title: Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3β inhibitor, SB216763.
abstract: OBJECTIVE
  Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3β (GSK-3β) in regulating mMPT. Using direct inhibition of GSK-3β with the GSK-3β inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential.
  
  
  METHODS
  Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O₂) followed by exposure to atmospheric oxygen (approximately 21% O₂). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability.
  
  
  RESULTS
  Treatment of HLE-B3 cells with SB216763 (12 µM), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment.
  
  
  CONCLUSIONS
  Inhibition of GSK-3β activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3β activity by the GSK-3β inhibitor SB216763 provides positive protection against mitochondrial depolarization.
authors.affiliation: Department of Cell Biology and Anatomy, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX, USA.
authors.forename: Morgan M
authors.initials: MM
authors.lastname: Brooks
authors.forename: Sudha
authors.initials: S
authors.lastname: Neelam
authors.forename: Rafal
authors.initials: R
authors.lastname: Fudala
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Cammarata
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D016477:Artifacts; D001562:Benzimidazoles / Q000494:pharmacology*; D002232:Carbocyanines / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D011419:Propidium / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence
chemical_list: D017304:Annexin A5; D001562:Benzimidazoles; D002232:Carbocyanines; D007211:Indoles; D008301:Maleimides; D047428:Protein Kinase Inhibitors; C417521:SB 216763; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D011419:Propidium; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D016650:Fluorescein-5-isothiocyanate