THE ROLE OF CALCIUM IN THE CYTOTOXCICTY OF CHLOROPHENOLS

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The goal of this proposal is to determine the membrane mechanism of action of chlorophenols. In this project, the role of calcium in the cytotoxicity of chlorophenols in excitable tissues, will be investigated. Single muscle fibers from the giant barnacle, will be used as a model preparation for these studies. Specific objectives of the project are: 1. To determine if exposure of barnacle muscle fibers to chlorophenols induce an increase in intracellular calcium levels in a dose and time dependent manner and whether the effect can be blocked by calcium channel antagonists. 2. To determine if the induced calcium accumulation activates the calcium messenger system, e. calcium/calmodulin protein kinase II. 3. To determine if the activated calcium messenger system results in the phosphorylation of specific membrane proteins, e.g., Na/Ca exchanger. 4. To train students in the techniques of working with single muscle cells to investigate the membrane mechanism of action of cytotoxic agents. Objective 1 will be carried out by 45Ca2+ uptake and Fura-2 fluorescence studies in the absence and presence of calcium channel antagonists. Objective 2 and 3 will be carried out by phosphorylation studies in the absence and presence of calmodulin inhibitors. Objective 4 will be carried out by recruiting two students under the MBRS program for participation in hands on research. Results obtained from using this preparation, will help provide insight to the physiological parameters modulated, and the consequent responses that are initiated early in the presence of chlorophenols, which may lead eventually to cell damage and death, especially in the excitable tissues. This may have an impact on mitigation of toxicity of chlorophenols.