Regulated vesicle trafficking of membrane transporters in hepatic epithelia  Raúl A. Marinelli, Pamela S. Tietz, Nicholas F. LaRusso  Journal of Hepatology  Volume 42, Issue 4, Pages 592-603 (April 2005) DOI: 10.1016/j.jhep.2005.01.001 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Hepatic epithelia contain membrane transporters in specialized intracellular vesicles. Specific transport activities can be regulated by trafficking to and insertion of these transporters into the plasma membrane in response to appropriate stimuli. When the stimulus is withdrawn, the transport proteins are retrieved by endocytosis and remain for a period of time in vesicles (i.e. early endosomes). The transporters may undergo re-fusion after re-stimulation or move to a non-recycling compartment (i.e. late endosomes) where they would be finally degraded by lysosomes. Some transporters might be targeted for proteolysis through the ubiquitin-proteasome pathway. Thus, the abundance of a transporter in the plasma membrane at any given time would result from the net balance between the rate of exocytic insertion and endocytic retrieval. Journal of Hepatology 2005 42, 592-603DOI: (10.1016/j.jhep.2005.01.001) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Localization of AQP-8 in hepatocytes by subcellular fractionation and immunoblotting: effect of dibutyryl cAMP (DBcAMP). Hepatocytes were incubated in the absence (controls) or presence of 100μM DBcAMP for 10min at 37°C, and subcellular fractionation was performed. (A) anti-AQP8 immunoblot of plasma and intracellular microsomal membranes (20μg total protein/lane). (B) Densitometric analysis of three separate experiments in each group (control and DBcAMP). Data are expressed in percent values as mean±S.E. *, P<0.05 for DBcAMP effect (Student's t test). Journal of Hepatology 2005 42, 592-603DOI: (10.1016/j.jhep.2005.01.001) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Effect of glucagon on the localization of AQP8 in hepatocytes by confocal immunofluorescence. Hepatocytes (A and B, singlets; C and D, couplets) in short term cultured were incubated at 37°C for 10min in the absence (controls) or presence of 1μM glucagon, fixed, permeabilized, and labeled with anti-QP8. Fluorescence localization was viewed by laser scanning confocal microscopy. (A and C), controls; (B and D), glucagon-treated. Magnification, ×600. Journal of Hepatology 2005 42, 592-603DOI: (10.1016/j.jhep.2005.01.001) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Polarized confluent mouse cholangiocytes were exposed to media alone (basal) (A and B) or media with dibutyryl cAMP (agonist) (A1 and B1). Agonist stimulation caused a redistribution of dual labeled immunogold and demonstrates a consistent and significant movement of vesicles containing colocalizated AQP1 with CFTR and AE2 with CFTR to the apical plasma membrane (arrowheads). Quantitation of the apical membrane-associated labeling (C and D) revealed a significant 2-fold or greater increase in the apical membrane-associated redistribution. Bar=200nm at a magnification of ×30,000. *P<0.05 (Student's t-test). Journal of Hepatology 2005 42, 592-603DOI: (10.1016/j.jhep.2005.01.001) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 (A) Immunoblotting for AQP1, CFTR, and AE2 on subcellular fractions prepared from microsomes (starting material), vesicles specifically associated with the magnetic beads (bound), and vesicles that did not bind to the beads (unbound) using AQP1 antibody. (B) Reverse vesicle immunoisolation using CFTR antibody. (C) Reverse vesicle immunoisolation using AE2 antibody. A 10% polyacrylamide gel was loaded with fractions for AQP1 (50μg of total protein/lane), CFTR (100μg of total protein/lane), and AE2 (50μg of total protein/lane). Data are expressed in arbitrary densitometry units as means±S.E. *P<0.05 (Student's t-test). Journal of Hepatology 2005 42, 592-603DOI: (10.1016/j.jhep.2005.01.001) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions