![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CARDIOVASCULAR
Vascular Biology Center (J.J.O, M.B.F, J.D.I.) and Department of Physiology (J.D.I.), Medical College of Georgia, Augusta, Georgia; and Department of Entomology and Cancer Research Center (I.-H.K., C.M., B.D.H.), University of California, Davis, California
Received February 24, 2006; accepted June 12, 2006.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). Mesenteric arteries were then incubated with increasing concentrations of the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA). AUDA resulted in a concentration-dependent relaxation of mesenteric arteries, with 10 µM resulting in a 48 ± 7% relaxation. Chain-shortened analogs of AUDA had an attenuated vasodilatory response. Interestingly, at 10 µM, the sEH inhibitors 1-cyclohexyl-3-dodecylurea, 12-(3-cyclohexylureido)dodecanoic acid, and 950 [adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea] were significantly less active, resulting in a 25 ± 8%, 10 ± 9%, and -8 ± 3% relaxation, respectively. Treatment of mesenteric arteries with tetraethylammonium, iberiotoxin, ouabain, or glibenclamide did not alter AUDA-induced relaxation. The AUDA-induced relaxation was completely inhibited when constricted with KCl. In separate experiments, denuding mesenteric resistance vessels did not alter AUDA-induced relaxation. Taken together, these data demonstrate that adamantyl-urea inhibitors have unique dilator actions on vascular smooth muscle compared with other sEH inhibitors and that these dilator actions depend on the adamantyl group and carbon chain length.
; Yu et al., 2004; Newman et al., 2005). Within these tissues, sEH metabolizes endogenous and/or exogenous epoxide-containing compounds to their corresponding diols and/or glycols by catalyzing the addition of water to the epoxide moiety (Morisseau and Hammock, 2005; Newman et al., 2005). An endogenously produced, biologically active group of epoxides that serve as a substrate for sEH are the epoxyeicosatrienoic acids (EETs). EETs are cytochrome P450 epoxygenase metabolites of arachidonic acid that have been identified as important regulatory molecules in the cardiovascular and renal circulations. EETs have been identified as potential endothelial-derived hyperpolarizing factors and have been reported to have specific anti-inflammatory properties (Campbell et al., 1996; Fisslthaler et al., 1999; Node et al., 1999; Falck et al., 2003). The EETs are metabolized by the sEH into their corresponding dihydroxyeicosatrienoic acids, thereby resulting in partial or complete loss of activity (Moghaddam et al., 1997; Zeldin, 2001; Spector et al., 2004).
As a result of the renal and cardiovascular protective effects afforded by the EETs, inhibiting their metabolism has identified the sEH enzyme as a potential therapeutic target in diseases like hypertension and inflammation. A number of sEH inhibitors have been developed, and their administration in animal models of disease has proved to have beneficial effects. In the spontaneously hypertensive rat, administration of the sEH inhibitor N,N'-dicyclohexylurea lowered blood pressure (Yu et al., 2000). Another sEH inhibitor, 1-cyclohexyl-3-dodecylurea, also lowered blood pressure in an animal model of angiotensin-dependent hypertension (Imig et al., 2002; Zhao et al., 2004). The sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA) lowered blood pressure and decreased renal damage associated with angiotensin-dependent, salt-sensitive hypertension (Imig et al., 2005). In addition to lowering blood pressure, it was demonstrated that sEH inhibition also inhibits the inflammatory response. Administration of 12-(3-adamantane-1-ylureido)-dodecanoic acid n-butyl ester to spontaneously hypertensive rats exposed to tobacco smoke significantly attenuated the tobacco smoke-induced infiltration of proinflammatory cells into the lung (Smith et al., 2005). The same sEH inhibitor was also reported to decrease lipopolysaccharide-induced tissue injury and mortality (Schmelzer et al., 2005).
