n-hydroxy-n--(4-butyl-2-methylphenyl)formamidine and Hypertension

In addition to being metabolized by cyclooxygenase and lipooxygenase to prostaglandins and leukotrienes, arachidonic acid can be metabolized to 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P450 enzymes omega-hydroxylases. As 20-HETE has both pro-hypertensive and antihypertensive actions, inhibitors of 20-HETE synthase may not be useful as antihypertensives in all forms of hypertension. However, 20-HETE synthase inhibitors can have cardioprotective and cerebroprotective effects in animal models, and can inhibit angiogenesis; therefore they may have clinical potential in these areas.

Topics: Amidines; Angiogenesis Inhibitors; Animals; Cerebrovascular Circulation; Enzyme Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Hypertension; Myocardial Reperfusion Injury; Oligonucleotides, Antisense

20-Hydroxyeicosatetraenoicacid (20-HETE) is an important mediator that regulates vascular tone and blood pressure (BP). Although various experimental animal hypertension models demonstrated that 20-HETE contributes to increased vascular resistance and BP, these effects have not been studied in Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertension model. In this study, we investigated the effects of 20-HETE on the vascular responsiveness and BP in an L-NAME-induced hypertension. Wistar Albino rats were used in this study. Hypertension was induced by the addition of L-NAME to drinking water for 5 weeks. The study was performed in three stages: first, BP changes were monitored in real time in the presence of 20-HETE enzymatic inhibitor, N-hydroxy-N´-(4-butly-2-methylphenyl)-formamidine (HET-0016) for 1 h. Second, vascular responses of the conduit and resistance arteries were investigated in the presence or absence of HET-0016 in the organ bath. Third, BP was monitored weekly in some hypertensive animals treated with HET-0016 and vascular responses were investigated at the end of the experiment. We demonstrated an increase in 20-HETE levels in the resistance arteries of hypertensive animals. 20-HETE inhibition by HET-0016 significantly decreased BP in L-NAME-induced hypertension model. In addition, HET-0016 treatment caused significant improvement in vascular dilator and constrictor responses in the conduit and resistance arteries. This study demonstrates an important role of 20-HETE in increasing BP and altering vascular responsiveness in L-NAME-induced hypertension model, which suggests a possible involvement of 20-HETE in essential hypertension development in humans.

Topics: Amidines; Animals; Blood Pressure; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Hypertension; Male; NG-Nitroarginine Methyl Ester; Rats; Rats, Wistar; Tissue Culture Techniques; Vascular Resistance; Vasoconstriction; Vasodilation

We previously generated cytochrome P450 4F2 (CYP4F2) transgenic mice and showed high 20-hydroxyeicosatetraenoic acid (20-HETE) production, which resulted in an elevation of blood pressure. However, it was unclear whether 20-HETE affected glucose metabolism. We measured fasting plasma glucose, insulin, hepatic CYP4F2 expression, and 20-HETE production by hepatic microsomes, and hepatic 20-HETE levels in transgenic mice. We also assessed glycogen phosphorylase (GP) activity and the cAMP/protein kinase A (PKA)-phosphorylase kinase (PhK)-GP pathway, as well as expressions of insulin receptor substrate 1 and glucose transporters in vivo and in vitro. The transgenic mice had overexpressed hepatic CYP4F2, high hepatic 20-HETE and fasting plasma glucose levels but normal insulin level. The GP activity was increased and the cAMP/PKA-PhK-GP pathway was activated in the transgenic mice compared with wild-type mice. Moreover, these alterations were eliminated with the addition of N-hydroxy-N'-(4-butyl-2 methylphenyl) formamidine, which is a selective 20-HETE inhibitor. The results were further validated in Bel7402 cells. In addition, the transgenic mice had functional insulin signaling, and 20-HETE had no effect on insulin signaling in Bel7402 cells, excluding that the observed hyperglycemia in CYP4F2 transgenic mice resulted from insulin dysfunction, because the target tissues were sensitive to insulin. Our study suggested that 20-HETE can induce hyperglycemia, at least in part, through the cAMP/PKA-PhK-GP pathway but not through the insulin-signaling pathway.

