Ko Y P, Lang H J, Loh S H, Chu K C, Wu M L
Department of Physiology, College of Medicine, National Taiwan University, Taipei, ROC.
Chin J Physiol. 1999 Dec 31;42(4):237-48.
It is still uncertain whether the Na+-dependent Cl--HCO3- exchanger (NCBE) is expressed in mammalian astrocytes. Using fluorescent indicators to monitor the intracellular pH (pHi) and intracellular Na+ or Cl- levels, the NCBE in cultured rat cerebellar astrocytes was examined in detail. In nominally bicarbonate-free (Hepes-buffered) medium, a marked pHi recovery from internal acid load was seen which could be blocked completely by 30 microM HOE 694, a specific Na+-H+ exchanger isoform 1(NHE-1) inhibitor, at a pHi above 6.9. These conditions were therefore used to block NHE activity in CO2/HCO3-buffered media when the NCBE was being studied at pHi above 6.9. After internal acid loading in completely Cl--free bicarbonate-buffered medium (containing HOE 694), the rates of pHi recovery and transient Na+ influx were considerably slowed, and the Cl--dependent acid extrusion was both Na+- and 4,4-diisothiocyano-stilbene-disulphonic acid (DIDS)-sensitive. Moreover, a HCO3-dependent Cl- efflux during internal acid injection was seen. These results suggest that the NCBE is present in astrocytes. Following repetitive internal acid loading by addition of 5% CO2 to internal Cl- depleted cells, a similar rate of pHi recovery was consistently seen, suggesting Cl--independent pHi regulation also occurred in astrocytes. Moreover, this pHi recovery was completely blocked in the absence of sodium or on addition of DIDS, confirming that the Na+-HCO3 cotransporter (NBC) is present. Thus, the present study provides evidence that both the NCBE and NBC play important roles in acid extrusion in cultured mammalian astrocytes.
尚不确定钠依赖型氯-碳酸氢根交换体（NCBE）是否在哺乳动物星形胶质细胞中表达。利用荧光指示剂监测细胞内pH值（pHi）以及细胞内钠离子或氯离子水平，对培养的大鼠小脑星形胶质细胞中的NCBE进行了详细研究。在名义上无碳酸氢根（Hepes缓冲）的培养基中，观察到从内部酸负荷中出现明显的pHi恢复，在pHi高于6.9时，这种恢复可被30微摩尔的HOE 694完全阻断，HOE 694是一种特异性的钠-氢交换体亚型1（NHE-1）抑制剂。因此，当在pHi高于6.9的条件下研究NCBE时，这些条件被用于在二氧化碳/碳酸氢根缓冲培养基中阻断NHE活性。在完全无氯的碳酸氢根缓冲培养基（含有HOE 694）中进行内部酸负荷后，pHi恢复速率和短暂的钠离子内流显著减慢，且依赖于氯离子的酸排出对钠离子和4,4-二异硫氰基芪-二磺酸（DIDS）敏感。此外，在内部酸注入期间观察到了依赖于碳酸氢根的氯离子外流。这些结果表明NCBE存在于星形胶质细胞中。通过向内部氯离子耗尽的细胞中添加5%二氧化碳进行重复的内部酸负荷后，始终观察到相似的pHi恢复速率，这表明星形胶质细胞中也发生了不依赖于氯离子的pHi调节。此外，在无钠或添加DIDS的情况下，这种pHi恢复被完全阻断，证实了钠-碳酸氢根共转运体（NBC）的存在。因此，本研究提供了证据，表明NCBE和NBC在培养的哺乳动物星形胶质细胞的酸排出中都发挥着重要作用。