Many of the renal and cardiovascular protective effects elicited with sEH inhibition are attributed to the measured decrease in blood pressure. To better understand the mechanism by which sEH inhibition decreases blood pressure, we turned our attention to the vasculature. In the present study, we investigated the hypothesis that a mechanism by which sEH inhibitors elicit their cardiovascular protective effects is via their direct action on the vasculature.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
In inhibitor experiments, tetraethylammonium (1 mM; Sigma-Aldrich), ouabain (100 µM; Sigma-Aldrich), and glibenclamide (1 µM; Sigma-Aldrich) were used postconstriction and for 10 to 20 min prior to dosing with AUDA. Iberiotoxin (IbTX; Tocris Bioscience, Ellisville, MO) was reconstituted with deionized water, and a 100 nM dose was given 15 min before constriction with U46619. [GenBank] N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) was dissolved in 50% ethanol, and a 1 µM dose was given 30 min before constriction with U46619. [GenBank] Potassium chloride (40 mM; Sigma-Aldrich) was used to constrict vessels 20 min prior to addition of AUDA. Endothelium-denuded vessels were prepared by inserting a human hair about which the vessel was rotated using a rolling motion atop a raised, crowned surface in a dish of Krebs physiological salt solution. To ensure that the vessel was no longer capable of an endothelium-related dilation, acetylcholine (100 µM for 5 min) was given. If an acetylcholine-induced relaxation was not observed, the vessel was rinsed and allowed to equilibrate for 10 min before constriction with U46619 [GenBank] and treatment with AUDA. At the end of the experimental protocol, sodium nitroprusside (100 µM) was added to the chamber to demonstrate that the endothelium-denuded vessel was capable of relaxation.
Western Blot Analysis. Mesenteric artery segments were obtained from five male Sprague-Dawley rats (250-310g) of the same size that would be used in the isolated mesenteric resistance vessel preparation. The vessels were homogenized in cell lysis buffer [0.05 M Tris-HCl, pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 0.1% NP-40, 1 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.8 µM aprotinin, 20 µM leupeptin, 40 µM bestatin, 15 µM pepstatin, and 14 µM E-64], and the homogenate centrifuged at 10,000g for 10 min at 4°C to sediment any unhomogenized material. The supernatant was removed, and the protein concentration was determined using a bicinchoninic acid protein assay (Pierce Biotechnology, Inc., Rockford, IL). Protein was solubilized in Laemmli sample buffer and resolved by electrophoresis using a 10% stacking Tris-glycine acrylamide gel. Protein was electrophoretically transferred to polyvinylidene difluoride membrane and blocked with 3% bovine serum albumin and 10% dry milk in phosphate-buffered saline containing 0.1% Tween 20 (PBS-T). After blocking, the membranes were washed several times with PBS-T and incubated with primary antibody for the sEH enzyme (1:2000; a gift from Dr. Bruce D. Hammock, University of California, Davis, CA). Membranes were washed with PBS-T and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (1:25,000; Pierce Biotechnology, Inc.). Protein was visualized using ECL Western Blotting substrate (Pierce Biotechnology, Inc.).
sEH Inhibitors. The synthesis and characterization of AUDA, CUDA, CDU, and AUBA are described elsewhere (Morisseau et al., 2002; Kim et al., 2004).
950. To a solution of adamantyl isocyanate (0.20 g, 1.13 mmol) in DMF (15 ml) was added a solution of 5-amino-1-pentanol (0.17 g, 1.69 mmol) in DMF (15 ml) at 0°C. After stirring for 12 h, an aqueous solution of 1 N HCl (40 ml) was added into the reaction at 0°C, and the mixture was stirred for 30 min. The solid product crystalized was filtered and washed with water (40 ml) and ethyl acetate (20 ml). The resulting solid was dried in the vacuum oven to give 1-adamantan-1-yl-3-(5-hydroxypentyl)urea as a white solid (I; 0.75 g, 100%). To a solution of diethylene glycol monoethyl ether (1 Eq) in tetrahydrofuran (30 ml) was added portion-wise triphenylphosphine (1.1 Eq) and carbon tetrabromide (1.1 Eq) at 0°C. After stirring for 12 h at room temperature, hexane (60 ml) was added to the reaction mixture. This crude mixture was filtered to get rid of triphenylphospine oxide, and the organic solvent dissolving the product was washed with water (60 ml), dried over MgSO4, and concentrated. The residue was purified using silica gel column chromatography (hexane only and hexane:ethyl acetate = 3:1) to give the corresponding bromide II (87%) as an oil. This bromide (1.5 Eq) was added portion-wise to a suspension of 95% sodium hydride (3 Eq) and I (1 Eq) in DMF at room temperature. After stirring for 12 h, water was poured into the reaction mixture, and the product was extracted with ether. The organic solution was dried over MgSO4 and concentrated. The residue was purified using silica gel column chromatography (hexane: ethyl acetate = 3:1) to afford 950 (52%) as a solid. 1H NMR 
(CDCl3) 1.22 (3H, t, J = 6.9 Hz), 1.37-1.43 (2H, m), 1.46-1.53 (2H, m), 1.56-1.61 (2H, m), 1.64-1.69 (6H, m), 1.95-1.99 (6H, m), 2.05-2.08 (3H, m), 3.11 (2H,q, J = 6.9 Hz), 3.46 (2H,t, J = 6.9 Hz), 3.48-3.67 (10H, m), 4.21 (1H, s), 4.26 (1H, s), LC-MS (ESI) m/z calculated for C22H40N2 O4 [M + H]+ 397.30, found [M + H]+ 397.31, mp 75°C, Anal. (C22H40N2O4) C,H,N.