Topics: Amidines; Animals; Blood Glucose; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytochrome P-450 Enzyme System; Glucose Transporter Type 1; Glycogen Phosphorylase; Hydroxyeicosatetraenoic Acids; Hyperglycemia; Hypertension; Insulin Receptor Substrate Proteins; Isoquinolines; Liver; Male; Mice; Mice, Transgenic; Phosphorylase Kinase; Phosphorylation; Signal Transduction; Sulfonamides

The objective of this study was to determine if acute inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis or reduced inactivation of epoxyeicosatrienoic acids (EETs) can correct L-N(G)-nitro-arginine-methyl-ester (L-NAME)-induced abnormal vascular reactivity in the perfused mesenteric bed and the carotid artery of spontaneously hypertensive rats (SHR). Administration of L-NAME in drinking water (80 mg/l) to SHR for 3 weeks resulted in abnormal vascular reactivity to norepinephrine and carbachol in the perfused mesenteric vascular bed and carotid artery, and significantly elevated mean arterial blood pressure (244 +/- 9 mm Hg) as compared to SHR controls drinking regular water (176 +/- 3 mm Hg). In the perfused mesenteric vascular bed, the impaired vascular responsiveness to norepinephrine was corrected by acute treatment with N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016), an inhibitor of 20-HETE formation, but not by 1-cyclohexyl-3-dodecyl urea (CDU), an inhibitor of soluble epoxide hydrolase. Treatment with either HET0016 or CDU did not improve impaired carbachol-induced vasodilation in the perfused mesenteric vascular bed. In the isolated carotid artery, treatment with HET0016 corrected the L-NAME-induced increase in norepinephrine-induced vasoconstriction, whereas only CDU treatment could improve impaired carbachol-induced vasodilation. Results of this study indicate that vascular function in a state of compromised nitric oxide formation is differentially modulated by 20-HETE and EETs, and that treatment with HET0016 or CDU may improve vascular function in a state of high blood pressure and endothelial dysfunction.

Topics: Amidines; Animals; Arachidonic Acids; Blood Pressure; Carbachol; Cardiovascular Physiological Phenomena; Carotid Arteries; Endothelial Cells; Hydroxyeicosatetraenoic Acids; Hypertension; Male; Norepinephrine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Urea; Vascular Diseases; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

We have shown previously that increased vascular endothelial expression of CYP4A2 leads to 20-hydroxyeicosatetraenoic (20-HETE)-dependent hypertension. The renin-angiotensin system is a key regulator of blood pressure. In this study, we examined possible interactions between 20-HETE and the renin-angiotensin system. In normotensive (110±3 mm Hg) Sprague-Dawley rats transduced with a lentivirus expressing the CYP4A2 cDNA under the control of an endothelial-specific promoter (VECAD-4A2), systolic blood pressure increased rapidly, reaching 139±1, 145±3, and 150±2 mm Hg at 3, 5, and 10 days after transduction; blood pressure remained elevated, thereafter, with maximum levels of 163±3 mm Hg. Treatment with lisinopril, losartan, or the 20-HETE antagonist 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid decreased blood pressure to control values, but blood pressure returned to its high levels after cessation of treatment. Endothelial-specific overexpression of CYP4A2 resulted in increased expression of vascular angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor and increased levels of plasma and tissue angiotensin II; all were attenuated by treatment with HET0016, an inhibitor of 20-HETE synthesis, or with 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid. In cultured endothelial cells, 20-HETE specifically and potently induced ACE expression without altering the expression of ACE2, angiotensinogen, or angiotensin II receptors. This is the first study to demonstrate that 20-HETE, a key constrictor eicosanoid in the microcirculation, induces ACE and angiotensin II type 1 receptor expression and increases angiotensin II levels, suggesting that the mechanisms by which 20-HETE promotes hypertension include activation of the renin-angiotensin system that is likely initiated at the level of ACE induction.

