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.

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.

Tunctan B ，Korkmaz B ，Sari AN ，Kacan M ，Unsal D ，Serin MS ，Buharalioglu CK ，Sahan-Firat S ，Cuez T ，Schunck WH ，Manthati VL ，Falck JR ，Malik KU ... -  《-》

5,14-HEDGE, a 20-HETE mimetic, reverses hypotension and improves survival in a rodent model of septic shock: contribution of soluble epoxide hydrolase, CYP2C23, MEK1/ERK1/2/IKKβ/IκB-α/NF-κB pathway, and proinflammatory cytokine formation.

We have previously demonstrated that a stable synthetic analog of 20-HETE, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), restores vascular reactivity, blood pressure, and heart rate in endotoxemic rats. The aim of this study was to determine whether decreased renal expression and activity of soluble epoxide hydrolase (sEH), MEK1, ERK1/2, IKKβ, IκB-α, and NF-κB as well as systemic and renal proinflammatory cytokine production associated with increased expression and activity of CYP2C23 contributes to the effect of 5,14-HEDGE to prevent hypotension, tachycardia, inflammation, and mortality in response to systemic administration of lipopolysaccharide (LPS). Blood pressure fell by 33 mmHg and heart rate rose by 57 beats/min in LPS (10 mg/kg, i.p.)-treated rats. Administration of LPS also increased mRNA and protein expression of sEH associated with a decrease in CYP2C23 mRNA and protein expression. Increased activity of sEH and p-MEK1, p-ERK1/2, p-IκB-α, NF-κB, and p-NF-κB protein levels as well as TNF-α and IL-8 production by LPS were also associated with a decreased activity of AA epoxygenases. These effects of LPS were prevented by 5,14-HEDGE (30 mg/kg, s.c.; 1 h after LPS). Treatment of endotoxemic mice with 5,14-HEDGE also raised the survival rate of animals from 84% to 98%. A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, 20-HEDE (30 mg/kg, s.c.; 1 h after LPS) prevented the effects of 5,14-HEDGE on blood pressure, heart rate, expression and/or activity of sEH, CYP2C23, and ERK1/2 as well as TNF-α and IL-8 levels in rats treated with LPS. These results suggest that decreased expression and/or activity of sEH and MEK1/ERK1/2/IKKβ/IκB-α/NF-κB pathway as well as proinflammatory cytokine production associated with increased CYP2C23 expression and antiinflammatory mediator formation participate in the protective effect of 5,14-HEDGE against hypotension, tachycardia, inflammation, and mortality in the rodent model of septic shock.

Tunctan B ，Korkmaz B ，Sari AN ，Kacan M ，Unsal D ，Serin MS ，Buharalioglu CK ，Sahan-Firat S ，Cuez T ，Schunck WH ，Falck JR ，Malik KU ... -  《PROSTAGLANDINS & OTHER LIPID MEDIATORS》

Effects of 5,14-HEDGE, a 20-HETE mimetic, on lipopolysaccharide-induced changes in MyD88/TAK1/IKKβ/IκB-α/NF-κB pathway and circulating miR-150, miR-223, and miR-297 levels in a rat model of septic shock.

Sari AN ，Korkmaz B ，Serin MS ，Kacan M ，Unsal D ，Buharalioglu CK ，Sahan Firat S ，Manthati VL ，Falck JR ，Malik KU ，Tunctan B ... -  《-》

Activation of MEK1/ERK1/2/iNOS/sGC/PKG pathway associated with peroxynitrite formation contributes to hypotension and vascular hyporeactivity in endotoxemic rats.

Increased production of inducible nitric oxide (NO) synthase (iNOS)-derived NO contributes to fall in blood pressure and vascular reactivity during endotoxemia. We investigated whether an increase in protein expression and activity of the enzymes involved in mitogen-activated protein kinase kinase 1 (MEK1)/extracellular signal-regulated kinase 1/2 (ERK1/2)/iNOS/soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway associated with peroxynitrite production would contribute to endotoxin-induced decrease in mean blood pressure (MAP) and vascular reactivity in rats. A selective iNOS inhibitor, 1,3-PBIT (10 mg/kg, i.p.), or a selective inhibitor ERK1/2 phosphorylation by MEK1, U0126 (5mg/kg, i.p.), prevented endotoxin (10mg/kg, i.p.)-induced decrease in MAP and vascular reactivity to norepinephrine (0.001-100 μM) in endothelium-intact and -denuded arteries associated with increased levels of nitrite (an index for NO production), cyclic GMP (an index for sGC activity), phosphorylated vasodilator stimulated phosphoprotein (an index for PKG activity), and nitrotyrosine (an index for peroxynitrite production). Endotoxin-induced increase in the phosphorylated MEK1 protein levels were not changed by 1,3-PBIT or U0126. U0126 prevented the endotoxin-induced increase in phosphorylated ERK1/2 and iNOS expressions. A selective sGC inhibitor, ODQ (3μM), prevented the endotoxin-induced decrease in the E(max) values and increase in the EC(50) values of norepinephrine in endothelium-intact aortic rings isolated from endotoxemic rats in vitro. ODQ also reversed the effect of endotoxin on the increase in the EC(50) values of norepinephrine in endothelium-denuded rings. A selective PKG inhibitor, KT5823 (1 μM), only prevented the endotoxin-induced decrease in the E(max) values of norepinephrine in arteries with endothelium. These results suggest that activation of MEK1/ERK1/2 pathway leading to an increase in iNOS protein expression and NO production associated with an increase in sGC and PKG activity and peroxynitrite formation results in hypotension and vascular hyporeactivity in endotoxemic rats. However, further study is needed to confirm the involvement of PKG to the fall in vascular reactivity in the rat model of endotoxemia.

Korkmaz B ，Buharalioglu K ，Sahan-Firat S ，Cuez T ，Tuncay Demiryurek A ，Tunctan B ... -  《-》

A synthetic analogue of 20-HETE, 5,14-HEDGE, reverses endotoxin-induced hypotension via increased 20-HETE levels associated with decreased iNOS protein expression and vasodilator prostanoid production in rats.

Cuez T ，Korkmaz B ，Buharalioglu CK ，Sahan-Firat S ，Falck J ，Malik KU ，Tunctan B ... -  《-》