Azilsartan is associated with increased circulating angiotensin-(1-7) levels and reduced renovascular 20-HETE levels.

BACKGROUND: Activation of angiotensin (ANG) II type 1 receptors (AT1R) promotes vasoconstriction, inflammation, and renal dysfunction. In this study, we addressed the ability of azilsartan (AZL), a new AT1R antagonist, to modulate levels of plasma ANG-(1-7) and renal epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE). METHODS: Sprague-Dawley rats were infused with ANG II (125 ng/min) or vehicle (VEH). AZL (3 mg/kg/day) or VEH was administered starting 1 day prior to ANG II or VEH infusion. On day 10, plasma was obtained for measurement of ANG-(1-7) and kidneys for isolation of microvessels for EET and 20-HETE determination and histological evaluation. RESULTS: Mean 24-hour blood pressure (BP) was not different between VEH and AZL treatment groups, whereas the BP elevation with ANG II infusion (121 ± 5 mm Hg) was completely normalized with AZL cotreatment (86 ± 3 mm Hg). The ANG II-induced renal damage was attenuated and cardiac hypertrophy prevented with AZL cotreatment. Plasma ANG-(1-7) levels (pg/ml) were increased with AZL treatment (219 ± 22) and AZL + ANG II infusion (264 ± 93) compared to VEH controls (74.62 ± 8). AZL treatment increased the ratio of EETs to their dihydroxyeicosatrienoic acid (DHET) metabolites and reduced 20-HETE levels. CONCLUSIONS: Treatment with AZL completely antagonized the elevation of BP induced by ANG II, prevented cardiac hypertrophy, attenuated renal damage, and increased ANG-(1-7) and EET/DHET ratio while diminishing 20-HETE levels. Increased ANG-(1-7) and EETs levels may emerge as novel therapeutic mechanisms contributing to the antihypertensive and antihypertrophic actions of AZL treatment and their relative role compared to AT1R blockade may depend on the etiology of the hypertension.

Pharmacological manipulation of arachidonic acid-epoxygenase results in divergent effects on renal damage.

Kidney damage is markedly accelerated by high-salt (HS) intake in stroke-prone spontaneously hypertensive rats (SHRSP). Epoxyeicosatrienoic acids (EETs) are epoxygenase products of arachidonic acid which possess vasodepressor, natriuretic, and anti-inflammatory activities. We examined whether up-regulation (clofibrate) or inhibition [N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH)] of epoxygenase would alter systolic blood pressure (SBP) and/or renal pathology in SHRSP on HS intake (1% NaCl drinking solution). Three weeks of treatment with clofibrate induced renal cortical protein expression of CYP2C23 and increased urinary excretion of EETs compared with vehicle-treated SHRSP. SBP and urinary protein excretion (UPE) were significantly lowered with clofibrate treatment. Kidneys from vehicle-treated SHRSP, which were on HS intake for 3 weeks, demonstrated focal lesions of vascular fibrinoid degeneration, which were markedly attenuated with clofibrate treatment. In contrast, 2 weeks of treatment with the selective epoxygenase inhibitor, MS-PPOH, increased UPE without significantly altering neither urinary EET levels nor SBP. Kidneys from vehicle-treated SHRSP, which were on HS intake for 11 days, demonstrated occasional mild damage whereas kidneys from MS-PPOH-treated rats exhibited widespread malignant nephrosclerosis. These results suggest that pharmacological manipulation of epoxygenase results in divergent effects on renal damage and that interventions to increase EET levels may provide therapeutic strategies for treating salt-sensitive hypertension and renal damage.

Role of the adenosine(2A) receptor-epoxyeicosatrienoic acid pathway in the development of salt-sensitive hypertension.

Activation of rat adenosine(2A) receptors (A(2A) R) dilates preglomerular microvessels, an effect mediated by epoxyeicosatrienoic acids (EETs). High salt (HS) intake increases epoxygenase activity and adenosine levels. A greater vasodilator response to a stable adenosine analog, 2-chloroadenosine (2-CA), was seen in kidneys obtained from HS-fed rats which was mediated by increased EET release. Because this pathway is antipressor, we examined the role of the A(2A) R-EET pathway in a genetic model of salt-sensitive hypertension, the Dahl salt-sensitive (SS) rats. Dahl salt resistant (SR) rats fed a HS diet demonstrated a greater renal vasodilator response to 2-CA. In contrast, Dahl SS rats did not exhibit a difference in the vasodilator response to 2-CA whether fed normal salt (NS) or HS diet. In Dahl SR but not Dahl SS rats, HS intake significantly increased purine flux, augmented the protein expression of A(2A) R and cytochrome P450 2C23 and 2C11 epoxygenases, and elevated the renal efflux of EETs. Thus the Dahl SR rat is able to respond to HS intake by recruiting EET formation, whereas the Dahl SS rat appears to have exhausted its ability to increase EET synthesis above the levels observed on NS intake. In vivo inhibition of the A(2A) R-EET pathway in Dahl SR rats fed a HS diet results in reduced renal EETs levels, diminished natriuretic capacity and hypertension, thus supporting a role for the A(2A) R-EET pathway in the adaptive natriuretic response to modulate blood pressure during salt loading. An inability of Dahl SS rats to upregulate the A(2A) R-EET pathway in response to salt loading may contribute to the development of salt-sensitive hypertension.

Increases in plasma trans-EETs and blood pressure reduction in spontaneously hypertensive rats.

Epoxyeicosatrienoic acids (EETs) are vasodilator, natriuretic, and antiinflammatory lipid mediators. Both cis- and trans-EETs are stored in phospholipids and in red blood cells (RBCs) in the circulation; the maximal velocity (V(max)) of trans-EET hydrolysis by soluble epoxide hydrolase (sEH) is threefold that of cis-EETs. Because RBCs of the spontaneously hypertensive rat (SHR) exhibit increased sEH activity, a deficiency of trans-EETs in the SHR was hypothesized to increase blood pressure (BP). This prediction was fulfilled, since sEH inhibition with cis-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid (AUCB; 2 mg·kg(-1)·day(-1) for 7 days) in the SHR reduced mean BP from 176 ± 8 to 153 ± 5 mmHg (P < 0.05), whereas BP in the control Wistar-Kyoto rat (WKY) was unaffected. Plasma levels of EETs in the SHR were lower than in the age-matched control WKY (16.4 ± 1.6 vs. 26.1 ± 1.8 ng/ml; P < 0.05). The decrease in BP in the SHR treated with AUCB was associated with an increase in plasma EETs, which was mostly accounted for by increasing trans-EET from 4.1 ± 0.2 to 7.9 ± 1.5 ng/ml (P < 0.05). Consistent with the effect of increased plasma trans-EETs and reduced BP in the SHR, the 14,15-trans-EET was more potent (ED(50) 10(-10) M; maximum dilation 59 ± 15 µm) than the cis-isomer (ED(50) 10(-9) M; maximum dilation 30 ± 11 µm) in relaxing rat preconstricted arcuate arteries. The 11,12-EET cis- and trans-isomers were equipotent dilators as were the 8,9-EET isomers. In summary, inhibition of sEH resulted in a twofold increase in plasma trans-EETs and reduced mean BP in the SHR. The greater vasodilator potency of trans- vs. cis-EETs may contribute to the antihypertensive effects of sEH inhibitors.

Inhibition of the adenosine2A receptor-epoxyeicosatrienoic acid pathway renders Dahl salt-resistant rats hypertensive.

