n-hydroxy-n--(4-butyl-2-methylphenyl)formamidine and 1-aminobenzotriazole

20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to the migration and proliferation of vascular smooth muscle cells (VSMC) in vitro, but there are few studies that address its effects on vascular remodeling in vivo. The present study determined whether inhibition of 20-HETE production attenuates intimal hyperplasia (IH) and vascular remodeling after balloon injury (BI). Sprague Dawley rats underwent BI of the common carotid artery and were treated with vehicle, 1-aminobenzotriazole (ABT, 50 mg/kg i.p. once daily), or HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (2 mg/kg s.c. twice daily) for 14 days. Fourteen days after BI and treatment, the animals underwent carotid angiography, and the arteries were harvested for morphometric, enzymatic and immunohistochemical analysis. There was a 96% reduction of angiographic stenosis in the rats treated with 1-ABT. There was a 61 and 66% reduction of the intima/media area ratios in the 1-ABT and HET0016 treated rats compared with the vehicle-treated group. 20-HETE levels were elevated in BI carotid arteries, and the levels were markedly suppressed in the groups treated with 1-ABT and HET0016 (P < 0.001). Immunostaining revealed that the expression of CYP4A enzyme was markedly increased in the neointima of BI arteries, and it colocalized with the expression of smooth muscle-specific actin, indicating increased proliferation of VSMC. An increase in the expression of CYP4A and the production of 20-HETE contributes to neointimal growth in BI rat carotid arteries. Systemic administration 1-ABT or HET0016 prevents the increase in 20-HETE levels and attenuates VSMC migration and proliferation, resulting in a marked reduction in IH and vascular remodeling after endothelial injury.

Topics: Amidines; Angioplasty, Balloon, Coronary; Animals; Carotid Artery Injuries; Carotid Artery, Common; Carotid Stenosis; Cell Movement; Cell Proliferation; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Disease Models, Animal; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Hyperplasia; Male; Muscle, Smooth, Vascular; Neointima; Rats; Rats, Sprague-Dawley; Triazoles; Tunica Intima

Endogenous renal dopamine is a major physiological regulator of renal ion transport; however its intracellular signaling pathways are not thoroughly understood. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE), the major cytochrome P450 (CYP4A) metabolite of arachidonic acid formed in the renal cortex, on the natriuretic response to dopamine in Sprague Dawley rats. Infusion of dopamine (1.5μg/kg/min, i.v.) increased urine flow (1.9 fold over basal), sodium excretion (UNaV, 2.7 fold), fractional sodium excretion (FENa, 3.3 fold) and proximal and distal delivery of sodium by 1.5- and 2-fold respectively. Administration of two inhibitors of the synthesis of 20-HETE, 1-aminobenzotriazole (ABT) and N-hydroxy-N'-(-4-butyl-2-methylphenyl)formamidine (HET0016) reduced the response to dopamine by 65%. Induction of the renal expression of CYP4A enzymes with clofibrate did not alter the response to dopamine. The natriuretic response to dopamine was lower in Dahl salt-sensitive rats in comparison to an SS.BN5 consomic strain in which transfer of chromosome 5 from Brown Norway to Dahl salt-sensitive rats upregulates the renal expression of CYP4A protein and the production of 20-HETE. Treatment with HET0016 blocked the renal effects of dopamine in SS.BN5 rats. We also examined the influence of 20-HETE in the natriuretic response to acute volume expansion that is in part mediated via the release of endogenous dopamine. The increase in urine flow, UNaV, FENa and distal FENa following volume expansion was markedly reduced in rats treated with ABT. These results suggest that 20-HETE plays at least a permissive role in the natriuretic response to dopamine.

