INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CYTOCHROMES P-450 (CYPs) are major catalysts of renal arachidonic acid metabolism, and CYP-derived eicosanoids have been implicated in the regulation of vascular tone and renal function (9, 41). The major products of CYP-catalyzed arachidonic acid metabolism are the -hydroxylated metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) and four regio- and stereoisomeric epoxyeicosatrienoic acids (EETs). EETs are further hydrated to the corresponding dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH) (53). The major CYP eicosanoid formed in rat and human renal microsomes is 20-HETE (8, 28). 20-HETE depolarizes vascular smooth muscle cells by inhibiting Ca²⁺-activated K⁺ channels and increases the conductance of L-type Ca²⁺ channels, both effects leading to Ca²⁺ entry and potent vasoconstriction (14, 56). Additional roles for 20-HETE include mediation of the myogenic response of small cerebral and renal arteries to elevations in transmural pressure, and the autoregulation of cerebral and renal blood flow, glomerular filtration rate, and tubular glomerular feedback (13, 22, 58). In renal tubules 20-HETE inhibits Na⁺-K⁺-ATPase, a Na⁺-K⁺-2Cl cotransporter, and 70-pS K⁺ channels, leading to natriuresis and diuresis (3, 30, 38). EETs and DHETs are generally considered vasodilatory, although they exhibit both vasodilatory and vasoconstrictive properties in vitro, depending on the vascular bed and species that are studied (20, 55, 57). A number of studies suggest that EETs are endothelium-derived hyperpolarizing factors that mediate vasodilation by activation of Ca²⁺-dependent K⁺ channels in vascular smooth muscle cells (6, 7, 10). Both EETs and DHETs can also mediate natriuresis and diuresis by inhibition of Na⁺-K⁺-ATPase in rat proximal tubules (38).

The effects of CYP eicosanoids in the regulation of vascular tone and renal function have promoted intense research interest in defining their roles in the control of blood pressure and the pathogenesis of hypertension. Age-dependent changes in CYP eicosanoid production are well documented in renal microsomes from the spontaneously hypertensive rat (SHR), and increased renal CYP4A3 mRNA and CYP4A-immunoreactive protein levels are consistent with the increased - and (-1)-hydroxylation of arachidonic acid in the prehypertensive SHR kidney relative to age-matched normotensive Wistar-Kyoto (WKY) rats (27, 37, 51). The increased 20-HETE formation in young SHRs is accompanied by a decreased diameter of interlobular arteries and afferent arterioles relative to the WKY rat (19). Importantly, this difference in the diameter of the interlobular and afferent arterioles of young SHR and WKY rats can be reduced in the presence of CYP inhibitors (19). The first in vivo evidence that CYP eicosanoids are important in the regulation of blood pressure was a decrease in renal CYP -hydroxylase activity and blood pressure after the treatment of SHRs with the heme oxygenase inducers stannous chloride and heme arginate (29, 42). More recently, mechanism-based CYP inhibitors and antisense oligonucleotides have been used to manipulate CYP eicosanoid production in vivo. Administration of CYP4A1 and CYP4A2 antisense oligonucleotides to SHR and Sprague-Dawley rats resulted in decreased levels of CYP4A mRNA and immunoreactive proteins and significant reduction of mean arterial blood pressure (MAP; 47, 48). Studies in our laboratory demonstrated that 1-aminobenzotriazole (ABT) is a potent inhibitor of renal arachidonic acid - and (-1)-hydroxylases with little effect on epoxygenases and that ABT has an antihypertensive effect in 7-wk-old SHRs (45). These studies support a role for CYP4A-mediated 20-HETE formation in the regulation of blood pressure.

The development of selective and potent mechanism-based inhibitors of the CYP -hydroxylases and epoxygenases is an important step for further study of the role of CYP eicosanoids in blood pressure regulation. Acetylenic fatty acids were designed as selective inhibitors of fatty acid -hydroxylases and are enzymatically converted to a ketene species that can alkylate the enzyme (4). Substitution of the carboxyl group with a sulfate or methylation at the -carbon is necessary for in vivo resistance to -oxidation (5). In the present study, sodium 10-undecynyl sulfate (10-SUYS) is characterized as a potent and selective inhibitor of renal -hydroxylation of arachidonic acid, which leads to degradation of CYP4A. Inhibition of renal microsomal 20-HETE formation and urinary 20-HETE excretion by 10-SUYS was associated with an acute reduction of blood pressure in 8-wk-old SHRs but had no effect in age-matched WKY rats and 14-wk-old SHRs. 10-SUYS attenuates the vasoconstrictor response to ANG II in rat renal interlobar arterioles, suggesting that the antihypertensive effect of this CYP inhibitor may be due to a decrease in vascular sensitivity to ANG II and other vasoconstrictors. These studies provide further evidence that 20-HETE contributes to the maintenance of elevated blood pressure in the SHR.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Micro-Renathane and RenaPulse tubing were purchased from Braintree Scientific (Braintree, MA). Ketamine HCl and acepromazine maleate were from Aveco (Fort Dodge, IA), and xylazine-20 was from Butler (Columbus, OH). [1-¹⁴C]arachidonic acid was obtained from Amersham Life Science (Arlington Heights, IL). HPLC solvents and ScintiVerse LC were from Fisher Scientific (Pittsburgh, PA). Nitrocellulose membranes were from Micron Separations (Westborough, MA). All other reagents were of the highest grade available and were purchased from Fisher Scientific or Sigma Chemical (St. Louis, MO).

