Inhibitor HET0016 Research Articles

20‐HETE contributes to hypertension in postmenopausal rats.

20‐HETE, a major metabolite of cytochrome P450 (CYP)‐4A, is a potent vasoconstrictor of renal arterioles, and has been shown to mediate hypertension in several animal models. The present study tested the hypothesis that 20‐HETE is involved in mediating hypertension in an animal model of postmenopausal hypertension, the old female spontaneously hypertensive rat (OF‐SHR). A CYP4A inhibitor of 20‐HETE production, 1‐aminobenzotriazole (ABT; 50 mg/kg per day i.p.), or vehicle, was administered to OF‐SHR, aged 16 mos, and young female SHR (YF‐SHR), aged 3 mos, (n=3–4 per group). Mean arterial pressure (MAP) was measured by radiotelemetry. ABT decreased MAP in OF‐SHR (164±5 vs. 151±6 mm Hg, p<0.05), whereas it had no effect in YF‐SHR (138±3 vs.136±1 mm Hg). CYP4A (isoform 2) mRNA expression was measured by real‐time RT‐PCR in kidney cortex and medulla of OF‐SHR and YF‐SHR. CYP4A2 mRNA expression was significantly upregulated in kidney cortex of OF‐SHR compared to YF‐SHR. In other studies administration of a specific 20‐HETE inhibitor, HET0016, also significantly decreased MAP in OF‐SHR. In summary, administration of the CYP4A inhibitor and a specific 20‐HETE inhibitor markedly attenuated hypertension in postmenopausal rats. Increases in renal vascular 20‐HETE constitute a new pathway that may be involved in mediating postmenopausal hypertension. (Supported by HL 51971).

20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK(1) cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK(1) cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release (P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 microM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 microM) also inhibited cytotoxicity significantly (P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase (P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells (P < 0.05). This was abolished in the presence of HET-0016 (P < 0.05) or MnTMPyP (P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.

The Roles of Intrarenal 20-Hydroxyeicosatetraenoic and Epoxyeicosatrienoic Acids in the Regulation of Renal Function in Hypertensive Ren-2 Transgenic Rats

Background: The present study was performed in hypertensive Ren-2 transgenic rats (TGR) and in normotensive Hannover Sprague-Dawley (HanSD) rats. First, the intrarenal protein expression of CYP4A, the enzyme catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), and of CYP2C23, the enzyme responsible for epoxyeicosatrienoic acid (EET) production, was evaluated. Second, the renal functional responses to inhibition of the intrarenal formation of 20-HETE and EETs were investigated. Methods: Renal hemodynamics and electrolyte excretion were evaluated in response to the administration of inhibitors of 20-HETE and EET formation into the renal artery. In renal cortical tissue, CYP4A and CYP2C23 protein expression was assessed by Western blot analysis. Urinary concentrations of 20-HETE and EETs were measured using a fluorescent HPLC assay. Results: TGR have higher kidney CYP4A protein expression and urinary 20-HETE excretion but significantly lower CYP2C23 protein expression and urinary EET excretion than HanSD. Intrarenal inhibition of 20-HETE and EET formation decreased sodium excretion in HanSD, whereas inhibition of 20-HETE increased urinary excretion of sodium in TGR without altering renal hemodynamics. Conclusions: Our data suggest that in TGR, deficient intrarenal synthesis of EETs combined with increased synthesis of 20-HETE with its stimulation of tubular sodium absorption may contribute to the development of hypertension in TGR.

Protective Effect of the 20-HETE Inhibitor HET0016 on Brain Damage after Temporary Focal Ischemia