Adenosine-induced renovasodilation in Dahl rats is mediated via activation of adenosine(2A) receptors (A(2A)Rs) and stimulation of epoxyeicosatrienoic acid (EET) synthesis. Unlike Dahl salt-resistant rats, salt-sensitive rats exhibit an inability to upregulate the A(2A)R-EET pathway with salt loading; therefore, we examined the effect of in vivo inhibition of the A(2A)R-EET pathway on blood pressure and the natriuretic response to salt-loading in Dahl salt-resistant rats. N-Methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 20 mg/kg per day), an epoxygenase inhibitor, or ZM241385 (ZM; 5 mg/kg per day), an A(2A)R antagonist, was given daily as an IV bolus dose for 3 days before and after placing rats on high salt intake (2% saline). After 3 days of high salt, systolic blood pressure per 24 hours increased from 108+/-2 mm Hg to 136+/-5 mm Hg and 140+/-4 mm Hg when treated with MS-PPOH or ZM, respectively (P<0.001). Plasma levels of EETs and dihydroxyeicosatrienoic acids during salt loading and MS-PPOH (29.3+/-1.8 ng/mL) or ZM treatment (9.8+/-0.5 ng/mL) did not increase to the same extent as in vehicle-treated rats (59.4+/-1.7 ng/mL; P<0.001), and renal levels of EETs+dihydroxyeicosatrienoic acids were 2-fold lower with MS-PPOH or ZM treatment. On day 3 of the high salt intake, MS-PPOH- and ZM-treated rats exhibited a positive Na(+) balance, and plasma Na(+) levels were significantly increased (163.3+/-1.2 and 158.1+/-4.5 mEq/L, respectively) compared with vehicle-treated rats (142.1+/-1 mEq/L), reflecting a diminished natriuretic capacity. These data support a role for the A(2A)R-EET pathway in the adaptive natriuretic response to modulate blood pressure during salt loading.

Failure to upregulate the adenosine2A receptor-epoxyeicosatrienoic acid pathway contributes to the development of hypertension in Dahl salt-sensitive rats.

Adenosine-activated renovascular dilatation in Sprague-Dawley (SD) rats is mediated by stimulating adenosine(2A) receptors (A(2A)R), which is linked to epoxyeicosatrienoic acid (EET) synthesis. The A(2A)R-EET pathway is upregulated by high salt (HS) intake in normotensive SD rats. Because this pathway is antipressor, we examined the role of the A(2A)R-EET pathway in Dahl salt-sensitive (SS) rats. Male Dahl salt-resistant (SR) and SS rats were fed either HS (8.0% NaCl) or normal salt (NS; 0.4% NaCl) diet for 7 days. On day 8, isolated kidneys were perfused with Krebs-Henseleit buffer containing indomethacin and N(G)-nitro-l-arginine methyl ester and preconstricted with phenylephrine. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1-20 microg) elicited dose-dependent dilation in both Dahl SR and SS rats. Dahl SR rats fed a HS diet demonstrated a greater renal vasodilator response to 10 microg of 2-CA, as measured by the reduction in renal perfusion pressure, than that of Dahl SR rats fed a NS diet (-104 +/- 6 vs. -77 +/- 7 mmHg, respectively; P < 0.05). In contrast, Dahl SS rats did not exhibit a difference in the vasodilator response to 2-CA whether fed NS or HS diet (96 +/- 6 vs. 104 +/- 13 mmHg in NS- and HS-fed rats, respectively). In Dahl SR but not Dahl SS rats, HS intake significantly increased purine flux, augmented the protein expression of A(2A)R and the cytochrome P-450 2C23 and 2C11 epoxygenases, and elevated the renal efflux of EETs. Thus the Dahl SR rat is able to respond to HS intake by recruiting EET formation, whereas the Dahl SS rat appears to have exhausted its ability to increase EET synthesis above the levels observed on NS intake, and this inability of Dahl SS rats to upregulate the A(2A)R-EET pathway in response to salt loading may contribute to the development of salt-sensitive hypertension.

Soluble epoxide hydrolase inhibitor, AUDA, prevents early salt-sensitive hypertension.

