V. Norz1,2, T. Spingler1,2, T. M. Gisinger1,2, V. M. Baratta2, M. J. Barahona2, J. Ollodart2, D. Mulligan2, J. P. Geibel2 1Paracelsus Medical University,Medicine,Salzburg, SALZBURG, Austria 2Yale University School Of Medicine,Surgery,New Haven, CT, USA
Introduction: Antibiotic-associated diarrhea (AAD) is a well-known complication of antibiotic administration, although the pathogenesis is not clearly elucidated. Symptoms range from mild gastrointestinal disturbances to fulminant gastroenteritis. Surgical patients who receive perioperative antibiotics are at higher risk. Antibiotic-associated diarrhea is thought to be due to an imbalance in the host microbiome, leading to pathogen overgrowth and increased intestinal secretion. We propose a novel pathway of AAD, whereby Penicillin G stimulates intestinal H, KATPase. Activation of H,KATPase leads to intraluminal fluid loss. Here, we demonstrate that L-arginine, a nitric oxide (NO) precursor, works synergistically with Penicillin G to activate H,KATPase. We corroborate this by showing how an inhibitor of the NO pathway, L-NAME, N (ω)-nitro-L-arginine methyl ester, eliminates the secretory effect of L-arginine and Penicillin G.
Methods: Rat distal colons were harvested and placed in solution for crypt isolation. Glands were maintained in a thermostatically controlled perfusion chamber and loaded with a pH indicator dye 2',7'-Bis(2-carboxyethyl)-5(6)- carboxyfluorescein,acetoxymethyl ester (BCECF) to measure intracellular pH in real time. Proton extrusion, a measure of acid secretion of the individual cells, was monitored by observing the recovery of pH, as previously described. A higher pH recovery rate indicates a higher rate of cellular secretion, i.e. diarrhea. Rat colonic crypts cells were perfused with the following solutions: 1.) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES + 5 mM Penicillin G sodium salt (Control) 2.) HEPES + 10 mM L-arginine 2.) HEPES + 5 mM Penicillin G + 10 mM L-arginine 3.) HEPES + 5 mM Penicillin G + 30 μM L-NAME. Statistical analysis of data was carried out with Graphpad Prism 7.0 software.
Results: The mean rate of pH recovery in the control was 0.00623 ± 0.00026 ΔpHi/min, p 0.0004. L-arginine administration increased H,KATPase activity 0.00097 ± 0.00011 Δ pHi/min, p 0.0002. L-arginine + Penicillin G administration led to a greater, statistically significant increase in H-KATPase activity (Figure 1, 0.00883 ± 0.00051 ΔpHi/min, p 0.0004). Exposing crypts to a combination of L-NAME and Penicillin G provoked a significant reduction in H,KATPase activity (0.00292 ± 0.0001 ΔpHi/min, p 0.0022).
Conclusion: It is well-known that antibiotic exposure disrupts the microbiome, leading to diarrhea. We propose a novel mechanism of antibiotic-associated diarrhea through Penicillin G’s activation of the H,KATPase. Maximum fluid secretion was found with Penicillin G and L-arginine, a nitric oxide precursor. In contrast, fluid secretion was significantly decreased when tissues were exposed to L-NAME, a known NO pathway inhibitor. This study may provide new therapeutic opportunities to address AAD in clinical settings by modulation of the colonic H,KATPase.