title: Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia.
abstract: Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K(ATP) channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia.
authors.affiliation: Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. zlacza@wfubmc.edu
authors.forename: Z
authors.initials: Z
authors.lastname: Lacza
authors.forename: M
authors.initials: M
authors.lastname: Puskar
authors.forename: J P
authors.initials: JP
authors.lastname: Figueroa
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: N
authors.initials: N
authors.lastname: Rajapakse
authors.forename: D W
authors.initials: DW
authors.lastname: Busija
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001921:Brain / Q000201:enzymology; D003429:Cross Reactions / Q000276:immunology; D004789:Enzyme Activation; D000860:Hypoxia / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D015854:Up-Regulation
chemical_list: D000906:Antibodies; D007093:Imidazoles; C031938:calmidazolium; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse