The survival of cells in the mammalian inner medulla requires an adaptive response to the hypertonic environment. This adaptation to changes in tonicity initially involves the activation of protein kinases and ultimately the upregulation of proteins that allow for cellular accumulation of inert osmolytes. It is now evident that a more complex array of both genes and proteins is involved in the adaptive response. The present proposal intends to examine the repertoire of genes and proteins that are both increased and decreased in cells adapted to live in hypertonic conditions (600 and 900 mOsm/kg) and in cells acutely (24-48 hours) exposed to increased tonicity. In this setting the role of various signaling pathways in the genetic regulation will be assessed by employing inhibitors of such pathways as well as transfected cells with both gain and loss of function mutations. The in-vitro results will also be extended to the study in-vivo in rodents in various states of water balance. Both genomic and proteomic approaches are contemplated to unveil as full a repertoire as the presently available techniques allow. In its second aim the proposal will examine the mechanism whereby the y subunit of Na-K-ATPase is upregulated by hypertonicity and how the protein provides a survival advantage in these conditions. More specifically, the transcriptional regulation of the gene will be studied. We hypothesize that the effect of JNK kinase to enhance y subunit synthesis is regulated at the transcriptional level. The hypothesis will be studied by employing inhibitors of JNK kinase, gain and loss of function transfectents of the kinase and knockout mice deficient in JNK1 and JNK2. The translational regulation will focus on the role of PI3 kinase in the process. The role of the y subunit in osmoprotection will be studied by comparing the effects of hypertonicity in wild type cells to that of cells rendered incapable of synthesizing the protein by a silencing RNA technique. This approach could be extended to the study of relevant genes unveiled in the genomics-proteomic studies. Effects on alteration in cell Na concentration, uptake of an inert osmolyte (inositol) and finally cell survival itself will be assessed. These experiments should define genes and proteins involved in osmoregulation and particularly unveil the role of the y subunit as a vital component in the adaption to the hypertonic environment of the mammalian inner medulla. ? ?
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