Membrane transport of C1 has an important role in cellular regulatory, absorptive and secretory processes. The techniques presently in use for measurement of C1 transport, including patch-clamp, microelectrodes and 36C1 tracer methods, have restricted sensitivities, time resolution and applicability. Based on the utility of fluorescence probes in examining physiological questions (fluoresceins to study pH, fura-2 to study Ca), we have been developing new fluorescence methods to study C1 transport in membrane vesicles, cells and intact epithelia. We have found that the fluorescence of several quinolinium derivatives is C1- sensitive and specific and responds rapidly to changes in (C1). The probes can be loaded non-invasively into vesicles and cells to provide a continuous record of (C1).
The aims of this grant request are: to optimize the chemical structure of the C1 indicator for use in measurement of cell (C1), to characterize the best C1 indicators to provide a prescription for their use in physiological measurements, and to use C1 indicators to characterize mechanisms of proximal tubule C1 transport. Using well-established methods in synthetic organic chemistry, the structure of the C1 indicator will be modified to maximize sensitivity, to optimize cell loading and trapping, and to produce the most favorable fluorescent properties including a wavelength shift in the emission spectrum with changes in (C1). Probes will be characterized in terms of their C1 sensitivity, specificity, response time, cell loading, toxicity and intracellular calibration. Mechanisms of C1 transport will be examined in isolated brush border and basolateral membrane vesicles from the renal proximal tubule, in isolate proximal tubule cells and in the intact proximal tubule. Cuvette fluorimetry and fluorescence microscopy will be used. The availability of a good fluorescent C1 indicator should have wide applications in transport physiology and facilitate the understanding of C1 transport mechanisms and C1 regulation in both epithelial and non-epithelial membranes.
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