DIGITAL FLUORESCENCE IMAGING SYSTEM

This facility is designed for the general purpose of correlating optical and electrophysiological measurements. The optical measurements that this equipment would permit include spatial measurements of intracellular pH and pCa, determination of membrane potentials in multicellular preparations by voltage sensitive dyes, quantitation of dye fluxes within and between cells resolution of cell volume and secretion changes and localization of antibody binding to subcellular compartments. Electrophysiological measurements will include resting and action potentials, junctional conductances and ion concentrations as measured by ion selective electrodes. Project 1 will evaluate gating mechanism and membrane trafficking of gap junctions in cardiac myocytes, astroglial cells and hepatocytes isolated from adult mammalian tissues. Sensitivity of junctional conductance to intracellular H and Ca will be determined. Fluorescent antibodies will be used to monitor internalization and possible reuse. Label applied intracellularly may determine whether formation is by redistribution of surface molecules or insertion from the cytoplasm. Other studies will concern drug effects on cardiac myocytes in respect to changes in intracellular ions and the concomitant changes in contractility. Contractility of hepatocyte canaliculi will also be evaluated. Project 2 will determine gap junction channel properties by quantitative comparison of dye fluxes and electrical conductance in various cell types. Problems addressed will include all-or-none channel closure and interactions of permeant molecules with channel walls. Project 3 will evaluate factors in death and growth of cultured sympathetic neurons. Changes in neuritic voltages, pH, pCa and redox state will be measured during normal growth and during injury caused by starvation and anoxia. Project 4 will study intracellular pH and intracellular communication between cells in the renal collecting ducts with particular attention to the mitochondria-rich cells that are presumed to secrete acid. These cells are surrounded by principal cells to which they may not be coupled. Comparison will be made to other regions of the kidney where coupling is presumed to occur. Project 5 will concern second messengers and membrane trafficking in cells and cell lines derived from kidney. Question to be addressed include temporal and spatial resolution of Ca generation and time course of response to antidiuretic hormone.