AUOA. To a suspension of 8-aminooctanoic acid (0.48 g, 3.0 mmol) in DMF (20 ml) was added 1-adamantyl-isocyanate (0.48 g, 2.7 mmol) at room temperature. The reaction mixture was stirred and heated at 100°C for 3 h. After cooling down to room temperature, 1 N HCl aqueous solution (25 ml) was added to the reaction and the mixture was stirred for 30 min. The solid crystalline product was filtered and washed with water (20 ml) and ethyl acetate (20 ml). The resulting solid was recrystallized from methanol. The obtained crystal was dried in a vacuum oven to give 0.90 g (98%) of 8-(3-adamantyl-ureido)-octanoic acid as a white solid: mp 140°C; 1H NMR (CDCl3/TMS) 1.37-1.40 (6H, m), 1.55-1.58 (2H, m), 1.70-1.77 (9H, m), 1.98-2.05 (5H, m), 2.10-2.16 (3H, m), 2.40 (2H,t, J = 7.4 Hz), 3.13 (2H,t, J = 6.5 Hz), 4.28 (1H, s), 4.47 (1H, s) ppm; LC-MS (TOF) m/z calculated for C19H33N2 O3 [M + H]+ 337.2491, found [M + H]+ 337.2508 Da.
AUHA. To a suspension of 6-aminohexanoic acid (0.98 g, 7.5 mmol) in DMF (15 ml) was added 1-adamantyl-isocyanate (0.89 g, 5.0 mmol) at room temperature. The reaction mixture was stirred and heated at 100°C for 3 h. After cooling down to room temperature, 1 N HCl aqueous solution (25 ml) was added to the reaction, and the mixture was stirred for 30 min. The solid crystalline product was filtered and washed with water (20 ml) and ethyl acetate (20 ml). The resulting solid was dried in a vacuum oven to give 1.05 g (68%) of 6-(3-adamantyl-ureido)-hexanoic acid as a white solid: mp 159°C; 1H NMR (CDCl3/TMS): 1.60-1.67 (13H, m), 1.90-1.96 (5H, m), 2.02-2.07 (3H, m), 2.36 (2H,t,J = 7.2 Hz), 3.09 (2H,t, J = 6.9 Hz), 4.15 (1H, s), 4.33 (1H, s) ppm; LC-MS (TOF) m/z calculated for C17H29N2 O3 [M + H]+ 309.2178, found [M + H]+ 309.2164 Da.
AADU. To a solution of 1,12-diaminododecane (1.0 g, 5.0 mmol) in DMF (20 ml), adamantyl-isocyanate (0.21 g, 1.1 mmol) at room temperature was added. The reaction mixture was stirred and heated at 100°C for 3 h. After cooling down to room temperature, 150 ml of water was added to the reaction, and the mixture was stirred for 30 min. The yellowish solid was filtered and washed with water (20 ml). The targeted product was purified by chromatography on a silica column equilibrated with ethyl acetate. Product was eluted with a 50:50 mixture of ethyl acetate:ammonia-saturated methanol. The solvent was evaporated, and the obtained crystal was dried in a vacuum oven to give 0.20 g (49%) of 1-adamantyl-3-(12-aminododecyl) urea as a white solid: mp 80°C; 1H NMR (CDCl3/TMS) 1.22-1.33 (19H, m), 1.42-1.49 (4H, m), 1.55-1.62 (12H, m), 2.06 (2H, s), 2.68 (2H,t, J = 7.2 Hz), 3.13 (2H,q, J = 6.6 Hz), 4.02 (1H, s), 4.10 (1H, s) ppm; LC-MS (TOF) m/z calculated for C23 H44 N3O [M + H]+ 337.1440, found [M + H]+ 378.1221 Da.
