Lubiprostone was obtained from R-Tech Ueno (Sanda, Japan). PEG 3350 (Miralax, Schering-Plough Health Care Products, Inc. Kenilworth NJ) was obtained from the North Carolina State University, College of Veterinary Medicine pharmacy. ³H-mannitol and ¹⁴C-inulin were obtained from Sigma Chemical (St. Louis, MO).

All studies were approved by the North Carolina State University Institutional Animal Care and Use Committee. Six to eight-week-old Yorkshire crossbred pigs of either sex were housed individually, and maintained on a commercial pelleted feed. Pigs were fasted for 24 h prior to experimental surgery. General anesthesia was induced with xylazine (1.5 mg/kg, IM), ketamine (11 mg/kg, IM), and 5% isoflurane vaporized in 1000 mL/L O₂ and was maintained with 2% isoflurane delivered via an endotracheal tube. Pigs were placed on a heating pad and ventilated with 1000 mL/L O₂ using a volume-limited, time-cycled ventilator (Hallowell, Pittsfield, MA). Lactated Ringers solution was administered iv at a maintenance rate of 15 mL/kg per hour. The ileum was approached via a ventral midline incision. Ileal segments were delineated by ligating the intestine at 10-cm intervals, and subjected to ischemia by occluding the local mesenteric blood supply for 45 min.

Following the 45-min ischemic period, tissues were harvested from the pig and the mucosa was stripped from the seromuscular layer in oxygenated (950 mL/L O₂/50 mL/L CO₂) Ringer’s solution (mmol/L: Na⁺, 154; K⁺, 6.3; Cl^-, 137; HCO₃^-, 24; pH 7.4) containing 5 μmol/L indomethacin to prevent endogenous prostaglandin production during the stripping procedure. Tissues were then mounted in 1.14 cm² aperture Ussing chambers, as described in previous studies. For Ussing chamber experiments, ileal tissues from one pig were mounted on multiple Ussing chambers and subjected to different in vitro treatments. Data means are representative of 6 Ussing chamber experiments (n = 6 animals). Tissues were bathed on the serosal and mucosal sides with 10 mL Ringer’s solution. The serosal bathing solution contained 10 mmol/L glucose, and was osmotically balanced on the mucosal side with 10 mmol/L mannitol. Bathing solutions were oxygenated (950 mL/L O₂/50 mL/L CO₂) and circulated in water-jacketed reservoirs. The spontaneous potential difference (PD) was measured using Ringer-agar bridges connected to calomel electrodes, and the PD was short-circuited through Ag-AgCl electrodes using a voltage clamp that corrected for fluid resistance. Transepithelial electrical resistance (TER) (Ω.cm²) was calculated from the spontaneous PD and short-circuit current (I_sc). If the spontaneous PD was between -1.0 and 1.0 mV, tissues were current-clamped at ± 100 μA for 5 s and the PD recorded. I_sc and PD were recorded at 15-min intervals over a 120-min experiment.

After tissues were mounted on Ussing chambers, tissues were allowed to acclimate for 30 min to achieve stable baseline measurements after which experimental treatments were added. Lubiprostone (1 μmol/L) or PEG 3350 (20 mmol/L) were added to the mucosal side of tissues and TER and I_sc were measured at 15-min intervals over a 120-min recovery period. The PEG 3350 dose was selected as it is the recommended oral dosage for laxative properties and thus would approximate the luminal concentrations attained in vivo.

To assess mucosal permeability after experimental treatments, 0.2 μCi/mL ³H-labeled mannitol (180 kDa) and 0.2 μCi/mL, ¹⁴C-labeled inulin (5000 kDa) were added to the mucosal side of tissues mounted in Ussing chambers. After a 15-min equilibration period, standards were taken from the mucosal side of each chamber and a 60-min flux period was established by taking 0.5 mL samples from the serosal compartment. The presence of ³H and ¹⁴C was established by measuring β-emission in a liquid-scintillation counter (LKB Wallac, Model 1219 Rack Beta, Perkin Elmer Life and Analytical Sciences, Inc., Boston, MA). Unidirectional mannitol fluxes from mucosa-to-serosa were determined using standard equations.

Tissues were taken at 0 and 120 min for routine histological evaluation. Tissues were sectioned (5 μm) and stained with hematoxylin and eosin. For each tissue, 3 sections were evaluated. Four well-oriented villi and crypts were identified in each section. Villus length was obtained using a micrometer in the eye piece of a light microscope.

All data were reported as mean ± SE. TER and I_sc data were analyzed by using an ANOVA for repeated measures. Radiolabeled flux data was analyzed by using a standard one-way ANOVA (Sigmastat, Jandel Scientific, San Rafael, CA). A Tukey’s test was used to determine differences between treatments following ANOVA.