Synthesis of 10-SUYS. 10-SUYS was synthesized according to a published method (5). Ethyl chloroformate (0.30 ml, 3 mmol) was added at 10°C to a stirred solution of 10-undecynoic acid (0.546 g, 3 mmol) and triethylamine (3 mmol) in 25 ml of anhydrous tetrahydrofuran (THF). The reaction mixture was stirred for 30 min at 10°C to form a white paste. The product mixture was filtered through a glass filter (medium pore) into an ice-chilled flask to yield the mixed anhydride in a colorless solution of THF. The filtrate was added over 30 min at 15°C to a stirred solution of NaBH₄ (12 mmol) in a water and THF mixture (1:4). Monitoring the reaction by thin-layer chromatography indicated the reaction was complete after 5-6 h at 15°C. The NaBH₄ was quenched with 2 N HCl to pH 5. The aqueous solution was extracted with ethyl acetate (3 × 20 ml), washed with 5% NaOH, H₂O, and brine, dried with MgSO₄, and concentrated under vacuum to a colorless oil. The crude alcohol was distilled under vacuum to give 0.419 g (83%) of pure 10-undecyn-1-ol [¹H NMR (CDCl₃)  (m, 14 H), 1.89 (t, 1 H), 2.14 (t, 2 H), 3.59 (t, 2 H)]. 10-Undecyn-1-ol (0.419 g, 2.49 mmol) and anhydrous pyridine (0.6 ml, 7.47 mmol) were dissolved in 10 ml of anhydrous dichloromethane under nitrogen and cooled to 10°C. Freshly distilled chlorosulfonic acid (0.165 ml, 2.49 mmol) diluted in 10 ml of dichloromethane was added dropwise to the alcohol over 20 min. The reaction was allowed to reach room temperature overnight while stirring. The solvent was removed under vacuum, and 10 ml of methanol was added. The remaining acid was neutralized with 10% NaOH to pH 7 and stirred overnight. The solvent was removed under vacuum, methanol was added, and the reaction mixture was stirred for another 2 h. The final product was filtered through a glass filter (medium pore), and the solvent was removed under vacuum to yield 0.531 g (2.19 mmol, 88%) of pure sodium 10-undecynyl sulfate [¹H NMR (D₂O) 0.98 (m, 14 H), 1.79 (m, 2 H), 1.93 (t, 1 H), 3.61 (t, 2 H)].

Animals and treatment. Male SHR (7 and 13 wk old) and WKY (7 wk old) rats were purchased from Charles River Laboratories (Wilmington, MA), and Sprague-Dawley (10-12 wk old) rats were from Harlan Sprague-Dawley Laboratories (Indianapolis, IN). Rats were housed in a controlled environment with 12:12-h light-dark cycles and a constant temperature (22°C) and fed standard laboratory chow and water ad libitum. Rats were allowed at least 3 days to acclimate to the housing conditions before use. All animal protocols were approved by the University of California San Francisco and the Medical College of Wisconsin Committee on Animal Research and fully conform with the American Physiological Society's "Guiding Principles for Research Involving Animals and Human Beings" (2).

Clinical chemistry analysis. Urinary Na⁺, K⁺, and creatinine levels were determined colorimetrically by the clinical laboratory of Moffitt Hospital at the University of California San Francisco.

Renal microsomal metabolism. Microsomes were prepared from the renal cortex of a single animal as described previously (45). Microsomal protein concentrations were measured with the Pierce bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL) with bovine serum albumin as the standard. Renal cortical and hepatic arachidonic acid metabolism were measured in incubations containing 75 µM arachidonic acid (0.2 µCi [1-¹⁴C]arachidonic acid), 0.5 mg/ml microsomal protein, 100 mM potassium phosphate buffer, pH 7.4, 10 mM MgCl₂, 8 mM sodium isocitrate, and 0.5 IU isocitrate dehydrogenase. The mixtures were preincubated at 37°C for 3 min, and the reaction was initiated by the addition of NADPH to a final concentration of 1 mM. The reaction was stopped after 30 min by the addition of 1 N HCl to a final pH of 3-3.5. Arachidonic acid and its metabolites were extracted and quantified by HPLC with radiometric detection as described previously (27). Epoxygenase activity is reported as the sum of EET and DHET formation.

In vitro inhibition of arachidonic acid metabolism. Microsomes prepared from the renal cortex or liver of untreated SHRs (5 mg/ml) were incubated with various concentrations of 10-SUYS or AIA, 1 mM NADPH, and an isocitrate dehydrogenase regenerating buffer for 30 min at 37°C. After inactivation, the microsomes were diluted 10-fold, and arachidonic acid metabolism was measured as described above with 0.5 mg/ml inactivated microsomal protein and 75 µM arachidonic acid (0.2 µCi [1-¹⁴C]arachidonic acid).

Effect of 10-SUYS on urinary eicosanoid excretion. Urine samples were collected on dry ice for 12 h before and after administration of 5 mg/kg 10-SUYS to 8-wk-old SHRs for determination of urinary eicosanoid excretion. Urine samples were stored at 80°C until detection of 20-HETE by fluorescent HPLC as described previously (31).

Western immunoblotting. Renal cortical microsomes (5-10 µg) were separated on a 8% SDS-polyacrylamide gel and transferred to nitrocellulose in 25 mM Tris/192 mM glycine/20% methanol using a semidry transfer system (Bio-Rad, Hercules, CA). Primary antibodies used in these studies were goat anti-rat CYP4A1 and goat anti-rat CYP2E1 antisera from Gentest (Woburn, MA), rabbit anti-rat CYP2C23 antisera, which was a gift from Dr. J. H. Capdevila (Vanderbilt University, Nashville, TN), and a rabbit anti-human CYP2J2 IgG from Dr. D. C. Zeldin (National Institute of Environmental Health Sciences, Research Triangle Park, NC). Western blots were incubated with a 1:1,000-fold (CYP4A1), 1:500-fold (CYP2E1), 1:10,000-fold (CYP2C23), or 1:3,000-fold (CYP2J2) dilution of the primary antibody followed by a 1:1,000-fold to 1:10,000-fold dilution of alkaline phosphatase-conjugated rabbit anti-goat IgG or horseradish peroxidase-conjugated goat anti-rabbit IgG. Immunoreactive proteins were visualized using an alkaline phosphatase conjugate substrate kit (Bio-Rad) or an enhanced chemiluminscence detection kit (Amersham Life Science). ScnImage software (Scion, Frederick, MD) was employed to quantify protein levels.

Effect of 10-SUYS on vasoconstrictor response to ANG II in renal arterioles. Experiments were performed on 10- to 12-wk-old male Sprague-Dawley rats. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and the kidneys were removed. Renal interlobar arteries were microdissected and mounted on glass micropipettes with 10-0 silk suture in a perfusion chamber filled with physiological salt solution (PSS) containing (in mM) 119 NaCl, 4.7 KCl, 1.17 MgSO₄, 1.6 CaCl₂, 12 NaHCO₃, 1.18 NaH₂PO₄, 0.03 EDTA, 10 glucose, and 10 HEPES (pH 7.4) and maintained at 37°C. The vessels were perfused with PSS and equilibrated with 95% O₂-5% CO₂ gas mixture. The inflow pipette was connected to a pressurized reservoir to maintain intraluminal perfusion pressure at 80 mmHg. Vessel diameter was recorded by a video system composed of a stereomicroscope, a TV camera (KP-130AU, Hitachi), a TV monitor, and a video measuring system (VIA-100, Boeckeler Instrument, Tucson, AZ). Endothelium of the vessel was removed by perfusing of the lumen of the vessel with a 5-ml bolus of air (11). After the air bolus was pushed through the lumen, perfusion with PSS was reestablished for 30 min before administration of phenylephrine (PE, 10⁷ M) followed by acetylcholine (ACh, 10³ M) to verify the success of deendotheliation. Any vessel that failed to constrict to PE or dilate to ACh after removal of the endothelium was not used in this study. The effect of 10-SUYS on the vasoconstrictor response to ANG II was assessed by comparing a cumulative concentration response to ANG II from 10⁹ M to 10⁶ M in the presence or absence of 10-SUYS (10⁵ M) in the bath. To eliminate the possibility that the effect of 10-SUYS was due to a desensitization of our vessels to the second cumulative dose response to ANG II, we repeated our study in the same preparation, except without 10-SUYS.

Effect of 10-SUYS on endothelial nitric oxide synthase activity. The effect of 10-SUYS on endothelial nitric oxide synthase (eNOS) activity was assayed via the oxyhemoglobin method by following the increase of absorbance at 401 nm. The oxidation of ferrous oxyhemoglobin to the ferric form has an isosbestic point at 411 nm, which was used as the baseline. The assay mixture contained 0.1 mg/ml BSA, 1 mM CaCl₂, 1 µM calmodulin, 0.4 mM dithiothreitol, 100 µM tetrahydrobiopterin, 100 U/ml catalase, 100 U/ml superoxide dismutase, 100 µM flavin adenine dinucleotide, 100 µM flavin mononucleotide, 100 µM NADPH, and 150 nM recombinant human eNOS (39) buffered by 200 mM HEPES at pH 7.8 in a total volume of 400 µl. In the presence of 100 µM L-arginine at 37°C, the increase in 401 nm was 25 arbitrary units (AU)/min, corresponding to a rate of 29 nmol ·min¹ · nmol¹ with an error of 6%. The increase in 401 nm was at steady state for ~2.5 min before substrate depletion caused a loss of activity. 10-SUYS was introduced to the eNOS assay at concentrations of 1, 5, 10, 50, 100, 500, and 1,000 µM. Activity of renal cortical NOS was too low to be detected with the oxyhemoglobin method.

Effect of 10-SUYS on cyclooxygenase activity. The effect of 10-SUYS on cyclooxygenase (COX) activity in vitro was measured in rat renal cortical microsomes. Renal cortex microsomes (0.5 mg) were added to 200 µl of 100 mM Tris · HCl buffer (pH 8.0) containing 1 mM glutathione, 1 mM epinephrine, and 1 µM hematin as cofactors. The mixture was preincubated with various concentrations of 10-SUYS or 10 µM indomethacin for 10 min at 37°C. After preincubation, the microsomes were then incubated with 50 µM arachidonic acid at 37°C for 10 min, the reaction was stopped by adding 50 µl of 0.2 N HCl, and then 50 µl of 0.2 N NaOH was added to each sample. The preparation was centrifuged for 5 min, and the supernatant was harvested and stored at 80°C until the measurement of PGE₂ content. The effect of in vivo administration of 10-SUYS on renal cortical COX activity was measured 6 h after a single intraperitoneal injection (5 mg/kg) to 8-wk-old SHRs. The amount of PGE₂ was measured using a commercial enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's protocol. The limit of detection for PGE₂ is 7.8 pg/ml.

Statistics. All measurements were performed on samples from individual rats, and results are expressed as means ± SE for 3-10 animals at a given time point or dosage. Statistical significance of differences between mean values was evaluated by an unpaired t-test or a one-way ANOVA followed by Bonferroni/Dunn multiple comparisons. Significance was set at a P value of <0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Inhibition of arachidonic acid metabolism by 10-SUYS. The specificity and potency of 10-SUYS as an inhibitor of arachidonic acid metabolism were first measured in vitro. To inactivate CYP enzymes, renal cortical and hepatic microsomes from untreated SHRs were incubated with 0.5-50 µM 10-SUYS in the presence of NADPH. Inactivated microsomes were diluted 10-fold before the measurement of arachidonic acid metabolism. 10-SUYS showed dose-dependent, potent, and selective inhibition of arachidonic acid - and (-1)-hydroxylase activity in renal cortical microsomes (Fig. 1A). The formation of 19- and 20-HETE was significantly inhibited at a concentration of 10-SUYS as low as 10 µM, whereas epoxygenase activity was not affected at concentrations up to 50 µM. The IC₅₀ for inhibition of 19- and 20-HETE formation is 15.7 ± 3.2 and 10.1 ± 2.6 µM, respectively. Arachidonic acid - and (-1)-hydroxylase activities were almost completely inhibited by 50 µM 10-SUYS. In contrast, 0.5-50 µM 10-SUYS did not significantly inhibit arachidonic acid metabolism in liver microsomes (Fig. 1B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Inhibition of arachidonic acid metabolism in microsomes after in vitro inactivation with sodium 10-undecynyl sulfate (10-SUYS). Renal cortical (A) and hepatic (B) microsomes from untreated spontaneously hypertensive rats (SHRs) were incubated with various concentrations of 10-SUYS in the presence of NADPH. 10-SUYS-inactivated microsomes were then used to measure 19-hydroxyeicosatetraenoic acid (19-HETE; ), 20-HETE (), and epoxyeicosatrienoic acids (EETs; ) formation from [1-14C]arachidonic acid. Values are expressed as percentage of control and are reported as means ± SE from 3-4 samples per concentration. * Significant differences between control and 10-SUYS treatment (P < 0.05). Control renal cortical formation rates (pmol · min1 · mg protein1) were 29.0 ± 0.8 (19-HETE), 183 ± 3.6 (20-HETE), and 39.3 ± 1.4 (EETs). Control hepatic formation rates (pmol · min1 · mg protein1) were 23.5 ± 0.1 (19-HETE), 44.9 ± 2.4 (20-HETE), and 151 ± 7.8 (EETs).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Inhibition of renal arachidonic acid metabolism after 10-SUYS administration. Male SHRs were treated with a single intraperitoneal dose of 10-SUYS, and kidneys were harvested 6 h after the dose. The formation of 19-HETE (), 20-HETE (), and EETs () was measured in renal cortical microsomes with [1-14C]arachidonic acid. Values are expressed as percentage of control and are reported as means ± SE from 3-4 animals per treatment group. Control animals were administered vehicle only. * Significant differences between control and 10-SUYS-treated rats (P < 0.05). Control formation rates (pmol · min1 · mg protein1) were 47.4 ± 2.7 (19-HETE), 290 ± 5.2 (20-HETE), and 119 ± 18.1 (EETs).

