Cardiac Electropharmacological Effects of Antidiarrheal Drug Loperamide and Its Antidote Naloxone in Anesthetized Guinea Pigs.

Cardiac electropharmacological effects of an antidiarrheal drug loperamide and its antidote naloxone were assessed in isoflurane-anesthetized guinea pigs. Intravenous administration of loperamide at 0.01-0.1 mg/kg did not affect parameters of electrocardiogram (ECG) or monophasic action potential (MAP) of the right ventricle. Additional administration of loperamide at 1 mg/kg prolonged the QT interval and MAP duration of the ventricle accompanied with increments of the PQ interval and QRS width. The potency of loperamide for QT-interval prolongation was about 100-times lower than that of dofetilide, in spite that similar inhibitory effects on the human Ether-a-go-go Related Gene (hERG) K+ channels have been reported between loperamide and dofetilide, implying lower accessibility of loperamide to the K+ channels. Intravenous administration of naloxone at 0.003-0.3 mg/kg, which effectively inhibits µ-opioid receptors, did not affect ECG parameters including QT interval or MAP duration. Furthermore, the loperamide-induced cardiac electrophysiological changes were not modified in the presence of naloxone at 0.3 mg/kg. These results suggest that loperamide has a potential to delay cardiac conduction and repolarization in the in vivo condition. Since naloxone did not modify ECG parameters and loperamide-induced ECG changes, naloxone is confirmed to possess acceptable cardiac safety when used as an antidote.

L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli.

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.