Hypertonicity inhibits lipopolysaccharide-induced nitric oxide synthase expression in smooth muscle cells by inhibiting nuclear factor kappaB.
The expression of inducible nitric-oxide synthase (iNOS) in vascular smooth muscle cells leads to prolonged vasorelaxation in vivo and contributes to the profound vasodilation induced by bacterial lipopolysaccharide (LPS) in septic shock. This induction of iNOS depends, in large part, on activation of nuclear factor (NF)-kappaB. Hypertonicity regulates the activity of NF-kappaB in different cell lines; as such, we propose that it should also regulate the expression of iNOS. Thus, the goal of this study was to determine whether hypertonicity regulates iNOS expression and function in smooth muscle cells and to elucidate the mechanism(s) underlying this process. Treatment of hamster ductus deferens (DDT1MF-2) cells and porcine aortic smooth muscle cells with either mannitol (50 mM) or NaCl (50 mM) reduced LPS-stimulated iNOS expression and nitric oxide release. Both of these agents also reduced the activation of NF-kappaB induced by LPS, tumor necrosis factor-alpha and interleukin-1beta in smooth muscle cells. This inhibitory action was caused by suppression of IkappaB-alpha phosphorylation, a prerequisite for ubiquitination and degradation of this protein, and showed additivity with N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132), an inhibitor of proteasomal degradation of IkappaB-alpha. Furthermore, exposure to mannitol inhibited the activity of IkappaB kinase, an enzyme involved in phosphorylation of IkappaB-alpha. Mannitol was unable to affect the induction of iNOS produced by overexpression of RelA in DDT1MF-2 cells, suggesting that this agent does not have additional downstream inhibitory actions on this activated NF-kappaB subunit. Taken together, these data suggest that these hypertonic solutions may prove useful as anti-inflammatory agents, especially against conditions associated with increased NF-kappaB activity.
Pingle
Sandeep C
SC
Department of Pharmacology, Southern Illinois University School of Medicine, Box 19230, Springfield, IL 62974-1222, USA.
Sanchez
Joseph F
JF
Hallam
Daniel M
DM
Williamson
Andrea L
AL
Maggirwar
Sanjay B
SB
Ramkumar
Vickram
V
D000818:Animals; D006224:Cricetinae; D004234:Diuretics, Osmotic / Q000494:pharmacology; D004347:Drug Interactions; D015870:Gene Expression / Q000187:drug effects; D051550:I-kappa B Kinase; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008353:Mannitol / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D013552:Swine
D004234:Diuretics, Osmotic; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D009569:Nitric Oxide; D008353:Mannitol; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D017346:Protein Serine-Threonine Kinases; D051550:I-kappa B Kinase