Schreiber R, Zhang F, Häussinger D
Klinik für Gastroenterologie, Heinrich Heine Universität, Düsseldorf, Germany.
Biochem J. 1996 Apr 15;315 ( Pt 2)(Pt 2):385-92. doi: 10.1042/bj3150385.
Short-term-cultivated rat hepatocytes and Kupffer cells were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran, in order to study the effects of aniso-osmotic exposure and NH4Cl on apparent vesicular pH (pHves) by single-cell fluorescence. Following a 2 h loading period with FITC-dextran in normo-osmotic (305 mosmol/l) medium, the apparent pHves was 6.01 +/- 0.05 (n = 39) in parenchymal cells and 4.94 +/- 0.04 (n = 76) in Kupffer cells. Under these conditions pHves in parenchymal cells, but not in Kupffer cells, was sensitive to changes in ambient osmolarity. Inhibition of vacuolar H(+)-ATPase by concanamycin A did not affect the osmosensitivity of pHves in parenchymal cells. However, the effects of anisotonicity on pHves were largely abolished in the presence of 4.4'-di-isothiocyanato-stibene-2,2'-disulphonic acid (DIDS) or when extracellular chloride was substituted for gluconate. In neither Kupffer cells, nor liver parenchymal cells did hypo-osmotic cell swelling cause an increase in intracellular Ca2+. With regard to vesicular acidification, the following differences were noted between parenchymal and Kupffer cells. (1) In Kupffer cells endocytosed FITC-dextran reached a strongly acidic compartment with a pH value of approx. 5 within 5 min, whereas it took 4-5 h in parenchymal cells. Modification of pHves by hypo-osmolarity in Kupffer cells was only observed in a short-lived "early' compartment with a pH value of approx. 6. (2) In contrast to pHves in parenchymal cells, pHves in Kupffer cells was very sensitive towards alkalinization by NH4Cl: addition of NH4Cl at 1 or 10 mM increased apparent pHves by 0.80 or 1.46 in Kupffer cells, but only by 0.18 or 0.56 in parenchymal cells. The low ammonia sensitivity of pHves in parenchymal cells was observed not only a the less acidic (pH approx. 6) endocytotic compartment which is reached by FITC-dextran within 2 h, but also in the stronger acidic compartment (pH approx. 5) which is reached after 4-5 h. (3) NH4Cl had no effect on the osmosensitivity of pHves in parenchymal cells, whereas in Kupffer cells pHves became sensitive to anisotonicity when NH4Cl was present. Osmosensitivity of pHves in Kupffer cells under these conditions, however, was not affected by genistein, DIDS or colchicine, whereas these compounds abolished the osmosensitivity of pHves in parenchymal cells. It is suggested that regulation of pHves by cell volume in liver parenchymal cells involves changes of vesicular chloride conductance. In addition, there are marked differences between Kupffer and parenchymal cells with respect to vesicular ammonia permeability and the kinetics of endocytotic membrane flow and acidification.
为了通过单细胞荧光研究等渗暴露和氯化铵对表观囊泡pH值（pHves）的影响，将短期培养的大鼠肝细胞和库普弗细胞用于内吞异硫氰酸荧光素（FITC）偶联的葡聚糖。在等渗（305 mosmol/l）培养基中用FITC - 葡聚糖加载2小时后，实质细胞中的表观pHves为6.01±0.05（n = 39），库普弗细胞中的表观pHves为4.94±0.04（n = 76）。在这些条件下，实质细胞中的pHves对环境渗透压的变化敏感，而库普弗细胞中的pHves则不敏感。 concanamycin A对液泡H(+) - ATP酶的抑制作用不影响实质细胞中pHves的渗透压敏感性。然而，在4,4'-二异硫氰酸根合芪-2,2'-二磺酸（DIDS）存在时或用葡萄糖酸盐替代细胞外氯化物时，各向异性对pHves的影响在很大程度上被消除。低渗性细胞肿胀在库普弗细胞和肝实质细胞中均未导致细胞内Ca2+增加。关于囊泡酸化，在实质细胞和库普弗细胞之间注意到以下差异。（1）在库普弗细胞中，内吞的FITC - 葡聚糖在5分钟内到达pH值约为5的强酸性区室，而在实质细胞中则需要4 - 5小时。低渗对库普弗细胞中pHves的改变仅在pH值约为6的短暂“早期”区室中观察到。（2）与实质细胞中的pHves相反，库普弗细胞中的pHves对氯化铵引起的碱化非常敏感：在库普弗细胞中加入1或10 mM的氯化铵使表观pHves增加0.80或1.46，但在实质细胞中仅增加0.18或0.56。实质细胞中pHves对氨的低敏感性不仅在FITC - 葡聚糖在2小时内到达的酸性较弱（pH约为6）的内吞区室中观察到，而且在4 - 5小时后到达的酸性较强（pH约为5）的区室中也观察到。（3）氯化铵对实质细胞中pHves的渗透压敏感性没有影响，而在库普弗细胞中，当存在氯化铵时，pHves对各向异性变得敏感。然而，在这些条件下库普弗细胞中pHves的渗透压敏感性不受染料木黄酮、DIDS或秋水仙碱的影响，而这些化合物消除了实质细胞中pHves的渗透压敏感性。提示肝实质细胞中细胞体积对pHves的调节涉及囊泡氯电导的变化。此外，在囊泡氨通透性以及内吞膜流动和酸化动力学方面，库普弗细胞和实质细胞之间存在明显差异。
