? ? Control of intracellular calcium (Ca2+) and reactive oxygen species (ROS) homeostasis is essential for cell survival and function. Elevation of intracellular free Ca2+ and ROS levels play a central role in lymphocyte activation in response to antigenic stimulation. ROS and Ca2+ exert a reciprocal regulation on each other: exogenous application of ROS leads to intracellular Ca2+ mobilization while increased cytoplasmic Ca2+ concentration induces ROS production by lymphocytes. In polycyclic aromatic hydrocarbons (PAH)-induced lymphocyte immune suppression, enhanced ROS production is associated with increased basal levels of intracellular Ca2+. Furthermore, these high basal levels of intracellular Ca2+ do not increase further in response to stimulation of the antigen receptor. Thus, the candidate hypothesizes that the crosstalk between Ca2+ and ROS is vital for lymphocyte activation and that the disruption of this crosstalk contributes to abnormal antigen receptor signaling pathways observed in immune suppression by PAH. Therefore, the candidate plans to investigate the crosstalk between Ca2+ and ROS signaling during normal B-cell receptor stimulation as well as the disruption of this crosstalk during immune suppression by PAH. Specifically, three specific aims will be pursued: (1) to define, using electrophysiology and Ca2+ imaging, the pharmacological and biophysical properties of the Ca2+ channels in B-cells that are activated by exogenous application of ROS, and determine whether members of the transient receptor potential channel proteins (TRPC) family of cation channels are involved. Based on preliminary experiments, TRPC3 and TRPC5 form ROS-sensitive cation channels when expressed in HEK293 cells; (2) to determine the role of intracellular Ca2+ rise in ROS production in B lymphocytes and which membrane-bound NADPH oxidase is necessary for mitogen-induced ROS production in B-cells; (3) to test if the chronic increase of intracellular Ca2+ induced by PAH is maintained by a ROS-dependent mechanism and/or by membrane depolarization. Preliminary data shows that both PAH and ROS induce Ca2+ entry in B-cells. A strong relationship between ROS and Ca2+ signaling defects in PAH-induced immune failure is expected. This effort will contribute to both a better understanding of normal and pathological lymphocyte immunity as well as to the discovery of new therapies for diseases such as immunodeficiency and cancer. ? ? ? ?
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