Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.
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(*).
Zanella
B
B
Dipartimento di Biochimica "G. Moruzzi,", Università degli Studi di Bologna, via Irnerio 48, Bologna, 40126, Italy. bzanella@biocfarm.unibo.it
Calonghi
N
N
Pagnotta
E
E
Masotti
L
L
Guarnieri
C
C
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
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