Topics: Amidines; Analysis of Variance; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Blotting, Western; Cells, Cultured; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Hydroxyeicosatetraenoic Acids; Hypertension; Lentivirus; Lisinopril; Losartan; Mass Spectrometry; Oligonucleotide Array Sequence Analysis; Peptidyl-Dipeptidase A; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Renin-Angiotensin System; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

20-hydroxyeicosatetraenoic acid (20-HETE) is a potent constrictor in small arteries and also has natriuretic properties. Urinary 20-HETE excretion is increased in adrenocorticotrophic hormone (ACTH)-induced hypertensive rats. In the present study, we investigated the effect of a specific enzyme inhibitor of 20-HETE production, N-hydroxy-N'-(4-butyl-2-methylphenyl) formamidine (HET0016), on glucocorticoid-induced hypertension in rats, a sodium-independent model.. Male Sprague-Dawley rats were treated with physiological saline (0.9% NaCl), ACTH (0.2 mg/kg per day) or dexamethasone (0.03 mg/rat per day) subcutaneously for 13 days. HET0016 (10 mg/kg per day) or its vehicle (10% lecithin in physiological saline) was coadministered (intraperitoneally) a day before (prevention study) or at day 8 of treatment (reversal studies). Systolic blood pressure was measured by the tail-cuff method.. Relative to physiological saline, systolic blood pressure was increased by ACTH (P < 0.001) and dexamethasone (P < 0.01). HET0016 reversed ACTH-induced (P < 0.01) but not dexamethasone-induced hypertension. HET0016 also prevented the development of hypertension induced by ACTH (P < 0.01). ACTH, but not dexamethasone, increased renal microsome 20-HETE formation and plasma F2-isoprostane concentrations. HET0016 inhibited renal 20-HETE formation but had no effect on plasma F2-isoprostane concentrations or renal cytochrome P450 4A1 expression.. Inhibition of 20-HETE production by HET0016 prevents and reverses ACTH-induced but not dexamethasone-induced hypertension. These results suggest that 20-HETE may play a role in the genesis of ACTH-induced hypertension but not in dexamethasone-induced hypertension.

Topics: Adrenocorticotropic Hormone; Amidines; Animals; Body Weight; Dexamethasone; F2-Isoprostanes; Hydroxyeicosatetraenoic Acids; Hypertension; Kidney; Male; Organ Size; Rats; Rats, Sprague-Dawley; Reactive Nitrogen Species; Systole

Hypertension is a major risk factor for stroke, but the factors that contribute to the increased incidence and severity of ischemic stroke in hypertension remain to be determined. 20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to be a potent constrictor of cerebral arteries, and inhibitors of 20-HETE formation reduce infarct size following cerebral ischemia. The present study examined whether elevated production of 20-HETE in the cerebral vasculature could contribute to the larger infarct size previously reported after transient middle cerebral artery occlusion (MCAO) in hypertensive strains of rat [spontaneously hypertensive rat (SHR) and spontaneously hypertensive stroke-prone rat (SHRSP)]. The synthesis of 20-HETE in the cerebral vasculature of SHRSP measured by liquid chromatography-tandem mass spectrometry was about twice that seen in Wistar-Kyoto (WKY) rats. This was associated with the elevated expression of cytochrome P-450 (CYP)4A protein and CYP4A1 and CYP4A8 mRNA. Infarct volume after transient MCAO was greater in SHRSP (36+/-4% of hemisphere volume) than in SHR (19+/-5%) or WKY rats (5+/-2%). This was associated with a significantly greater reduction in regional cerebral blood flow (rCBF) in SHR and SHRSP than in WKY rats during the ischemic period (78% vs. 62%). In WKY rats, rCBF returned to 75% of control following reperfusion. In contrast, SHR and SHRSP exhibited a large (166+/-18% of baseline) and sustained (1 h) postischemic hyperperfusion. Acute blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016; 1 mg/kg) reduced infarct size by 59% in SHR and 87% in SHRSP. HET0016 had no effect on the fall in rCBF during MCAO but eliminated the hyperemic response. HET0016 also attenuated vascular O2*- formation and restored endothelium-dependent dilation in cerebral arteries of SHRSP. These results indicate the production of 20-HETE is elevated in the cerebral vasculature of SHRSP and contributes to oxidative stress, endothelial dysfunction, and the enhanced sensitivity to ischemic stroke in this hypertensive model.

