Formation of cardiac fibers in Matrigel matrix.

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.

Plasmalemmal vacuolar H+-ATPase is decreased in microvascular endothelial cells from a diabetic model.

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.

Plasmalemmal V-H(+)-ATPases regulate intracellular pH in human lung microvascular endothelial cells.

The lung endothelium layer is exposed to continuous CO(2) transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na(+)/H(+) exchanger and HCO(3)(-)-dependent H(+)-transporting mechanisms regulate intracellular pH (pH(cyt)) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H(+)-ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na(+)/H(+) exchanger and HCO(3)(-)-based H(+)-transporting mechanisms, to maintain pH(cyt) homeostasis. Immunocytochemical studies revealed V-H(+)-ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na(+) and HCO(3)(-) that were similar to those observed in the presence of either Na(+), or Na(+) and HCO(3)(-). The Na(+)- and HCO(3)(-)-independent pH(cyt) recovery was inhibited by bafilomycin A(1), a V-H(+)-ATPase inhibitor. These studies show a Na(+)- and HCO(3)(-)-independent pH(cyt) regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases.

Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity.

Tumor 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(+)-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 V-ATPase 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(+)/H(+) exchanger and HCO(3)(-)-based H(+)-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.