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Modeled Microgravity Alters Distribution of Lymphocyte Signaling Kinases Associated with Lipid Rafts.  A. Rivera1, H. Maldonado2 and L. A. Cubano1 1 Dept. of Physiology and 2 Pharmacology, Universidad Central del Caribe, Bayamуn, PR.

   Immune suppression has been observed both in space and in ground-based models.  Lymphocyte activation upon T-cell receptor engagement is mediated by lateral mobility and spatial reorganization of proteins within the membrane, in distinct cholesterol/sphingolipid-rich microdomains or lipid rafts.  The cascade of biochemical events is initiated by kinase phosphorylation and leads to activation of second messenger pathways, subsequent proliferation and cytokine production.  Since signaling molecules are targeted to lipid rafts, we hypothesize that the immune suppression seen in spaceflown cells is consequent to improper segregation of signaling molecules. Our objective was to determine the effects of modeled microgravity on kinase distribution between raft and non-raft membrane fractions in Jurkat cells. To test this hypothesis we examined the effect of simulated microgravity on calcium mobilization, a marker for activation that leads to proliferation and interleukin-2 production.  We also performed Western blot analysis of caveolin enriched subcellular fractions from Jurkat cells before and after exposure to modeled microgravity. Jurkat cells were incubated in high-aspect ratio vessels for different times and calcium mobilization was measured using fura-2 fluorescence.  Jurkat cells in normal gravity showed an increase in intracellular calcium upon application of anti-CD3, acetylcholine or ATP.  Cells exposed to modeled microgravity for 24 hrs exhibited substantially reduced or no calcium signaling upon application of activators.  Western blot analysis of Jurkat cells after exposure to modeled microgravity showed removal of caveolin and the src kinase lck from light-density, Triton insoluble membrane fractions.  The effect of modeled microgravity could be mimicked by membrane cholesterol sequestration using methyl beta cyclodextrin.  These results support the involvement of lipid rafts in the altered lymphocyte function observed in microgravity.

Supported in part by 2G12RR03035 and S06-GM50695