Mayer S E, Sanders-Bush E
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600.
J Neurochem. 1993 Apr;60(4):1308-16. doi: 10.1111/j.1471-4159.1993.tb03291.x.
The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na(+)-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO3- plus 95% O2/5% CO2. Intracellular pH (pHi) was determined from the steady-state distribution of [14C]benzoate. Recovery after acidification with NH4Cl was rapid (t1/2 = 5 min) and was dependent on external Na+ (EC50 = 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na+/H+ antiport, blocked 80% of recovery when [Na+] was 5 mM with IC50 values of 100 nM. However, neither drug blocked recovery in normal [Na+]. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl-/HCO3- antiports, blocked recovery of pHi in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na+ channel, nor with dimethylbenzamil, an inhibitor of Na+/Ca2+ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of 22Na+ uptake was very rapid (227 pmol/micrograms of DNA/min at pH 6.2), was dependent on external [Na+] with an EC50 value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of 22Na+ uptake by hexamethyleneamiloride was 60% with an IC50 value of 76 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
研究了兔脉络丛上皮（CPE）原代培养物中酸负荷恢复的机制，重点关注钠依赖性反向转运体。细胞在分别用HEPES或HCO₃⁻加95% O₂/5% CO₂缓冲至pH 7.38的盐溶液中孵育。通过[¹⁴C]苯甲酸的稳态分布来测定细胞内pH（pHi）。用氯化铵酸化后的恢复迅速（t₁/₂ = 5分钟），且依赖于细胞外钠离子（EC₅₀ = 12 mM）。钠/氢反向转运体的强效抑制剂六甲胺基氨氯吡脒和乙基异丙基氨氯吡脒，当[Na⁺]为5 mM时，可阻断80%的恢复，IC₅₀值为100 nM。然而，在正常[Na⁺]条件下，这两种药物均未阻断恢复。4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS），一种氯/碳酸氢根反向转运体的抑制剂，在存在碳酸氢根时以剂量相关方式阻断pHi的恢复，但在存在HEPES时则无此作用。对钠通道具有高亲和力的氨氯吡脒类似物苯甲酰胺以及钠/钙交换抑制剂二甲基苯甲酰胺均未产生抑制作用。碳酸酐酶抑制剂乙酰唑胺也未改变酸化后的恢复。在用尼日利亚菌素和氯化钾进行pH钳制的CPE中，²²Na⁺摄取的初始速率非常快（在pH 6.2时为227 pmol/μg DNA/分钟），依赖于细胞外[Na⁺]，EC₅₀值为8 mM，且与培养基的pH呈负相关。六甲胺基氨氯吡脒对²²Na⁺摄取的最大抑制率为60%，IC₅₀值为
