1. Volume 127, Issue 3, Pages 802-815 (September 2004)
ClC-2 chloride secretion mediates prostaglandin-induced recovery of barrier function in ischemia-injured porcine ileum 
Adam J. Moeser, Melissa M. Haskell, Donnie E. Shifflett, Dianne Little, Bruce D. Schultz, Anthony T. Blikslager 
Gastroenterology 
Volume 127, Issue 3, Pages (September 2004)
DOI: /j.gastro
Copyright © 2004 American Gastroenterological Association Terms and Conditions

2. Figure 1
Electrical responses of ischemia-injured porcine ileal mucosa to treatment with PGE2 and Cl− secretory regulators. Values represent means ± SE; n = 6. (A) Ischemic tissues bathed in indomethacin (5 × 10−6 mol/L) showed marked increases in TER in the presence of 16,16-dimethyl-PGE2 (pge2) (10−6 mol/L). PGE2 was added after a 30-minute equilibration period. Pretreatment with the basolateral Na+-K+-2Cl− transport inhibitor bumetanide (10−4 mol/L) impaired the PGE2 response, whereas treatment with the MEK inhibitor PD-098,059 (10−4 mol/L) augmented the effect of PGE2 (P < 0.05). (B, C) A significant (P < 0.05) increase in Cl− secretion, indicated by both Isc and the absolute change in short-circuit current (▵Isc), was observed in response to treatment with PGE2. PGE2-induced increases in Isc were inhibited fully by pretreating tissues with bumetanide and enhanced in the presence of PD-098,059. A and B, •, Control; ▿, ischemia/Indo; ■, ischemia/PGE2; ◊, ischemia/Indo/PGE2; ▴, ischemia/Indo/PGE2/bumetanide; hexagon, ischemia/Indo/PD098059; •, ischemia/Indo/PGE2/PD *P < 0.05 vs. all other treatments; #P < 0.05 vs. Indo and PGE2.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

3. Figure 2
Histologic appearance of ischemia-injured porcine ileal mucosa. (A) Nonischemic control tissue. (B) Forty-five minutes of intestinal ischemia resulted in lifting and sloughing of epithelium from the tips of villi. (C) After a 60-minute recovery period in an Ussing chamber, in the presence of indomethacin (5 × 10−6 mol/L), villi have contracted and epithelial restitution is nearly complete. (D) After a 60-minute recovery period in an Ussing chamber, in the presence of PGE2 (1 × 10−6 mol/L). (E) After a 60-minute recovery period in the presence of indomethacin and PGE2. (F) Ischemia-injured ileal mucosa at the end of the 240-minute recovery period in an Ussing chamber, in the presence of indomethacin (5 × 10−6 mol/L), has undergone complete restitution. Bar = 100 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

4. Figure 3
Evaluation of the effects of furosemide on TER and 3H-mannitol fluxes of in vivo recovered ischemia-injured porcine ileal mucosa. Values represent means ± SE, n = 6. (A) Pigs subjected to intestinal ischemia and treated with furosemide (1 mg/kg, intravenously) exhibited impaired recovery of TER (Ω · cm2) compared with both nonischemic control and non-furosemide-treated ischemic-injured tissues (*P < 0.05). (B) Pigs subjected to intestinal ischemia and administered furosemide exhibited increased 3H-mannitol mucosal-to-serosal fluxes compared with both nonischemic control and non-furosemide-treated ischemic-injured tissues (*P < 0.05). There was a trend toward increased mannitol flux in nonischemic control tissues from furosemide-treated pigs compared with control tissues from saline-treated pigs, but this was not significant (P = 0.08). ■, saline; □, furosemide.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

5. Figure 4
Electrical responses of ischemia-injured porcine ileal mucosa to treatment with PGE2 and Cl− channel inhibitors. Values represent means ± SE; n = 6. (A) Ischemic tissues bathed in indomethacin (5 × 10−6 mol/L) showed marked increases in TER in the presence of 16,16-dimethyl-PGE2 (pge2) (10−6 mol/L), added after a 30-minute equilibration period. Pretreatment with DNDS, 3 × 10−4 mol/L, augmented TER (P < 0.05) whereas N-(4-methylphenylsulfonyl)-N′-(4-trifluoromethylphenyl) urea (DASU-02, 3 × 10-4 mol/L) was without effect on PGE2-induced increases in TER (P > 0.05). (B, C) A significant increase in Cl− secretion, indicated by both Isc and ▵Isc, was observed in response to treatment with PGE2. Application of DNDS and DASU-02 significantly inhibited PGE2-stimulated Isc (P < 0.05). A and B, •, Ischemia; ▿, ischemia/Indo; ■, ischemia/Indo/PGE2; ◊, ischemia/DASU; ▴, ischemia/Indo/PGE2/DASU; hexagon, ischemia/DNDS; •, ischemia/Indo/PGE2/DNDS. *P < 0.05 vs. all treatments; #P < 0.05 vs. Indo and PGE2.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

