Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H+-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells

Vacuolar H+-ATPases (V-ATPases) are a family of electrogenic ATP-driven proton pumps which function in almost every eukaryotic cell. V-ATPases are required to maintain proton gradients between intracellular compartments and for proton secretion from the plasma membrane of certain specialized cells (reviewed in references 32, 42, and 43). In the intracellular compartments, V-ATPases acidify early and late endosomes, lysosomes, and Golgi-derived secretory vesicles, providing the motive force and optimal pH for internalization and dissociation of ligand-receptor complexes, pH-driven secretion, and activation of lysosomal enzymes for protein processing and degradation (43, 48). Plasma membrane V-ATPases are essential for acid secretion and bicarbonate transport in the proximal tubule and collecting duct of the kidney, pH homeostasis in macrophages and neutrophils, acidification of the extracellular environment by certain tumor cells, and K+ secretion in insect midgut cells, (24, 43). V-ATPase-dependent acidification of the extracellular compartment between the osteoclast ruffled membrane and the bone surface is crucial for bone remodeling (23). V-ATPase is a large, multisubunit protein. It is composed of at least 13 subunits with a total molecular mass of about 900 kDa and has two domains, the V1 domain (catalytic, membrane-associated, 640 kDa) and Vo domain (membrane spanning, 260 kDa). The V1 domain is responsible primarily for ATP hydrolysis. It consists of eight different subunits (A to H) in a stoichiometry of A3B3CDEFG2H1-2. V1 attaches to the proton-translocating Vo domain, which is composed of five subunits (a, b, c, c′, and c") in a stoichiometry of abc′c"c4 (20, 32, 43). Regulation of V-ATPase function in response to physiological stimuli is thought to be a multilevel process. It includes control of the expression of V-ATPase subunit genes (34, 62), intracellular targeting and translocation from vesicles to the plasma membrane (2, 9, 10), and reversible dissociation of the Vo and V1 domains, entailing inactivation of the pump (30, 31, 55). Several extrinsic regulatory factors have been reported to control V-ATPase-mediated proton transport. They include bicarbonate concentration and the closely related parameters pH and pCO2, mineralocorticoid hormones, endothelin, angiotensin II (reviewed in references 23 and 24), and cytokines, such as interleukin-1 (IL-1) (7) and IL-4 and -13 (59). The underlying intracellular signaling mechanisms remain largely unknown. Involvement of G proteins and protein kinases A and C has been implicated in several studies (23, 24). The E subunit of V-ATPase is able to interact directly with the Dbl homology domain of the guanine nucleotide exchange factor mSOS, linking V-ATPase with the small G protein Rac1 (39). A recent study demonstrated the role of bicarbonate-activated soluble adenylyl cyclase and cyclic AMP-dependent signal transduction in pH-dependent V-ATPase recycling (45). Among the most intriguing functions of V-ATPase is its ability to respond to extracellular glucose. In the yeast Saccharomyces cerevisiae, glucose deprivation produced rapid and reversible dissociation of the V-ATPase into V1 and Vo domains (30). In S. cerevisiae, this mechanism regulates the balance between the conservation of cytoplasmic ATP and maintenance of intracellular pH homeostasis (31). In human kidney, subunit E of V-ATPase interacts directly with the glycolytic enzyme aldolase (35, 36) and subunit a interacts with phosphofructokinase-1 (54), thereby providing a coupling mechanism with the glycolytic pathway. The kidney has the most V-ATPase activity among mammalian tissues, and within the kidney, V-ATPase is most abundant in epithelial cells of the initial segments of the proximal tubule and in intercalated cells of the collecting duct (24). Therefore, in kidney epithelia, the response of V-ATPase to glucose may represent a pivotal regulatory mechanism. The mechanism by which glucose regulates V-ATPase remains unclear. In S. cerevisiae, V-ATPase assembly in response to glucose did not involve the Ras-cyclic AMP pathway, Snf1p (the homolog of the mammalian fuel sensor protein kinase AMPK), protein kinase C, or the general stress response protein Rts1p (homolog of the mammalian B-regulatory subunit of protein phosphatase A2) (44). These results showed that the reversible disassembly of the V-ATPase in S. cerevisiae does not require any known glucose-induced signaling pathway (31) On the other hand, V-ATPase assembly depends on the Skp1-containing adaptor complex RAVE, suggesting involvement of intracellular signaling (49, 51). Furthermore, glucose-induced activation of plasma membrane V-ATPase in S. cerevisiae is likely to be activated via phosphatidylinositol-type signaling involving protein kinase C (6). In the ruffled border of osteoclasts, V-ATPase is localized along with phosphatidylinositol 3-kinase (PI3K) (40). Wortmannin, a specific inhibitor of PI3K, inhibited the formation of ruffled border and osteoclastic bone resorption (40, 41), suggesting involvement of PI3K-dependent signaling in the control of V-ATPase. However, the biochemical mechanism of the glucose effects on V-ATPase in mammalian cells remains unknown. In the present study, we demonstrated that glucose is a potent regulator of V-ATPase in kidney proximal tubular cells in vitro. We showed that glucose stimulates acidification of intracellular compartments by V-ATPase and that this effect is mediated by PI3K. Glucose removal diminished V-ATPase-dependent acidification, decreased ATP hydrolytic activity of V-ATPase, induced its disassembly into Vo and V1 domains, and led to translocation of both V1 and Vo from plasma membrane and submembrane vesicles to intracellular vesicles (V1 and Vo) and the cytoplasm (V1). Glucose stimulation induces V-ATPase activity, reassembly, and translocation to the vesicular and plasma membranes and fast acidification of intracellular compartments. Glucose flux through the glycolytic pathway is not required to provide a signal for V-ATPase assembly. Active PI3K is required for the glucose-induced changes in V-ATPase assembly and trafficking. Adenoviral expression of the constitutively active p110α catalytic subunit of PI3K mimics the effect of glucose in cells maintained in glucose-free conditions. These results suggest that, in renal epithelial cells, glucose is a physiological regulator of acidification of intracellular compartments and V-ATPase activity, assembly, and trafficking and that its effects are mediated by the PI3K signaling pathway.