Cytochrome P450 metabolism of arachidonic acid produces the potent vasoconstrictive metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE). Recent studies have implicated 20-HETE as a vasoconstrictive mediator in hemorrhagic stroke. The purpose of this study was to determine the effect of the 20-HETE inhibitor, HET0016, on lesion volume and cerebral blood flow (CBF) after temporary middle cerebral artery occlusion (MCAO) in rats. Plasma pharmacokinetics and tissue concentrations of HET0016 were determined after a 10 mg/kg intraperitoneal dose. Separate rats were treated with HET0016 or vehicle before 90 mins of MCAO. Lesion volume was assessed by 2,3,5-triphenyl-tetrazolium-chloride and cerebral flow was determined using laser Doppler flow. The effect of MCAO on in vitro microsomal formation of mono-oxygenated arachidonic acid metabolites was also determined. Results show that HET0016 has a short biologic half-life, distributes into the brain, and is associated with a 79.6% reduction in 20-HETE concentration in the cortex. Lesion volume was greatly reduced in HET0016-treated (9.1%+/-4.9%) versus vehicle-treated (57.4%+/-9.8%; n=6; P<0.001) rats. An attenuation of the observed decrease in CBF was observed in HET0016-treated (180 mins 89.2%+/-6.2%; 240 mins 88.1%+/-5.7% of baseline flow) versus vehicle control (180 mins 57.6%+/-19.0%; 240 mins 53.8%+/-20.0% of baseline flow; n=6; P<0.05). Brain cortical microsomal formation rate of 20-HETE was also reduced at 24 h in the ipsilateral hemisphere after MCAO. These data support a significant role for 20-HETE in the pathogenesis of ischemic stroke.

Increased renal vascular reactivity to ANG II after unilateral nephrectomy in the rat involves 20-HETE

This study examined the role of intrarenal ANG II in the renal vascular reactivity changes occurring in the remaining kidney undergoing adaptation following contralateral nephrectomy. Renal blood flow responses to intrarenal injections of ANG II (0.25 to 5 ng) were measured in anesthetized euvolemic male Wistar rats 1, 4, 12, and 24 wk after uninephrectomy (UNX) or sham procedure (SHAM). At week 4, renal vasoconstriction induced by 2 ng ANG II was greater in UNX (69 +/- 5%) than in SHAM rats (50 +/- 3%; P < 0.01). This response was inhibited, by 50 and 66%, and by 20 and 25%, in SHAM and UNX rats, after combined injections of ANG II and losartan, or PD-123319 (P < 0.05), respectively. Characteristics of ANG II receptor binding in isolated preglomerular resistance vessels were similar in the two groups. After prostanoid inhibition with indomethacin, renal vasoconstriction was enhanced by 42 +/- 8% (P < 0.05), only in SHAM rats, whereas after 20-HETE inhibition with HET0016, it was reduced by 53 +/- 16% (P < 0.05), only in UNX rats. These differences vanished after concomitant prostanoid and 20-HETE inhibition in the two groups. After UNX, renal cortical protein expression of cytochrome P-450 2c23 isoform (CYP2c23) and cyclooxygenase-1 (COX-1) was unaltered, but it was decreased for CYP4a and increased for COX-2. In conclusion, renal vascular reactivity to ANG II was significantly increased in the postuninephrectomy adapted kidney, independently of protein expression, but presumably involving interactions between 20-HETE and COX in the renal microvasculature and changes in the paracrine activity of ANG II and 20-HETE.

Interaction of nitric oxide and 20‐HETE during cortical functional hyperemia

Nitric oxide (NO) is postulated to modulate rather than mediate increased cerebral blood flow (CBF) during sensory activation. We tested whether this modulation can be explained by NO inhibition of synthesis of the vasoconstrictor, 20-HETE, which may counteract opening of KCa channels by EETs. In anesthetized rats, the increase in CBF during whisker stimulation was reduced from 27±10% (±SD; n=8) to 18±6% by the neuronal NO synthase inhibitor 7-nitroindazole (7-NI 40 mg/kg). Subsequent subdural superfusion of the 20-HETE synthesis inhibitor HET0016 (1 μM) restored the CBF response to 25±12%. HET0016 alone had only a small effect on the evoked CBF response (21±5 to 24±5%; n=8). Simultaneous administration of 7-NI and HET0016 resulted in no change in the CBF response (25±5 to 24±6%; n=8). Subsequent superfusion of the EET antagonist 14,15-EEZE (30 μM) reduced the response to 19±5%. However, when 7-NI decreased the CBF response from 26±7 to 17±3%, subsequent superfusion of 14,15-EEZE had no further effect (17±5%). Thus, the inhibitory effect of 7-NI on the cortical CBF response to sensory activation requires 20-HETE synthesis. The ability of an EET antagonist to reduce the response is lost in the presence of 7-NI, but is restored when 20-HETE synthesis is simultaneously inhibited. These results suggest that increased NO synthesis during functional activation acts to suppress 20-HETE synthesis and permit vasodilation by EETs. (Supported by NIH HL59996 and GM31278)

