basolateral Na+ transport, putatively through modulation of Na,K-ATPase cell surface expression by Na+ apical entry. AMPK and/or p38 kinase signaling pathways may control Na, K-ATPase endocytosis involved in cross-talk between ENaC and Na,K-ATPase. In this study, we describe a cross-talk between apical ENaC and basolateral Na,K-ATPase in a physiologic context. We identified p38 kinase-regulated endocytosis and degradation of cell surface Na,K-ATPase as a key player in this cross-talk. Results Enhanced Apical Na+ Delivery Increases Na,K-ATPase Activity and Expression in Isolated Rat Cortical Collecting Ducts To investigate whether ENaC-mediated Na+ entry is coordinated with Na,K-ATPase-dependent Mouse monoclonal antibody to DsbA. Disulphide oxidoreductase (DsbA) is the major oxidase responsible for generation of disulfidebonds in proteins of E. coli envelope. It is a member of the thioredoxin superfamily. DsbAintroduces disulfide bonds directly into substrate proteins by donating the disulfide bond in itsactive site Cys30-Pro31-His32-Cys33 to a pair of cysteines in substrate proteins. DsbA isreoxidized by dsbB. It is required for pilus biogenesis Na+ exit investigation of coordination between apical ENaC and basolateral Na,K-ATPase that occurs independently of variations Antitumor agent-2 of aldosterone levels. Higher apical Na+ entry ENaC in rats fed with the normal Na+ diet compared with rats fed the low-Na+ diet was associated with an increase in Na,K-ATPase activity (Figure 1B). The observed stimulation of Na,K-ATPase activity was associated with a proportional increase of the Na,K-ATPase -subunit expression assessed by Western blotting in total lysates of isolated CCDs (Figure 1, C and D). Therefore, the stimulation of Na,K-ATPase activity most likely relies on an increased number of active Na,K-ATPase units at the plasma membrane. In rat CCDs, Na,K-ATPase activity measured as ouabain-sensitive currents was upregulated by exogenous aldosterone under a Na+-rich diet.13 This effect was eliminated by inhibition of ENaC-mediated Na+ entry with coinfused amiloride,13 suggesting cross-talk between ENaC and Na,K-ATPase. Here our results show that, its hydrolysis by Na,K-ATPase and thus results in an elevated cytosolic AMP-to-ATP ratio.