In stroke-prone spontaneously hypertensive rats (SHRSP) end-organ damage is markedly accelerated by high-salt (HS) intake. Since epoxyeicosatrienoic acids (EETs) possess vasodepressor and natriuretic activities, we examined whether a soluble epoxide hydrolase (sEH) inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), to inhibit the metabolism of EETs, would protect against pathologic changes in SHRSP. Seven-week-old male SHRSP were treated as follows: normal salt (NS), NS + AUDA, HS and HS + AUDA. Systolic blood pressure (SBP) (205 +/- 4 v 187 +/- 7 mmHg) and proteinuria (3.7 +/- 0.2 v 2.6 +/- 0.2 mg/6 h), but not plasma EETs (11.0 +/- 0.9 v 9.7 +/- 1.1 ng/ml), were significantly increased at 9 weeks of age in HS v NS SHRSP. HS was associated with fibrinoid degeneration and hypertrophy of arterioles in the kidney and perivascular fibrosis and contraction band necrosis in the heart. AUDA ameliorated these early salt-dependent changes in saline-drinking SHRSP and increased plasma levels of EETs but did not affect water and electrolyte excretion. sEH inhibition may provide a therapeutic strategy for treating salt-sensitive hypertension and its sequelae.

Contribution of epoxyeicosatrienoic acids to flow-induced dilation in arteries of male ERalpha knockout mice: role of aromatase.

We studied the roles of estrogen receptors (ER) and aromatase in the mediation of flow-induced dilation (FID) in isolated arteries of male ERalpha-knockout (ERalpha-KO) and wild-type (WT) mice. FID was comparable between gracilis arteries of WT and ERalpha-KO mice. In WT arteries, inhibition of NO and prostaglandins eliminated FID. In ERalpha-KO arteries, N(omega)-nitro-L-arginine methyl ester (L-NAME) inhibited FID by approximately 26%, whereas indomethacin inhibited dilations by approximately 50%. The remaining portion of the dilation was abolished by additional administration of 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) or iberiotoxin, inhibitors of epoxyeicosatrienoic acid (EET) synthesis and large-conductance potassium channels, respectively. By using an electrophysiological technique, we found that, in the presence of 10 dyne/cm(2) shear stress, perfusate passing through donor vessels isolated from gracilis muscle of ERalpha-KO mice subjected to L-NAME and indomethacin elicited smooth muscle hyperpolarization and a dilator response of endothelium-denuded detector vessels. These responses were prevented by the presence of iberiotoxin in detector or PPOH in donor vessels. Gas chromatography-mass spectrometry (GC-MS) analysis indicated a significant increase in arterial production of EETs in ERalpha-KO compared with WT mice. Western blot analysis showed a significantly reduced endothelial nitric oxide synthase expression but enhanced expressions of aromatase and ERbeta in ERalpha-KO arteries. Treatment of ERalpha-KO arteries with specific aromatase short-interfering RNA for 72 h, knocked down the aromatase mRNA and protein associated with elimination of EET-mediation of FID. Thus, FID in male ERalpha-KO arteries is maintained via an endothelium-derived hyperpolarizing factor/EET-mediated mechanism compensating for reduced NO mediation due, at least in part, to estrogen aromatized from testosterone.

Adenosine2A receptor vasodilation of rat preglomerular microvessels is mediated by EETs that activate the cAMP/PKA pathway.

