TRANSPORT MEDIATED REGULATION OF CELLULAR MYOINOSITOL

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Myo-inositol is a critical constituent of mammalian cells. In addition to serving as the precursor of membrane phosphoinositides, important in the signal transduction processes which involve calcium and protein kinase C, this cyclic polyol compound plays a key role in cellular osmoregulation. Myo-inositol is "the idiogenic osmole" or organic osmolyte which accumulates in brain tissue during hyperosmolar states. In most cells, myo-inositol levels are maintained by energy -dependent transport. During adaptation to hypertonic stress, it is the regulation of the number of plasma membrane transporters as evidenced by Vmax measurements which determine the new set point for intracellular myo-inositol levels. The synthesis of the transporter protein, in turn, is exerted at the gene transcriptional level at least in some mammalian kidney, brain and ocular cells. We have recently cloned the human osmoregulatory Na+/myo-inositol cotransporter (SMIT) gene. The gene is expressed in many human tissues including the brain. We have localized the gene to the distal portion of the long arm of chromosome 21. Patients with Down Syndrome may have increased levels of myo-inositol in any cell, during fetal or post-natal life, if the cell is unable to dampen the expression of 3 gene copies to that which would be appropriate for 2. The SMIT gene may play a role in the pathogenesis of Down Syndrome, especially the neurological dysfunction. In this study, we propose to complete the sequencing of the human SMIT gene using two newly isolated bacteriophage PI clones; delineate the expression of SMIT gene in different human tissues via in situ hybridization and immunohistochemistry; measure the level of myo- inositol and alternate osmolytes in the brain of patients with Down Syndrome and control subjects, and correlate the tissue levels with the expression of the SMIT genes and determine the effect of hypertonic or hypotonic stress on the normal and trisomic 21 cultured fibroblasts by assessing viability, osmolyte levels and SMIT mRNA levels.