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

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

The thromboxane receptor (TPr) and multiple TPr ligands, including thromboxane A(2) (TxA(2)) and prostaglandin H(2), are elevated during vascular and atherothrombotic diseases. How TPr stimulation causes vascular injury remains poorly defined. This study was conducted to investigate the mechanism by which TPr stimulation leads to vascular injury.. Exposure of bovine aortic endothelial cells to either [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(d'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1] heptan-2-yl]-5'-heptenoic acid (IBOP) or U46619, 2 structurally related TxA(2) mimetics, for 24 hours markedly increased the release of superoxide anions (O(2)(·-)) and peroxynitrite (ONOO(-)) but reduced cyclic GMP, an index of nitric oxide bioactivity. IBOP also significantly suppressed activity of endothelial nitric oxide synthase (eNOS), increased enzyme-inactive eNOS monomers, and reduced levels of tetrahydrobiopterin, an essential eNOS cofactor. IBOP- and U46619-induced increases in O(2)(·-) were accompanied by the membrane translocation of the p67(phox) subunit of NAD(P)H oxidase. Pharmacological or genetic inhibition of either NAD(P)H oxidase or TPr abolished IBOP-induced O(2)(·-) formation. Furthermore, TPr activation significantly increased protein kinase C-ζ (PKC-ζ) in membrane fractions and PKC-ζ phosphorylation at Thr410. Consistently, PKC-ζ inhibition abolished TPr activation-induced membrane translocation of p67(phox) and O(2)(·-) production. Finally, exposure of isolated mouse aortae to IBOP markedly increased O(2)(·-) in wild-type but not in those from gp91(phox) knockout mice.. We conclude that TPr activation via PKC-ζ-mediated NAD(P)H oxidase activation increases both O(2)(·-) and ONOO(-), resulting in eNOS uncoupling in endothelial cells.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Apoptosis; Biopterins; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Cell Survival; Cells, Cultured; Cyclic GMP; Cytochrome P-450 Enzyme System; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Peroxynitrous Acid; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Transport; Receptors, Immunologic; Receptors, Thromboxane A2, Prostaglandin H2; RNA Interference; Signal Transduction; Superoxides; Time Factors; Tyrosine

The 4-amino analogue of tetrahydrobiopterin (4-ABH(4)) is a potent pterin-site inhibitor of nitric oxide synthases (NOS). Although 4-ABH(4) does not exhibit selectivity between purified NOS isoforms, a pronounced selectivity of the drug towards inducible NOS (iNOS) is apparent in intact cells. This work was carried out to investigate the potential iNOS selectivity of 4-ABH(4) in isolated pig pulmonary and coronary arteries. Endothelium-dependent relaxations of pig pulmonary and coronary artery strips to bradykinin or calcium ionophore A23187 were inhibited by 4-ABH(4) in a concentration-dependent manner. Half-maximal inhibition was observed at 60 - 65 microM (pulmonary artery) and 200 - 250 microM 4-ABH(4) (coronary artery). Pig coronary artery strips precontracted with 0.1 microM 9, 11-dideoxy-9, 11-methanoepoxy-prosta-glandin F(2alpha) (U46619) showed a time-dependent relaxation (monitored for up to 18 h) upon incubation with 1 microg ml(-1) lipopolysaccharide (LPS). Addition of 10 microM 4-ABH(4) 1 h after LPS led to a pronounced inhibition of the LPS-triggered relaxation, whereas the pterin antagonist had no effect when given> or =4 h after LPS. Incubation of pulmonary and coronary artery strips with 1 microg ml(-1) LPS attenuated contractile responses to norepinephrine (1 microM) and U46619 (0.1 microM). This hyporeactivity of the blood vessels to vasoconstrictor agents was inhibited by 4-ABH(4) in a concentration-dependent manner [IC(50)=17.5+/-5.9 microM (pulmonary artery) and 20.7+/-3 microM (coronary artery)]. The effect of 0.1 mM 4-ABH(4) was antagonized by coincubation with 0.1 mM sepiapterin, which is known to supply intracellular BH(4) via a salvage pathway. These results demonstrate that 4-ABH(4) is a fairly selective inhibitor of iNOS in an in vitro model of endotoxaemia, suggesting that this drug and/or related pterin-site NOS inhibitors may be useful to increase blood pressure in severe infections associated with a loss of vascular responsiveness to constrictor agents caused by endotoxin-triggered iNOS induction in the vasculature.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biopterins; Bradykinin; Calcimycin; Coronary Vessels; Dose-Response Relationship, Drug; Endothelium, Vascular; Endotoxins; Enzyme Inhibitors; Hydrazines; In Vitro Techniques; Lipopolysaccharides; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Nitrogen Oxides; Norepinephrine; Pteridines; Pterins; Pulmonary Artery; Swine; Vasoconstriction; Vasoconstrictor Agents; Vasodilation