Dilation of rat preglomerular microvessels (PGMV) by activation of adenosine A2A receptors (A2AR) is coupled to epoxyeicosatrienoic acid (EET) release. We have investigated the commonality of this signal transduction pathway, i.e., sequential inhibition of G(salpha), adenylyl cyclase, PKA, and Ca2+-activated K+ (KCa) channel activity, to the vasoactive responses to A2AR activation by a selective A2A agonist, CGS-21680, compared with those of 11,12-EET. Male Sprague-Dawley rats were anesthetized, and microdissected arcuate arteries (110-130 microm) were cannulated and pressurized to 80 mmHg. Vessels were superfused with Krebs solution containing NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin and preconstricted with phenylephrine. We assessed the effect of 3-aminobenzamide (10 microM), an inhibitor of mono-ADP-ribosyltranferases, on responses to 11,12-EET (3 nM) and CGS-21680 (10 microM) and found that both were inhibited by approximately 70% (P<0.05), whereas the response to SNP (10 microM) was unaffected. Furthermore, 11,12-EET (100 nM), like cholera toxin (100 ng/ml), stimulated ADP-ribose formation in homogenates of arcuate arteries compared with control. SQ-22536 (10 microM), an inhibitor of adenylyl cyclase activity, and myristolated PKI (14-22) amide (5 microM), an inhibitor of PKA, decreased activity of 11,12-EET and CGS-21680. Incubation of 11,12-EET (3 nM-3 microM) with PGMV resulted in an increase in cAMP levels (P<0.05). The responses to both 11,12-EET and CGS-21680 were significantly reduced by superfusion of iberiotoxin (100 nM), an inhibitor of KCa channel activity. Thus in rat PGMV activation of A2AR is coupled to EET release upstream of adenylyl cyclase activation and EETs stimulate mono-ADP-ribosyltransferase, resulting in Gsalpha protein activation.

Adenosine inhibits ENaC via cytochrome P-450 epoxygenase-dependent metabolites of arachidonic acid.

We used the patch-clamp technique to examine the effect of adenosine on epithelial sodium channel (ENaC) activity in rat cortical collecting duct (CCD). Application of adenosine inhibits ENaC activity, and the effect of adenosine was mimicked by cyclohexyladenosine (CHA), an A(1) adenosine-receptor agonist that reduced channel activity from 1.32 to 0.64. The inhibitory effect of CHA on ENaC was mimicked by cyclopentyladenosine (CPA), which reduced channel activity from 1.1 to 0.55. In contrast, application of CGS-21680, an A(2a) adenosine-receptor agonist, had no effect on ENaC and increased channel activity from 0.96 to 1.22. This suggests that the inhibitory effect of adenosine analogs resulted from stimulation of the A(1) adenosine receptor. Inhibition of PLC with U-73122 failed to abolish the effect of CHA on ENaC. In contrast, the inhibitory effect of CHA on ENaC was absent in the presence of the PLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)). This suggests a role of arachidonic acid (AA) in mediating the effect of adenosine on ENaC. To determine the metabolic pathway of AA responsible for the effect of adenosine, we examined the effect of CHA in the presence of indomethacin or N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH). Inhibition of cytochrome P-450 (CYP) epoxygenase with MS-PPOH blocked the effect of CHA on ENaC. In contrast, CHA reduced ENaC activity in the presence of indomethacin. This suggests that CYP epoxygenase-dependent metabolites of AA mediate the effect of adenosine. Because 11,12-epoxyeicosatrienoic acid (11,12-EET) inhibits ENaC activity in the CCD (Wei Y, Lin DH, Kemp R, Yaddanapudi GSS, Nasjletti A, Falck JR, and Wang WH. J Gen Physiol 124: 719-727, 2004), we examined the role of 11,12-EET in mediating the effect of adenosine on ENaC. Addition of 11,12-EET inhibited ENaC channels in the CCD in which adenosine-induced inhibition was blocked by AACOCF3. We conclude that adenosine inhibits ENaC activity by stimulation of the A(1) adenosine receptor in the CCD and that the effect of adenosine is mediated by 11,12-EET.

Exaggerated response to adenosine in kidneys from high salt-fed rats: role of epoxyeicosatrienoic acids.

