title: 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: OBJECTIVE
  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.
  
  
  METHODS
  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.
authors.affiliation: 1 Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool , Liverpool, United Kingdom .
authors.forename: Michael
authors.initials: M
authors.lastname: Chvanov
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Tao
authors.initials: T
authors.lastname: Jin
authors.forename: Li
authors.initials: L
authors.lastname: Wen
authors.forename: Jane
authors.initials: J
authors.lastname: Armstrong
authors.forename: Vicky
authors.initials: V
authors.lastname: Elliot
authors.forename: Ben
authors.initials: B
authors.lastname: Alston
authors.forename: Alex
authors.initials: A
authors.lastname: Burdyga
authors.forename: David N
authors.initials: DN
authors.lastname: Criddle
authors.forename: Robert
authors.initials: R
authors.lastname: Sutton
authors.forename: Alexei V
authors.initials: AV
authors.lastname: Tepikin
mesh_terms: D061354:Acinar Cells / Q000187:drug effects / Q000648:ultrastructure; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D008297:Male; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry / Q000494:pharmacology*; D009952:Ornithine / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology*; D010179:Pancreas / Q000166:cytology / Q000187:drug effects; D019283:Pancreatitis, Acute Necrotizing / Q000378:metabolism; D011710:Pyocyanine / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D015335:Molecular Probes; D016877:Oxidants; D017382:Reactive Oxygen Species; D012235:Rhodamines; C000602114:dihydrorhodamine B octadecyl ester; D011710:Pyocyanine; D009952:Ornithine; D002118:Calcium