Tong C K, Brion L P, Suarez C, Chesler M
Department of Physiology and Neuroscience, New York University School of Medicine, New York, New York 10016, USA.
J Neurosci. 2000 Nov 15;20(22):8247-53. doi: 10.1523/JNEUROSCI.20-22-08247.2000.
We tested the hypothesis that extracellular membrane-bound carbonic anhydrase (CA) type IV is responsible for the regulation of interstitial pH (pH(o)) transients in brain. Rat hippocampal slices were incubated in phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the link of CA IV to the external face of plasma membranes. Then evoked alkaline pH(o) shifts were studied in a recording chamber, using pH microelectrodes. Incubation fluid was saved for later analysis. The ability to buffer a rapid alkaline load was reduced markedly in PI-PLC-treated tissue as compared with adjacent, paired control slices. The effect of benzolamide (a poorly permeant CA inhibitor) on evoked pH(o) shifts was diminished greatly in the PI-PLC-treated tissue, consistent with the washout of interstitial CA. Treatment of the incubation fluid with SDS abolished nearly all of the CA activity in fluid from controls, whereas an SDS-insensitive component remained in the fluid from PI-PLC-treated slices. These data suggested that CA type II (which is blocked by SDS) leaked from injured glial cells in both slice preparations, whereas CA type IV (which is insensitive to SDS) was liberated selectively into the fluid from PI-PLC-treated tissue. Western blot analysis was consistent with this interpretation, demonstrating a predominance of CA IV in the incubation fluid from PI-PLC-treated tissue and variable amounts of CA II in fluid from PI-PLC-treated and control slices. These results demonstrate that interstitial CA activity brain is attributable principally to membrane-bound CA IV.
细胞外膜结合型碳酸酐酶IV负责调节脑间质pH值（pH(o)）的瞬变。将大鼠海马切片置于磷脂酰肌醇特异性磷脂酶C（PI-PLC）中孵育，该酶可切断碳酸酐酶IV与质膜外表面的连接。然后，使用pH微电极在记录室中研究诱发的碱性pH(o)变化。收集孵育液以备后续分析。与相邻的配对对照切片相比，PI-PLC处理的组织缓冲快速碱性负荷的能力明显降低。在PI-PLC处理的组织中，苯磺酰胺（一种渗透性差的碳酸酐酶抑制剂）对诱发的pH(o)变化的作用大大减弱，这与间质碳酸酐酶的洗脱一致。用SDS处理孵育液可消除对照液中几乎所有的碳酸酐酶活性，而PI-PLC处理切片的孵育液中仍保留对SDS不敏感的成分。这些数据表明，II型碳酸酐酶（被SDS阻断）在两种切片制备中均从受损的神经胶质细胞中泄漏，而IV型碳酸酐酶（对SDS不敏感）则选择性地释放到PI-PLC处理组织的孵育液中。蛋白质印迹分析与该解释一致，表明PI-PLC处理组织的孵育液中以IV型碳酸酐酶为主，而PI-PLC处理切片和对照切片的孵育液中II型碳酸酐酶含量各不相同。这些结果表明，脑间质碳酸酐酶活性主要归因于膜结合型碳酸酐酶IV。
