INTRACELLULAR H+ AND CA++ IN MYXICOLA AXON

长期目标是更好地理解该法规。 F细胞内游离钙，[Ca~(2+)]i和游离质子，[H]~i， 这两种离子浓度的相互依赖，细胞内 这些离子的扩散，它们在细胞内信号传递中的作用，以及 细胞内不同位置之间的扩散(尤其是钙的扩散) 影响细胞内的生理通讯。三个独立的 但相关项目将研究(A)细胞质扩散 钙和肌醇(1，4，5)三磷酸，IP3的系数，(B) 药物确定的细胞内钙缓冲能力的研究 (C)外源性流动钙络合剂对45Ca的影响 在细胞器中的积累，包括对分配的影响 ~(45)Ca在细胞器间的相互依赖关系。 实现这些目标的方法如下：(A) 扩散系数将使用以下模型系统来确定 将挤出的神经细胞质放置在塑料毛细管中。无线电标记的 3[H]-IP3将被添加到轴浆的末端，并在定时的 扩散期，毛细血管和轴浆会被快速冰冻， 切片放在切片机上，切片放入单独的闪光片中 用来计数的瓶子。在其他实验中，钙或IP3将加到1上 轴浆末端，“迷你”钙电极放置在指定位置 要监测的头寸[钙]。(B)耐光性“笼式”钙络合剂 作为Nitr-5、Nitr-7或DM-Nitrophen将与萃取物混合 细胞质，并暴露在紫外线下，同时监测游离钙 “迷你”钙电极。内源性钙缓冲能力将是 在不同的钙、pH条件下及添加或添加后测定 移动钙缓冲器的移除。(C)使用45Ca和分离的样品 粘液巨轴突的细胞质或用通透性的大鼠脑 突触体内，将测量~(45)Ca的积累。3[H[-菊粉或 3[H]-甘露醇可作为表面滞留45Ca的标志物。 细胞内钙的储存，如内质网或 线粒体将被用抑制剂和代谢来操纵 底物。具体地说，流动钙络合剂的影响，如 EGTA或BAPTA对~(45)Ca摄取速率和分配的影响 将对钙隔离细胞器进行评估，并对 观察线粒体对非线粒体隔离的操纵作用。

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.