Poulsen J H, Machen T E
Department of Molecular and Cell Biology, 231 LSA, University of California, Berkeley, CA 94720-3200, USA.
Pflugers Arch. 1996 Jul;432(3):546-54. doi: 10.1007/s004240050168.
Regulation of intracellular pH (pHi) was studied in cultured bovine tracheal epithelial cells using microspectrofluorimetry of the fluorescent indicator 2',7'-biscarboxyethyl- 5(6)-carboxyfluorescein (BCECF). The cells, which were grown on coverslips and superfused in a chamber on the stage of a microscope, were acidified by NH4Cl-prepulses, and pHi recovery was measured (in DeltapH/min) at approximately pHi 6.7. In HCO3-free solutions the recovery rate was 0.14 pH/min, and addition of amiloride or Na-free solution reduced this rate to 0.02-0.03 pH/min. In HCO3/CO2-buffered Ringer's, the rate of recovery was 0.32 pH/min, and amiloride or Na-free reduced the rate to 0.08-0.10 pH/min. This residual Na-independent and HCO3-dependent pHi recovery was studied by using inhibitors of HCO3 and H transporters. Bafilomycin (inhibits H-ATPases) at 100 nM did not significantly affect pHi recovery, while 100 microM SCH28080 (inhibits H,K-ATPase) had a variable inhibitory effect (25-75%), indicating that a gastric-like H, K-ATPase, but not electrogenic H pump, may contribute in a minor way to the recovery from acidification. Cl-free solution and 500 microM H2DIDS (dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, blocks anion exchange and the outwardly rectifying Cl channel, ORCC), both blocked apparent anion exchange activity, but had no effect on the recovery; 100 microM DNDS (4-4''-dinitro-2-2'-stilbenedisulfonate blocks the ORCC but not the cystic fibrosis transmembrane conductance regulator, CFTR) had no effect on pHi recovery; DPC (diphenylamine carboxylate, blocks the CFTR and the ORCC) caused a complete and reversible inhibition of the recovery. When [K] was increased ten fold to depolarize the cell's membrane potential, the magnitude of the pHi recovery (though not the rate) was enhanced. Thus, the HCO3-dependent, Na- and Cl-independent, DPC-blockable pHi recovery may be largely due to an influx of HCO3 via CFTR Cl channels. Under physiological conditions, when the electrochemical gradient for HCO3 is likely to be outwardly rather than inwardly directed, the CFTR (or another HCO3-permeable channel) may mediate HCO3 secretion and contribute to regulation of pH of the periciliary fluid.
利用荧光指示剂2',7'-双羧乙基-5(6)-羧基荧光素（BCECF）的显微分光荧光测定法，研究了培养的牛气管上皮细胞内pH值（pHi）的调节。细胞生长在盖玻片上，并在显微镜载物台上的小室中进行灌流，通过氯化铵预脉冲使其酸化，并在大约pHi 6.7时测量pHi恢复情况（以ΔpH/分钟计）。在无HCO3的溶液中，恢复速率为0.14 pH/分钟，加入氨氯吡咪或无钠溶液可将该速率降至0.02 - 0.03 pH/分钟。在HCO3/CO2缓冲的林格氏液中，恢复速率为0.32 pH/分钟，氨氯吡咪或无钠溶液可将速率降至0.08 - 0.10 pH/分钟。通过使用HCO3和H转运体抑制剂研究了这种残余的不依赖钠和依赖HCO3的pHi恢复情况。100 nM的巴弗洛霉素（抑制H - ATP酶）对pHi恢复没有显著影响，而100 μM的SCH28080（抑制H,K - ATP酶）具有可变的抑制作用（25 - 75%），表明类似胃的H,K - ATP酶而非电生性H泵可能在酸化恢复中起较小作用。无氯溶液和500 μM的H2DIDS（二氢 - 4,4'-二异硫氰酸根合芪 - 2,2'-二磺酸，阻断阴离子交换和外向整流Cl通道，ORCC）均阻断了明显的阴离子交换活性，但对恢复没有影响；100 μM的DNDS（4 - 4'' - 二硝基 - 2 - 2'-芪二磺酸阻断ORCC但不阻断囊性纤维化跨膜电导调节因子，CFTR）对pHi恢复没有影响；DPC（二苯胺羧酸盐，阻断CFTR和ORCC）导致恢复完全且可逆地受到抑制。当[K]增加10倍使细胞膜电位去极化时，pHi恢复的幅度（尽管不是速率）增强。因此，依赖HCO3、不依赖钠和氯且可被DPC阻断的pHi恢复可能主要归因于HCO3通过CFTR Cl通道的内流。在生理条件下，当HCO3的电化学梯度可能是外向而非内向时，CFTR（或另一种HCO3可通透的通道）可能介导HCO3分泌并有助于调节纤毛周液的pH值。
