Dynamics of pH Regulation in the Brain

描述（由申请人提供）：现在有强有力的证据表明，脑细胞外pH值在一个快速的时间尺度上由一种或多种形式的细胞外碳酸酐酶（ECA）控制。ECA的一个主要功能是调节碱性间质pH值变化的表现。这些pH值变化在100ms内出现，并伴有同步神经活动、癫痫发作、扩张性抑郁和脑缺血。这些pH值变化的速度足以通过它们对NMDA受体的影响来调节突触传递。一种形式的碱中毒不需要碳酸氢盐，而是由进入细胞的净质子通量引起的。ECA的作用是通过催化二氧化碳的水合作用来快速缓冲pH值的变化。第二种形式的碱中毒是由碳酸氢盐通过GABA-A阴离子通道流出引起的。ECA不能缓冲pH值的变化。相反，它通过催化碳酸脱水来产生碱性转变。ECA的第三个作用是促进乳酸的运输。本实验室最近证明，分离的星形胶质细胞和神经元上的表面碳酸酐酶是通过单羧酸运输机制流入乳酸所必需的。因此，乳酸盐与H+的共转运显然需要表面CA以足够的速率提供质子。由于pH微电极的时间分辨率较差，ECA在缓冲和产生碱性pH变化中的作用仍然不清楚。使用荧光素-葡聚糖探针，我们将分辨率提高了两个数量级。这种光学记录技术将用于在活性依赖碱性瞬态上升100毫秒期间执行ECA功能的第一次定量分析。实验将确定ECA是否是这一时期间质pH调节的唯一和充分手段，并将阐明gaba能碱性转移的幅度、时间过程和ECA依赖性。通过利用乳酸转运对ECA的依赖性，可以识别和量化单个星形胶质细胞和神经元的表面CA活性。后续实验将利用血糖和缺氧模型的大鼠海马切片，探讨ECA在组织中乳酸的运输和利用中的作用。这些项目将阐明ECA如何调节间质pH和促进乳酸运输。因此，这项研究将深入了解氢离子在正常、癫痫、缺血性和创伤后环境中作为脑功能调节剂的作用。

DESCRIPTION (provided by applicant): There is now strong evidence that brain extracellular pH is governed on a fast time scale by one or more forms of extracellular carbonic anhydrase (ECA). A principal function of ECA is to regulate the manifestation of alkaline interstitial pH shifts. These pH changes arise within 100 ms, and accompany synchronous neural activity, seizure, spreading depression and brain ischemia. The speed of these pH changes is sufficient to modulate synaptic transmission, through their effect on NMDA receptors. One form of alkalosis does not require bicarbonate, and arises from a net proton flux into cells. ECA functions to rapidly buffer this pH change by catalyzing the hydration of carbon dioxide. A second form of alkalosis arises from the efflux of bicarbonate across GABA-A anion channels. ECA does not buffer this pH change. Rather it acts to generate the alkaline shift by catalyzing the dehydration of carbonic acid. A third role of ECA is to facilitate the transport of lactate. This laboratory has recently demonstrated that surface carbonic anhydrase on isolated astrocytes and neurons is necessary for the influx of lactic acid by the monocarboxylate transport mechanism. Thus, the cotransport of lactate with H+ apparently requires surface CA to supply protons at an adequate rate. The role of ECA in the buffering and generation of alkaline pH shifts has remained obscure, owing to the poor temporal resolution of pH microelectrodes. Using a fluorescein-dextran probe, we have improved the resolution by two orders of magnitude. This optical recording technique will be used to perform the first quantitative analysis of ECA function during the 100 ms rise of an activity-dependent alkaline transient. Experiments will determine whether ECA is the sole and sufficient means of interstitial pH regulation in this period, and will elucidate the magnitude, time course and ECA-dependence of GABAergic alkaline shifts. By capitalizing on the ECA dependence of lactate transport, the surface CA activity on individual astrocytes and neurons will be identified and quantified. Corollary experiments will address the role of ECA in the transport and utilization of lactate in tissue, using rat hippocampal slices in models of aglycemia and hypoxia. These projects will elucidate how ECA functions to regulate interstitial pH and facilitate lactate transport. This research will thereby provide insights into the role of the hydrogen ion as a modulator of brain function, in the normal, as well as the epileptic, ischemic and post-traumatic setting.