V. M. Baratta1, T. M. Gisinger1,2, M. J. Barahona1, J. Ollodart1, D. Mulligan1, J. P. Geibel1,3 1Yale University School Of Medicine,Department Of Surgery,New Haven, CT, USA 2Paracelsus Medical University,Department of Medicine,Salzburg, SALZBURG, Austria 3Yale University School Of Medicine,Department of Cellular and Molecular Physiology,New Haven, CT, USA
Introduction: One of the barriers to successful small bowel procurement is the sensitivity of the intestinal mucosa to ischemia. During procurement, the University of Wisconsin Universal Organ Preservation (UW) solution is used for in situ flushing and cold storage to minimize progression to ischemia. In addition, donor intestines undergo selective bowel decontamination with antibiotics and antifungals. Recently, we demonstrated that exposure of the small intestine to Penicillin G can protect from ischemic injury in a rat model. This finding may be helpful during the procurement process for intestinal transplantation. Here, we demonstrate that the protective effect is partly mediated by activation of an H,KATPase, independent of the Nitric Oxide (NO) pathway.
Methods: The small bowel segments were harvested from rats and perfused with an ex-vivo intestinal perfusion device. Each intestinal segment was maintained at 37°C and perfused both from the luminal and basolateral side. As previously described, FITC-Inulin (fluorescein isothiocyanate-inulin), was used to assess the ischemic conditions of the colonic grafts in real-time. Small bowel segments were perfused with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES-Ringer) solution. To create an ischemic environment, HEPES-Ringer was pre-saturated with 100% N2 and exposed to the extraluminal components of all rat segments. For the experimental tissues, the intraluminal components were perfused with 5 mM Penicillin G and 10 μM SCH-28080, a known H,KATPase inhibitor. The intraluminal components of the control group were exposed to 5 mM Penicillin G. To test the Nitric Oxide (NO)-dependent mechanism, we used L-NAME (N(ω)-nitro-L-arginine methyl ester), an inhibitor of NO synthesis. The intraluminal compartments of the experimental tissue were exposed to 30 μM L-NAME with 5 mM Penicillin G, while control tissues were exposed to 5 mM Penicillin G.
Results: The small bowel samples exposed to Penicillin G and SCH-28080 exhibited a significant decrease in FITC-Inulin fluorescence, compared with the control colonic tissue exposed to Penicillin G, (39.83 ± 2.601 μM FITC-Inulin vs 47.37 ± 0.7288 μM FITC-Inulin, respectively p 0.0163). We observed no statistically significant difference in the FITC-Inulin concentration between tissues exposed to L-NAME with Penicillin G versus tissues exposed to only Penicillin G.
Conclusion: Our study unveils the mechanism of Penicillin G’s protective effect from ischemia. Our results indicate that Penicillin G’s protective effect against ischemia is through stimulation of the H,KATPase and is not NO-dependent. Therefore, Penicillin G not only has its well-known antimicrobial properties, but also appears to modulate a transport protein. In the future, Penicillin G may be implemented during the early procurement phase of intestinal transplantation to minimize ischemia.