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Background: Epoxyeicosatrienoic acids (EETs) derived from cytochrome P450 (CYP)-dependent metabolism of arachidonic acid are increased in the plasma of women with preeclampsia as compared with normal pregnancy and are significantly higher in fetal than in maternal plasma and erythrocytes. We hypothesized that differences in EET synthesis or metabolism in the feto-placental unit contributed to the observed differences in circulating EETs., Method: To evaluate EETs, formation as well as the expression of relevant CYP isoforms and the metabolizing enzyme, soluble epoxide hydrolase (sEH), biopsies of placenta were collected from 19 normal pregnancy and 10 preeclampsia at the time of cesarean section delivery. EETs were extracted from tissue homogenates and analyzed by liquid chromatography coupled with tandem mass spectrometry., Results: Both cis-EETs and trans-EETs were detected in the placenta. Concentration of total EETs was higher in the placenta from preeclampsia compared with normal pregnancy (2.37 ± 1.42 ng/mg vs. 1.20 ± 0.72 ng/mg, mean ± SD, P < 0.01), especially the 5,6-, 8,9- and 11,12-EETs, measured in a subgroup of tissue samples (normal pregnancy = 10, preeclampsia = 5). By immunohistochemistry, sEH, CYP2J2, CYP4A11 were present in placental villi with different pattern distribution, whereas CYP2C8 was not detectable. Neither were CYP2J2, CYP4A11, and CYP2C8 detected in the umbilical cord. Western blot analysis of placenta homogenates showed reduced expression of sEH in preeclampsia as compared with normal pregnancy., Conclusion: Increased EETs in the placenta and umbilical cord are associated with the presence of CYP2J2, whereas reduced expression of sEH in preeclampsia may be the key factor of increased EETs in the placenta.

Background: Epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are cytochrome P450 metabolites of arachidonic acid posited to act in the circulatory adaptation to pregnancy and the development of preeclampsia. Red blood cells (RBCs) may function as major contributors of cis- and trans-EETs., Methods: We performed paired analyses of EETs, dihydroxyeicosatrienoic acids (DHETs), and 20-HETE in RBCs, plasma, and urine from preeclamptic and normotensive pregnant and nonpregnant women. Blood from fetal and maternal circulation was collected. EETs, DHETs, and 20-HETE were analyzed by gas chromatography and liquid chromatography mass spectrometry. Vascular function and inflammation indices were analyzed., Results: Plasma EET is higher in normotensive (median, range; 9.9, 6.3-25.2ng/mL n = 29) and preeclamptic (10.9, 6.0-48.0ng/mL, n = 19) women than in nonpregnant controls (7.3, 3.7-10.2ng/mL, n = 19) and correlate with RBC EETs, C-reactive protein, and arterial stiffness. Renal production of EETs, measured as urinary DHETs, was reduced in preeclamptic (4.5, 1.6-24.5ng/mg creatinine) compared to normotensive (11.4, 1.6-44.5ng/mg creatinine) pregnancies. EETs are 3- to 5-fold greater in fetoplacental than in maternal circulation (RBCs 36.6, 13.1-69.4 vs. 12.5, 6.4-12.0ng/10(9) cells; plasma 31.6, 8.5-192.6 vs. 12.0, 6.8-48.0ng/mL). Both cis- and trans-EETs are present in fetal RBCs., Conclusions: RBCs contribute to elevated levels of EETs in the fetoplacental circulation. EETs may modulate systemic and fetoplacental hemodynamics in normal and preeclamptic pregnancies. Decreased renal EET generation may be associated with the development of maternal renal dysfunction and hypertension in preeclampsia.

