Sheldon Claire, Church John
Department of Anatomy and Physiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Neurophysiol. 2002 May;87(5):2209-24. doi: 10.1152/jn.2002.87.5.2209.
The effects of anoxia on intracellular pH (pH(i)) were examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H(+)-sensitive fluorophore, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. During perfusion with HCO/CO(2)- or HEPES-buffered media (pH 7.35) at 37 degrees C, 5- or 10-min anoxic insults were typified by an intracellular acidification on the induction of anoxia, a subsequent rise in pH(i) in the continued absence of O(2), and a further internal alkalinization on the return to normoxia. The steady-state pH(i) changes were not consequent on changes in Ca(2+) and, examined in the presence of HCO, were not significantly affected by (DIDS). In the absence of HCO, the magnitude of the postanoxic alkalinization was attenuated when external Na(+) was reduced by substitution with N-methyl-D-glucamine (NMDG(+)), but not Li(+), suggesting that increased Na(+)/H(+) exchange activity contributes to this phase of the pH(i) response. In contrast, 100-500 microM Zn(2+), a known blocker of H(+)-conductive pathways, reduced the magnitudes of the internal alkalinizations that occurred both during and following anoxia. The effects of NMDG(+)-substituted medium and Zn(2+) to reduce the increase in pH(i) that occurred after anoxia were additive. Consistent with the steady-state pH(i) changes, rates of pH(i) recovery from internal acid loads imposed immediately after anoxia were increased, and the application of Zn(2+) and/or perfusion with NMDG(+)-substituted medium slowed pH(i) recovery. Reducing extracellular pH from 7.35 to 6.60, or reducing ambient temperature from 37 degrees C to room temperature, also attenuated the increases in steady-state pH(i) observed during and after anoxia and reduced rates of pH(i) recovery from acid loads imposed in the immediate postanoxic period. Finally, inhibition of the cAMP/protein kinase A second-messenger system reduced the magnitude of the rise in pH(i) after anoxia in a manner that was dependent on external Na(+); conversely, activation of the system with isoproterenol increased the postanoxic alkalinization, an effect that was attenuated by pretreatment with propranolol, Rp-cAMPS, or when NMDG(+) (but not Li(+)) was employed as an external Na(+) substitute. The results suggest that a Zn(2+)-sensitive acid efflux mechanism, possibly a H(+)-conductive pathway activated by membrane depolarization, contributes to the internal alkalinization observed during anoxia in adult rat CA1 neurons. The rise in pH(i) after anoxia reflects acid extrusion via the H(+)-conductive pathway and also Na(+)/H(+) exchange, activation of the latter being mediated, at least in part, through a cAMP-dependent signaling pathway.
使用对H⁺敏感的荧光团2',7'-双-(2-羧乙基)-5-(及-6)-羧基荧光素加载急性分离的成年大鼠海马CA1神经元，研究缺氧对细胞内pH（pH(i)）的影响。在37℃下用HCO₃⁻/CO₂或HEPES缓冲液（pH 7.35）灌注期间，5或10分钟的缺氧损伤表现为缺氧诱导时细胞内酸化、持续缺氧时pH(i)随后升高以及恢复正常氧合时进一步的细胞内碱化。稳态pH(i)变化并非由Ca²⁺变化引起，且在HCO₃⁻存在下检测时，不受（二异丙基氟磷酸）（DIDS）显著影响。在无HCO₃⁻时，用N-甲基-D-葡萄糖胺（NMDG⁺）替代降低外部Na⁺后，缺氧后碱化的幅度减弱，但用Li⁺替代则不然，这表明增加的Na⁺/H⁺交换活性促成了pH(i)反应的这一阶段。相反，100 - 500 μM Zn²⁺（一种已知的H⁺传导途径阻滞剂）降低了缺氧期间及之后发生的细胞内碱化幅度。NMDG⁺替代培养基和Zn²⁺降低缺氧后pH(i)升高的作用是相加的。与稳态pH(i)变化一致，缺氧后立即施加的细胞内酸负荷的pH(i)恢复速率增加，而应用Zn²⁺和/或用NMDG⁺替代培养基灌注会减缓pH(i)恢复。将细胞外pH从7.35降至6.60，或将环境温度从37℃降至室温，也减弱了缺氧期间及之后观察到的稳态pH(i)升高，并降低了缺氧后立即施加酸负荷时的pH(i)恢复速率。最后，抑制cAMP/蛋白激酶A第二信使系统以依赖外部Na⁺的方式降低了缺氧后pH(i)升高的幅度；相反，用异丙肾上腺素激活该系统增加了缺氧后碱化，普萘洛尔、Rp-cAMPS预处理或当使用NMDG⁺（而非Li⁺）作为外部Na⁺替代物时，这种作用会减弱。结果表明，一种对Zn²⁺敏感的酸外排机制，可能是由膜去极化激活的H⁺传导途径，促成了成年大鼠CA1神经元缺氧期间观察到的细胞内碱化。缺氧后pH(i)升高反映了通过H⁺传导途径以及Na⁺/H⁺交换进行的酸外排，后者的激活至少部分通过cAMP依赖性信号通路介导。
