Nuclear factor-kappa B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits

Citation
Yt. Xuan et al., Nuclear factor-kappa B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits, CIRCUL RES, 84(9), 1999, pp. 1095-1109
Citations number
65
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cardiovascular & Hematology Research
Journal title
CIRCULATION RESEARCH
ISSN journal
0009-7330 → ACNP
Volume
84
Issue
9
Year of publication
1999
Pages
1095 - 1109
Database
ISI
SICI code
0009-7330(19990514)84:9<1095:NFBPAE>2.0.ZU;2-K
Abstract
Although it is recognized that late preconditioning (PC) results from upreg ulation of cardioprotective genes, the specific transcription factor(s) tha t govern this genetic adaptation remains unknown. The aim of this study was to test the hypothesis that the development of late PC is mediated by nucl ear factor-kappa B (NF-kappa B) and to elucidate the mechanisms that contro l the activation of NF-kappa B after an ischemic stimulus in vivo. A total of 152 chronically instrumented, conscious rabbits were used. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles, which elicits late PC, induced rapid activation of NF-kappa B, as evidenced by a marked increase in p65 content (+164%; Western immunoblotting) and NF-kappa B DNA binding activity (+306%; electrophoretic mobility shift assay) in nuclear e xtracts isolated 30 minutes after the last reperfusion. These changes were attenuated 2 hours after ischemic PC and resolved by 4 hours. Competition a nd supershift assays confirmed the specificity of the NF-kappa B DNA comple x signals. The mobility of the NF-kappa B DNA complex was shifted by anti-p 65 and anti-p50 antibodies but not by anti-c-Rel antibodies, indicating tha t the subunits of NF-kappa B involved in gene activation after ischemic PC consist of p65-p50 heterodimers. Pretreatment with the NF-kappa B inhibitor diethyldithiocarbamate (DDTC; 150 mg/kg IP 15 minutes before ischemic PC) completely blocked the nuclear translocation and increased DNA binding acti vity of NF-kappa B. The same dose of DDTC completely blocked the cardioprot ective effects of late PC against both myocardial stunning and myocardial i nfarction, indicating that NF-kappa B activation is essential for the devel opment of this phenomenon in vivo. The ischemic PC-induced activation of NF -kappa B was also blocked by pretreatment with N-omega-nitro-L-arginine (L- NA), a nitric oxide synthase (NOS) inhibitor, N-2-mercaptopropionyl glycine (MPG), a reactive oxygen species (ROS) scavenger, chelerythrine, a protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor ( all given at doses previously shown to block late PC), indicating that isch emic PC activates NF-kappa B via formation of NO and ROS and activation of PKC- and tyrosine kinase-dependent signaling pathways. A subcellular redist ribution and increased DNA binding activity of NF-kappa B quantitatively si milar to those induced by ischemic PC could be reproduced pharmacologically by giving the NO donor diethylenetriamine/NO (DETA/NO) (at a dose previous ly shown to elicit late PC), demonstrating that NO in itself can activate N F-kappa B in the heart. Taken together, these results provide direct eviden ce that activation of NF-kappa B is a critical step in the signal transduct ion pathway that underlies the development of the late phase of ischemic PC in conscious rabbits. The finding that four different pharmacological mani pulations (L-NA, MPG, chelerythrine, and lavendustin A) produced similar in hibition of NF-kappa B suggests that this transcription factor is a common downstream pathway through which multiple signals elicited by ischemic stre ss (NO, ROS, PKC, tyrosine kinases) act to induce gene expression. To our k nowledge, this is the first demonstration that NO can promote NF-kappa B ac tivation in the heart, a finding that identifies a new biological function of NO and may have important implications for various pathophysiological co nditions in which NO is involved and for nitrate therapy.