The long-term objective is to determine the factors which control cell volume regulation in epithelia. Cell volume is measured by viewing the tissue with a differential interference microscope, and images are displayed with a video camera on a monitor. With the aid of a computer and a stepper motor, optical sections are obtained as the microscope is focused through the tissue; video images are recorded on tape at 1 um intervals. Subsequently, the images are recalled to the monitor, and at each level a computer-driven cursor is used to trace the perimeter of a particular cell on the screen. From the coordinates of the cursor positions, the area of each section may be calculated, and from this plus the known distance between sections, the volume of the cell may be determined. Intracellular electrophysiological techniques will be utilized to determine cell membrane voltages and resistances; cell Ca++ activity will be measured with the intracellular fluorescent dye quin-2, and cell K+ and C1- activities with ion-selective microelectrodes; finally, ionic substitutions will be utilized to determine specific membrane conductances. In frog urinary bladder, we plan to study the volume regulatory decrease (VRD) that follows hypotonic swelling. We shall (1) evaluate the role of calcium in this process by measuring cell calcium activity during swelling and VRD, (2) determine the voltage dependence of VRD by clamping the membrane potentials at different values and measuring cell volume during VRD, (3) measure, in Necturus urinary bladder, basolateral membrane specific K+ and C1- conductances and cell ion activities to determine the driving forces and fluxes that bring about VRD; in addition, we shall study the role of apical membrane permeability in volume regulation by varying the former with nystatin and then swelling the cells isosmotically with KC1. In Necturus gallbladder we shall measure cell volume and VRD after isosmotic swelling with high mucosal K+. We will determine (1) the role of transepithelial sodium transport in this process by varying the transport rate with inhibitors, (2) the relationships between VRD and ion fluxes by measuring cell volume and Ca++ activity and, in parallel experiments, cell K+ and Cl- activities during the swelling-VRD sequence, (3) the role of the K+ and C1- transmembrane gradients in VRD, and (4) the role of initial cell volume itself in determining the responses to swelling. Since cells must maintain normal cell volume to survive, we need to learn the factors by which cells do this in the face of external events which tend to change volume.
| Finn, A L; Tsai, L M; Falk, R J (1989) Monoclonal antibodies to the apical chloride channel in Necturus gallbladder inhibit the chloride conductance. Proc Natl Acad Sci U S A 86:7649-52 |
