Volume 123, Issue 1, Pages 281-290 (July 2002) Intracellular distribution and functional importance of vesicle-associated membrane protein 2 in gastric parietal cells  Serhan Karvar, Xuebiao Yao, Joseph G. Duman, Kevin Hybiske, Yuechueng Liu, John G. Forte  Gastroenterology  Volume 123, Issue 1, Pages 281-290 (July 2002) DOI: 10.1053/gast.2002.34217 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Localization of GFP–VAMP-2 in gastric parietal cells. Primary cultures of gastric parietal cells were infected with adenovirus containing recombinant GFP–VAMP-2. After 36 hours, cells were treated to effect various physiologic states and examined for general morphology and GFP fluorescence. Each cell is shown as imaged by DIC and by the corresponding GFP fluorescence. (A) Rest: Resting cells treated with 100 μmol/L cimetidine. (B) Stim: Cells stimulated with 100 μmol/L histamine plus 30 μmol/L IBMX. (C) Stim+SCH: Cells stimulated as above in the presence of 5 μmol/L SCH-28080. Apical membrane vacuoles (arrows), clearly seen in all images, are enlarged in the stimulated state compared to the resting state or when stimulated in the presence of SCH-28080. GFP–VAMP-2 fluorescence appears to be broadly distributed throughout the cytoplasm in the resting state and more heavily distributed to the apical membrane vacuoles in the stimulated state, with or without SCH-28080. The bar marker is 20 μm. Gastroenterology 2002 123, 281-290DOI: (10.1053/gast.2002.34217) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Intracellular sorting of GFP–VAMP-2. Cultured parietal cells infected with GFP–VAMP-2 adenovirus were continuously monitored by fluorescence microscopy in a microscope stage chamber at 37°C. (A) Cells were stimulated at time 0 with 100 μmol/L histamine and 30 μmol/L IBMX. (B) Cells stimulated at time 0 as in A but in the presence of 5 μmol/L SCH-28080. At zero and early times GFP–VAMP-2 is located throughout the cytoplasm. After 10–12 minutes, GFP–VAMP-2 fluorescence tends to diminish in the general cytoplasm and accumulate to the apical membrane vacuoles. In the stimulated cells there is clearly also a swelling of the vacuoles with time; the vacuole size does not change in the presence of SCH-28080. The bar marker is 10 μm. Gastroenterology 2002 123, 281-290DOI: (10.1053/gast.2002.34217) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of TeTx on gastric acid secretion. Rabbit isolated gastric glands were permeabilized with SLO and then incubated for 20 minutes with cimetidine (resting) or stimulated in the presence of cAMP/ATP (stimulated). The stimulated glands were divided into 3 groups: (1) maintained as a control (ctrl), (2) treated with 40 nmol/L TeTx plus 1 mmol/L DTT (TeTx), or (3) treated with 1 mmol/L DTT alone (DTT). Acid secretion was measured by [14C]AP accumulation. To account for differences among gland preparations to the next the AP uptake of the stimulated control was normalized to 100% for each experiment. Values are the mean ± standard error of 7 independent gland preparations, each with duplicate determinations. Gastroenterology 2002 123, 281-290DOI: (10.1053/gast.2002.34217) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Comparison of GFP–VAMP-2 and H+-K+-ATPase distribution. Confocal images of gastric parietal cells infected with GFP–VAMP-2 adenovirus. After 36 hours, cultures were held in a resting state (rest) by the addition of 100 μmol/L cimetidine. Other cultures were stimulated with 100 μmol/L histamine and 30 μmol/L IBMX either in the absence (stim) or presence of 5 μmol/L SCH-28080 (stim+SCH). After 30 minutes the cells were fixed, immunostained for H+-K+-ATPase, and examined by laser confocal microscopy. Digital images are shown for GFP–VAMP-2 (VAMP-2), detected using a FITC filter, and H+-K+-ATPase (H,K-ATPase), detected by rhodamine-labeled secondary antibody. The third panel of each experimental treatment is an image constructed by indicating only those pixels that were positive for both GFP–VAMP-2 and H+-K+-ATPase (congruent pixels). A high degree of colocalization of signals for GFP–VAMP-2 and H+-K+-ATPase is seen under all physiologic conditions. For cells stimulated in the presence of 5 μmol/L SCH-28080, H+-K+-ATPase, along with a substantial amount of GFP–VAMP-2, was cleared from the cytoplasm and was translocated to the apical membrane vacuoles. Gastroenterology 2002 123, 281-290DOI: (10.1053/gast.2002.34217) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Treatment of SLO-permeabilized glands with TeTx inhibits acid secretion in a dose-dependent manner. Gastric glands were permeabilized with SLO and then incubated with increasing concentrations of TeTx for 20 minutes in the presence of 0.1 mmol/L cAMP and 1 mmol/L ATP (cAMP/ATP) as described in Materials and Methods. Acid secretion was measured by [14C]AP accumulation ratio. In this single experiment, the results shown are the mean ± range from triplicate samples at each concentration. The dotted line shows the AP accumulation ratio for nonstimulated, resting gastric glands treated with cimetidine. Gastroenterology 2002 123, 281-290DOI: (10.1053/gast.2002.34217) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Cleavage of VAMP-2 by TeTx. SLO-permeabilized gastric glands (A) and purified tubulovesicle membranes (B) were treated with the indicated concentrations of TeTx for 30 minutes. The respective preparations were solubilized, developed by SDS-PAGE and analyzed by Western blot. In each gel the extreme right hand lane included a standard membrane preparation from rabbit brain (br) as a positive control for VAMP-2. The blots were first probed using a rabbit polyclonal antibody against VAMP-2 and these data are shown in the lower panels of A and B. The blots were stripped and reprobed using a monoclonal anti–H+-K+-ATPase β-subunit antibody and shown in the upper panels of A and B. The left hand strip in each panel shows the position of the pre-stained molecular weight standards. (A) For SLO-permeabilized glands (15 μg protein/lane) there was a dose-dependent diminution, or partial proteolysis, of VAMP-2 (V2) by TeTx. (B) The purified tubulovesicles (10 μg protein/lane) were more sensitive to TeTx with complete proteolysis of VAMP-2 (V2) occurring at 20 nmol/L TeTx. In both cases the β-subunit of H+-K+-ATPase (HK) was insensitive to TeTx. Gastroenterology 2002 123, 281-290DOI: (10.1053/gast.2002.34217) Copyright © 2002 American Gastroenterological Association Terms and Conditions