Long-term objectives are to gain a better understanding of the regulation f intracellular free calcium, [Ca2+]i, and free protons, [H+]i, the interdependence of these two ion concentrations, the intracellular diffusion of these ions, their role in intracellular signaling, and of how diffusion (especially of calcium) among different intracellular loci influences physiological communication within the cell. Three separate but related projects will examine (a) the cytoplasmic diffusion coefficients of Ca and inositol (1,4,5) trisphosphate, IP3, (b) the calcium buffering capacity of pharmacologically-defined intracellular Ca stores, and (c) the influence of exogenous mobile Ca chelators on 45Ca accumulation into organelles, including effect on the partitioning of 45Ca among organelles and the interdependence of these organelles. Methods to be used for accomplishing these goals are as follows: (a) The diffusion coefficients will be determined using a model system of extruded neural cytoplasm placed in a plastic capillary. Radiolabeled 3[H]-IP3 will be added to the end of the axoplasm and, after a timed period of diffusion, the capillary and axoplasm will be flash frozen, sliced on a microtome, and slices placed into individual scintillation vials for counting. In other experiments, Ca or IP3 will be added to one end of the axoplasm in which """"""""mini"""""""" Ca electrodes are placed at specified positions to monitor [Ca2+]. (b) Photolabile """"""""caged"""""""" Ca chelators such as a Nitr-5, Nitr-7, or DM-Nitrophen will be mixed with extracted cytoplasm and exposed to UV light while monitoring free calcium with """"""""mini"""""""" Ca electrodes. The endogenous Ca buffering capacity will be determined under different conditions of Ca, pH and after addition or removal of mobile Ca buffers. (c) Using 45Ca and isolated samples of cytoplasm from the Myxicola giant axon or using permeabilized rat brain synaptosomes, the 45Ca accumulation will be measured. 3[H[-inulin or 3[H]-mannitol will be used as a marker for superficially retained 45Ca. Stores of intracellular calcium such as endoplasmic reticulum or mitochondria will be manipulated with inhibitors and with metabolic substrates. Specifically, the influence of mobile Ca chelators, such as EGTA or BAPTA, on the rate of uptake and the partitioning of 45Ca into Ca-sequestering organelles will be evaluated, and the influence of mitochondrial manipulation on nonmitochondrial sequestration examined.
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