Topics: Amidines; Animals; Cytochrome P-450 CYP4A; Dopamine; Glomerular Filtration Rate; Hydroxyeicosatetraenoic Acids; Kidney Cortex; Liver; Male; Natriuretic Agents; Rats; Rats, Inbred Dahl; Rats, Sprague-Dawley; Triazoles

1 This study examined the role of 20-hydroxyeicosatetraenoic (20-HETE) in altering vascular function in streptozotocin (STZ)-induced diabetic rats. 2 The expression of CYP4A protein and the formation of 20-HETE were elevated in the kidney, but not in the renal or mesenteric vasculature, of diabetic animals. The vasoconstrictor responses to norepinephrine (NE), endothelin-1 (ET-1), and angiotensin II (Ang II) were significantly enhanced in the isolated perfused mesenteric vascular bed and renal artery segments of diabetic rats. Chronic treatment of the diabetic rats with 1-aminobenzotriazole (ABT, 50 mg kg(-1) alt(-1) diem) or N-hydroxy-N'-(4-butyl-2-methylphenyl) formamidine (HET0016, 2.5 mg kg(-1) day(-1)) attenuated the responses to these vasoconstrictors in both vascular beds. 3 The synthesis of 20-HETE in renal microsomes was reduced by >80% confirming that the doses of ABT and HET0016 were sufficient to achieve system blockade. Addition of HET0016 (1 microM) in vitro also normalized the enhanced vascular responsiveness of renal and mesenteric vessels obtained from diabetic animals to NE and inhibited the formation of 20-HETE by >90% while having no effect on the formation of epoxides. Vasodilator responses to carbachol and histamine were reduced in the mesenteric vasculature, but not in renal arteries, of diabetic rats. Treatment of the diabetic animals with HET0016 improved vasodilator responses in both vascular beds. Vascular sensitivity to exogenous 20-HETE was elevated in the mesenteric bed of diabetic animals compared to controls. 4 These results suggest that 20-HETE contributes to the elevation in vascular reactivity in diabetic animals. This effect is not due to increased vascular expression of CYP4A but may be related to either enhanced agonist-induced release of substrate (arachidonic acid) by the CaMKII/Ras-GTPase system and/or elevated vascular responsiveness to 20-HETE by the CaMKII/Ras-GTPase system and/or elevated vascular responsiveness to 20-HETE.

Topics: Amidines; Angiotensin II; Animals; Blood Glucose; Body Weight; Carbachol; Cytochrome P-450 CYP4A; Diabetes Mellitus, Experimental; Endothelin-1; Enzyme Inhibitors; Histamine; Hydroxyeicosatetraenoic Acids; Kidney; Male; Mesenteric Arteries; Microsomes; Nitroprusside; Norepinephrine; Perfusion; Rats; Rats, Wistar; Renal Artery; Triazoles; Vasoconstriction; Vasodilation

This study examined the role of changes in renal interstitial pressure on the renal levels of cytochrome P450 metabolites of arachidonic acid and compared the effects of inhibition of the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids with 1-aminobenzotriazole on the pressure-natriuretic response versus that seen after administration of HET0016, a more selective inhibitor of the formation of 20-HETE. Renal interstitial pressure rose by 3.4+/-0.3 mm Hg, and the levels of 20-HETE in renal cortical tissue doubled when renal perfusion pressure was increased from 100 to 160 mm Hg. Removal of the renal capsule prevented the increase in renal interstitial pressure and 20-HETE levels after an elevation in renal perfusion pressure. Urine flow and sodium excretion increased 5-fold when renal perfusion pressure was increased from 106 to 160 mm Hg. The administration of 1-aminobenzotriazole (50 mg/kg, IP) or HET0016 (10 mg/kg IV bolus plus 1 mg/kg per hour of infusion) decreased the pressure-natriuretic response by 50% and inhibited the renal formation of 20-HETE and epoxyeicosatrienoic acids by 90% and 50%, respectively. Administration of a lower dose of HET0016 (1 mg/kg per hour, IV) selectively reduced the formation of 20-HETE by 80% without inhibiting renal epoxygenase activity and blunted the pressure-natriuretic response by 42%. These results indicate that elevations in renal perfusion pressure increase 20-HETE levels in the kidney secondary to a rise in renal interstitial pressure. They also suggest that 20-HETE, rather than epoxyeicosatrienoic acids, modulates the pressure-natriuretic response, because selective blockade of the formation of 20-HETE with HET0016 blunts the response to the same extent as that seen after inhibition of the formation of 20-HETE and epoxyeicosatrienoic acids with 1-aminobenzotriazole.

