title: Contribution of iNOS/sGC/PKG pathway, COX-2, CYP4A1, and gp91(phox) to the protective effect of 5,14-HEDGE, a 20-HETE mimetic, against vasodilation, hypotension, tachycardia, and inflammation in a rat model of septic shock.
abstract: We have previously demonstrated that a stable synthetic analog of 20-hydroxyeicosatetraenoic acid (20-HETE), N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), prevents vascular hyporeactivity, hypotension, tachycardia, and inflammation in rats treated with lipopolysaccharide (LPS) and mortality in endotoxemic mice. These changes were attributed to decreased production of inducible nitric oxide (NO) synthase (iNOS)-derived NO, cyclooxygenase (COX)-2-derived vasodilator prostanoids, and proinflammatory mediators associated with increased cyctochrome P450 (CYP) 4A1-derived 20-HETE and CYP2C23-dependent antiinflammatory mediator formation. The aim of this study was to determine whether decreased expression and activity of iNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG), COX-2, gp91(phox) (NOX2; a superoxide generating NOX enzyme), and peroxynitrite production associated with increased expression of COX-1 and CYP4A1 and 20-HETE formation in renal and cardiovascular tissues of rats contributes to the effect of 5,14-HEDGE to prevent vasodilation, hypotension, tachycardia, and inflammation in response to systemic administration of LPS. Mean arterial pressure fell by 28mmHg and heart rate rose by 47beats/min in LPS (10mg/kg, i.p.)-treated rats. Administration of LPS also increased mRNA and protein expression of iNOS and COX-2 associated with a decrease in COX-1 and CYP4A1 mRNA and protein expression. Increased NOS activity, iNOS-heat shock protein 90 complex formation (an index for iNOS activity), protein expression of phosphorylated vasodilator stimulated phosphoprotein (an index for PKG activity), gp91(phox), p47(phox) (NOXO2; organizer subunit of gp91(phox)), and nitrotyrosine (an index for peroxynitrite production) as well as cGMP (an index for sGC activity), 6-keto-PGF1α (a stable metabolite PGI2) and PGE2 levels (indexes for COX activity), and nitrotyrosine levels by LPS were also associated with decreased CYP hydroxylase activity as measured by 20-HETE formation from arachidonic acid in renal microsomes of LPS-treated rats. These effects of LPS, except iNOS mRNA and COX-1 protein expression, were prevented by 5,14-HEDGE (30mg/kg, s.c.; 1h after LPS). A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (30mg/kg, s.c.; 1h after LPS) reversed the effects of 5,14-HEDGE, except iNOS and COX-1 mRNA and protein expression as well as expression of CYP4A1 mRNA. These results suggest that increased CYP4A1 expression and 20-HETE formation associated with suppression of iNOS/sGC/PKG pathway, COX-2, and gp91(phox) participate in the protective effect of 5,14-HEDGE against vasodilation, hypotension, tachycardia, and inflammation in the rat model of septic shock.
authors.affiliation: Department of Pharmacology, Faculty of Pharmacy, Mersin University, 33169 Mersin, Turkey. tunctanb@yahoo.com
authors.forename: Bahar
authors.initials: B
authors.lastname: Tunctan
authors.forename: Belma
authors.initials: B
authors.lastname: Korkmaz
authors.forename: Ayse Nihal
authors.initials: AN
authors.lastname: Sari
authors.forename: Meltem
authors.initials: M
authors.lastname: Kacan
authors.forename: Demet
authors.initials: D
authors.lastname: Unsal
authors.forename: Mehmet Sami
authors.initials: MS
authors.lastname: Serin
authors.forename: C Kemal
authors.initials: CK
authors.lastname: Buharalioglu
authors.forename: Seyhan
authors.initials: S
authors.lastname: Sahan-Firat
authors.forename: Tuba
authors.initials: T
authors.lastname: Cuez
authors.forename: Wolf-Hagen
authors.initials: WH
authors.lastname: Schunck
authors.forename: Vijaya L
authors.initials: VL
authors.lastname: Manthati
authors.forename: John R
authors.initials: JR
authors.lastname: Falck
authors.forename: Kafait U
authors.initials: KU
authors.lastname: Malik
mesh_terms: D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D006162:Guanylate Cyclase / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology; D055666:Lipopeptides / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D009928:Organ Specificity; D030421:Peroxynitrous Acid / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D020011:Protective Agents / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012772:Shock, Septic / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D000071756:Soluble Guanylyl Cyclase; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: C494667:20-hydroxyeicosa-6(Z),15(Z)-dienoic acid; D015815:Cell Adhesion Molecules; C000717351:Cyp2c23 protein, rat; D018841:HSP90 Heat-Shock Proteins; D006893:Hydroxyeicosatetraenoic Acids; D055666:Lipopeptides; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; C533498:N-(20-hydroxyeicosa-5,14-dienoyl)glycine; D010750:Phosphoproteins; D020011:Protective Agents; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D000098662:Vasodilator-Stimulated Phosphoprotein; D030421:Peroxynitrous Acid; D003577:Cytochrome P-450 Enzyme System; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D000090762:Cytochrome P-450 CYP2J2; D051546:Cyclooxygenase 2; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase
keywords: 1,3-PBIT; 20-HEDE; 20-HETE; 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid; 20-hydroxyeicosatetraenoic acid; 5,14-HEDGE; AA; COX; COX-2; CYP; CYP4A1; EET; ELISA; ERK; Endotoxin; HR; Hypotension; IKK; IL; IκB; IκB kinase; LPS; MAP; MAPK; MEK; N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine; NADPH; NF-κB; NO; PG; PKG; RT-PCR; TNF; VASP; arachidonic acid; cDNA; cGMP; complementary deoxyribonucleic acid; cyclic guanosine monophosphate; cyclooxygenase; cyctochrome P450; eNOS; endothelial nitric oxide synthase; enzyme-linked immunosorbent assay; epoxyeicosatrienoic acid; extracellular signal-regulated kinase; gp91(phox)/NOX2; heart rate; heat shock protein; hsp; i.p.; iNOS; iNOS/sGC/PKG pathway; inducible nitric oxide synthase; inhibitor of κB; interleukin; intraperitoneally; lipopolysaccharide; mRNA; mean arterial pressure; messenger ribonucleic acid; mitogen-activated protein kinase; mitogen-activated protein kinase kinase 1; nicotinamide adenine dinucleotide phosphate; nitric oxide; nuclear factor-κB; p-VASP; phenylene-1,3-bis(ethane-2-isothiourea) dihydrobromide; phosphorylated vasodilator stimulated phosphoprotein; prostaglandin; protein kinase G; reverse transcription-polymerase chain reaction; s.c.; sEH; sGC; soluble epoxide hydrolase; soluble guanylyl cyclase; subcutaneously; tumor necrosis factor; vasodilator stimulated phosphoprotein