Effect of 10-SUYS on blood pressure. The effect of a single dose of 10-SUYS (5 mg/kg) on blood pressure, heart rate, and arachidonic acid metabolism was measured over 5 days in SHRs and WKY rats. Administration of 10-SUYS resulted in an acute reduction of blood pressure in 8-wk-old SHRs. MAP was significantly decreased within 3 h after 10-SUYS administration. The decrease in MAP was maximal and averaged 17.9 ± 3.2 mmHg 6 h after 10-SUYS administration (P < 0.05), and MAP returned to baseline levels by 9-24 h after the dose (Fig. 3A). Administration of 2.5 ml/kg saline to SHRs had no effect on MAP, and no significant change in heart rate was observed during the 5 days after a single dose of 10-SUYS to SHRs (data not shown). In contrast, there was no significant change in MAP during the 5 days after a single dose of 10-SUYS to WKY rats (Fig. 3B). Arachidonic acid -hydroxylase activity was inhibited to a similar degree in renal cortical microsomes from SHRs and WKY rats. In the SHR, the formation of 20-HETE in renal cortical microsomes was inhibited 66% 6 h after 10-SUYS treatment (P < 0.01) and gradually returned to 63% of control by 96 h after the dose (Fig. 4A). Both 19- and 20-HETE formation were inhibited 50% 6 h after a single dose of 10-SUYS to WKY rats. In contrast, epoxygenase activity was not affected by 10-SUYS administration to either SHRs or WKY rats (Table 1). The effect of 10-SUYS on MAP was only apparent in young SHRs, despite similar inhibition of -/(-1)-hydroxylase activity in renal microsomes from 8- and 14-wk-old rats (Table 1). A single dose of 10-SUYS had no effect on MAP in 14-wk-old SHRs, although renal cortical 20-HETE formation was inhibited 55% (Table 1).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of 10-SUYS on blood pressure in 8-wk-old SHRs and Wistar-Kyoto (WKY) rats. Mean arterial blood pressure (MAP) and heart rate were measured through a femoral catheter for 3-5 days before and for various times after a single dose (5 mg/kg) of 10-SUYS to male 8-wk-old SHRs and WKY rats. The change in MAP in the SHRs (A) and WKY rats (B) after 10-SUYS administration was calculated and is expressed as mean ± SE from 4-10 animals per time point. Baseline MAP is 131 ± 14.9 mmHg for the SHRs and 102 ± 2.1 mmHg for the WKY rats. MAP decreased an average of 17.9 mmHg in 8-wk-old SHRs 6 h after 10-SUYS administration. * Significant difference from baseline (P < 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effect of 10-SUYS on renal arachidonic acid metabolism and urinary 20-HETE excretion. A: time course of effect of a single dose of 10-SUYS on renal arachidonic acid metabolism. Male SHRs (8 wk old) were killed at various times after a single dose (5 mg/kg) of 10-SUYS. The formation of 19-HETE (), 20-HETE (), and EETs () was measured in renal cortical microsomes with [1-14C]arachidonic acid. Values are expressed as percentage of control and are reported as means ± SE from 3-5 animals per time point. Control animals were administered vehicle only. * Significant differences between control and 10-SUYS-treated rats (P < 0.05). Control formation rates (pmol · min1 · mg protein1) are 63.3 ± 4.9 (19-HETE), 362 ± 29.6 (20-HETE), and 56.8 ± 9.4 (EETs). B: effect of a single dose (5 mg/kg) of 10-SUYS on urinary 20-HETE excretion. Urine samples were collected for 12 h before (control) and after a single dose (5 mg/kg) of 10-SUYS to 8-wk-old SHRs. Urinary 20-HETE levels were measured by fluorescent HPLC. Values are expressed as means ± SE from 4 animals. * Significant difference between control and 10-SUYS treatment (P < 0.05).

CYP -/(-1)-hydroxylase and epoxygenase expression. Western blots were used to determine the protein levels of major CYPs involved in arachidonic acid - and (-1)-hydroxylation (CYP4A, CYP2E1) and epoxygenation (CYP2C23 and CYP2J). Goat anti-rat CYP4A antibody was made against clofibrate-induced rat liver and reported to cross-react with CYP4A1, CYP4A2, and CYP4A3. The rabbit anti-human CYP2J2 antibody has been shown to cross-react with rat CYP2J proteins but not with members of the rat CYP1A, -2A, -2B, -2C, -2E, and -4A subfamilies (36, 50) and detects three immunoreactive proteins in rat renal microsomes (Fig. 5A). CYP4A protein levels were decreased 32-51% and CYP2E1 levels 48-69% during the 48-h period after 10-SUYS administration. In contrast, the levels of the two major epoxygenases, CYP2C23 and CYP2J, were not affected (Fig. 5). The decreased renal CYP4A levels are consistent with decreased renal arachidonic acid -hydroxylase activity. Loss of immunoreactive CYP2E1 protein might be partly responsible for the observed inhibition of 19-HETE formation.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of 10-SUYS on expression of renal arachidonic acid -hydroxylases and epoxygenases. Male 8-wk-old SHRs were killed at various times after a single dose (5 mg/kg) of 10-SUYS. A: renal cortical microsomal proteins (5 µg) were separated on a 8% SDS-polyacrylamide gel, transferred to nitrocellulose, and blotted with antisera against rat CYP4A1, rat CYP2E1, rat CYP2C23, or human CYP2J2. Immunoreactive proteins were detected by alkaline phosphatase staining (CYP4A1) or chemiluminescence (CYP2E1, CYP2C23, and CYP2J2). Blots shown are representative of results from 3 to 6 animals per time point. B: ScnImage software (Scion, Frederick, MD) was employed to quantify protein levels of CYP4A1 (open bars), CYP2E1 (solid bars), CYP2C23 (hatched bars), and CYP2J (gray bars). Values are expressed as percentage of control (0 h) and are reported as means ± SE. * Significant differences between control and 10-SUYS treatment (P < 0.05).

