CHARACTERIZATION OF THE SWELLING-INDUCED ALKALINIZATION OF ENDOCYTOTIC VESICLES IN FLUORESCEIN ISOTHIOCYANATE-DEXTRAN-LOADED RAT HEPATOCYTES

Short-term cultivated rat hepatocytes were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran and the apparent vesicular pH (pH(ves.)) 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 pH(ves.) 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 pH(ves) 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 pH(ves). 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 osmosensitivity of pH(ves.) is apparently restricted to early endocytotic compartments. Aniso-osmotic regulation of apparent pH(ves.) 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 pH(ves.). Dibutyryl-cyclic AMP (cAMP) or vanadate (0.5 mmol/l) were without effect on apparent pH(ves.) 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 pH(ves.), but did not affect apparent pH(ves.) It is concluded that regulation of pH(ves.) 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.