15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and acetovanillone

Treatment of human pulmonary artery smooth muscle cells (HPASMCs) with the thromboxane A2 receptor antagonist, SQ29548 inhibited U46619 stimulation of phospholipase D (PLD) and NADPH oxidase activities in the cell membrane. Pretreatment with apocynin inhibited U46619 induced increase in NADPH oxidase activity. The cell membrane contains predominantly PLD2 along with PLD1 isoforms of PLD. Pretreatment with pharmacological and genetic inhibitors of PLD2, but not PLD1, attenuated U46619 stimulation of NADPH oxidase activity. U46619 stimulation of PLD and NADPH oxidase activities were insensitive to BFA and Clostridium botulinum C3 toxin; however, pretreatment with secinH3 inhibited U46619 induced increase in PLD and NADPH oxidase activities suggesting a major role of cytohesin in U46619-induced increase in PLD and NADPH oxidase activities. Arf-1, Arf-6, cytohesin-1 and cytohesin-2 were observed in the cytosolic fraction, but only Arf-6 and cytohesin-1 were translocated to the cell membrane upon treatment with U46619. Coimmunoprecipitation study showed association of Arf-6 with cytohesin-1 in the cell membrane fraction. In vitro binding of GTPγS with Arf-6 required the presence of cytohesin-1 and that occurs in BFA insensitive manner. Overall, BFA insensitive Arf6-cytohesin1 signaling axis plays a pivotal role in U46619-mediated activation of PLD leading to stimulation of NADPH oxidase activity in HPASMCs.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetophenones; ADP Ribose Transferases; ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Antioxidants; Botulinum Toxins; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Cell Membrane; Fatty Acids, Unsaturated; Gene Expression Regulation; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hydrazines; Myocytes, Smooth Muscle; NADPH Oxidases; Phospholipase D; Primary Cell Culture; Protein Synthesis Inhibitors; Pulmonary Artery; Receptors, Thromboxane A2, Prostaglandin H2; Signal Transduction; Triazoles; Vasoconstrictor Agents

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose-alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0-1000 μmol/L) was measured in response to 5-25 mmol/L D-glucose (0-36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose-increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose-increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2•-) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2•- generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose-increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose-reduced BH4 level. Insulin and tempol blocked the high D-glucose-increased p42/44mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O2•-/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetophenones; Arginine; Biopterins; Calcitonin Gene-Related Peptide; Cationic Amino Acid Transporter 1; Cyclic N-Oxides; Gene Expression Regulation; Glucose; Human Umbilical Vein Endothelial Cells; Humans; Insulin; Mitogen-Activated Protein Kinase 1; Nitric Oxide; Nitric Oxide Synthase Type III; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Spin Labels; Tissue Culture Techniques; Transcription Factors; Umbilical Veins; Vasoconstrictor Agents

To investigate whether superoxide mediates angiotensin (Ang) II-induced vasoconstriction.. Human coronary arteries (HCAs), porcine femoral arteries (PFA) and porcine coronary arteries (PCAs) were mounted in organ baths and concentration-response curves to Ang II, the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) and the NAD(P)H oxidase substrate NADH were constructed in the absence and presence of superoxide inhibiting and activating drugs. Extracellular superoxide was measured using cytochrome c reduction.. Ang II constricted both HCAs and PFAs. In HCAs, the NAD(P)H inhibitors diphenyleneiodonium (DPI) and apocynin, and the xanthine oxidase (XO) inhibitor allopurinol, but not the superoxide dismutase (SOD) mimetic tempol or the SOD inhibitor diethyldithiocarbamate (DETCA), reduced this constriction. Catalase potentiated Ang II in HCAs, indicating a vasodilator role for H2O2. DPI, tempol and SOD did not affect Ang II in PFAs. DPI, apocynin and allopurinol relaxed preconstricted HCAs. Although the relaxant effects of the NO donor SNAP in PCAs was reduced by DETCA, indicating that superoxide-induced constrictions depend on NO inactivation, the apocynin-induced relaxations were NO independent. Moreover, NADH relaxed all vessels, and this effect was blocked by KCl but not DPI or NO removal. Xanthine plus XO also relaxed HCAs and PCAs. Incubation of human or porcine arteries with Ang II or NADH did not result in detectable increases of extracellular superoxide within 1 h.. Acute vasoconstriction by Ang II is not mediated via superoxide generated through NAD(P)H oxidase and/or XO activation. Such activation, if occurring, rather results in the generation of the vasodilator H2O2.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetophenones; Adolescent; Adult; Angiotensin II; Animals; Catecholamines; Child; Child, Preschool; Coronary Vessels; Cyclic N-Oxides; Dopamine Agonists; Dose-Response Relationship, Drug; Endothelin-1; Enzyme Inhibitors; Female; Femoral Artery; Free Radical Scavengers; Humans; Imidazolines; Male; Myocardial Contraction; NAD; NADP; Nitroprusside; Oxidants; S-Nitroso-N-Acetylpenicillamine; Spin Labels; Superoxide Dismutase; Superoxides; Swine; Time Factors; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Xanthine Oxidase