title: The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers.
abstract: We report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca(2+) marker in enzymatically dissociated mouse FBD muscle fibers. Using laser scanning confocal microscopy and the dyes Mitotracker Green (MTG), di-8-ANEPPS and endoplasmic reticulum tracker green (ERTG), we determined the relative position of mitochondria, transverse tubules and sarcoplasmic reticulum in the sarcomere. Comparison with electron micrographies showed that mitochondria are mostly present at both sides of Z lines and near the triads located at the A-I band border. CaOr-5N fluorescence was mainly distributed in mitochondria, highly co-localised with MTG and basically excluded from the A band space. ERTG localised mostly between the two t-tubules present in each sarcomere. We studied the effect of the protonophore FCCP using CaOr-5N to measure mitochondrial Ca(2+) and JC-1 dye to measure mitochondria inner membrane potential (DeltaPsi(m)). After FCCP treatment, the CaOr-5N fluorescence diminished by about 33% in 80 s, while JC-1 fluorescence diminished by 36% in 200 s. Our results show the loss of Ca(2+) from mitochondria when DeltaPsi(m) is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial [Ca(2+)](m).
authors: Laboratorio de Fisiología Celular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas IVIC, Apartado 21827, Caracas 1020A, Venezuela.
authors: Pura
authors: P
authors: Bolaños
authors: Alis
authors: A
authors: Guillen
authors: Héctor
authors: H
authors: Rojas
authors: Simona
authors: S
authors: Boncompagni
authors: Carlo
authors: C
authors: Caputo
mesh_terms: D000447:Aldehydes; D000818:Animals; D001562:Benzimidazoles; D002118:Calcium / Q000378:metabolism*; D002232:Carbocyanines; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D005456:Fluorescent Dyes; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018613:Microscopy, Confocal; D008931:Mitochondria, Muscle / Q000378:metabolism* / Q000648:ultrastructure; D018485:Muscle Fibers, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D009930:Organic Chemicals; D011726:Pyridinium Compounds; D012518:Sarcomeres / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors; D014475:Uncoupling Agents / Q000494:pharmacology
chemical_list: C089669:1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine; D000447:Aldehydes; D001562:Benzimidazoles; C093686:CaOrange-5N; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011726:Pyridinium Compounds; D014475:Uncoupling Agents; C111472:mitotracker green FM; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002118:Calcium
references: Cell Calcium. 2006 Nov-Dec;40(5-6):513-25
references: 17067669
references: J Neurosci. 1994 Jan;14(1):348-56
references: 8283242
references: J Neurosci. 2000 Oct 1;20(19):7290-6
references: 11007886
references: Pflugers Arch. 2002 Feb;443(4):508-19
references: 11907817
references: Methods Enzymol. 1995;260:417-28
references: 8592464
references: J Physiol. 2003 Aug 15;551(Pt 1):179-90
references: 12815178
references: Pflugers Arch. 2008 Jan;455(4):733-43
references: 17676335
references: Biochem J. 2000 Apr 15;347(Pt 2):593-600
references: 10749691
references: Hum Reprod. 2003 Nov;18(11):2429-40
references: 14585897
references: Am J Physiol Cell Physiol. 2001 Jul;281(1):C114-22
references: 11401833
references: Am J Physiol Cell Physiol. 2006 Apr;290(4):C1199-208
references: 16306125
references: Eur J Biochem. 2000 Feb;267(3):877-84
references: 10651826
references: Cell Tissue Res. 1985;241(2):251-6
references: 4028126
references: FEBS J. 2006 Jan;273(1):96-108
references: 16367751
references: Cell Calcium. 2003 Apr;33(4):247-56
references: 12618145
references: J Biol Chem. 2006 Jan 20;281(3):1547-54
references: 16216882
references: J Gen Physiol. 2001 Jul;118(1):79-82
references: 11429445
references: J Physiol. 2001 Jul 1;534(Pt 1):87-97
references: 11432994
references: J Physiol. 1986 Mar;372:261-92
references: 3487641
references: J Physiol. 1994 Dec 1;481 ( Pt 2):251-71
references: 7738824
references: Am J Physiol Renal Physiol. 2005 Jun;288(6):F1092-102
references: 15625081
references: Am J Physiol. 1996 Dec;271(6 Pt 1):E1044-50
references: 8997224
references: J Physiol. 2003 Mar 1;547(Pt 2):453-62
references: 12562922
references: J Physiol. 1998 May 15;509 ( Pt 1):59-65
references: 9547381
references: J Muscle Res Cell Motil. 1999 Aug;20(5-6):555-67
references: 10555074
references: J Physiol. 2000 Nov 15;529 Pt 1:69-81
references: 11080252
references: Am J Physiol Heart Circ Physiol. 2005 Apr;288(4):H1820-8
references: 15563537
references: Invest Ophthalmol Vis Sci. 2005 Dec;46(12):4541-7
references: 16303946
references: J Muscle Res Cell Motil. 2001;22(3):265-75
references: 11763199
references: Physiol Rev. 1999 Oct;79(4):1127-55
references: 10508231
references: J Biol Chem. 1993 Oct 15;268(29):21939-45
references: 8408050
references: J Physiol. 2000 Nov 15;529 Pt 1:37-47
references: 11080249
references: Biophys J. 1996 Feb;70(2):896-916
references: 8789107
references: J Physiol. 2005 Jun 15;565(Pt 3):855-72
references: 15845582
references: J Physiol. 1999 Jul 15;518 ( Pt 2):433-47
references: 10381590
references: Am J Physiol Cell Physiol. 2006 Nov;291(5):C840-50
references: 16723510
references: J Cell Biol. 2004 Aug 16;166(4):527-36
references: 15314066
references: Pflugers Arch. 2002 May;444(1-2):236-43
references: 11976937
references: J Physiol. 2005 May 1;564(Pt 3):775-90
references: 15746171
references: J Muscle Res Cell Motil. 2004;25(4-5):315-28
references: 15548860
references: Biochim Biophys Acta. 2005 May 15;1744(1):19-28
references: 15878394
references: J Physiol. 1998 Mar 1;507 ( Pt 2):379-403
references: 9518700
references: J Physiol. 2003 Aug 15;551(Pt 1):125-38
references: 12813151
references: Nature. 2004 Jan 22;427(6972):360-4
references: 14737170
references: Cell Calcium. 1999 Nov;26(5):193-9
references: 10643557