title: Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential.
abstract: Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (DeltaPsim) and the production of reactive oxygen species. Fluorescent probes were used to assess DeltaPsim (JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide), reactive oxygen species [DCF, 5- (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] and [Ca2+][Fluo-3, glycine N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-(acetyloxy)methyl ester] in human kidney cells (HK-2 cells) and in a line of human small cell carcinoma cells (GLC4 cells), because these do not express cyclosporin A-sensitive P-glycoprotein. We used transfected GLC4 cells expressing P-glycoprotein as control for GLC4 cells. NIM811 (N-methyl-4-isoleucine-cyclosporin) and PSC833 (SDZ-PSC833) were applied as selective mitochondrial permeability transition pore and P-glycoprotein blockers, respectively. To study the effect of cyclosporin A on mitochondrial function, we isolated mitochondria from fresh pig livers. Cyclosporin A and PSC833 induced a more than two-fold increase in JC-1 fluorescence in HK-2 cells, whereas NIM811 had no effect. None of the three substances induced a significant increase in JC-1 fluorescence in GLC4 cells. Despite this, cyclosporin A, NIM811 and PSC833 induced a 1.5-fold increase in DCF fluorescence (P<0.05) and a two-fold increase in Fluo-3 fluorescence (P<0.05). Studies in isolated mitochondria showed that blockage of mitochondrial permeability transition pores by cyclosporin A affected neither DeltaPsim, ATP synthesis, nor respiration rate. The mitochondrial permeability transition pore blockers cyclosporin A and NIM811, but also the non-mitochondrial permeability transition pore blocker PSC833, induced comparable degrees of reactive oxygen species production and cytosolic [Ca2+]. Neither mitochondria, effects on P-glycoprotein nor inhibition of calcineurin therefore play a role in cyclosporin A-induced oxidative stress and disturbed Ca2+ homeostasis.
authors.affiliation: Department of Internal Medicine, University Medical Center Groningen, University of Groningen, the Netherlands.
authors.forename: Marco
authors.initials: M
authors.lastname: van der Toorn
authors.forename: Henk F
authors.initials: HF
authors.lastname: Kauffman
authors.forename: Margaretha
authors.initials: M
authors.lastname: van der Deen
authors.forename: Dirk-Jan
authors.initials: DJ
authors.lastname: Slebos
authors.forename: Gerard H
authors.initials: GH
authors.lastname: Koëter
authors.forename: Rijk O B
authors.initials: RO
authors.lastname: Gans
authors.forename: Stephan J L
authors.initials: SJ
authors.lastname: Bakker
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000814:Aniline Compounds; D000818:Animals; D001562:Benzimidazoles; D002118:Calcium / Q000378:metabolism; D002232:Carbocyanines; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D066298:In Vitro Techniques; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000502:physiology; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors; D051336:Mitochondrial Membranes / Q000187:drug effects* / Q000502:physiology; D000083162:Mitochondrial Permeability Transition Pore; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D013552:Swine; D014966:Xanthenes
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000814:Aniline Compounds; D001562:Benzimidazoles; D002232:Carbocyanines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007166:Immunosuppressive Agents; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D017382:Reactive Oxygen Species; D014966:Xanthenes; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C059715:Fluo-3; D016572:Cyclosporine; D002118:Calcium