Statistical Analysis. All data are presented as mean ± S.E.M. In experiments using AUDA, statistical significance between concentrations was determined using an analysis of variance followed by a least significance difference test to identify individual differences. The statistical significance between experimental groups was determined using a two-way analysis of variance and Bonferroni post tests to compare replicate means. P 
0.05 was considered statistically significant.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
). To determine whether or not the sEH enzyme is expressed in mesenteric resistance vessels from male Sprague-Dawley rats, vessels were obtained, and the expression of the sEH enzyme was measured by Western blot. The sEH enzyme was identified in the mesenteric resistance vessels from Sprague-Dawley rats (Fig. 2). However, compared with the kidney cortex from Sprague-Dawley rats, sEH protein expression in mesenteric resistance vessels was less per microgram of total protein. The unique structure of adamantyl-urea inhibitors of the sEH enzyme imparts specific vasoactive properties that are not observed with other sEH inhibitors that could contribute to the reported blood pressure-lowering effects and renal and cardiovascular protective properties. Adamantyl-urea inhibitors are 1,3-disubstituted ureas with an adamantyl group on one side of the urea pharmacophore and a saturated 11-carbon aliphatic chain on the other (Table 1). To investigate the structural elements of adamantyl-urea inhibitors that are responsible for the observed vasoactive actions, structural analogs of AUDA were prepared. These analogs, the structures of which are provided in Table 1, were designed to identify the contributions of the adamantyl group, the carboxylic acid functional group, and the length of the aliphatic carbon chain on vascular reactivity. Changing the adamantyl group of AUDA to a cyclohexyl group, which is the structure of the sEH inhibitor CUDA, significantly attenuated the vasodilatory response (Fig. 3). CUDA resulted in a 10 ± 5% relaxation of mesenteric resistance vessels at the 10 µM dose anda52 ± 13% relaxation at the 100 µM dose. Replacing the carboxylic acid at the end of the aliphatic chain to a primary amine, AADU, resulted in concentration-dependent dilations of mesenteric resistance vessels that were not different from that of AUDA (Fig. 3). At 10 µM, AADU induced a 55 ± 14% increase in vessel diameter, whereas AUDA induced a 48 ± 7% increase in vessel diameter. Finally, shortening the aliphatic side chain from 12 carbons to eight carbons, AUOA, or to four carbons, AUBA, significantly attenuated the vasodilatory response (Fig. 4). AUOA resulted in a -2 ± 4% relaxation, and AUBA resulted in a 3 ± 11% relaxation of mesenteric resistance vessels at the 10 µM dose. Interestingly, shortening the aliphatic side chain from 12 carbons to six carbons, AUHA, altered the tendency for the chain-shortened analogs to decrease activity (Fig. 4). At 10 µM, AUHA induced a 22 ± 9% relaxation of mesenteric resistance vessels. After treatment with the highest dose of AUDA or its analogs, acetylcholine (10 µM) was added to the isolated mesenteric resistance vessel preparation to ensure endothelial integrity. Acetylcholine increased vessel diameter to 242.5 ± 5.0 µm, a 106 ± 3% dilation.
