The long term goal of this work is to establish the molecular mechanism by which the corneal endothelium pumps fluid and electrolytes. We proposed to single out and characterize membrane proteins involved in transport of water and electrolytes and cell volume regulation, with special attention to the facilitative glucose transporter (GT), and to integrate our findings into a model of endothelial function. We will investigate how cells regulate the osmolarity of the solution transported by the endothelium, and by which message is the cell likely to translate a volume change in a change of the activity of a membrane effector. We will measure cell volume by the intensity of light scattered by cultured endothelial cells . We will utilize time-shared recordings of the fluorescent emissions of marker-dyes trapped in cultured cells to measure simultaneously a cell electrolyte such as H+ ,Na+ , or Ca++, and the cell volume. We will investigate the route of fluid transport across across in-vitro rabbit corneal endothelial, and across endothelial cells grown on permeable substrates. We will attempt to define up to which passage do they maintain that function. Isoenzymes will be assayed for to characterize cells in different passages. We will investigate the hypotheses that a Na+ current recirculating across a circuit which includes the intercellular junctions can generate enough electroosmotic viscous drag on water so as to yield isotonic transport. We will quantify the effects of replacement of ambient Na+ pump. and the effects of drugs such as protamine and heparin on fluid movements induced by electric currents across cultured cell layers and in vivo rabbit preparations. We will investigate whether there is an apical entry path for Na+. We will measure the time course of radiolabeled Na+ and K+ uptakes by cultured endothelial cells,and the effect on them of amiloride and derivatives; and of inhibitors of Na+ channels, Na+-H+ exchange, Na+ -Ca+ exchange, K+ channels and the Na+ pump. We will attempt to identify and characterize cell membrane proteins using SDS PAGE. We will investigate which form of the GT is present in endothelial cells and their fractions using immunoblots, and will attempt to determine GT numbers by cytochalasin B binding. We will investigate whether the GT is located in the apical membrane as part of an osmosensing loop. We will attempt to reconstitute GT into vesicles so as to determine its molecular permeability to water. We will determine whether other membrane proteins such as the anion transporter can be recognize by antibodies. We will attempt to identify which isoforms of the Na+ -K+ /ATpase subunits are present in cultured endothelia using specific antisera, and will study the number, type,and activity of the Na+ -K+ /ATpase sites in cultured endothelial cells of several species. Using Northern blots, we plan to determine whether mRNA encoding either brain, liver or muscle glucose transporter of the RBC anion transporter can be detected in cultured endothelia.
| Sanchez, J M; Cacace, V; Kusnier, C F et al. (2016) Net Fluorescein Flux Across Corneal Endothelium Strongly Suggests Fluid Transport is due to Electro-osmosis. J Membr Biol 249:469-73 |
| Diecke, Friedrich P J; Ma, Li; Iserovich, Pavel et al. (2007) Corneal endothelium transports fluid in the absence of net solute transport. Biochim Biophys Acta 1768:2043-8 |
| Ma, Li; Kuang, Kunyan; Smith, Randall W et al. (2007) Modulation of tight junction properties relevant to fluid transport across rabbit corneal endothelium. Exp Eye Res 84:790-8 |