Renal 20-HETE inhibition attenuates changes in renal hemodynamics induced byl-NAME treatment in pregnant rats

We previously reported that inhibition of nitric oxide (NO) synthesis by N-nitro-L-arginine methyl ester (L-NAME) during late pregnancy leads to increased production of renal vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 (CYP) 4A-derived vasoconstrictor, in pregnant rats. However, the effect of upregulation of vascular 20-HETE production on renal function after NO inhibition is not known. To test the hypothesis that increased gestational vascular 20-HETE synthesis after NO inhibition is involved in mediating blood pressure and renal functional changes, we first determined the IC(50) value of the effect of nitroprusside (SNP), a NO donor, on renal 20-HETE production in cortical microsomes. We then divided pregnant rats and age-matched virgin rats into a vehicle control group, an L-NAME treatment group (0.25 mg/ml in drinking water), and a group treated with L-NAME plus N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; CYP4A-selective inhibitor, 10 mg.kg(-1).day(-1) iv). After 4 days of treatment, we measured blood pressure, renal blood flow (RBF), renal vascular resistance (RVR), and glomerular filtration rate (GFR) in each group. The addition of SNP (IC(50) = 22 microM) decreased renal cortical 20-HETE production. In pregnant rats, L-NAME treatment led to significantly higher mean arterial pressure (MAP) and RVR, and lower RBF and GFR. Combined treatment with DDMS and L-NAME significantly attenuated the increases in MAP and RVR and the decrease in GFR, but not the reduction in RBF induced by L-NAME treatment. L-NAME and L-NAME plus DDMS had no significant impact on renal hemodynamics in virgin rats. In addition, chronic treatment with DDMS selectively inhibited cortical 20-HETE production without a significant effect on CYP4A expression in L-NAME-treated pregnant rats. In conclusion, NO effectively inhibits renal cortical microsomal 20-HETE production in female rats. In pregnant rats, the augmentation of renal 20-HETE production after NO inhibition is associated with increased MAP and RVR, whereas decreased GFR is negated by treatment of a selective and competitive CYP4A inhibitor. These results demonstrate that the interaction between renal 20-HETE and NO is important in the regulation of renal function and blood pressure in pregnant rats.

Inhibition of 20-HETE abolishes the myogenic response during NOS antagonism in the ovine fetal pulmonary circulation

Mechanisms that maintain high pulmonary vascular resistance (PVR) and oppose vasodilation in the fetal lung are poorly understood. In fetal lambs, increased pulmonary artery pressure evokes a potent vasoconstriction, suggesting that a myogenic response contributes to high PVR in the fetus. In adult systemic circulations, the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to modulate the myogenic response, but its role in the fetal lung is unknown. We hypothesized that acute increases in pulmonary artery pressure release 20-HETE, which causes vasoconstriction, or a myogenic response, in the fetal lung. To address this hypothesis, we studied the hemodynamic effects of N-methylsufonyl-12,12-dibromododec-11-enamide (DDMS), a specific inhibitor of 20-HETE production, on the pulmonary vasoconstriction caused by acute compression of the ductus arteriosus (DA) in chronically prepared fetal sheep. An inflatable vascular occluder around the DA was used to increase pulmonary artery pressure under three study conditions: control, after pretreatment with nitro-L-arginine (L-NA; to inhibit shear-stress vasodilation), and after combined treatment with both L-NA and a specific 20-HETE inhibitor, DDMS. We found that DA compression after L-NA treatment increased PVR by 44 +/- 12%. Although intrapulmonary DDMS infusion did not affect basal PVR, DDMS completely abolished the vasoconstrictor response to DA compression in the presence of L-NA (44 +/- 12% vs. 2 +/- 4% change in PVR, L-NA vs. L-NA + DDMS, P < 0.05). We conclude that 20-HETE mediates the myogenic response in the fetal pulmonary circulation and speculate that pharmacological inhibition of 20-HETE might have a therapeutic role in neonatal conditions characterized by pulmonary hypertension.

20-HETE and the kidney: resolution of old problems and new beginnings.