|
|
|
|
; Yang et al., 2005; Ye et al., 2005, 2006). To investigate the possible roles of these channels on AUDA-induced vascular reactivity, mesenteric resistance vessels were treated with ouabain, an inhibitor of the Na+/K+-ATPase, or glibenclamide, an inhibitor of the KATP channel. In experiments using ouabain, the starting vessel diameter measured 216.4 ± 9.7 µm and measured 51.8 ± 2.7 µm after treatment with U46619
[GenBank]
(n = 5). Treatment with ouabain did not significantly alter the vessel diameter, 79.2 ± 20.8 µm. AUDA-induced relaxation was not affected by ouabain treatment (Fig. 7). At the 10 µM dose, AUDA induced a 42 ± 12% increase in vessel diameter (Fig. 7). In experiments using glibenclamide, the starting vessel diameter measured 194.2 ± 15.8 µm and measured 102.3 ± 14.0 µm after treatment with U46619
[GenBank]
(n = 4). Treatment with glibenclamide resulted in a 28 ± 4% increase in vessel diameter. Glibenclamide treatment did tend to inhibit increases in vessel diameter induced by AUDA; however, these decreases were not statistically significant (Fig. 7). To determine whether adamantyl-urea inhibitors induce vasodilation indirectly via their actions on the endothelium or directly via their actions on the vascular smooth muscle, experiments were conducted using mesenteric resistance vessels where the endothelium was disrupted. The average vessel diameter measured 168.4 ± 12.9 µm and measured 85.0 ± 8.6 µm after treatment with U46619. [GenBank] Treatment of the preconstricted, denuded vessel with acetylcholine did not statistically alter the vessel diameter, which averaged 93.9 ± 14.3 µm. Disrupting the endothelium did not affect the AUDA-induced relaxation (Fig. 8). At the 10 µM dose, AUDA elicited 48.1 ± 7.0% dilation in nondenuded vessels and 39.5 ± 15.4% dilation in denuded vessels. The addition of sodium nitroprusside (10 µM) to the denuded mesenteric resistance vessel at the end of the experiment increased the vessel diameter to 163.6 ± 10.4 µm, a 158 ± 29% dilation.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Newman et al., 2005). EETs, cytochrome P450 epoxygenase metabolites of arachidonic acid, are endogenously produced epoxides that serve as substrates for the sEH enzyme. The metabolism of EETs by sEH into the corresponding dihydroxyeicosatrienoic acids results in partial or complete loss of activity (Moghaddam et al., 1997; Zeldin, 2001; Spector et al., 2004). The EETs have been identified as important regulatory molecules in the cardiovascular and renal circulations. As a result, the sEH enzyme has been identified as a therapeutic target for diseases like hypertension and inflammation. Inhibitors of the sEH enzyme have been successfully employed in animal models of hypertension where sEH inhibition was reported to decrease blood pressure and decrease hypertension-induced renal damage. These renal and cardiovascular protective effects are attributed, at least in part, to the blood pressure-lowering effects of the sEH inhibitor. Within the present study, we addressed the hypothesis that a mechanism by which sEH inhibitors elicit their cardiovascular protective effects is via their direct actions on the vasculature.
We demonstrated, using isolated mesenteric resistance vessels, that treatment with adamantyl-urea inhibitors of the sEH enzyme resulted in a concentration-dependent relaxation of the preconstricted vessel. The dilator responses observed in mesenteric resistance vessels occur at concentrations that are above the reported IC50 for the sEH enzyme. This response was also demonstrated with the sEH inhibitor CDU but was not observed with the more sterically hindered sEH inhibitor 950. These data suggest that the vasoactive properties measured with adamantyl-urea inhibitors are unique to their structure and are independent of their ability to inhibit the sEH enzyme itself. Adamantyl-urea inhibitors are 1,3-disubstituted ureas with an adamantyl group on one side of the urea pharmacophore and an 11-carbon aliphatic chain on the other (Table 1). To investigate what structural elements of adamantyl-urea inhibitors are responsible for these unique vasoreactive responses, analogs of AUDA were prepared (Table 1). Each of the analogs of AUDA had IC50s for the human sEH enzyme in the nanomolar range.
The sEH inhibitor CUDA exhibited significantly decreased vasoreactivity in mesenteric vessels compared with that measured with AUDA. The structural difference between CUDA and AUDA is that CUDA has a cyclohexyl group on one side of the urea pharmacophore, whereas AUDA has an adamantyl group. These data would suggest that the adamantyl group is an important structural feature that is unique to adamantyl-urea inhibitors and is responsible for the observed vasoactive properties. Interestingly, the adamantyl functional group was determined to be an important structural feature for the inhibition of the sEH enzyme itself (Morisseau et al., 2002). CUDA shares some structural similarity to CDU in that both sEH inhibitors have a cyclohexyl group occupying one side of the urea pharmacophore. This could explain why CDU exhibits a similar reactivity as CUDA in this preparation. However, in addition to having a cyclohexyl group on one side of the urea pharmacophore, the structure of CDU also differs from AUDA in that it has an aliphatic chain terminating with a methyl group instead of a carboxylic acid. This could explain why at the highest concentration, CDU demonstrated similar reactivity to AUDA.
