MECHANISM OF NA+-DEPENDENT METABOLITE TRANSPORT

这一建议旨在快速发展新的方法

This proposal is aimed at the development of new methodology for rapid noninvasive measurement of cellular membrane potentials and use of these methods to characterize the Delta Psi dependence of a variety of plasma membrane related transport events. Isolated intestinal epithelial cells prepared from chickens will continue to be the primary model experimental system. The work described falls in three broad categories: 1) establishment of the relationship between unidirectional influx of tetraphenylphosphonium (TPP+) and Delta Psi for use as a calibration curve in defining unknown membrane potentials, 2) a systematic study of the quantitative relationship between Delta Psi and Na+-dependent sugar or amino acid transport processes, and 3) identification of the cellular "signals" and mode of action of those agents which are important in allowing intestinal tissue to convert from its normal absorptive functional state for salt and water to a secretory state resulting in the symptoms of diarrhea. The Delta Psi-sugar transport relationships will be used to discriminate between transport models involving Delta Psi-dependent translocation vs. Delta Psi-dependent Na+ binding events as a means of evaluating a "Na+-well" conceptual model which has not yet been tested experimentally. The possibility that Na+-dependent transport systems can be used as intrinsic sensors for noninvasive quantitative measurement of membrane potentials will also be evaluated. Finally, the methods established will be applied to a characterization of the specific ion flux pathways altered in activity by Ca++, cAMP, and certain hormones or drugs with particular emphasis on correlating changes in cellular transport activity with concomitant changes in the appropriate "signal" molecule or ion. The endeavor offers an opportunity for providing some of the first mechanistic insight to the quantitative role which membrane potentials play in driving Na+-dependent transport systems as well as insight to the mechanisms by which the intestinal epithelium converts from absorptive to secretory activity for salt and water and the regulation of the ion transport pathways involved. This insight carries significant clinical ramifications because of the enormous impact diarrheal diseases have as world-wide health problems.