Novel Lipophilic Probe for Detecting Near-Membrane Reactive Oxygen Species Responses and Its Application for Studies of Pancreatic Acinar Cells: Effects of Pyocyanin and L-Ornithine

Abstract

The aim of this study was to develop a fluorescent reactive oxygen species (ROS) probe, which is preferentially localized in cellular membranes and displays a strong change in fluorescence upon oxidation. We also aimed to test the performance of this probe for detecting pathophysiologically relevant ROS responses in isolated cells. We introduced a novel lipophilic ROS probe dihydrorhodamine B octadecyl ester (H2RB-C18). We then applied the new probe to characterize the ROS changes triggered by inducers of acute pancreatitis in pancreatic acinar cells. We resolved ROS changes produced by L-ornithine, L-arginine, cholecystokinin-8, acetylcholine, taurolithocholic acid 3-sulfate, palmitoleic acid ethyl ester, and the bacterial toxin pyocyanin. Particularly prominent ROS responses were induced by pyocyanin and L-ornithine. These ROS responses were accompanied by changes in cytosolic Ca(2+)concentration ([Ca(2+)]i), mitochondrial membrane potential (ΔΨ), and NAD(P)H concentration. The study describes a novel sensitive lipophilic ROS probe. The probe is particularly suitable for detecting ROS in near-membrane regions and therefore for reporting the ROS environment of plasma membrane channels and pumps. In our experimental conditions, the novel probe was more sensitive than 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-H2DCF) and dihydrorhodamine123 (H2R123) and allowed us to resolve ROS responses to secretagogues, pyocyanin, and L-ornithine. Changes in the fluorescence of the new probe were particularly prominent in the peripheral plasma membrane-associated regions. Our findings suggest that the new probe will be a useful tool in studies of the contribution of ROS to the pathophysiology of exocrine pancreas and other organs/tissues.