Effect of 10-SUYS on urinary ion excretion and renal function in the SHR. It has been reported that 20-HETE inhibits Na⁺-K⁺-ATPase, a Na⁺-K⁺-2Cl cotransporter, and 70-pS K⁺ channels in renal tubules and results in natriuresis and diuresis (3, 30, 38). Urine volume and urinary Na⁺, K⁺, and creatinine levels were measured before and after 10-SUYS treatment to assess its effect on renal function. Despite significant inhibition of renal cortical 20-HETE formation and urinary 20-HETE excretion after 10-SUYS treatment (Fig. 4), there was no effect on urinary ion excretion and renal function within either 6 or 24 h after 10-SUYS administration (Table 2).

Effect of 10-SUYS on vasoconstrictor response to ANG II in renal arterioles. To investigate the mechanism of the antihypertensive effect of 10-SUYS, its effect on the vasoconstrictor response to ANG II in renal arterioles was evaluated. Baseline inner diameter of the renal interlobar artery used in this study averaged 159 ± 11 µm. The baseline diameter of the vessels in the two studies with 10-SUYS or vehicle was similar. Therefore, the control baseline value was pooled and presented together in Fig. 6. The diameter of these vessels fell to 89 ± 9 µm after addition of PE to the bath and rose to 156 ± 12 µm after administration of ACh. Removal of the endothelium markedly impaired the vasodilator response to ACh. After removal of the endothelium, vessel diameters averaged 159 ± 12, and decreased to 86 ± 9 and 85 ± 6 µm after addition of PE and ACh, respectively, to the bath. Under control conditions, a cumulative concentration of ANG II, from 10⁹ to 10⁶ M, dose dependently reduced the diameter of the renal interlobar arteries from 4 ± 1 to 24 ± 2% (Fig. 6). 10-SUYS (10⁵ M) alone did not alter the baseline vessel diameter; however, it significantly attenuated the vasoconstrictor response to ANG II compared with vehicle control in the renal interlobar arteriole. In contrast, vehicle treatment did not alter the second cumulative concentration response to ANG II compared with the first in the denuded renal interlobar arteriole.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of 10-SUYS on vasoconstrictor response to ANG II in renal interlobar arterioles. Cumulative concentration-response curves depict the effects of ANG II on the inner diameter of isolated, perfused rat renal interlobar arterioles. Paired experiments were performed under control conditions () and in the presence of 10 µM 10-SUYS () or vehicle (). Results are expressed as the percent reduction in the inner diameter of the renal interlobar artery. * Significant difference compared with vehicle treatment (P < 0.05).

