Hypoxia inactivates inducible nitric oxide synthase in mouse macrophages by disrupting its interaction with alpha-actinin 4.
Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-gamma induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 +/- 14 micro M, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 +/- 1.7 micro M, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and alpha-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.
Daniliuc
Sharon
S
Immunology Research Unit and Ischemia-Shock Research Laboratory, Carmel Medical Center, Rappaport Family Institute for Research in the Medical Sciences, and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.
Bitterman
Haim
H
Rahat
Michal A
MA
Kinarty
Amalia
A
Rosenzweig
Doron
D
Lahat
Nitza
N
Nitza
Lahat
L
D000185:Actinin / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D003571:Cytochalasin B / Q000494:pharmacology; D019281:Dimerization; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000502:physiology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000037:antagonists & inhibitors / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000201:enzymology / Q000378:metabolism
C491752:Actn4 protein, mouse; D008840:Microfilament Proteins; D009573:Nitrites; D000185:Actinin; D009569:Nitric Oxide; D003571:Cytochalasin B; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D010100:Oxygen