title: Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.
abstract: This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).
authors.affiliation: Dipartimento di Biochimica "G. Moruzzi,", Università degli Studi di Bologna, via Irnerio 48, Bologna, 40126, Italy. bzanella@biocfarm.unibo.it
authors.forename: B
authors.initials: B
authors.lastname: Zanella
authors.forename: N
authors.initials: N
authors.lastname: Calonghi
authors.forename: E
authors.initials: E
authors.lastname: Pagnotta
authors.forename: L
authors.initials: L
authors.lastname: Masotti
authors.forename: C
authors.initials: C
authors.lastname: Guarnieri
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D019793:Fluorescein / Q000737:chemistry; D005470:Fluorometry; D007202:Indicators and Reagents / Q000737:chemistry; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000737:chemistry* / Q000201:enzymology; D009569:Nitric Oxide / Q000032:analysis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: C414689:4,5-diaminofluorescein diacetate; D007202:Indicators and Reagents; D020033:Protein Isoforms; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D000109:Acetylcholine; D019793:Fluorescein