Mechanisms underlying the anti-inflammatory effects of the Ca2+/calmodulin antagonist CV-159 in cultured vascular smooth muscle cells.
CV-159 is a unique dihydropyridine Ca(2+) antagonist with an anti-calmodulin (CaM) action. A pathogenic feature of atherosclerosis is vascular inflammatory change. In the present study, we examined whether CV-159 exerts protective effects on smooth muscle inflammatory responses. After pretreatment of rat mesenteric arterial smooth muscle cells (SMCs) with CV-159 (0.1 - 10 microM, 30 min), TNF-alpha (10 ng/ml) was applied for 20 min or 24 h. CV-159 inhibited TNF (24 h)-induced vascular cell adhesion molecule (VCAM)-1 as determined by Western blotting. CV-159 inhibited TNF (20 min)-induced phosphorylation of Akt (Ser473) and NF-kappaB p65 (Ser536). An Akt inhibitor, LY294002, and an NF-kappaB inhibitor, pyrrolidine dithiocarbamate, inhibited TNF-induced VCAM-1. An antioxidant drug, N-acetyl-L-cysteine (NAC) inhibited TNF-induced VCAM-1. NAC also inhibited TNF-induced phosphorylation of Akt and NF-kappaB. Furthermore, CV-159 inhibited TNF-induced reactive oxygen species (ROS) production as determined fluorometrically using dichlorodihydrofluorescein diacetate. A CaM inhibitor, W-7, and a calcium/CaM-dependent protein kinase type II inhibitor, KN93, inhibited TNF-induced VCAM-1. W-7 and KN93 inhibited TNF-induced phosphorylation of Akt but not NF-kappaB. The present results indicate that in vascular SMCs, CV-159 inhibits TNF-induced VCAM-1 through inhibition of NF-kappaB and Akt phosphorylation. CV-159 prevents NF-kappaB phosphorylation by inhibiting ROS, while it prevents Akt phosphorylation by inhibiting both ROS and CaM.
Usui
Tatsuya
T
Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Aomori, Japan.
Yamawaki
Hideyuki
H
Kamibayashi
Masato
M
Okada
Muneyoshi
M
Hara
Yukio
Y
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002318:Cardiovascular Diseases / Q000188:drug therapy; D002478:Cells, Cultured; D004095:Dihydropyridines / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009994:Osmolar Concentration; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000975:Antioxidants; D002147:Calmodulin; D004095:Dihydropyridines; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; C055634:CV 159; D051057:Proto-Oncogene Proteins c-akt; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2