Characterization of the swelling-induced alkalinization of endocytotic vesicles in fluorescein isothiocyanate-dextran-loaded rat hepatocytes

Abstract

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.

Flow cytometric analysis of vesicular pH in rat hepatocytes after ethanol administration

“…These latter results indicate that a sustained alkalinization of hepatic lysosomes by continuous ethanol administration would indeed affect the proteolytic capacity of this organelle in vivo and would account in part for the ethanol-elicited reduction in hepatic protein catabolism. [1][2][3] These studies also showed the effectiveness of flow cytometry in comparison with single-cell fluorescence recordings 21,23 for measuring the pH of prelysosomal particles such as endosomes. In accordance with our findings with lysosomes, we observed that ethanol administration caused a significant alkalinization of hepatic endosomes compared FIG.…”

“…Sixteen hours after its intravenous injection, FITC-dextran was detected in an acidic cellular compartment, the pH of which corresponded to that of lysosomes. 16,[19][20][21]23 Comparison of the intralysosomal pH in cells from control and ethanol-fed rats indicated that longterm ethanol administration caused a consistent increase of lysosomal pH that was 0.13-0.2 units higher than that in controls (Fig. 2).…”

Calcium Mobilization Evoked by Hepatocellular Swelling Is Linked to Activation of Phospholipase Cγ

“…In liver, selected downstream responses to cell swelling, such as endosomal alkalinization and stimulation of bile flow, have been linked to activation of tyrosine kinases (17,18). We therefore reasoned that PLC␥, an effector of both receptor and non-receptor tyrosine kinase-mediated pathways, could be involved in volume-sensitive hepatocellular Ca 2ϩ signaling.…”

Second International Ringberg Conference: “Cell Biology and Molecular Basis of Liver Transport”

“…55 Transcytosed proteins takis inhibited by pertussis toxin, cholera toxin, erbstatin, and genistein (but not by daidzein). 51 ing the indirect route pass through a subapical membrane compartment before being integrated into the canalicular It is generally assumed that regulatory volume increase (RVI) in rat hepatocytes is mediated via Na / /H / exchange membrane. This compartment lacks any basolateral membrane protein and is enlarged if the common bile duct is and Na / /K / -ATPase: activation of Na / /H / exchange leads to an increase in cell Na / ; Na / is then exchanged for K / by ligated.…”