Effect of 10-SUYS on eNOS and COX activity. Products of COX metabolism of arachidonic acid and nitric oxide also influence vascular tone and renal tubular transport mechanisms, and modulation of these pathways by 10-SUYS could influence the observed phenotype in the SHR. To address this possibility, the effect of 10-SUYS on eNOS and COX activity was measured. No significant effect on eNOS activity was observed until concentrations of 1 mM 10-SUYS (37% inhibition, Fig. 7A). These results suggest that 10-SUYS would have no effect on eNOS activity in vivo. Similarly, 10-SUYS did not affect COX activity in renal cortical microsomes at concentrations up to 500 µM (Fig. 7B). As expected, 10 µM indomethacin significantly inhibited PGE₂ formation by 48%. Furthermore, 6 h after a single dose (5 mg/kg) of 10-SUYS to SHRs, no change in renal COX activity was observed (data not shown). These results demonstrate that the antihypertensive effect of 10-SUYS is not related to its effect on NOS or COX activity.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Effect of 10-SUYS on endothelial nitric oxide synthase (eNOS) activity and cyclooxygenase (COX) activity. A: eNOS inhibition by 10-SUYS was assayed via the oxyhemoglobin method by following the increase of absorbance at 401 nm as described in MATERIALS AND METHODS. *Significant difference compared with vehicle treatment (P < 0.05). B: renal cortical microsomes from untreated SHRs were preincubated with various concentrations of 10-SUYS, and then PGE2 formation from arachidonic acid was measured as an index of COX activity. Values are expressed as percentage of control and are reported as means ± SE from 3 samples per treatment. Control renal cortical PGE2 formation rate was 7.95 ng · min1 · mg protein1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present report 10-SUYS has been characterized as a potent and specific mechanism-based inhibitor of arachidonic acid -hydroxylation that leads to a loss of CYP4A apoprotein, decreased urinary 20-HETE excretion, attenuation of the vasoconstrictor response to ANG II, and an acute reduction of blood pressure in 8-wk-old SHRs. As a result of the potent effects of CYP eicosanoids in the regulation of vascular tone and renal function, there is considerable interest in understanding their roles in the regulation of blood pressure and the pathogenesis of hypertension. Until recently, these efforts were limited to in vitro or in situ studies because of the lack of selective inhibitors of CYP eicosanoid formation with in vivo activity. Heme oxygenase inducers are nonspecific CYP inhibitors and decrease CYP-mediated - and (-1)-hydroxylation of arachidonic acid and blood pressure in 7-wk-old SHRs to the levels found in age-matched WKY rats (29, 42). However, these results are confounded because heme oxygenase inducers also stimulate the formation of the vasodilatory gas carbon monoxide and may influence nitric oxide synthase activity and produce vasodilatory factors via the cGMP pathway (35). Dibromo-olefinic fatty acids are relatively specific inhibitors of arachidonic acid -/(-1)-hydroxylation and have been extensively used to characterize 20-HETE biological effects in vitro and in situ (17, 21, 46, 54). The most potent inhibitor of arachidonic acid -hydroxylation to date is N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET-0016) (33). HET-0016 inhibits 20-HETE formation in rat renal microsomes with an IC₅₀ of 35 nM, while epoxygenation is relatively resistant to inhibition. Recently, HET-0016 and dibromododecenyl methyl sulfimide have been used in vivo to implicate 20-HETE in the vasoconstrictor response to ANG II and changes in cerebral blood flow after subarachnoid hemorrhage (1, 24).

Selective inhibition of arachidonic acid -hydroxylation in vivo has also been achieved with antisense therapy and mechanism-based inhibition by ABT. CYP4A1 and CYP4A2 antisense oligonucleotides inhibit 20-HETE synthesis in both the vasculature and renal tubules and have an antihypertensive effect in Sprague-Dawley rats and SHRs (47, 48). Antisense oligonucleotides have also been used to demonstrate that CYP4A1 plays an important role in mesenteric vascular reactivity (48). ABT is a mechanism-based inhibitor of renal - and (-1)-hydroxylation of arachidonic acid and causes a loss of CYP4A apoprotein, a decrease in urinary 20-HETE excretion, and reduction in blood pressure in the SHR and in an ANG II model of hypertension (1, 31, 45). However, ABT is a broad-spectrum CYP inhibitor as evident from the large loss in hepatic and renal total CYP content after its administration to rats (26, 32, 34, 45).

Acetylenic fatty acids were designed to be more specific mechanism-based fatty acid -/(-1)-hydroxylase inhibitors. These compounds are enzymatically converted to ketenes, which can alkylate fatty acid -hydroxylases (4). Alkylation of CYP -hydroxylases apparently targets these enzymes for degradation, as is evident from the dose- and time-dependent loss of CYP4A protein in this study. One such inhibitor, 17-octadecynoic acid, has been used in situ to establish the role of 20-HETE in mediation of the myogenic response of renal and cerebral arteries and in the autoregulation of renal blood flow and tubular glomerular feedback (22, 58, 59). However, the use of these acetylenic fatty acids is limited in vivo to intrarenal infusions (44) because of their rapid metabolic degradation by -oxidation and extensive protein binding. The replacement of the carboxyl group in 10-undecynoic acid with a sulfate yields 10-SUYS, a compound resistant to -oxidation and therefore of use in vivo (5). In a previous study administration of 50 mg/kg of 10-SUYS to Sprague-Dawley rats decreased the hepatic lauric acid -hydroxylase activity by 50% with little effect on (-1)-hydroxylase activity. The catalytic inactivation of lauric acid -hydroxylases by 10-SUYS was not tied to destruction of a significant portion of total CYP because these isoforms comprise only a small fraction of the total CYP pool (5). These studies formed the basis for our use of 10-SUYS to investigate the role of 20-HETE formation in blood pressure regulation in the SHR.

Both in vitro and in vivo measurements showed that 10-SUYS selectively inhibited arachidonic acid - and (-1)-hydroxylation in a dose-dependent manner. Western blots clearly demonstrate a loss of CYP4A-immunoreactive protein in kidneys of 10-SUYS-treated rats, similar to that observed after treatment of SHRs with ABT (45). CYP4F isoforms have also been identified as fatty acid -hydroxylases (23, 25, 28), and the possibility that inhibition of 20-HETE formation by 10-SUYS and ABT is due to inactivation of both CYP4A and CYP4F isoforms will require further study.