6. Figure 5
Electrical responses of ischemia-injured porcine ileal mucosa to treatment with PGE2 and the ClC-2 Cl− channel inhibitor, ZnCl2. Values represent means ± SE; n = 6. (A) Ischemic tissues bathed in indomethacin (5 × 10−6 mol/L) showed marked increases in TER in the presence of 16,16-dimethyl-PGE2 (pge2) (10−6 mol/L). PGE2 was added after a 30-minute equilibration period. Pretreatment with ZnCl2 (300 μmol/L) inhibited PGE2-induced increases in TER (P < 0.05). (B and C) A significant (P < 0.05) increase in Cl− secretion, indicated by both Isc and ▵Isc, was observed in response to treatment with PGE2. Application of ZnCl2 inhibited PGE2-stimulated Isc (P < 0.05). A and B, •, ischemia/Indo; ▿, ischemia/Indo/PGE2; ■, ischemia/Indo/ZnCL2; ♦, ischemia/Indo/PGE2/ZnCL2. *P < 0.05 vs. all treatments; #P < 0.05 vs. Indo and PGE2.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

7. Figure 6
Western analyses of ClC-2 and CFTR in ischemia-injured porcine ileal mucosa. Blots are representative of experiments on 3 separate animals. For each blot, lanes were loaded with equal concentrations of protein. (A) ClC-2 protein was recognized as a single band of approximately 97–100 kilodaltons in molecular mass in nonischemic (cont) and ischemic (isch) ileal mucosal lysates. Densitometric analysis revealed a significant increase in ClC-2 protein expression in ischemic tissue compared with nonischemic control tissue (n = 3; P < 0.05). (B) Mucosal lysates were immunoprecipitated (ip) with preimmune serum (lane 1) or anti-ClC-2 (lanes 2 and 3). Precipitated protein-antibody complexes were subjected to Western blotting (wb) and probed for occludin (lanes 2 and 3). Occludin was detected as a protein of approximately 65 kilodaltons in molecular mass in ClC-2 immunoprecipitated extracts in both nonischemic control tissues (cont) and ischemic-injured tissues (isch), but not in lysates immunoprecipitated with pre-immune serum. (C) CFTR was recognized as a protein of approximately 170–180 kilodaltons in molecular mass in nonischemic (cont) and ischemic (isch) tissue. Densitometric analysis revealed a significant decrease in CFTR protein expression in ischemia-injured tissues compared with nonischemic control tissue (n = 3; P < 0.05).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

8. Figure 7
Immunogold labeling of ClC-2 in porcine ileal mucosa. (A) Immunogold-negative control in which uninjured tissues were incubated in the absence of primary antibody. Microvilli are visible in the upper portion of the figure (solid black arrows). The region of the tight junction is apparent near the apex of the interepithelial space (white arrow), with the subjunctional paracellular space below the tight junction (open black arrow) (B) Immunogold labeling of ClC-2 in uninjured porcine ileal mucosa revealed deposition of gold particles (arrows) predominantly in the region of the tight junction. (C) Immunogold-negative control in which ischemic-injured tissues were incubated in the absence of primary antibody. (D) Immunogold labeling of ClC-2 in ischemic-injured porcine ileal mucosa revealed clustering of gold particles predominantly in the region of the tight junction (arrows). All images are the same magnification (A, Bar = 5 μm), and are representative of multiple sections from n = 3 animals.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

9. Figure 8
ClC-2 immunofluorescence in porcine ileal mucosa. (A) Negative control in which tissues were incubated in the absence of ClC-2 primary antibody. (B) Immunofluorescence labeling showing ClC-2 expression in the interepithelial region of the epithelium in mucosal villi (arrows) of uninjured tissues with a similar but less-intense pattern of fluorescence in the crypts (asterisk). (C) Immunofluorescence labeling of ClC-2 expression in ischemic-injured tissues, showing a similar pattern of fluorescence as B in the interepithelial region of the epithelium in mucosal villi (arrows) and crypts (asterisk). (A) Bar = 100 μm. All images are representative of multiple sections from n = 3 animals.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions