INTERACTIONS BETWEEN H+ AND CA+ IN AXON

长期目标是更好地了解法规 细胞内游离钙（Ca 2+）i和游离质子（H+）i的浓度， 神经细胞中这两种离子浓度之间的相互依赖性。 具体的目的是研究，无论是在孤立的轴浆和完整的轴突 Myxicola巨大轴突的（Ca 2+）i调节机制在四个位点： (a)质膜，（B）细胞器，（c）细胞器，如 通过ATP水解螯合Ca的内质网，和（d）细胞 这些成分通过纯粹的物理化学过程结合Ca。 的 方法将是生理上区分钙结合位点的类别 通过研究琥珀酸依赖性结合（线粒体）， ATP依赖性结合和能量非依赖性结合。 的描述 在分离的轴质中，这些Ca缓冲位点将由三种类型组成 （a）轴浆的Ca含量将被确定为 与之平衡的游离钙的函数。 (b)同位素 分离的轴浆（保持在聚乙烯中）的Ca 40之间的自交换 管）和含有相同游离Ca的相邻溶液的Ca 45将被 测定了 已经迁移到轴质中的Ca 45的分布将是 通过冷冻样品，切片轴浆圆柱形塞， 在25微米厚的圆盘中，并测定放射性。 结果将 通过产生Ca扩散系数的方法分析， Ca结合反应的性质。 (c)Ca的净扩散进入 将用离子敏感微电极监测轴浆， 使用扩散反应方程分析的行为。 在完整的轴突中， 从注入的Ca负荷中恢复的动力学将被检查为 载荷相对于质膜的位置的函数。 将这些结果与分离轴浆的结果进行比较， 质膜在钙调节中的作用的量度。 换句 实验中，将研究pH对Na-Ca交换的影响。 而 如果pHi是变化的，则pCai将通过使用外源性调节剂保持恒定。 非pH依赖性Ca缓冲系统注入轴突。 的抑制 Na+依赖性Ca流出和Ca+依赖性Na流出的比率将是 动力学检查，以确定是否H+和 Ca ~（2+）或H ~+和Na ~+之间的竞争性、非竞争性或混合性。

Long-term objectives are to gain a better understanding of the regulation of both intracellular free calcium (Ca2+)i and free protons (H+)i and the interdependence between these two ion concentrations in nerve cells. Specific aims are to study, both in isolated axoplasm and in intact axons of the Myxicola giant axon, (Ca2+)i regulatory mechanisms at four loci: (a) the plasma membrane, (b) the mitochondrion, (c) organelles such as the endoplasmic reticulum that sequester Ca by ATP hydrolysis, and (d) the cell constituents that bind Ca by purely physio-chemical processes. The approach will be to physiologically distinguish classes of Ca binding sites in situ by studying succinate-dependent binding (mitochondria), ATP-dependent binding, and energy-independent binding. A description of these Ca buffering sites in isolated axoplasm will be made from three types of measurements: (a) The Ca content of the axoplasm will be determined as a function of the free Ca with which it is in equilibrium. (b) Isotopic self-exchange between Ca40 of isolated axoplasm (held in a polyethelene tube) and Ca45 of an adjacent solution containing the same free Ca will be measured. The profile of Ca45 that has migrated into the axoplasm will be determined by freezing the sample, slicing the cylindrical plug of axoplasm in 25 um thick disks, and assaying the radioactivity. Results will be analyzed by a method which yields the Ca diffusion coefficient and properties of the Ca binding reactions. (c) Net diffusion of Ca into axoplasm will be monitored with ion-sensitive microelectrodes and the behavior analyzed using the diffusion-reaction equation. In intact axons, the kinetics of recovery from injected Ca loads will be examined as a function of the location of the load with respect to the plasma membrane. Comparison of these results with results in isolated axoplasm will provide a measure for the role of the plasmalemma in Ca regulation. In other experiments, the effect of pHi on Na-Ca exchange will be studied. While pHi is varied, pCai will be held constant through the use of an exogenous pH-independent Ca buffering system injected into the axon. The inhibition of Na-o-dependent Ca efflux and Ca-o-dependent Na efflux by H+i will be examined kinetically to determine whether the interaction between H+ and Ca2+ or between H+ and Na+ is competitive, uncompetitive, or mixed.