A single dose of 10-SUYS or ABT caused a similar reduction in MAP in SHRs. The fact that these two inhibitors are not only structurally different but that they inhibit CYPs by distinct mechanisms (heme vs. apoprotein alkylation) strongly suggests that their antihypertensive effect is associated with their ability to inhibit 20-HETE formation. The 8- to 39-mmHg decrease in MAP after a single 5 mg/kg dose of 10-SUYS was associated with a selective and potent inhibition of arachidonic acid -hydroxylation measured in renal cortical microsomes, the rapid destruction of renal cortical CYP4A-immunoreactive proteins, and a significant decrease in renal 20-HETE excretion. The lack of effect of a saturated analog of 10-SUYS or a broad-spectrum mechanism-based CYP inhibitor provides strong evidence that the antihypertensive effect of 10-SUYS requires inactivation of the CYP -hydroxylases and is not related to inhibition of the formation of other CYP products involved in blood pressure regulation. The possibility that 10-SUYS is modulating the formation of COX eicosanoids or NO production has also been eliminated.

The antihypertensive effect of 10-SUYS and the ability of this inhibitor to attenuate the vasoconstrictive response to a known agonist are consistent with inhibition of the formation of the potent vasoconstrictor 20-HETE in vivo. An increased formation of 20-HETE in the SHR kidney relative to age-matched normotensive WKY rats has been associated with increased renal interlobular and afferent arteriolar resistance (12, 15, 19). On the other hand, the natriuretic and diuretic effects of 20-HETE would oppose the vascular effects of this eicosanoid and are considered antihypertensive (3, 30, 38). The overall effect of 20-HETE on blood pressure will be a function of the relative rates of 20-HETE production and the inhibitor concentration in the renal vasculature and tubules. Our results suggest that in the short term the effects of 10-SUYS on renal tubular 20-HETE formation are not sufficient to retain salt and water and counteract the antihypertensive effects of blockade of the influence of 20-HETE on vascular tone. A similar effect was previously reported after treatment of SHRs with ABT (45).

The effect of a single dose of 10-SUYS on blood pressure was acute while the effect on renal arachidonic acid -hydroxylase activity was sustained. One possible explanation for this discrepancy is that renal mechanisms not involving the CYP eicosanoids are operative in response to the acute 10-SUYS antihypertensive effect. The time course of this response is consistent with a change in blood volume and a role for the kidneys (18). The present study suggests that changes in the COX and NOS systems do not contribute to any compensatory response. Alternatively, complete recovery of CYP -hydroxylase activity may not be necessary to maintain basal 20-HETE levels and normal vascular tone and tubular function. To address this possibility, in vivo tissue levels of 20-HETE should be correlated with the duration of the 10-SUYS antihypertensive effect. Inhibition of 20-HETE formation by 10-SUYS may also trigger the release of 20-HETE from phospholipid pools, effectively maintaining normal tissue levels of 20-HETE within a short period of time after treatment despite inhibition of CYP-mediated arachidonic acid -hydroxylation.

The ability of 10-SUYS to lower blood pressure is likely due to inhibition of 20-HETE production in the vasculature as well as in the tubules. An antihypertensive effect was only apparent in 8-wk-old SHRs and not in age-matched WKY rats or 14-wk-old SHRs with established hypertension. In all cases, inhibition of renal cortical 20-HETE formation was similar. A similar pattern was also found with ABT and in earlier studies with the heme oxygenase inducer stannous chloride (29, 42). During the period when blood pressure rises rapidly in the SHR (5-9 wk of age), 20-HETE formation in renal microsomes is elevated relative to age-matched WKY rats (27, 37), and there is a decreased diameter of interlobular and afferent arterioles that can be reversed by blockade of renal 20-HETE formation (12, 15, 19). The elevation of medullary vascular resistance associated with decreased papillary blood flow contributes to an upward shift of the pressure-natriuresis relationship in the early developmental phase of hypertension in SHRs, and 20-HETE has been implicated in this response (40). This is consistent with the finding in the present study that mechanism-based inhibition of renal 20-HETE formation only results in blood pressure reduction in young SHR with apparent changes in renal function.

In summary, studies with potent and selective mechanism-based inhibitors of CYP arachidonic acid -hydroxylase activity have provided compelling evidence that renal 20-HETE formation is an important mediator of blood pressure and vascular tone in the SHR. Further studies are needed to investigate the effect of chronic treatment with 10-SUYS and ABT on blood pressure and to determine whether administration of CYP inhibitors to prehypertensive SHRs can prevent the development of hypertension. Such studies would help define the critical period during which 20-HETE plays a pivotal role in the resetting of the pressure-natriuresis relationship in the SHR. The development of isoform-selective mechanism-based CYP4A and CYP4F inhibitors would contribute to our understanding of the relative contribution of the individual CYP isoforms to 20-HETE formation in the renal vasculature and tubules. The antihypertensive effect of tight binding sEH inhibitors in SHRs and reduction of blood pressure in mice genetically deficient in sEH were reported recently (43, 52). Dual inhibition of 20-HETE formation and EET hydrolysis might lead to further decreases in blood pressure than inhibition of either pathway alone. The potential for selective modulation of vascular 20-HETE formation for the management of human essential hypertension is compelling.