When the negatively charged carboxylic acid at the end of the aliphatic carbon chain of AUDA was changed to a positively charged amine group (Fig. 3, AADU), the vascular reactivity of AUDA was not altered. These data suggest that the terminal functional group at the end of the aliphatic carbon chain is not an important structural feature for the vascular reactivity observed with adamantyl-urea inhibitors. Interestingly, altering this functional group also had little effect on the IC50 for the human sEH enzyme. This finding would also suggest that the inability of CDU to induce similar vasodilations was mainly the result of the cyclohexyl functional group, not the methyl group at the end of the aliphatic carbon chain. Changing the length of the aliphatic carbon chain, however, did alter the observed vasoreactivity of adamantyl-urea inhibitors. AUOA, which shortened the length of the aliphatic carbon chain from 12 to eight carbons, significantly attenuated the vasoactive properties of AUDA. The same result was observed with AUBA, which shortened the length of the aliphatic carbon chain from 12 to four carbons. AUHA, which has an aliphatic carbon chain length of six carbons, demonstrated a slightly attenuated vasoreactivity compared with AUDA. The human sEH enzyme IC50s for AUOA, AUHA, and AUBA did not directly correspond to the observed vasoreactivity. Of the chain-shortened analogs, AUOA has the lowest IC50 for the human sEH but demonstrated very little vasoreactivity compared with AUDA. AUBA had the highest IC50 for the human sEH enzyme and also had the least amount of vasoactivity. AUHA had an intermediate IC50 for the human sEH enzyme, and although the responses look attenuated compared with those measured with AUDA, they are not statistically different. Each 
bond that comprises the aliphatic carbon side chain can freely rotate giving the side chain significant flexibility. Therefore, it is possible that the compounds with the 12-carbon (AUDA) and six-carbon (AUHA) side chains are able to assume a configuration that allows them to induce the observed relaxation. Conversely, these configurations might not be possible in the eight-carbon (AUOA) or four-carbon (AUBA) side chains. In addition, 950, which has a carbon side chain that is significantly more sterically hindered by the addition of oxygen atoms to the carbon side chain and therefore cannot assume conformations that are available to AUDA, does not induce changes in vessel diameter. Taken together, these data suggest that the length of the aliphatic carbon chain and its flexibility are important structural features of adamantyl-urea inhibitors that are responsible for the observed vasoreactive properties.
Inhibitors of the sEH enzyme increase endogenous EET levels by decreasing the metabolism of the EETs to less active diols. If the mechanism by which adamantyl-urea inhibitors induce vasorelaxation is by increasing endogenous EET levels, then it would be expected that inhibiting EET synthesis would attenuate the AUDA-induced response. However, pretreatment of mesenteric resistance vessels with the epoxygenase inhibitor MS-PPOH did not alter AUDA-induced increases in vessel diameter. One interpretation of these data is that the mechanism by which AUDA induces vascular reactivity is not only due to increasing EET levels but may have effects independent of sEH inhibition.
One mechanism by which the EETs exert their renal and cardiovascular effects is via activation of calcium-activated potassium channels in the endothelium and smooth muscle cells, resulting in a decrease in the membrane potential (Chataigneau et al., 1998; Wu et al., 2000). Neither TEA, a nonspecific potassium channel blocker, nor IbTX, a specific calcium-activated potassium channel blocker, attenuated the observed AUDA-induced dilation (Fig. 4). These data suggest that a possible mechanism by which adamantyl-urea inhibitors exert their vasoactive effects is not through their action on these specific potassium channels. In addition, these data suggest that the dilation observed with AUDA may not due to the inhibition of sEH and the resulting increase in vascular EET levels.
The EETs have also been reported to alter the activity of other channels that might participate in the observed vaso-reactivity, the Na+/K+-ATPase and the KATP channel (Pratt et al., 2001; Yang et al., 2005; Ye et al., 2005, 2006). The observed AUDA-induced vasorelaxation was not altered with ouabain treatment; however, glibenclamide did tend to attenuate the AUDA-induced response, although this decrease did not reach statistical significance. Therefore, the mechanism by which adamantyl-urea inhibitors induce increases in vessel diameter is not by their actions on the Na+/K+-ATPase or the KATP channel.
Denuded mesenteric resistance vessels were used to determine whether adamantyl-urea inhibitors are acting on the endothelium or directly on the smooth muscle to induce the observed vasodilation. Disrupting the endothelium did not affect AUDA-induced relaxation at the lower concentrations tested. However, disrupting the endothelium did result in a more robust relaxation in response to 100 µM AUDA compared with vessels where the endothelium remained intact. These data provide evidence that AUDA acts directly on the vascular smooth muscle in an endothelium-independent manner to induce its vascular reactivity. In addition, AUDA-induced relaxation was completely abolished when mesenteric resistance vessels were pretreated with KCl. This finding provides additional support that AUDA acts on a vascular smooth muscle cell membrane channel. Therefore, it is possible that AUDA is perturbing the function of ligand- and/or ion-gated channel or channels present in the smooth muscle membrane.
