cells thrive in a hypoxic microenvironment with an acidic extracellular pH. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. We hypothesize that vacuolar H ϩ -ATPases (V-ATPases) that normally reside in acidic organelles are also located at the cell surface, thus regulating cytosolic pH and exacerbating the migratory ability of metastatic cells. Immunocytochemical data revealed for the first time that VATPase is located at the plasma membrane of human breast cancer cells: prominent in the highly metastatic and inconspicuous in the lowly metastatic cells. The V-ATPase activities in isolated plasma membranes were greater in highly than in lowly metastatic cells. The proton fluxes via V-ATPase evaluated by fluorescence spectroscopy in living cells were greater in highly than in lowly metastatic cells. Interestingly, lowly metastatic cells preferentially used the ubiquitous Na ϩ /H ϩ exchanger and HCO 3 Ϫ -based H ϩ -transporting mechanisms, whereas highly metastatic cells used plasma membrane V-ATPases. The highly metastatic cells were more invasive and migratory than the lowly metastatic cells. V-ATPase inhibitors decreased the invasion and migration in the highly metastatic cells. Altogether, these data indicate that V-ATPases located at the plasma membrane are involved in the acquisition of a more metastatic phenotype. metastasis; intracellular pH; migration; sodium ion/hydrogen ion exchanger; bicarbonate transport MAINTENANCE OF CYTOSOLIC pH (pH cyt ) is crucial to normal cell function because many cellular processes have a narrow pH optimum (38). Tumor cells possess high-glycolytic activity and produce acidic metabolites (39). Moreover, tumor cells often exist in a hypoxic microenvironment with a lower extracellular pH (pH ex ) than that of surrounding normal cells (10,15,40). Acidic pH ex and hypoxic environment are not permissive for cell growth and have been associated with apoptosis (14, 25). To minimize the potentially toxic reduction in pH cyt that would accompany growth in a chronically acidic environment, tumor cells must upregulate the proton extrusion mechanism(s) that maintains pH cyt . The ability to upregulate proton extrusion may be essential for tumor cell survival. The influence of pH cyt on many cellular functions has been studied with respect to cell growth (16), cell motility (27), tumorigenesis (36), metastasis (45), apoptosis (14), and drug resistance in cancer cells (28, 50).Four major types of pH cyt regulatory mechanisms have been identified in tumor cells: Na ϩ /H ϩ exchangers, bicarbonate (HCO 3 Ϫ ) transporters, proton-lactate symporters, and proton pumps (11,38,42). Recently, the vacuolar H ϩ -ATPase (VATPase) has emerged as a novel and important pH cyt regulatory system in some specialized cells, including tumor (25,26,28). This proton pump is ubiquitously expressed (33, 35), not only in vacuolar membranes but also in plasma membranes (26, 58) of eukaryotic cells. The V-ATPase is a multi-subunit enzyme complex composed of a me...

Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3−-based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and cell migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascular endothelial cells.

We report a simple in vitro model of cardiac tissue that mimics three-dimensional (3-D) environment and mechanical load conditions and, as such, may serve as a convenient method to study stem cell engraftment or address developmental questions such as cytoskeleton or intercalated disk maturation. To create in vitro cardiac fibers we used Matrigel, a commercially available basement membrane preparation. A semisolid pillow from concentrated Matrigel was overlaid with a suspension of rat neonatal cardiomyocytes in a diluted Matrigel solution. This created an environment in which the multicellular fibers continuously contracted against a mechanical load. The described approach allows continuous structural and functional monitoring of 20-300-micron-thick cardiac fibers and provides easy access to epitopes for immunostaining purposes.

Angiogenesis requires invasion of extracellular matrix (ECM) proteins by endothelial cells and occurs in hypoxic and acidic environments that are not conducive for cell growth and survival. We hypothesize that angiogenic cells must exhibit a unique system to regulate their cytosolic pH in order to cope with these harsh conditions. The plasmalemmal vacuolar type H(+)-ATPase (pmV-ATPase) is used by cells exhibiting an invasive phenotype. Because angiogenesis is impaired in diabetes, we hypothesized that pmV-ATPase is decreased in microvascular endothelial cells from diabetic rats. The in vitro angiogenesis assays demonstrated that endothelial cells were unable to form capillary-like structures in diabetes. The proton fluxes were slower in cells from diabetic than normal model, regardless of the presence or absence of Na(+) and HCO(3) (-) and were suppressed by V-H(+)-ATPase inhibitors. Immunocytochemical data revealed that pmV-ATPases were inconspicuous at the plasma membrane of cells from diabetic whereas in normal cells were prominent. The pmV-ATPase activity was lower in cells from diabetic than normal models. Inhibition of V-H(+)-ATPase suppresses invasion/migration of normal cells, but have minor effects in cells from diabetic models. These novel observations suggest that the angiogenic abnormalities in diabetes involve a decrease in pmV-ATPase in microvascular endothelial cells.

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