dizocilpine-maleate and diphenyleneiodonium

Homocysteine (Hcy) is known as an independent risk factor for atherosclerosis. C-reactive protein (CRP) directly participates in initiation and progression of atherosclerosis. However, there is no direct evidence to demonstrate pro-inflammatory effect of Hcy on vascular smooth muscle cells (VSMCs) through CRP. In the present study, we examined the effect of Hcy on CRP expression and investigated the related mechanism in VSMCs.. Protein expression and secretion were detected by Western blot and ELISA, respectively. mRNA expression was detected by RT-PCR. Superoxide anion was detected by lucigenin chemiluminometry and the immunofluorescence staining was observed by a fluorescence microscope. The results revealed that Hcy significantly induced mRNA and protein expressions of CRP in VSMCs both in vitro and in vivo, and anti-IL-1β or anti-IL-6 neutralizing antibody alone or in combination partially reduced Hcy-induced CRP expression. Hcy increased the expression of NR1 subunit of N-methyl-d-aspartate receptor (NMDAr), and MK-801 alleviated Hcy-induced CRP expression in VSMCs. Further studies showed that Hcy-stimulated superoxide anion generation in VSMCs. Nevertheless, pretreatment of the cells with MK-801, TTFA and DPI significantly reduced Hcy-stimulated superoxide anion generation, and antioxidant NAC decreased Hcy-induced CRP expression in VSMCs. Additionally, PD98059, SB205380 or PDTC antagonized Hcy-induced CRP expression, and MK-801, NAC, PD98059 or SB205380 inhibited Hcy-activated phosphorylations of ERK1/2 and p38.. The present study demonstrates that Hcy is able to initiate an inflammatory response in VSMCs by stimulating CRP production, which is mediated through NMDAr-ROS-ERK1/2/p38-NF-κB signal pathway. These findings provide new evidence for a role of Hcy in pathogenesis of atherosclerosis.

Topics: Animals; Atherosclerosis; C-Reactive Protein; Cells, Cultured; Dizocilpine Maleate; Gene Expression Regulation; Homocysteine; Hyperhomocysteinemia; Interleukins; Male; MAP Kinase Signaling System; Methionine; Mitogen-Activated Protein Kinases; Myocytes, Smooth Muscle; NF-kappa B; Onium Compounds; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Signal Transduction; Superoxides; Thenoyltrifluoroacetone

Binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) was significantly inhibited by the addition of several different compounds related to generation of nitric oxide (NO) at 100 microM in rat brain synaptic membranes. These included sodium nitroprusside, diphenyliodonium (DI), diphenyleneiodonium (DPI) and methylene blue. However, neither S-nitroso-N-acetylpenicillamine nor S-nitroso-L-glutathione inhibited binding at 100 microM. Both DI and DPI inhibited binding in a concentration-dependent manner at a concentration range of over 1 microM, while further addition of spermidine (SPD) significantly attenuated the potency of DPI to inhibit binding without affecting that of DI. In contrast, SPD induced significant potentiation of the ability of unlabelled MK-801 to displace [3H]MK-801 binding in a fashion sensitive to antagonism by the novel polyamine antagonist bis-(3-aminopropyl)nonanediamine. This novel polyamine antagonist also prevented the reversing effect of SPD on inhibition by DPI of [3H]MK-801 binding. Moreover, DPI competitively exacerbated the ability of SPD to potentiate [3H]MK-801 binding in the presence of both L-glutamic acid and glycine at maximally effective concentrations. On the other hand, SPD was effective in reversing the inhibition by DPI in cerebellar, but not hippocampal, synaptic membranes. These results suggest that both DI and DPI may modulate synaptic responses mediated by the N-methyl-D-aspartate receptor through inhibition of opening processes of the ion channel in a manner irrespective of generation of NO radicals in particular situations. Possible involvement of the polyamine domain in the inhibition by DPI is also suggested.

Topics: Animals; Biphenyl Compounds; Brain; Diamines; Dizocilpine Maleate; Drug Evaluation, Preclinical; Drug Synergism; Enzyme Inhibitors; Free Radicals; Male; Nitric Oxide Synthase; Onium Compounds; Radioligand Assay; Rats; Rats, Wistar; Spermidine; Synaptic Membranes