s-nitro-n-acetylpenicillamine and diphenyleneiodonium

We reported that insulin stimulates NAD(P)H oxidase activity but not migration of cultured rat vascular smooth muscle cells (VSMCs). Because angiotensin II (Ang II) increases NAD(P)H oxidase activity in these cells, we wished to determine whether insulin stimulates migration of Ang II-treated VSMCs by synergistically stimulating enzyme activity.. Cultured rat VSMC superoxide anion (O2-) production, cyclic GMP production, and migration were measured by lucigenin luminescence, immunoassay, and wound closure rate, respectively. Nitric oxide (NO) scavenging was measured by inhibition of NO-induced fluorescence of 4-5-diaminofluorescin.. Insulin (1 nmol/L) did not affect and Ang II (100 nmol/L) stimulated VSMC migration by 65% (P < .05), but together stimulated it by 150% (P < .05 versus Ang II) by a mechanism inhibited by the NAD(P)H oxidase inhibitors, diphenyleneiodonium (DPI) or gp91ds-tat. Insulin and Ang II stimulated O2- production by 34% and 35%, respectively (both P < .05), but together synergistically stimulated it by 143% (P < .05 versus insulin or Ang II) in a DPI or gp91ds-tat-sensitive manner. Neither insulin nor Ang II measurably affected NO scavenging, but together reduced NO availability by 46% in a DPI-sensitive manner (P < .05) and significantly inhibited NO-stimulated cyclic GMP production.. Insulin synergestically stimulates NAD(P)H oxidase activity in Ang II-treated cultured rat VSMCs causing increased migration.

Topics: Aminoquinolines; Angiotensin II; Animals; Cell Movement; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Glycoproteins; Guanylate Cyclase; Insulin; Male; Muscle, Smooth, Vascular; NADPH Oxidases; Nitric Oxide Donors; Onium Compounds; Penicillamine; Rats; Rats, Sprague-Dawley; Superoxides; Vasoconstrictor Agents

Regardless of the underlying pathological mechanisms oxidative stress seems to be present in all forms of hypertension. Thus, we tested the hypothesis that chronic presence of high pressure itself elicits increased arterial O(2)(.-) production. Hypertension was induced in rats by abdominal aortic banding (Ab). Rats with Ab had elevated pressure in vessels proximal and normal pressure in vessels distal to the coarctation, yet both vascular beds were exposed to the same circulating factors. Compared to normotensive hind limb arteries (HLAs) hypertensive forelimb arteries (FLAs) exhibited 1) impaired dilations to acetylcholine and the nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine that were restored by administration of superoxide dismutase; 2) an increased production of O(2)(.-) (measured by lucigenin chemiluminescence and ethidium bromide fluorescence) that was inhibited or reduced by superoxide dismutase, the NAD(P)H oxidase inhibitors diphenyleneiodonium and apocynin, or the protein kinase C (PKC) inhibitors chelerythrine and staurosporine or by the angiotensin-converting enzyme (ACE) inhibitor captopril; and 3) increased ACE activity. In organ culture, exposure of isolated arteries of normotensive rats to high pressure (160 mmHg, for 24 hours) significantly increased O(2)(.-) production compared to that in arteries exposed to 80 mmHg. High pressure-induced O(2)(.-) generation was reduced by inhibitors of ACE and PKC. Incubation of cultured arteries with angiotensin II elicited significantly increased O(2)(.-) generation that was inhibited by chelerythrine. Thus, we propose that chronic presence of high pressure itself can elicit arterial oxidative stress, primarily by activating directly a PKC-dependent NAD(P)H oxidase pathway, but also, in part, via activation of the local renin-angiotensin system.

Topics: Acetophenones; Acetylcholine; Alkaloids; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Arteries; Benzophenanthridines; Captopril; Enzyme Inhibitors; Hypertension; Male; Models, Biological; NADPH Oxidases; Nitric Oxide Donors; Nitric Oxide Synthase; Onium Compounds; Organ Culture Techniques; Oxidative Stress; Penicillamine; Phenanthridines; Protein Kinase C; Rats; Rats, Wistar; Renin-Angiotensin System; Staurosporine; Superoxide Dismutase; Vasoconstrictor Agents; Vasodilator Agents

Treatment of human hepatoma cells (HepG2) with NO-donors for 24 h inhibited hypoxia-induced erythropoietin (EPO) gene activation. NO was found to increase the production of reactive oxygen species (ROS), the putative signaling molecules between a cellular O2-sensor and hypoxia inducible factor 1 (HIF-1). HIF-1 is the prime regulator of O2-dependent genes such as EPO. NO-treatment for more than 20 h reduced HIF-1-driven reporter gene activity. In contrast, immediately after the addition of NO, ROS levels in HepG2 cells decreased below control values for as long as 4 h. Corresponding to these lowered ROS-levels, HIF-1 reporter gene activity and EPO gene expression transiently increased but were reduced when ROS levels rose thereafter. Our findings of a bimodal effect of NO on ROS production shed new light on the involvement of ROS in the mechanism of O2-sensing and may explain earlier conflicting data about the effect of NO on O2-dependent gene expression.

Topics: Acridines; Anaerobiosis; Carcinoma, Hepatocellular; DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Hydrogen Peroxide; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; NADH, NADPH Oxidoreductases; NADPH Oxidases; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nuclear Proteins; Onium Compounds; Oxygen; Penicillamine; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Spermine; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured