The mode of action of gamma-aminobutyric acid (GABA) on intracellular pH (pHi) and surface pH (pHs) was studied in crayfish muscle fibres using H(+)-selective microelectrodes. The extracellular HCO3- concentration was varied (0-30 mM) at constant pH (7.4). 2. GABA (5 x 10(-6)-10(-3) M) produced a reversible fall in pHi which showed a dependence on the concentrations of both GABA and HCO3-. The fall in pHi was associated with a transient increase in pHs and it was inhibited by a K(+)-induced depolarization. 3. In the presence of 30 mM-HCO3-, a near-saturating concentration of GABA (0.5 mM) produced a mean fall in pHi of 0.43 units. This change in pHi accounted for about two-thirds of the GABA-induced decrease (from -66 to -29 mV) in the sarcolemmal H+ driving force, while the rest was due to the simultaneous depolarization. 4. The apparent net efflux of HCO3- (JHCO3e) produced by a given concentration of GABA was estimated on the basis of the instantaneous rate of change of pHi. In the presence of 30 mM-HCO3-, JHCO3e following exposure to 0.5 mM-GABA had a mean value of 8.0 mmol l-1 min-1. Under steady-state conditions (at plateau acidosis), the intracellular acid load produced by 0.5 mM-GABA was about 25% of that seen at the onset of the application. 5. The GABA-induced HCO3- permeability, calculated on the basis of the flux data, showed a concentration dependence similar to that of the GABA-activated conductance described in previous work. 6. The GABA-induced increase in pHs was immediately blocked by both a membrane-permeant inhibitor of carbonic anhydrase (acetazolamide, 10(-6) M) and by a poorly permeant inhibitor (benzolamide, 10(-6) M). 7. Application of acetazolamide (10(-4) M) for 5 min or more produced a decrease of up to 60% in the maximum rate of fall of pHi at GABA concentrations higher than 20 microM. 8. The recovery of the GABA-induced acidosis was associated with a fall in pHs. The recovery was completely blocked in solutions devoid of Na+ or of Cl-, as well as by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, 10(-5) M). This indicates that the maintenance of a non-equilibrium H+ gradient at plateau acidosis and the recovery of pHi are attributable to Na(+)-dependent Cl(-)-HCO3- exchange. 9. We conclude that the effects of GABA on pHi and pHs are due to electrodiffusion of HCO3- across postsynaptic anion channels.(ABSTRACT TRUNCATED AT 400 WORDS)
摘要
运用氢离子选择性微电极，在小龙虾肌肉纤维中研究了γ-氨基丁酸（GABA）对细胞内pH值（pHi）和表面pH值（pHs）的作用方式。在恒定pH值（7.4）条件下，改变细胞外碳酸氢根离子（HCO3-）浓度（0 - 30 mM）。2. GABA（5×10⁻⁶ - 10⁻³ M）可使pHi出现可逆性下降，这显示出对GABA和HCO3-浓度均有依赖性。pHi的下降与pHs的短暂升高相关，且受到钾离子诱导的去极化作用抑制。3. 在存在30 mM - HCO3-时，接近饱和浓度的GABA（0.5 mM）可使pHi平均下降0.43个单位。pHi的这种变化约占GABA诱导的肌膜氢离子驱动力下降（从 - 66 mV降至 - 29 mV）的三分之二，其余则归因于同时发生的去极化作用。4. 根据pHi的瞬时变化速率估算给定浓度GABA产生的表观碳酸氢根离子净外流（JHCO3e）。在存在30 mM - HCO3-时，暴露于0.5 mM - GABA后JHCO3e的平均值为8.0 mmol·l⁻¹·min⁻¹。在稳态条件下（平台期酸中毒时），0.5 mM - GABA产生的细胞内酸负荷约为施加初期的25%。5. 根据通量数据计算得出的GABA诱导的HCO3-通透性，显示出与先前工作中描述的GABA激活电导相似的浓度依赖性。6. 碳酸酐酶的膜通透性抑制剂（乙酰唑胺，10⁻⁶ M）和低通透性抑制剂（苯并酰胺，10⁻⁶ M）均可立即阻断GABA诱导的pHs升高。7. 施加乙酰唑胺（10⁻⁴ M）5分钟或更长时间，在GABA浓度高于20 μM时，可使pHi最大下降速率降低多达60%。8. GABA诱导的酸中毒的恢复与pHs下降相关。在无钠离子或无氯离子的溶液中，以及在4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS，10⁻⁵ M）存在时，恢复完全受阻。这表明在平台期酸中毒时维持非平衡氢离子梯度以及pHi的恢复归因于钠离子依赖性氯离子-碳酸氢根离子交换。9. 我们得出结论，GABA对pHi和pHs的作用是由于HCO3-通过突触后阴离子通道进行电扩散所致。（摘要截取自400字）