Porcine ileum was subjected to 45 min of acute mesenteric ischemia and mounted on Ussing chambers for measurement of TER and I_sc over a 120-min recovery period. Ischemic-injured mucosa had significantly lower starting TER values (by approximately 40%) compared with non-ischemic control tissue (Figure 1), indicating significant impairment of intestinal barrier function induced by ischemia. Application of 1 μmol/L lubiprostone to the mucosal side of ischemic-injured mucosa induced rapid elevations in TER that attained non-ischemic control tissues within 15 min of its addition and TER continued to increase 45 min post-treatment. Lubiprostone stimulated rapid elevations in I_sc (an index of electrogenic ion transport) that peaked at 15 min post-treatment (peak ΔI_sc = 66 μA/cm²) and remained elevated throughout the remainder of the experiment. Mucosal addition of 20 mmol/L PEG 3350 stimulated a transient increase in TER measured 15 min after treatment; however, TER returned to ischemic control levels within 30 min post-treatment. PEG 3350 stimulated significant elevations in I_sc (peak ΔI_sc = 35 μA/cm²) compared with ischemic control tissues. In non-ischemic ileal tissues, lubiprostone and PEG 3350 induced similar elevations in TER (ΔTER = 30% ± 7% and 36% ± 8% in lubiprostone and PEG 3350-treated tissues) (data not shown).

Mucosal-to-serosal flux of both medium molecular weight (³H-mannitol, 180 kDa) and large molecular weight (¹⁴C-Inulin, 5000 kDa) paracellular probes in ileal tissues mounted in Ussing chambers were conducted as an alternative measurement of mucosal permeability. In line with TER responses, ischemic tissues had greater (P < 0.01) serosal-to-mucosal fluxes of both ³H-mannitol and ¹⁴C-inulin (Figure 2). Lubiprostone treatment significantly decreased the fluxes of ³H-mannitol and ¹⁴C-inulin (P < 0.05). Alternatively, ischemic tissues treated with PEG 3350 displayed significantly increased fluxes of permeability markers compared with all other treatments (P < 0.05).

Control (non-ischemic) tissues treated with lubiprostone or PEG 3350 had no identifiable histopathological findings compared with non-treated control tissues (not shown). Ileal tissues subjected to ischemia displayed classic histological ischemic lesions characterized by sloughing of the surface epithelium of the apical villus (Figure 3). The epithelial layer was completely restituted after 60 min of mounting tissues on Ussing chambers (not shown); this effect has been demonstrated in our previous studies[4,5]. At 120 min post-ischemia on Ussing chambers, villus contraction was evident demonstrated by villi that were reduced in height and increased in width compared with non-ischemic control tissues (Figure 4). Laxative agents had a significant effect on intestinal villous length in ischemic-injured tissues in this study. Lubiprostone-treated tissues had greater villous lengths compared with ischemic-injured controls when measured at 120 min post-ischemia. In contrast, PEG 3350-treated ischemic tissues had significantly reduced villous height compared with ischemic-injured control tissues.

A number of important intestinal diseases, including ischemia/reperfusion injury, are characterized by damage to the epithelium lining the gut. Mechanisms are in place to rapidly repair epithelial defects, including epithelial migration (restitution). More recently, studies have shown the importance of the interepithelial tight junctions in recovery of the epithelial barrier. Studies have shown that prostaglandins and prostones increase the rate of epithelial recovery via re-assembly of tight junctions.

The prostone lubiprostone, a new medication on the market indicated for treatment of chronic constipation and irritable bowel syndrome has its effect on chloride channels (ClC-2) within tight junctions. These channels are involved in secretion of chloride in the intestine, but to a far lesser extent than the chloride channel that is genetically disrupted in patients with cystic fibrosis (CFTR). Recent studies have shown that ClC-2, when activated by prostaglandins or prostones such as lubiprostone, also play an important role in re-assembly of tight junctions, resulting in increases in the speed of epithelial repair.

Stimulation of one of the minor chloride channels, ClC-2, is an innovative way to induce low level secretion into the gut, thereby serving as a laxative. This is in contrast to traditional laxatives such as Polyethylene Glycol 3350 (PEG), which result in increased fluid in the intestinal lumen because of its osmotic properties. Other effects of ClC-2 activation by lubiprostone have recently been discovered, particularly the ability to increase mucosal repair. A comparison of lubiprostone and PEG showed that only lubiprostone facilitated the repair of the mucosa. This increased repair could not be seen at the histological level. This is consistent with prior studies showing that enhanced repair is noted at the level of tight junctions.

When treating constipation, the choice of medication includes laxatives such as PEG and the ClC-2 activator lubiprostone. The present study suggests that use of lubiprostone will also hasten the recovery of injured gut mucosa in patients with more severe intestinal disease. Further basic science research followed by clinical trials will be needed to determine the validity of these findings.

The term ClC-2 is used to describe a chloride channel in the gut epithelium that is localized to interepithelial tight junctions. The term prostone refers to a new group of compounds which are distinct from prostaglandins and specifically activate ClC-2.

The present study was performed using porcine tissues; it demonstrates that lubiprostone stimulates recovery of barrier function in ischemic intestinal tissues whereas the PEG laxative had deleterious effects on mucosal repair. This is an interesting study and well written manuscript.