Taken together, these data provide support for the hypothesis that the adamantyl-urea inhibitors of the sEH enzyme have unique vasodilator actions compared with other sEH inhibitors and that these actions depend on specific structural elements, the adamantyl group, and carbon chain length. AUDA does not act on potassium channels, the Na+/K+-ATPase, or the KATP channel, known EET effector proteins; however, the effects of AUDA could still be mediated by other membrane potential-sensitive mechanisms. In addition, AUDA was found to act directly on the vascular smooth muscle to induce vasodilation, although this mechanism requires further investigation.
|
Footnotes |
|---|
ABBREVIATIONS: sEH, soluble epoxide hydrolase; EET, epoxyeicosatrienoic acid; AUDA, 12-(3-adamantan-1-yl-ureido)dodecanoic acid; CUDA, 12-(3-cyclohexyl-ureido)dodecanoic acid; AUOA, 8-(3-adamantyl-ureido)-octanoic acid; AUHA, 6-(3-adamantyl-ureido)-hexanoic acid; AUBA, 4-(3-adamantan-1-yl-ureido)butanoic acid; AADU, 1-adamantyl-3-(12-aminododecyl)urea; 950, adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea; DMSO, dimethyl sulfoxide; CDU, 1-cyclohexyl-3-dodecylurea; IbTX, iberiotoxin; MS-PPOH, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide; PBS-T, phosphate-buffered saline containing 0.1% Tween 20; TEA, tetraethylammonium; DMF, dimethyl formamide; E-64, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane; LC, liquid chromatography; MS, mass spectrometry; TOF, time-of-flight; U46619
[GenBank]
, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F2.
Address correspondence to: Dr. Jeffrey J. Olearczyk, The Medical College of Georgia, Vascular Biology Center, 1120 15th Street, Augusta, GA 30912. E-mail: jolearczyk{at}mail.mcg.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Campbell WB, Gebremedhin D, Pratt PF, and Harder DR (1996) Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 78: 415-423.
Chataigneau T, Feletou M, Duhault J, and Vanhoutte PM (1998) Epoxyeicosatrienoic acids, potassium channel blockers and endothelium-dependent hyperpolarization in the guinea-pig carotid artery. Br J Pharmacol 123: 574-580.[CrossRef][Medline]
Enayetallah AE, French RA, Barber M, and Grant DF (2006) Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues. J Histochem Cytochem 54: 329-335.
Falck JR, Reddy LM, Reddy YK, Bondlela M, Krishna UM, Ji Y, Sun J, and Liao JK (2003) 11,12-epoxyeicosatrienoic acid (11,12-EET): structural determinants for inhibition of TNF-alpha-induced VCAM-1 expression. Bioorg Med Chem Lett 13: 4011-4014.[CrossRef][Medline]
Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, and Busse R (1999) Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature (Lond) 401: 493-497.[CrossRef][Medline]
Imig JD, Zhao X, Capdevila JH, Morisseau C, and Hammock BD (2002) Soluble epoxide hydrolase inhibition lowers arterial blood pressure in angiotensin II hypertension. Hypertension 39: 690-694.
Imig JD, Zhao X, Zharis C, Olearczyk JJ, Pollock DM, Newman JW, Kim I-H, Watanabe T, and Hammock BD (2005) An orally active epoxide hydrolase inhibitor lowers blood pressure and provides renal protection in salt-sensitive hypertension. Hypertension 46: 1-7.
