Glucocorticoids, Gc, play an important role in regulating cellular glutamine metabolism as well as in acid base homeostasis. Renal base generation depends upon glutamine oxidation, a reaction catalyzed by the enzyme glutamate dehydrogenase, GDH. Both Gc administration and acidosis increase GDH activity and accelerate renal ammoniagenesis. In contrast adrenalectomy leads to metabolic acidosis reflecting limited base generation and impaired glutamine oxidation. An important question therefore entails differentiating the role of Gc and acidosis. This proposal focuses on a proximal tubule cell line (LLCPK-F+) and the molecular mechanisms underlying the ammoniagenic response induced by Gc. This response will be related to the level of functional glucocorticoid receptors, GR, as well as GDH messenger RNA content and its expressed activity. Preliminary studies suggest that the failure to observe a Gc response may be due to a subthreshold level of GR; initial studies therefore will focus on the up-regulation of GR employing cis-acting cAMP analogues and blocking trans-acting proteins with cycloheximide. A major thrust of this proposal will be to insert a functional GR gene into the LLCPK-F+ genome. By engineering a vector containing the GR cDNA fused to a Gc sensitive promoter and the gene conveying resistance to neomycin, selection of stably transfected cells can be made from a media containing the antibiotic. Since the GR gene now responds positively to its product in the presence of Gc, cellular GR levels can be set ensuring maximal GR- complexes for testing genomic GDH responsiveness. Thus the relationship between gene expression and GR population as well as receptor occupancy should be observable over a range from subthreshold to supramaximal. A cDNA coding for GDH will be used to construct probes in order to monitor message levels in response to Gc. Primary versus secondary effects of the GR complex on GDH expression will be differentiated using cycloheximide to block secondary trans-acting proteins. The levels of glutamine synthetase mRNA whose regulatory gene contains GREs will serve as a positive control for the GDH response in these stably transfected cells. The integrated response of the cultured proximal tubule cells therefore provides insight into molecular mechanisms underlying glucocorticoid responsiveness in a physiological context. Monitoring 11-hydroxysteroid dehydrogenase, 11-HSD, activity in these cells will provide information of metabolic modulation of receptor occupancy using natural glucocorticoids. Alterations in 11-HSD activity can be related to GR populations in vivo as well as in cultured cells. Furthermore having established these relationships the affect of acidosis on Gc induced GDH expression can be differentiated in relation to ammoniagenesis. Understanding these mechanisms on the cellular level should greatly facilitate the elucidation of these effectors on interorgan glutamine metabolism. Finally, these studies may provide a basis for the modulation of interorgan nutrition in clinically relevant conditions, i.e., catabolic states such as diabetes, starvation, trauma and chronic renal disease.
