Shuttleworth T J, Wood C M
Department of Physiology, University of Rochester School of Medicine, New York 14642.
Am J Physiol. 1992 Jan;262(1 Pt 1):C221-8. doi: 10.1152/ajpcell.1992.262.1.C221.
The fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein (BCECF) was used to determine changes in intracellular pH (pHi) associated with activation of secretion in isolated cells from the salt-secreting avian nasal gland. A correction procedure overcoming artifacts due to BCECF leakage is described. Resting pHi averaged 7.15 +/- 0.03 and was unaffected by the nominal removal of medium HCO3- or by the addition of the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) but was significantly reduced by amiloride (7.07 +/- 0.02). Muscarinic activation of secretion resulted in a rapid intracellular acidification that was compensated by mechanisms which raised pHi to restore approximately resting levels within 5 min. The principal mechanism involved was amiloride-sensitive and independent of any sustained intracellular Ca2+ concentration change. Recovery of pHi was also aided by HCO3(-)-dependent and DIDS-sensitive mechanisms not seen in the resting cell. The direction of the latter was pHi-dependent, with DIDS further decreasing pHi in acidified cells and increasing pHi in alkalinized cells. This suggests that the DIDS-sensitive pathways are activated under conditions where pHi has been shifted away from resting levels in either direction and act primarily to restore resting pHi.
使用荧光pH敏感染料2'，7'-双（2-羧乙基）-5（6）-羧基荧光素（BCECF）来测定来自分泌盐分的鸟类鼻腺的分离细胞中与分泌激活相关的细胞内pH（pHi）变化。描述了一种克服由于BCECF泄漏导致的假象的校正程序。静息pHi平均为7.15±0.03，不受名义上去除培养基HCO3-或添加阴离子交换抑制剂4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）的影响，但被氨氯吡咪显著降低（7.07±0.02）。毒蕈碱介导的分泌激活导致细胞内迅速酸化，随后通过一些机制进行补偿，这些机制会提高pHi，使其在5分钟内恢复到大约静息水平。主要涉及的机制对氨氯吡咪敏感，且与任何持续的细胞内Ca2+浓度变化无关。pHi的恢复还得益于静息细胞中未观察到的HCO3-依赖性和DIDS敏感机制。后者的作用方向取决于pHi，DIDS会使酸化细胞中的pHi进一步降低，使碱化细胞中的pHi升高。这表明DIDS敏感途径在pHi偏离静息水平的任何方向的情况下被激活，主要作用是恢复静息pHi。