Kim IH, Morisseau C, Watanabe T, and Hammock BD (2004) Design, synthesis, and biological activity of 1,3-disubstituted ureas as potent inhibitors of the soluble epoxide hydrolase of increased water solubility. J Med Chem 47: 2110-2122.[CrossRef][Medline]
Moghaddam MF, Grant DF, Cheek JM, Greene JF, Williamson KC, and Hammock BD (1997) Bioactivation of leukotoxins to their toxic diols by epoxide hydrolase. Nat Med 3: 562-566.[CrossRef][Medline]
Morisseau C, Goodrow MH, Newman JW, Wheelock CE, Dowdy DL, and Hammock BD (2002) Structural refinement of inhibitors of urea-based soluble epoxide hydrolases. Biochem Pharmacol 63: 1599-1608.[CrossRef][Medline]
Morisseau C and Hammock BD (2005) Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles. Annu Rev Pharmacol Toxicol 45: 311-333.[CrossRef][Medline]
Newman JW, Morisseau C, and Hammock BD (2005) Epoxide hydrolases: their roles and interactions with lipid metabolism. Prog Lipid Res 44: 1-51.[CrossRef][Medline]
Node K, Huo Y, Ruan X, Yang B, Spiecker M, Ley K, Zeldin DC, and Liao JK (1999) Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science (Wash DC) 285: 1276-1279.
Pratt PF, Li P, Hillard CJ, Kurian J, and Campbell WB (2001) Endothelium-independent, ouabain-sensitive relaxation of bovine coronary arteries by EETs. Am J Physiol 280: H1113-H1121.
Schmelzer KR, Kubala L, Newman JW, Kim IH, Eiserich JP, and Hammock BD (2005) Soluble epoxide hydrolase is a therapeutic target for acute inflammation. Proc Natl Acad Sci USA 102: 9772-9777.
Smith KR, Pinkerton KE, Watanabe T, Pedersen TL, Ma SJ, and Hammock BD (2005) Attenuation of tobacco smoke-induced lung inflammation by treatment with a soluble epoxide hydrolase inhibitor. Proc Natl Acad Sci USA 102: 2186-2191.
Spector AA, Fang X, Snyder GD, and Weintraub NL (2004) Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog Lipid Res 43: 55-90.[CrossRef][Medline]
Wang P, Meijer J, and Guengerich FP (1982) Purification of human liver cytosolic epoxide hydrolase and comparison to the microsomal enzyme. Biochemistry 21: 5769-5776.[CrossRef][Medline]
Wu SN, Li HF, and Chiang HT (2000) Actions of epoxyeicosatrienoic acid on largeconductance Ca(2+)-activated K(+) channels in pituitary GH(3) cells. Biochem Pharmacol 60: 251-262.[CrossRef][Medline]
Yang W, Gauthier KM, Reddy LM, Sangras B, Sharma KK, Nithipatikom K, Falck JR, and Campbell WB (2005) Stable 5,6-epoxyeicosatrienoic acid analog relaxes coronary arteries through potassium channel activation. Hypertension 45: 681-686.
Ye D, Zhou W, and Lee HC (2005) Activation of rat mesenteric arterial KATP channels by 11,12-epoxyeicosatrienoic acid. Am J Physiol 288: H358-H364.
Ye D, Zhou W, Lu T, Jagadeesh SG, Falck JR, and Lee HC (2006) Mechanism of rat mesenteric arterial KATP channel activation by 14,15-epoxyeicosatrienoic acid. Am J Physiol 290: H1326-H1336.
Yu Z, Davis BB, Morisseau C, Hammock BD, Olson JL, Kroetz DL, and Weiss RH (2004) Vascular localization of soluble epoxide hydrolase in the human kidney. Am J Physiol 286: F720-F726.
Yu Z, Xu F, Huse LM, Morisseau C, Draper AJ, Newman JW, Parker C, Graham L, Engler MM, Hammock BD, Zeldin DC, and Kroetz DL (2000) Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids. Circ Res 87: 992-998.
Zeldin DC (2001) Epoxygenase pathways of arachidonic acid metabolism. J Biol Chem 276: 36059-36062.
Zhao X, Yamamoto T, Newman JW, Kim IH, Watanabe T, Hammock BD, Stewart J, Pollock JS, Pollock DM, and Imig JD (2004) Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage. J Am Soc Nephrol 15: 1244-1253.
This article has been cited by other articles:
|
|
 |
|
  M. J. Corenblum, V. E. Wise, K. Georgi, B. D. Hammock, P. A. Doris, and M. Fornage Altered Soluble Epoxide Hydrolase Gene Expression and Function and Vascular Disease Risk in the Stroke-Prone Spontaneously Hypertensive Rat Hypertension, February 1, 2008; 51(2): 567 - 573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Spector and A. W. Norris Action of epoxyeicosatrienoic acids on cellular function Am J Physiol Cell Physiol, March 1, 2007; 292(3): C996 - C1012. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, P < 0.05, different from other drugs at the same dose.
