SODIUM REGULATION OF NEUROHORMONE SECRETION

拟议研究的长期目标是了解 钠调节神经激素/神经递质的机制 在静息条件下和响应刺激释放。 的 胞浆钠离子升高对促进神经递质的重要性 破伤风刺激后的分泌是很好的。 电流 模型表明，Na+的易化作用的结果， 改变Ca 2+调节。 然而，最近我们发现， 细胞内Na+（[Na+]i）调节静息加压素的速率 (AVP)离体神经垂体神经末梢在Ca ~（2+）作用下的分泌 夹紧条件。 这导致了一个非传统的假设，即Na+ 它本身可以调节分泌，Na+ 可以调节Ca 2+诱导的（即刺激的）分泌。 拟议 实验将利用神经末梢的 下丘脑-神经垂体系统，具有独特的解剖结构 允许解析分泌性肿瘤的分子事件的优点 在神经末梢的过程中，要比任何 其他神经末梢 具体目标是：1）利用时间分辨 膜电容（Cm）测量，在全细胞膜片钳下 单个神经末梢，以确定Na+的需求， 胞吐作用 将通过荧光测量细胞内[Na+] 谱 这种方法将提供一个定量评价， [Na+]i对胞吐的速率和程度的影响，并允许确定 胞吐释放位点所需的[Na+]i。2)使用离子 神经末梢群体的替代方案， AVP释放的放射免疫测定，我们将确定在何种程度上， Na+调节Ca 2+诱导的分泌。3)利用完整的神经叶 准备，我们将调查的生理模式的影响， 动作电位对[Na+]i变化幅度和时间过程的影响。 然后我们将测量单个神经末梢的分泌活动 通过使用Cm测量的类似变化引起的。 我们将尝试 使用荧光光谱法定量内源性缓冲能力 和Na+电流的测量。4)我们将试图确定 Na+诱导的AVP分泌利用独特的分泌机制或共享 与Ca 2+诱导的分泌机制相同。 配体调节 受体相互作用或通过针对特定颗粒蛋白的抗体 Na+诱导的分泌方式类似于Ca 2+诱导的分泌 将被确定。 监管的相似性可能表明 机制 所提出的实验对于描述正常的 神经末梢的生理学，因为它们与学习，记忆和 神经系统疾病和了解基本细胞 机制等

The long range goal of the proposed research is to understand the mechanism(s) of sodium regulation of neurohormone/neurotransmitter release under resting conditions and in response to stimulation. The importance of elevated cytosolic Na+ to facilitation of neurotransmitter secretion following tetanic stimulation is well established. Current models suggest that the facilitatory action of Na+ results from alteration of Ca2+ regulation. Recently, however, we showed that intracellular Na+ ([Na+]i) regulates the rate of resting vasopressin (AVP) secretion from isolated neurohypophysial nerve endings under Ca2+ clamp conditions. This has led to the unconventional hypothesis that Na+ itself can regulate secretion and to the remarkable possibility that Na+ may modulate Ca2+-induced (i.e. stimulated) secretion. The proposed experiments will utilize nerve endings of the hypothalamo-neurohypophysial system, which posses unique anatomical advantages allowing resolution of the molecular events of the secretory process in nerve endings, to be studied in greater detail than in any other nerve endings. The specific aims are: 1) to utilize time-resolved membrane capacitance (Cm) measurements, under whole cell patch clamp on individual nerve endings to determine the Na+ requirements for exocytosis. Intracellular [Na+] will be measured by fluorescence spectroscopy. This approach will provide a quantitative evaluation of [Na+]i on the rate and extent of exocytosis and allow determination of the [Na+]i required at an exocytotic release site. 2) Using ionic substitution protocols on populations of nerve endings and radioimmunoassay for AVP release, we will determine the extent to which Na+ modulates Ca2+-induced secretion. 3) Using the intact neural lobe preparation we will investigate the effect of physiologic patterning of action potentials on the amplitude and time course of changes in [Na+]i. We will then measure, on single nerve endings, the secretory activity induced by similar changes using Cm measurements. We will attempt to quantitate the endogenous buffer capacity using fluorescence spectroscopy and measurements of Na+ currents. 4) We will attempt to determine if Na+-induced AVP secretion utilizes unique secretory mechanisms or shares mechanisms in common with Ca2+-induced secretion. Regulation by ligand receptor interactions or by antibodies against specific granule proteins of Na+-induced secretion in a manner similar to Ca2+ induced secretion will be determined. Similarity of regulation may suggest commonality of mechanism. The proposed experiments are crucial to describe the normal physiology of nerve endings both as they relate to learning, memory and disease of the nervous system and in understanding basic cellular mechanisms.