INTRACELLULAR H+ AND CA++ IN MYXICOLA AXON

长期目标是更好地了解法规 f细胞内游离钙[Ca2 +] i和游离质子[H +] i， 这两种离子浓度的相互依赖性， 这些离子的扩散，它们在细胞内信号传导中的作用， 细胞内不同位点间的扩散（尤其是钙） 影响细胞内的生理交流。 三个独立 但相关项目将研究（a）细胞质扩散 钙和肌醇（1，4，5）三磷酸盐，IP 3系数，（B） 药理学定义的细胞内钙的钙缓冲能力 外源性移动的Ca螯合剂对~（45）Ca 积累到细胞器，包括对分配的影响 45Ca之间的细胞器和这些细胞器的相互依赖性。 实现这些目标的方法如下： 扩散系数将使用以下模型系统来确定： 挤压的神经细胞质置于塑料毛细管中。 放射标记 3 [H]-IP3将被添加到轴质的末端，并且在定时的 在扩散期间，毛细血管和轴浆将被快速冷冻， 在切片机上切片，并将切片置于单独的闪烁器中， 用于计数的小瓶。 在其他实验中，Ca或IP3将被添加到一个 轴浆的末端，其中"迷你" Ca电极被放置在指定的位置 监测[Ca2 +]。 (b)光不稳定的"笼状" Ca螯合剂，例如 因为Nitr-5、Nitr-7或DM-Nitrophen将与提取的 细胞质并暴露于UV光，同时监测游离钙 "迷你"钙电极。 内源性钙缓冲能力将是 在不同的Ca、pH和添加后条件下测定，或 除去移动的Ca缓冲液。 (c)使用45Ca和分离的 细胞质从Myxicola巨大轴突或使用透化大鼠脑 在突触体中，将测量45Ca积累。 3 [H]-菊粉或 3 [H]-甘露醇将用作表面保留的45 Ca的标记物。 细胞内钙的储存，如内质网或 线粒体将用抑制剂和代谢酶来操纵。 印刷受体. 具体而言，移动的Ca螯合剂，如 EGTA或BAPTA对45Ca摄取速率和分配至 将评估钙螯合细胞器，并评估 线粒体操纵非线粒体隔离检查。

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.