Topics: Amidines; Animals; Arachidonic Acids; Blood Pressure; Enzyme Inhibitors; Hydrostatic Pressure; Hydroxyeicosatetraenoic Acids; Kidney Cortex; Male; Natriuresis; Rats; Triazoles

The present study evaluated the contribution of 20-hydroxyeicosatetraenoic acid (20-HETE) and its interaction with nitric oxide (NO) in cyclosporin A-induced nephrotoxicity and hypertension.. The treatment of rats with cyclosporin A (25 mg/kg) for 7 days increased the renal microsomal conversion of arachidonic acid (AA) to 20-HETE (93 +/- 6%, P < 0.05), increased systolic blood pressure (SBP), reduced the urinary excretion of nitrite (53 +/- 8%, P < 0.05), induced renal damage as indicated by a marked increase in protein excretion (163 +/- 14%, P < 0.05), increased renal vasoconstrictor responses to AA (82 +/- 5%, P < 0.05) but not endothelin-1 or phenylephrine, and decreased vasodilator responses to bradykinin (42 +/- 10%, P < 0.05) and sodium nitroprusside (SNP; 56 +/- 13%, P < 0.05) in the renal preglomerular vessel treated with indomethacin and NO synthase inhibitor. The pretreatment of rats with HET0016 (10 mg/kg) or 1-aminobenzotriazole (50 mg/kg), inhibitors of cytochrome P450 (CYP450) activity, attenuated or prevented cyclosporin A-induced increases in 20-HETE production, SBP, and protein excretion, as did L-arginine (4 g/l), a substrate for NO synthase. L-Arginine but not HET0016 or 1-aminobenzotriazole blunted the cyclosporin A-induced decrease in nitrite excretion. Similarly, L-arginine blunted the enhanced vasoconstriction by AA as did HET0016 or 1-aminobenzotriazole. However, cyclosporin A-blunted dilator responses to bradykinin and SNP were not affected by L-arginine, HET0016, or 1-aminobenzotriazole.. These data suggest that cyclosporin A-induced nephrotoxicity can be accounted for by reduced NO production and a consequent increase in 20-HETE. The cyclosporin A-induced nephrotoxicity is thus an ideal model for evaluating NO/CYP450 interactions.

Topics: Amidines; Animals; Arginine; Blood Pressure; Bradykinin; Cyclosporine; Cytochrome P-450 Enzyme System; Endothelin-1; Hydroxyeicosatetraenoic Acids; Kidney; Male; Microsomes; Nitric Oxide; Nitroprusside; Phenylephrine; Rats; Rats, Sprague-Dawley; Triazoles

The renal regulatory role of cytochrome P450 dependent metabolites of arachidonic acid (AA), vasodilator epoxyeicosatrienoic acids (EETs) and vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE), was examined in anaesthetised rats. We measured renal artery flow (RBF), cortical (CBF) and medullary (MBF) perfusion (laser-Doppler) and medullary tissue nitric oxide (NO, selective electrode), after non-selective inhibition of CYP-450 pathway with 1-aminobenzotriazole (ABT, 10 mg/kg i.v.) or after selective inhibition of 20-HETE synthesis with HET0016 (Taisho Co, Yoshino-cho, Japan), infused into renal artery at 0.3 mg/kg/h or into renal medulla at rates increasing from 0.15 to 1.5 mg/kg/h. ABT caused significant (by 13.7%) decrease in RBF without changing MBF. Renal arterial HET0016 increased MBF (not RBF or CBF) from 152+/-12 to 174+/-12 perfusion units (+16%, P<0.001), while medullary tissue nitric oxide was significantly increased (P<0.001). After renal medullary HET0016, renal perfusion indices were significantly higher than after HET0016 solvent (beta-cyclodextrin). Total renal blood flow seems to be under vasodilator control of EETs whereas renal medullary perfusion under tonic suppression by 20-HETE. The data document, for the first in the whole kidney studies, the functional antagonism of 20-HETE and NO.