Cytochrome P-450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) dilate rat preglomerular microvessels when adenosine(2A) receptors (A(2A)R) are stimulated. As high salt (HS) intake increases epoxygenase activity and adenosine levels, we hypothesized that renal adenosine responses would be greater in HS-fed rats. Male Sprague-Dawley rats were fed either HS (4.0% NaCl) or normal salt (NS; 0.4% NaCl) diet. On day 8, isolated kidneys were perfused with Krebs' buffer containing indomethacin (10 microM) and L-NAME (200 microM) and preconstricted to approximately 150 mmHg with infusion of phenylephrine (10(-7) M). Renal effluents were extracted for analysis of eicosanoids by gas chromatography-mass spectrometry. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1-10 microg) resulted in dose-dependent dilation; at 10 microg, perfusion pressure (PP) was lowered to a greater extent in the kidneys of HS rats compared with NS rats (-60 +/- 4 vs. -31 +/- 8 mmHg; P < 0.05) and the area of response was increased (27 +/- 6 vs. 9 +/- 4 mm(2); P < 0.05), as was EET release (132 +/- 23 vs. 38 +/- 18 ng; P < 0.05). HS treatment increased A(2A)R and CYP2C23 protein expression. A selective epoxygenase inhibitor, MS-PPOH (12 microM), significantly reduced the response to 2-CA in HS rats; PP, area of response, and EET release decreased by 40, 70, and 81%, respectively, whereas lesser changes were evident in NS kidneys. Thus the greater vasodilator response to 2-CA seen in kidneys obtained from HS-fed rats was mediated by increased EET release. As EETs are renal vasodilator and natriuretic eicosanoids, interactions between adenosine and EETs may contribute to the adaptive response to HS intake.

Epoxyeicosatrienoic acids are released to mediate shear stress-dependent hyperpolarization of arteriolar smooth muscle.

We hypothesized that shear stress stimulates the release of epoxyeicosatrienoic acids (EETs) from arteriolar endothelium, which directly hyperpolarize smooth muscle. To test this hypothesis, a perfusion system, consisting of two separate, serially connected chambers (A and B), was used. A donor vessel, isolated from gracilis muscle of female NO-deficient mice and rats, was cannulated in chamber A. In chamber B, an endothelium-denuded detector vessel isolated from mesentery of these animals was cannulated. In the presence of indomethacin, 5, 10, and 20 dyne/cm2 shear stress elicited dilation of donor vessels, followed by dilation of detector vessels. Changes in membrane potential of the detector vessel smooth muscle cells in response to the perfusate from 5 and 10 dyne/cm2 shear stress-stimulated donor vessels was also recorded (by approximately -12 to -15 and -20 to -30 mV, respectively). Exposing detector vessels to 30 mmol/L KCl or pretreating them with iberiotoxin abolished their hyperpolarization and dilation to the flow of perfusate. Pretreatment of donor vessels with PPOH, an inhibitor of cytochrome P-450/epoxygenase, eliminated dilator responses in both donor and detector vessels, as well as the hyperpolarization of detector vessels. GC-MS analysis showed increasing release of EETs into the perfusate collected from 1, 5, and 10 dyne/cm2 shear stress-stimulated arterioles, which was abolished by PPOH. Thus, EETs, released from endothelial cells of donor vessels stimulated with shear stress, hyperpolarize smooth muscle of downstream detector vessels, confirming their identity as endothelium-derived hyperpolarizing factors and suggesting that gap junctional communication may not be necessary for shear stress-stimulated EDHF-mediated vasodilation.

Purinoceptors in renal microvessels: adenosine-activated and cytochrome P450 monooxygenase-derived arachidonate metabolites.

Cytochrome P450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) dilate rat preglomerular microvessels (PGMVs) when adenosine 2A receptors (A(2A)R) are stimulated. As high salt intake increases epoxygenase activity and adenosine levels, we hypothesized that renal adenosine responses would be greater in high salt-fed rats. We have obtained evidence supporting this hypothesis in rats fed a high salt diet for 7 days. Stimulation of adenosine receptors with 2-chloroadenosine in kidneys obtained from rats on high salt (4%) intake produced an increase in EET release that was several-fold greater than in kidneys of rats on normal salt (0.4% NaCl) diets, which was associated with a sharp decline in renovascular resistance. Under conditions of high salt intake, an associated upregulation of A(2A)R and 2C23 protein expression was observed. As EETs are renal vasodilator and natriuretic eicosanoids, the antipressor response to salt loading may operate through an A(2A)R - EET mechanism. These findings expand the role of adenosine-related mechanisms in protecting renal function.

Renal COX-2, cytokines and 20-HETE: tubular and vascular mechanisms.

