Regulation of vesicular pH in liver macrophages and parenchymal cells by ammonia and anisotonicity as assessed by fluorescein isothiocyanate-dextran fluorescence

Abstract

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.