2-2--(hydroxynitrosohydrazono)bis-ethanamine and 3-(5--hydroxymethyl-2--furyl)-1-benzylindazole

The aim of the study was to evaluate the effect of Nitric oxide (NO) on redox changes and fat accumulation in hepatocytes. AML-12 hepatocytes were exposed to the NO donor Diethylenetriamine-NONOate (DETA-NO). DETA-NO led to a dose- and time-dependent increase in lipid accumulation in the cells, measured by Nile red fluorescence. Exposure of the cells to 1mM DETA-NO for 24h increased reactive oxygen species production, mainly peroxides. At the same time, NO induced elevation of reduced glutathione (GSH) and a mild activation of the antioxidant transcription factors Hypoxia-inducible factor 1α (HIF1α) and NF-E2 related factor 2 (Nrf-2). We used 100 μM YC-1 to inhibit HIF1α activity and induce activation of soluble Guanylate Cyclase (sGC). YC-1 alone did not affect fat accumulation, and only moderately increased the expression of Nrf-2-targeted genes Heme oxygenase 1 (Hmox1), NAD(P)H dehydrogenase (quinone 1) (Nqo1) and Glutathione S-transferase α1 (Gstα1). However, YC-1 abolished the negative effect of NO on fat accumulation when administered together. Strikingly, YC-1 potentiated the effect of NO on Nrf-2 activation, thus increasing dramatically the antioxidant properties of NO. Moreover, YC-1 intensified the effect of NO on the expression of peroxisome-proliferator-activated receptor-gamma co-activator 1α (PGC1α) and mitochondrial biogenesis markers. This study suggests that YC-1 may shift the deleterious effects of NO into the beneficial ones, and may improve the antioxidant properties of NO.

Topics: Animals; Antioxidants; Glutathione; Heme Oxygenase-1; Hepatocytes; Humans; Indazoles; Mice; NF-E2-Related Factor 2; Nitric Oxide; Nitroso Compounds; Transforming Growth Factor alpha

This study was designed to compare the effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), a nitric oxide (NO)-independent soluble guanylate cyclase activator, and diethylenetriamine-NONOate (DETA/NO), a NO donor, on spontaneous contractions and the levels of cyclic GMP (cGMP) of myometrial strips isolated from timed-pregnant rats. Myometrial strips were obtained from timed-pregnant Wistar albino rats (n=10) and were mounted in organ baths and tested for changes in isometric tension in response to YC-1 and DETA/NO. We also evaluated the effect of YC-1 and DETA/NO on the levels of cGMP in myometrial strips obtained from timed-pregnant rat uterine horns (n=20). YC-1 (10(-9)-3x10(-5) M) and DETA/NO (10(-7)-10(-4) M) concentration-dependently decreased the amplitude and frequency of spontaneous contractions of myometrial strips isolated from term-pregnant rats. The inhibitions of the amplitude and frequency of spontaneous contractions by YC-1 and DETA/NO were antagonized with methylene-blue (10(-5) M). Antagonistic effect of methylene-blue (10(-5) M) was more on DETA/NO responses than that of YC-1 (P<0.05). In addition, YC-1-stimulated myometrial strips showed more elevation in myometrial cGMP than that of DETA/NO (P<0.05). We demonstrated that YC-1 and DETA/NO induce relaxations in the amplitude and frequency of spontaneous contractions of myometrial strips with different potencies. We also found that YC-1 and DETA/NO-induced relaxations are associated with significant increases in cGMP. These results might suggest that the relaxant effects of YC-1 and DETA/NO on the rat myometrium could be due to the stimulation of the soluble guanylate cyclase and cGMP may play a role for the maintenance of uterine quiescence during pregnancy.

Topics: Animals; Cyclic GMP; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Indazoles; Male; Methylene Blue; Muscle Relaxation; Myometrium; Nitric Oxide Donors; Pregnancy; Rats; Rats, Wistar; Time Factors; Triazenes; Uterine Contraction

NO and cGMP have antigrowth and anti-inflammatory effects on the vessel wall in response to injury. It is well established that after vascular injury proinflammatory cytokines are involved in vascular wall remodeling. The purpose of this study was to ascertain the signaling mechanisms involved in cGMP-dependent protein kinase (PKG) suppression by inflammatory cytokines in primary bovine aortic vascular smooth muscle cells (VSMC). Interleukin (IL)-Ibeta, tumor necrosis factor (TNF)-alpha, and LPS decreased the mRNA and protein levels of PKG in VSMC. IL-Ibeta, TNF-alpha, and LPS increased inducible nitric oxide synthase (iNOS) expression and cGMP production. Treatment of cells with selective inhibitors of iNOS or soluble guanylate cyclase (sGC) reversed the downregulation of PKG expression induced by cytokines and LPS. The NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) and 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), a NO-independent sGC activator, decreased PKG mRNA and protein expression in bovine aortic VSMC. Cyclic nucleotide analogs [8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP) and 8-(4-chlorophenylthio)adenosine 3,5'-cyclic monophosphate (CPT-cAMP)] also suppressed PKG mRNA and protein expression. However, CPT-cAMP was more effective than CPT-cGMP in decreasing PKG mRNA levels. Selective inhibition of PKA with the Rp isomer of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphorothioate (Rp-8p-CPT cAMPS) prevented the downregulation of PKG by LPS. In contrast, the Rp isomer of 8-(4-chlorophenylthio)guanosine 3,5'-cyclic monophosphorothioate (Rp-8p-CPT cGMPS; inhibitor of PKG) had no effect on LPS-induced inhibition of PKG mRNA and protein expression. These studies suggest that cross-activation of PKA in response to iNOS expression by inflammatory mediators downregulates PKG expression in bovine aortic VSMC.

Topics: Animals; Aorta; Cattle; Cells, Cultured; Cyclic GMP-Dependent Protein Kinases; Down-Regulation; Enzyme Activators; Enzyme Inhibitors; Humans; Indazoles; Interleukin-1; Lipopolysaccharides; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitric Oxide Donors; Nitroso Compounds; Nucleotides, Cyclic; Oxadiazoles; Quinoxalines; Recombinant Proteins; Tumor Necrosis Factor-alpha