Research in the last decade has revealed that inositol plays an important role in protecting cells from hypertonic stress. The renal medulla is the only tissue in mammals that normally undergoes large changes in tonicity. The changes are intrinsic to the mechanism for producing concentrated or dilute urine. The stress is the result of the high concentration of intracellular potassium that rapidly occurs to balance extracellular tonicity. Over a period of time, however, the cells in the medulla lower their concentration of potassium to the same level as in isotonic cells in other organs by accumulating small organic solutes, such as inositol, that do not perturb cell function the way high concentrations of potassium do. In some abnormal states, such as chronic hypernatremia, brain also accumulates inositol as a non-perturbing osmolyte. In hypertonic culture medium cells from many tissues including kidney, brain, endothelium, and eye, accumulate inositol. The accumulation is the result of increased activity of the sodium/inositol cotransporter that brings insitol into cells against enormous (over 500-fold) concentration gradients. We have cloned the cDNA for the cotransporter and demonstrated that hypertonicity increases transcription of its gene. We have also found evidence for regulation at other steps. We propose to study the mechanisms involved in the regulation starting at the level of transcription. Enhancers are DNA sequence elements that increase the rate of transcription of specific genes in response to specific stimuli typically mediated by proteins that bind to the enhancer. We will identify the enhancer based on its ability to stimulate transcription of reporter genes in response to hypertonicity. The protein(s) that interacts with the enhancer will be cloned and antibodies to the protein will be raised. We will examine how hypertonicity regulates the enhancer binding protein at the level of mRNA expression, binding/dissociation to the enhancer, phosphorylation, and interaction with other proteins. We have identified a post-transcriptional mechanism of regulation of the activity of the cotransporter. We will identify the sequence of the mRNA that is required for the regulation of RNA turnover by tonicity. The cytoplasmic protein(s) that interacts with this sequence and changes the RNA stability will be identified. To explore the third, post-translational, level of regulation, we will determine phosphorylation sites involved in the inhibition of transport by protein kinase C and protein kinase A. Information obtained from these studies should provide insight into how cells sense hypertonic stress and how the signal is transduced.
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