Topics: Amidines; Animals; Arachidonic Acid; Blood Pressure; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Hemodynamics; Hydroxyeicosatetraenoic Acids; Kidney; Kidney Cortex; Kidney Medulla; Laser-Doppler Flowmetry; Male; Nitric Oxide; Rats; Rats, Wistar; Renal Circulation; Triazoles; Vasoconstrictor Agents; Vasodilation

Diabetes causes accelerated vascular dysfunction through mechanisms that are poorly understood. This study examined the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII), Ras-GTPase and 20-hydroxyeicosatetraenoic acid (20-HETE) in the development of abnormal reactivity to vasoactive agents in the carotid artery of diabetic rats. The vasoconstrictor response induced by endothelin-1 (ET-1) was significantly increased, whereas vasodilator response to carbachol was significantly reduced in the carotid artery segments of the STZ-diabetic rats. In contrast, the vasoconstrictor response to depolarization of the carotid arterial rings with 50mM KCl was similar in control and diabetic animals. Chronic intraperitoneal administration of KN-93 (5 mg/kg/alt diem), an inhibitor of CaMKII, FPTIII (1.5 mg/kg/alt diem), an inhibitor of Ras-GTPase, and inhibitors of 20-HETE formation 1-aminobenzotriazole (ABT, 50 mg/kg/alt diem) and N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016, 2.5mg/kg/day), produced significant normalization of the altered agonist-induced vasoconstrictor and vasodilator responses without affecting blood glucose levels. All the inhibitors were administered for 4 weeks starting from the day 1 of diabetes induction. Inhibition of CaMKII, Ras-GTPase or 20-HETE formation did not affect the agonist-induced vasoconstrictor and vasodilator responses in the non-diabetic control animals. These data indicate that chronic blockade of CaMKII, Ras-GTPase or the production of 20-HETE normalizes the altered vascular reactivity to ET-1 and carbachol in the carotid artery of STZ-induced diabetic rats.

Topics: Amidines; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carotid Arteries; Carotid Artery Diseases; Diabetes Mellitus, Experimental; Hydroxyeicosatetraenoic Acids; Monomeric GTP-Binding Proteins; Organophosphonates; Protein Kinase Inhibitors; Rats; Rats, Wistar; Streptozocin; Sulfonamides; Triazoles; Vascular Diseases; Vasoconstriction; Vasodilation

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

The present study evaluated whether inhibition of guanylyl cyclase (GC) with 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) and methylene blue (MB) or inhibition of the renal metabolism of arachidonic acid by cytochrome P-450 (CYP450) enzymes with 1-aminobenzotriazole (ABT) and N-hydroxy-N'-(4 butyl-2-methyl phenyl)formamidine (HET0016) alters the renal tubular and vascular effects of a nitric oxide (NO) donor in vivo. Intrarenal infusion of ODQ or MB at a dose of 170 nmol. kg(-1). min(-1) lowered renal blood flow (RBF) by 30 and 15%, respectively; glomerular filtration rate (GFR) by 26 and 18%, respectively; and sodium and water excretion by approximately 35%. In rats pretreated with nitro-L-arginine methyl ester (37 nmol. kg(-1). min(-1)) to block the endogenous production of NO, intrarenal infusion of the NO donor S-nitroso-N-acetylcysteine (S-NO-NAC; 50 nmol. kg(-1). min(-1)) increased RBF (18%), sodium (73%), and water excretion (61%). ODQ or MB administration blocked the effect of S-NO-NAC on RBF but not the diuretic and natriuretic response. Pretreatment of rats with ABT or HET0016 also abolished the renal vasodilatory response to the NO donor and reduced its diuretic and natriuretic effect. These results indicate that both activation of GC and inhibition of CYP450 enzymes contribute to the renal vascular actions of NO, whereas the natriuretic and diuretic actions of NO appear to be largely CYP450 dependent.

Topics: Acetylcysteine; Amidines; Animals; Blood Pressure; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glomerular Filtration Rate; Guanylate Cyclase; Infusions, Parenteral; Kidney; Male; Methylene Blue; Natriuresis; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Oxadiazoles; Rats; Rats, Sprague-Dawley; Renal Circulation; Triazoles