cyclic-gmp and calpeptin

Cinnamyl alcohol (CAL) is known as an antipyretic, and a recent study showed its vasodilatory activity without explaining the mechanism. Here we demonstrate the vasodilatory effect and the mechanism of action of CAL in rat thoracic aorta. The change of tension in aortic strips treated with CAL was measured in an organ bath system. In addition, vascular strips or human umbilical vein endothelial cells (HUVECs) were used for biochemical experiments such as Western blot and nitrite and cyclic guanosine monophosphate (cGMP) measurements. CAL attenuated the vasoconstriction of phenylephrine (PE, 1 μM)-precontracted aortic strips in an endothelium-dependent manner. CAL-induced vasorelaxation was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 10⁻⁴ M), methylene blue (MB; 10⁻⁵ M) and 1 H-[1,2,4]-oxadiazolole-[ 4,3-a] quinoxalin-10one, (ODQ; 10⁻⁶ or 10⁻⁷ M) in the endothelium-intact aortic strips. Atrial natriuretic peptide (ANP; 10⁻⁸ or 10⁻⁹ M) did not affect the vasodilatory effect of CAL. The phosphorylation of endothelial nitric oxide synthase (eNOS) and generation of nitric oxide (NO) were stimulated by CAL treatment in HUVECs and inhibited by treatment with L-NAME. In addition, cGMP and PKG1 activation in aortic strips treated with CAL were also significantly inhibited by L-NAME. Furthermore, CAL relaxed Rho-kinase activator calpeptin-precontracted aortic strips, and the vasodilatory effect of CAL was inhibited by the ATP-sensitive K⁺ channel inhibitor glibenclamide (Gli; 10⁻⁵ M) and the voltage-dependent K⁺ channel inhibitor 4-aminopyridine (4-AP; 2 × 10⁻⁴ M). These results suggest that CAL induces vasorelaxation by activating K⁺ channels via the NO-cGMP-PKG pathway and the inhibition of Rho-kinase.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dipeptides; Human Umbilical Vein Endothelial Cells; Humans; In Vitro Techniques; Male; Methylene Blue; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxadiazoles; Phenylephrine; Phosphorylation; Potassium Channel Blockers; Potassium Channels; Propanols; Quinoxalines; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Signal Transduction; Vasoconstriction; Vasodilation

Nitric oxide is an important messenger that regulates mast cell activity by modifications to gene expression and intracellular pathways associated with exocytosis and adhesion. Integrin interactions with extracellular matrix components modulate an array of cell activities, including mediator production and secretion. To investigate the molecular mechanisms underlying NO regulation of mast cell function, we studied its effects on adhesion of a human mast cell line (HMC-1) to fibronectin (FN). The NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine strongly down-regulated the adhesion of HMC-1 to FN. Inhibitors of soluble guanylate cyclase and protein kinase G did not alter the response of cells to NO. A peroxynitrite scavenger did not affect modulation of adhesion by NO, nor could the effect of NO be mimicked by the peroxynitrite-producing compound 3-morpholinosydnonimine. NO donors inhibited the cysteine protease, calpain, while calpain inhibitors mimicked the effect of NO and led to a decrease in the ability of HMC-1 cells to adhere to FN. Thus, NO is an effective down-regulator of human mast cell adhesion. The mechanism for this action does not involve peroxynitrite or activation of soluble guanylate cyclase. Instead, a portion of NO-induced down-regulation of adhesion may be attributed to inhibition of the cysteine protease, calpain, an enzyme that has been associated with control of integrin activation in other cell types. The inhibition of calpain is most likely mediated via nitrosylation of its active site thiol group. Calpain may represent a novel therapeutic target for the regulation of mast cell activity in inflammatory disorders.

Topics: Calpain; Cell Adhesion; Cyclic GMP; Dipeptides; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Fibronectins; Humans; Leupeptins; Mast Cells; Nitric Oxide; Nitric Oxide Donors; Peroxynitrous Acid; Protein Binding; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione; Tumor Cells, Cultured