Our initial studies on renal cyclooxygenase (COX)-2 expression and activity addressed the critical role of angiotensin II (Ang II) in increasing tumor necrosis factor alpha (TNF) that eventuated in expression of COX-2 in the medullary thick ascending limb (mTAL) of the nephron. COX-2 supplanted the dominant oxygenase, the cytochrome P450 (CYP) enzyme, omega-hydroxylase, that synthesized 20-hydroxyeicosatetraenoic acid (20-HETE). These findings served as the basis for additional studies on: 1) the role of glucocorticoids in regulating COX-2 expression and activity in the mTAL; and 2) the utilization of the same signaling pathways in response to stimulation of the mTAL calcium receptor (CaR). These studies of mTAL COX-2 expression which are addressed in the first part of this chapter are followed by explorations of the expression of COX-2 in preglomerular microvessels (PGMV) and the relationship of COX-2 to 20-HETE, the principal eicosanoid of PGMV. The third and last component of this chapter explores the signaling events, focusing on COX-2, which are set in motion by diabetes.

Cytochrome P-450 inhibition attenuates hypertension induced by reductions in uterine perfusion pressure in pregnant rats.

The present study tested the hypothesis that cytochrome P-450 (CYP) metabolites of arachidonic acid (AA) are involved in mediating hypertension and renal vasoconstriction during chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats. 1-aminobenzotriazole (ABT), a CYP enzyme inhibitor (25 mg/kg per day), or vehicle (saline 0.9%) was administered for 7 days to normal pregnant (NP) rats and to pregnant rats with chronic RUPP. RUPP rats infused with vehicle showed significantly (P<0.01) higher mean arterial pressure (MAP) (130+/-2 versus 106+/-1 mm Hg), renal vascular resistance (RVR) (22.6+/-1.8 versus 16.3+/-1.1 mm Hg/mL per minute) and lower (P<0.05) glomerular filtration rate (GFR) (1.6+/-0.1 versus 2.3+/-0.1 mL/min) than NP rats. ABT decreased (P<0.01) MAP in RUPP rats (111+/-1 mm Hg), whereas it had no effect in NP rats (108+/-2 mm Hg). CYP inhibition also attenuated the differences in renal hemodynamics observed between NP and RUPP rats. After treatment with ABT, RVR and GFR were similar in RUPP rats (19.3+/-1.5 mm Hg/mL per minute and 2.0+/-0.2 mL/min, respectively) and NP rats (16.3+/-2.4 mm Hg/mL per minute and 2.4+/-0.2 mL/min). The effects of CYP enzymes inhibitor in RUPP rats were associated with a reduction (P<0.05) of 20-HETE formation (32%) and a decreased (P<0.05) expression (33%) of CYP4A protein in renal cortex. In contrast, renal epoxygenase activity did not change in these animals. These results suggest that 20-HETE contributes to hypertension and renal vasoconstriction induced by chronic RUPP in pregnant rats.

Estrogen elicits cytochrome P450--mediated flow-induced dilation of arterioles in NO deficiency: role of PI3K-Akt phosphorylation in genomic regulation.

