This proposal is to study the mechanisms by which tetanus toxin (TT), one of the most potent microbial toxins known, inhibits secretion. Clinically, intoxication with this toxin causes a sometimes fatal spastic paralysis which results from the preferential inhibition of neurotransmitter secretion from inhibitory neurons. The mechanisms of action of TT are unknown and have been difficult to study because of the lack of an easily accessible, well characterized model system. The experiments proposed are the direct outgrowth of our recent discovery that TT inhibits the secretion of lysosomal enzymes from a model secretory cell, the human macrophage. After additional studies to further characterize the secretion inhibition by TT on these cells including the kinetics, reversibility, stimulus specificity and effects of tetanus toxin fragments on macrophage secretion, we propose studies to examine the mechanism of action of TT. Since a rise in the concentration of intracellular free calcium is critical to secretion from both neurons and macrophages and recent evidence indicates that TT interferes with calcium homeostasis in neurons, we will specifically examine the effects of TT on intracellular calcium ion transport in intact macrophages. For these studies we will utilize macrophages loaded with calcium sensitive fluorescent and luminescent probes and examine the changes of intracellular calcium concentration in control and TT-treated macrophages during secretion. We will also further investigate our recent discovery that microsomes from macrophages and lysosomes from human neutrophils contain an ATP-dependent calcium uptake pump whose activity is markedly stimulated by TT and its toxic B fragment. These studies may provide an intracellular target enzyme system for the mechanism of action of TT and also provide a biologically relevant tool to better understand the general process of stimulus secretion coupling.
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