The protean properties of 20-hydroxyeicosatetraenoic acid (HETE), vasoactivity, mitogenicity, and modulation of transport in key nephron segments, serve as the basis for the essential roles of 20-HETE in the regulation of the renal circulation and electrolyte excretion and as a second messenger for endothelin-1 and mediator of selective renal effects of ANG II. Renal autoregulation and tubular glomerular feedback are mediated by 20-HETE through constriction of preglomerular arterioles, responses that are maintained by 20-HETE inhibition of calcium-activated potassium channels. 20-HETE modulates ion transport in the proximal tubules and the thick ascending limb by affecting the activities of Na+-K+-ATPase and the Na+-K+-2Cl- cotransporter, respectively. The range and diversity of activity of 20-HETE derives in large measure from COX-dependent transformation of 20-HETE to products affecting vasomotion and salt and water excretion. Nitric oxide (NO) exerts a negative modulatory effect on 20-HETE formation; inhibition of NO synthesis produces marked perturbation of renal function resulting from increased 20-HETE production. 20-HETE is an essential component of interactions involving several hormonal systems that have central roles in blood pressure homeostasis, including angiotensins, endothelins, NO, and cytokines. 20-HETE is the preeminent renal eicosanoid, overshadowing PGE2 and PGI2. This review is intended to provide evidence for the physiological roles for cytochrome P-450-derived eicosanoids, particularly 20-HETE, and seeks to extend this knowledge to a conceptual framework for overall cardiovascular function.

C001 Inhibition of 20-HETE lowers blood pressure in rats with angiotensin II (ANG II) induced hypertension following no inhibition

C001 Inhibition of 20-HETE lowers blood pressure in rats with angiotensin II (ANG II) induced hypertension following no inhibition

Inhibition of 20-HETE production contributes to the vascular responses to nitric oxide.

Nitric oxide (NO) inhibits a variety of heme-containing enzymes, including NO synthase and cytochrome P4501A1 and 2B1. The present study examined whether NO inhibits the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504A enzymes and whether blockade of the production of this substance contributes to the vascular effects of NO. Sodium nitroprusside (SNP; 10(-5), 10(-4), and 10(-3) mol/L) reduced the production of 20-HETE by renal microsomes incubated with arachidonic acid to 71 +/- 5%, 29 +/- 4%, and 4 +/- 2% of control, respectively (n = 5). Similar results were obtained with the use of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (n = 3). To determine whether inhibition of 20-HETE contributes to the vasodilatory effects of NO, the effects of dibromo-dodecenyl-methylsulfimide (DDMS), a selective inhibitor of the formation of 20-HETE, on the response to SNP (10(-7) to 10(-3) mol/L) were examined in rat renal arterioles preconstricted with phenylephrine (n = 5). SNP increased vascular diameter in a concentration-dependent manner to 82 +/- 4% of control. After DDMS (25 mumol/L), SNP (10(-3) mol/L) increased vascular diameter by only 17 +/- 3%. The effects of DDMS on the mean arterial pressure (MAP) and renal blood flow (RBF) responses to infusion of an NO donor and a synthase inhibitor were also examined in thiobutabarbital-anesthetized, Sprague-Dawley rats. Infusion of MAHMA NONOate at 1, 3, 5, and 10 nmol/min reduced MAP by 16 +/- 2, 30 +/- 3, 40 +/- 5, and 48 +/- 5 mm Hg and lowered renal vascular resistance (RVR) by 15 +/- 3%, 26 +/- 2%, 30 +/- 3%, and 34 +/- 4% of control. After DDMS (10 mg/kg, n = 7 rats), the MAP and RVR responses to 1-hexamine, 6-(2-hydroxy-1-methyl-2-nitrohydrazino)N-methyl (MAHMA NONOate) averaged only 20% of those seen during control. In other experiments, MAP increased by 32 +/- 4% and RBF fell to 56 +/- 5% of control after administration of N-nitro-L-arginine (L-NArg) (10 mg/kg IV). After DDMS (10 mg/kg, n = 7 rats), MAP increased by only 19 +/- 4% and RBF fell by only 7 +/- 4% after L-NArg. These results indicate that NO inhibits cytochrome P4504A enzymes and that inhibition of the production of 20-HETE contributes to the vasodilatory effects of NO.