This study investigated the mechanisms responsible for the estrogen-dependent, cytochrome P450 (CYP)-mediated dilator responses to shear stress in arterioles of NO-deficient female rats and mice. Flow-induced dilation (FID) was assessed in isolated arterioles from N(G)-nitro-L-arginine methyl ester (L-NAME)-treated male and ovariectomized female rats before and after overnight incubation with 17beta-estradiol (17beta-E2, 10(-9) mol/L). In control conditions, prostaglandins (PGs) mediated FID, because indomethacin (INDO) abolished the responses. After incubation of the vessels with 17beta-E2, the basal tone of arterioles was significantly reduced and FID was augmented. INDO did not affect the dilation of the vessels incubated with 17beta-E2. Dilations of these vessels, however, were eliminated by PPOH and miconazole, inhibitors of CYP/epoxygenase. Simultaneous incubation of the vessels with 17beta-E2 plus ICI, 182,780, an estrogen receptor antagonist, or wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation or the transcriptional inhibitor DRB, prevented the reduced arteriolar tone and the enhanced CYP-mediated FID caused by incubation of vessels with 17beta-E2. Western blot analysis indicated a significantly increased phospho-Akt level in arterioles incubated with 17beta-E2 compared with those without 17beta-E2. The enhanced phospho-Akt in response to 17beta-E2 was localized, by immunohistochemistry, to arteriolar endothelial cells. Moreover, GC-MS analysis indicated a significantly increased production of epoxyeicosatrienoic acids, vasodilator metabolites of CYP/epoxygenase, in arterioles incubated with 17beta-E2, a response that was prevented by ICI 182780 and wortmannin, respectively. Thus, estrogen, via a receptor-dependent, PI3K/Akt-mediated pathway, transcriptionally upregulates CYP activity, leading to an enhanced arteriolar response to shear stress.

TNFalpha regulates renal COX-2 in the rat thick ascending limb (TAL).

We have examined cyclooxygenase (COX)-2-dependent mechanisms in preglomerular microvessels and the thick ascending limb (TAL). These renal structures are linchpins in the regulation of the renal circulation and extracellular fluid volume. Cytochrome P450 monooxygenases are the principal oxygenases in the TAL segment; however, COX-2 can be expressed in the TAL, as when challenged by angiotensin II. Glucocorticoids also affect the expression and activity of oxygenases in the TAL. Before adrenalectomy, <2% TAL cells expressed COX-2; after, >30% of TAL cells expressed COX-2. Recruitment of COX-2 is initiated in the renal cortex and proceeds to the medulla associated with: (1) COX-2 mRNA accumulation; (2) increased COX-2 expression; and (3) a two-fold increase in PGE2 production by cortical microsomes. These changes were nullified by dexamethasone. COX-2 mRNA, protein expression and PGE2 synthesis in the TAL are also increased in response to increased extracellular Ca2+. The Ca2+ sensing receptor is G-protein coupled and responds to changes in extracellular Ca2+ concentration by increasing protein kinase C activity to produce expression of COX-2. Thus, multiple signaling pathways contribute to COX-2 expression in TAL cells.

5,6-epoxyeicosatrienoic acid mediates the enhanced renal vasodilation to arachidonic acid in the SHR.

We have shown a cytochrome P450-dependent renal vasodilator effect of arachidonic acid in response to inhibition of cyclooxygenase and elevation of perfusion pressure, which was enhanced in the spontaneously hypertensive rat (SHR) and linked to increased production of and/or responsiveness to epoxyeicosatrienoic acids (EETs). In the SHR, vasodilation elicited by low doses of arachidonic acid was attenuated by the nitric oxide synthase inhibitor Nw-nitro-L-arginine (50 micromol/L), whereas the responses to high doses were unaffected. Inhibition of epoxygenases with miconazole (0.3 micromol/L) in the presence of Nw-nitro-L-arginine greatly reduced the renal vasodilator response to all doses of arachidonic acid. Tetraethylammonium (10 mmol/L), a nonselective K+ channel blocker, abolished the nitric oxide-independent renal vasodilator effect of arachidonic acid as well as the vasodilator effect of 5,6-EET, confirming that EET-dependent vasodilation involves activation of K+ channels. Under conditions of elevated perfusion pressure (200 mm Hg) and cyclooxygenase inhibition, 5,6-EET, 8, 9-EET, and 11,12-EET caused renal vasodilatation in both SHR and Wistar-Kyoto rats (WKY), whereas 14,15-EET produced vasoconstriction. 5,6-EET was the most potent renal vasodilator of the EET regioisomers in the SHR by a factor of 4 or more. In the SHR, 5,6-EET- and 11,12-EET-induced renal vasodilatation was >2-fold greater than that registered in WKY. Thus, the augmented vasodilator responses to arachidonic acid in the SHR is through activation of K+ channels, and 5,6-EET is the most likely mediator.