Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases:: Studies in cell populations, single cells and subcellular compartments.

Changes in pH(in) and [Ca2+](cyt) are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanisms might be altered. To study these ions, we employ fluorescence spectroscopy (cell populations attached to coverslips), and cell imaging spectroscopy/confocal microscopy (single cells and discrete subcellular compartments). pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (En) compartments by overnight incubation of cells with dextran-conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar-type H+-ATPases (pmV-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental model, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ ([Ca2+](cyt)) homeostasis in both VSM and microvascular endothelial cells; (b) E/L, compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V-ATPases are important for pH(in) regulation in microvascular endothelial cells.