T. M. Gisinger1,2, V. M. Baratta1, 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: Ischemic colitis is one of the most common types of intestinal ischemia. Currently, many patients with ischemic colitis are treated with empiric antibiotics, even though the mechanism of action is not fully understood. Previously, we established that Penicillin G has a protective effect from ischemia in the rat colon. In this study, we demonstrate that the protective effect is mediated through stimulation of the colonic H,KATPase, independent of the Nitric Oxide (NO) pathway.
Methods: The colonic segments were harvested from Sprague Dawley male rats and perfused with an ex-vivo intestinal perfusion device. Each colon was maintained at 37°C and perfused both from the luminal and basolateral side. FITC-Inulin, fluorescein isothiocyanate, was used to assess the ischemic conditions of the colonic grafts in real-time. Colonic 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 colonic segments. For the experimental colonic tissues, the intraluminal compartments were perfused with 5 mM Penicillin G and 10 μM SCH-28080, a known colonic H,KATPase inhibitor. The intraluminal components of the control group were exposed to 5 mM Penicillin G. To test the NO-dependent mechanism, we used L-NAME, N(ω)-nitro-L-arginine methyl ester, a NO synthesis inhibitor. 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 colon 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, (36.39 ± 2.721 μM FITC-Inulin vs 43.62 ± 1.569 μM FITC-Inulin, respectively, p 0.0401, Figure 1). 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 acts by stimulating the colonic 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 on the colonic cell membrane. A better understanding of Penicillin G’s effects on colonic tissue may help further guide the clinical management of ischemia in the gastrointestinal tract.
