Schreiber R, Häussinger D
Medizinische Klinik, Heinrich-Heine-Universität, Düsseldorf, Germany.
Biochem J. 1995 Jul 1;309 ( Pt 1)(Pt 1):19-24. doi: 10.1042/bj3090019.
Short-term cultivated rat hepatocytes were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran and the apparent vesicular pH (pHves) was measured by single-cell fluorescence. After 2 h of exposure to FITC-dextran, the apparent pH in the vesicular compartments accessible to endocytosed FITC-dextran was 6.01 +/- 0.05 (n = 39) in normo-osmotic media. Hypo-osmotic exposure increased, whereas hyper-osmotic exposure decreased apparent pHves. by 0.18 +/- 0.02 (n = 26) and 0.12 +/- 0.01 (n = 23) respectively. Incubation of the cells with unlabelled dextran for 2h before a 2-h FITC-dextran exposure had no effect on apparent pHves and its osmosensitivity. When, however, hepatocytes were exposed to unlabelled dextran for 5 h after a 2 h exposure to FITC-dextran, in order to allow transport of endocytosed FITC-dextran to late endocytotic/lysosomal compartments, apparent pHves. decreased to 5.38 +/- 0.04 (n = 12) and the apparent pH in the vesicular compartment containing the dye was no longer sensitive to aniso-osmotic exposure. These findings indicate that the osomosensitivity of pHves. is apparently restricted to early endocytotic compartments. Aniso-osmotic regulation of apparent pHves. in freshly FITC-loaded hepatocytes was not accompanied by aniso-osmolarity-induced changes of the cytosolic free calcium concentration, and neither vasopressin nor extracellular ATP, which provoked a marked Ca2+ signal, affected apparent pHves. Dibutyryl-cyclic AMP (cAMP) or vanadate (0.5 mmol/l) were without effect on apparent pHves. and its osmosensitivity. However, pertussis toxin-treatment or genistein (but not daidzein) or the erbstatin analogue methyl 2,5-dihydroxycinnamate fully abolished the osmo-sensitivity of apparent pHves., but did not affect apparent pHves. It is concluded that regulation of pHves. by cell volume occurs in early endocytotic compartments, but probably not in lysosomes, and is mediated by a G-protein and tyrosine kinase-dependent, but Ca2+- and cAMP-independent mechanism.
将短期培养的大鼠肝细胞用于内吞异硫氰酸荧光素（FITC）偶联的葡聚糖，并通过单细胞荧光测量表观囊泡pH值（pHves）。在暴露于FITC-葡聚糖2小时后，在等渗培养基中，内吞的FITC-葡聚糖可进入的囊泡区室中的表观pH值为6.01±0.05（n = 39）。低渗暴露会使表观pHves升高，而高渗暴露则会使其降低，分别降低0.18±0.02（n = 26）和0.12±0.01（n = 23）。在2小时的FITC-葡聚糖暴露前，将细胞与未标记的葡聚糖孵育2小时，对表观pHves及其渗透压敏感性没有影响。然而，当肝细胞在暴露于FITC-葡聚糖2小时后再暴露于未标记的葡聚糖5小时，以便使内吞的FITC-葡聚糖转运至晚期内吞/溶酶体区室时，表观pHves降至5.38±0.04（n = 12），并且含有染料的囊泡区室中的表观pH值不再对非等渗暴露敏感。这些发现表明，pHves的渗透压敏感性显然仅限于早期内吞区室。新鲜加载FITC的肝细胞中表观pHves的非等渗调节并未伴随着非等渗诱导的胞质游离钙浓度变化，并且加压素和细胞外ATP（它们会引发明显的Ca2 +信号）均未影响表观pHves。二丁酰环磷腺苷（cAMP）或钒酸盐（0.5 mmol / l）对表观pHves及其渗透压敏感性没有影响。然而，百日咳毒素处理或染料木黄酮（而非黄豆苷元）或埃布他汀类似物2,5-二羟基肉桂酸甲酯完全消除了表观pHves的渗透压敏感性，但不影响表观pHves。得出的结论是，细胞体积对pHves的调节发生在早期内吞区室中，但可能不在溶酶体中，并且是由G蛋白和酪氨酸激酶依赖性但Ca2 +和cAMP非依赖性机制介导的。
