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

Antioxid Redox Signal. 2015 Feb 20;22(6):451-64. doi: 10.1089/ars.2013.5589. Epub 2014 Apr 30.

Abstract

Aims: 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.

Results: 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.

Innovation: 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.

Conclusions: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acinar Cells / drug effects
  • Acinar Cells / ultrastructure
  • Animals
  • Calcium / metabolism
  • Cell Membrane / metabolism
  • Male
  • Mice
  • Molecular Probes / chemistry
  • Molecular Probes / pharmacology*
  • Ornithine / pharmacology*
  • Oxidants / pharmacology*
  • Pancreas / cytology
  • Pancreas / drug effects
  • Pancreatitis, Acute Necrotizing / metabolism
  • Pyocyanine / pharmacology*
  • Reactive Oxygen Species / metabolism*
  • Rhodamines / chemistry
  • Rhodamines / pharmacology*

Substances

  • Molecular Probes
  • Oxidants
  • Reactive Oxygen Species
  • Rhodamines
  • dihydrorhodamine B octadecyl ester
  • Pyocyanine
  • Ornithine
  • Calcium