The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

All cells, especially those of epithelial origin undergo moment to moment changes in their ability to absorb and/or secrete salts and water thereby regulating the composition of body fluids. The mechanisms involve changes of the density of ionic channels and carrier proteins within the apical and basolateral plasma membranes of the epithelial cells. It is recognized that changes of channels densities may involve not only activation of quiescent or dormant channels within the plasma membranes but also fusion of channel containing vesicles with the plasma membranes with activation/deactivation of channels mediated by hormonal second messenger systems, and by nonhormonal signal transduction pathways.

Our work has focused on 1) developing new methods of blocker-induced noise analysis of the apical Na channels of tight epithelial as a means of measuring directly and noninvasively the Na channel density, channel open probability and single channel current, and the changes that occur during hormonal and autoregulatory (nonhormonal) activation of epithelial Na channels (ENaCs), and 2) developing new methods of dielectric spectroscopy (electrical impedance analysis) to measure changes of electrical capacitance and hence the changes of area of apical and basolateral membranes that accompany changes of channel density.

During the past year we have used these new methods to study how prostaglandins (PGE2) acting through the second messenger cyclic-AMP regulate apical membrane Na+ entry into cell cultured A6 epithelia in terms of epithelial Na channel density and channel open probabilities and have identified that stimulation of Na transport is due to increase of the densities of functional channel ENaCs.

An exciting new direction of research has begun with investigation of the role of phosphatidyl-3-kinase (PI3-K) in regulation of transport. This family of kinases is directly involved in phosphorylating the headgroups of phosphatidyl inositol lipids of the plasma membranes and in this regard represents a link between regulation of channel densities and open probabilities by lipids as well as proteins associated with the channels. Our data has indicated that this kinase(s) is intimately involved in maintaining baseline rates of Na+ transport. Specific PI3-kinase inhibitors (LY294002) and wortmannin are being used to sort out the various kinases and their roles in regulation of the channels. As these kinases are central in the pathways of regulation of transport, it will be of great interest to know their roles and relationships to hormonal regulation of transport by aldosterone, ADH, insulin and other hormones.