Clonidine-induced enhancement of iNOS expression involves NF-kappaB.
BACKGROUND
Up-regulation of inducible nitric oxide synthase (iNOS) plays a crucial role in initiating systemic inflammatory response during sepsis. Clonidine, a widely used anti-hypertension agent and an effective adjunct to anesthesia/sedation and pain management, has been shown to enhance iNOS expression, but the mechanisms underlying its action remain unstudied. Among the possible mechanisms, enzyme induction and enzyme stability are two most likely ones. Endotoxin-induced iNOS induction is regulated by nuclear factor-kappaB (NF-kappaB). Stability of iNOS mRNA is regulated by RNA stabilizing factor, e.g., Hu antigen R (HuR), and RNA destabilizing factors, e.g., AU-rich element/poly(U) binding factor-1 (AUF-1) and tristetraprolin (TTP). We sought to elucidate which of these enzymes is involved in the clonidine-induced enhancement of iNOS expression.


METHODS
Confluent murine macrophages were randomized to receive 1x phosphated buffer saline, clonidine (100 mum), lipopolysaccharide (LPS, 100 ng/mL), or LPS plus clonidine (100 microm). Expression of iNOS and stability of iNOS mRNA were then measured. Expression of the aforementioned relevant enzymes in each group was also analyzed.


RESULTS
Clonidine significantly enhanced LPS-induced iNOS expression. Clonidine also significantly enhanced LPS-induced NF-kappaB activation by enhancing the nuclear translocation of NF-kappaB as well as increasing the NF-kappaB-DNA binding activity. However, clonidine did not affect iNOS mRNA stability. The LPS-induced expression of AUF-1 and TTP but not HuR was significantly enhanced by clonidine.


CONCLUSIONS
NF-kappaB is involved in the clonidine-induced enhancement of iNOS expression in endotoxin-activated macrophages. Clonidine exerts its action on iNOS expression through increasing enzyme induction instead of enzyme stability.
Su
Nuan-Yen
NY
Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan.
Tsai
Pei-Shan
PS
Huang
Chun-Jen
CJ
D000322:Adrenergic Agonists / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D003000:Clonidine / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D015854:Up-Regulation
D000322:Adrenergic Agonists; D016328:NF-kappa B; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D003000:Clonidine