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值在快速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的主要功能是调节碱性间隙pH变化的表现。这些pH值变化在100 ms内出现，并伴随同步神经活动、癫痫发作、扩散性抑制和脑缺血。这些pH变化的速度足以通过它们对NMDA受体的作用来调节突触传递。一种形式的质子交换不需要碳酸氢盐，并且由净质子流入细胞引起。ECA通过催化二氧化碳的水合作用来快速缓冲这种pH变化。第二种形式的碳酸氢盐通过GABA-A阴离子通道流出引起。ECA不缓冲这种pH变化。相反，它的作用是通过催化碳酸的脱水来产生碱转移。ECA的第三个作用是促进乳酸盐的运输。本实验室最近证明，表面碳酸酐酶对孤立的星形胶质细胞和神经元是必要的流入乳酸的单羧酸转运机制。因此，乳酸与H+的共转运显然需要表面CA以足够的速率提供质子。ECA在缓冲和产生碱性pH值变化的作用仍然模糊，由于pH微电极的时间分辨率差。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被用来执行ECA功能的第一个定量分析，在100毫秒的活性依赖性碱性瞬变上升。实验将确定ECA是否是唯一的和足够的手段间质pH调节在这一时期，并将阐明GABA能碱性位移的幅度，时间过程和ECA依赖性。通过利用乳酸转运的ECA依赖性，将鉴定和定量单个星形胶质细胞和神经元上的表面CA活性。推论实验将解决ECA在组织中的乳酸的运输和利用中的作用，使用大鼠海马脑片在模型中的血糖和缺氧。这些项目将阐明ECA如何调节间质pH值和促进乳酸转运。因此，这项研究将提供对氢离子作为脑功能调节剂的作用的见解，在正常情况下，以及癫痫，缺血和创伤后环境。

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.