Topics: Amidines; Animals; Blood Pressure; Cerebral Arteries; Cerebrovascular Circulation; Cytochrome P-450 CYP4A; Disease Models, Animal; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Hypertension; Infarction, Middle Cerebral Artery; Isoenzymes; Male; Oxidative Stress; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Reactive Oxygen Species; Severity of Illness Index; Stroke; Time Factors; Up-Regulation

Epidemiological evidence suggests a role for sex-dependent mechanisms in the pathophysiology of hypertension. It has been shown that 5alpha-dihydrotestosterone (DHT) administration (56 mg/kg of body weight per day IP for 14 days) increases blood pressure, cytochrome P450 4A expression, and 20-hydroxyeicosatetraenoic acid synthesis in rats. We examined whether increased vascular 20-hydroxyeicosatetraenoic acid synthesis underlies endothelial dysfunction and hypertension in DHT-treated male Sprague-Dawley rats by using HET0016, a selective cytochrome P450 4A inhibitor. Coadministration of HET0016 (10 mg/kg per day IP for 14 days) to DHT-treated rats markedly reduced DHT-induced interlobar arterial production of 20-hydroxyeicosatetraenoic acid (14.3+/-1.5 versus 1.5+/-0.5 ng/mg of protein per hour; P<0.05), superoxide anion (246+/-47 versus 31+/-8 cpm/microg of protein), and the levels of gp91-phox, p47-phox, and 3-nitrosylated proteins. Moreover, the maximal relaxing response to acetylcholine in phenylephrine-preconstricted renal interlobar arteries from DHT-treated rats (42.8+/-4.8%) significantly (P<0.05) increased in the presence of HET0016 (81.5+/-10.8%). Importantly, the administration of HET0016 negated DHT-induced hypertension; systolic blood pressure was reduced from 146+/-2 mm Hg in DHT-treated rats to 130+/-1 mm Hg (P<0.05). The results strongly implicate vascular cytochrome P450 4A-derived 20-hydroxyeicosatetraenoic acid in the development of androgen-induced endothelial dysfunction and hypertension.

Topics: Acetylcholine; Amidines; Animals; Blood Pressure; Cytochrome P-450 CYP4A; Dihydrotestosterone; Drug Synergism; Endothelium, Vascular; Hydroxyeicosatetraenoic Acids; Hypertension; Male; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidases; Oxidative Stress; Rats; Rats, Sprague-Dawley; Renal Artery; Superoxides; Vasodilation; Vasodilator Agents

This study examined whether chronic blockade of epoxyeicosatrienoic acids (EETs) and/or 20-hydroxyeicosatetraenoic acid (20-HETE) formation promotes development of salt-sensitive hypertension. Changes in blood pressure, renal cytochrome P450 metabolism of arachidonic acid, and 20-HETE excretion in response to a high salt diet were measured in rats chronically treated with 1-aminobenzotriazole (ABT, 50 mg/kg per day) to block EETs and 20-HETE formation or N-hydroxy-N'-(4-butyl-2 methylphenyl) formamidine (HET0016, 10 mg/kg per day) that selectively reduces 20-HETE formation. ABT reduced blood pressure in rats fed a low salt (0.4% NaCl) diet, but blood pressure rose by 20 mm Hg after these rats were switched to a high salt (8% NaCl) diet for 10 days. HET0016 had no effect on blood pressure in rats fed a low salt diet; however, blood pressure rose by 18 mm Hg after the rats were fed a high salt diet. 20-HETE formation in kidney homogenates rose by 30% and epoxygenase activity doubled when rats were fed a high salt diet. Chronic treatment with ABT and HET0016 inhibited the renal formation of 20-HETE by approximately 90%. Renal epoxygenase activity decreased by 76% in ABT-treated rats and was not significantly altered in rats treated with HET0016. 20-HETE excretion rose from 470+/-21 to 570+/-41 ng/d when the rats were switched from the low to the high salt diet. 20-HETE excretion fell by 68% and 85% in rats that were chronically treated with ABT and HET0016. These results suggest that chronic blockade of the formation of 20-HETE promotes the development of salt-sensitive hypertension in rats.

Topics: Amidines; Animals; Arachidonic Acid; Blood Pressure; Hydroxyeicosatetraenoic Acids; Hypertension; Kidney; Male; Rats; Rats, Sprague-Dawley; Sodium Chloride; Triazoles