The induction of renal cyclooxygenase-2 (COX-2) in diabetes has been implicated in the renal functional and structural changes in models where hypertension or uninephrectomy was superimposed. We examined the protective effects of 3 mo treatment of streptozotocin-diabetic rats with a highly selective COX-2 inhibitor (SC-58236) in terms of albuminuria, renal hypertrophy, and the excretion of TNF-α and TGF-β, which have also been implicated in the detrimental renal effects of diabetes. SC-58236 treatment (3 mg·kg(-1)·day(-1)) of diabetic rats resulted in reduced urinary excretion of PGE(2), 6-ketoPGF(1α), and thromboxane B(2), all of which were increased in the diabetic rat compared with age-matched nondiabetic rats. However, serum thromboxane B(2) levels were unchanged, confirming the selectivity of SC-58236 for COX-2. The renal protective effects of treatment of diabetic rats with the COX-2 inhibitor were reflected by a marked reduction in albuminuria, a reduction in kidney weight-to-body weight ratio, and TGF-β excretion and a marked decrease in the urinary excretion of TNF-α. The protective effects of SC-58236 were independent of changes in plasma glucose levels or serum advanced glycation end-product levels, which were not different from those of untreated diabetic rats. In an additional study, the inhibition of COX-2 with SC-58236 for 4 wk in diabetic rats resulted in creatinine clearance rates not different from those of control rats. These results confirm that the inhibition of COX-2 in the streptozotocin-diabetic rat confers renal protection and suggest that the induction of COX-2 precedes the increases in cytokines, TNF-α, and TGF-β.

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.

Cyclooxygenase (COX)-2 expression is increased in the kidney of rats made diabetic with streptozotocin and associated with enhanced release of prostaglandins stimulated by arachidonic acid (AA). Treatment of diabetic rats with nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide synthase or with tempol to reduce superoxide prevented these changes, suggesting the possibility that peroxynitrite (ONOO) may be the stimulus for the induction of renal COX-2 in diabetes. Consequently, we tested the effects of an ONOO decomposition catalyst, 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron(III) (FeTMPyP), which was administered for 3-4 wk after the induction of diabetes. FeTMPyP treatment normalized the twofold increase in the expression of nitrotyrosine, a marker for ONOO formation, in the diabetic rat and prevented the increase in renal COX-2 expression without modifying the two- to threefold increases in renal release of prostaglandins PGE(2) and 6-ketoPGF(1α) in response to AA. FeTMPyP treatment of diabetic rats reduced the elevated creatinine clearance and urinary excretion of TNF-α and transforming growth factor (TGF)-β, suggesting a renoprotective effect. Double immunostaining of renal sections and immunoprecipitation of COX-2 and nitrotyrosine suggested nitration of COX-2 in diabetic rats. In cultured human umbilical vein endothelial cells (HUVECs) exposed to elevated glucose (450 mg/dl) or ONOO derived from 3-morpholinosydnonimine (SIN-1), expression of COX-2 was increased and was prevented when endothelial cells were treated with FeTMPyP. These results indicate that elevated glucose increases the formation of ONOO, which contributes to the induction of renal COX-2 in the diabetic rat.

We examined the hypothesis that vascular and renal dysfunction caused by angiotensin II (Ang II) through increased levels of blood pressure, inflammatory cytokines, and oxidative stress in Sprague-Dawley rats can be prevented by lentiviral-mediated delivery of endothelial heme oxygenase (HO)-1. We targeted the vascular endothelium using a lentiviral construct expressing human HO-1 under the control of the endothelium-specific promoter VE-cadherin (VECAD-HO-1) and examined the effect of long-term human HO-1 expression on blood pressure in Ang II-mediated increases in blood pressure and oxidant stress. A bolus injection of VECAD-HO-1 into the renal artery resulted in expression of human HO-1 for up to 6-9 weeks. Sprague-Dawley rats were implanted with Ang II minipumps and treated with lentivirus carrying either the HO-1 or green fluorescent protein. Renal tissue from VECAD-HO-1-transduced rats expresses human HO-1 mRNA and proteins without an effect on endogenous HO-1. Infusion of Ang II increased blood pressure (p < 0.001) but decreased vascular relaxation in response to acetylcholine, endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (peNOS) levels, and renal and plasma levels of adiponectin (p < 0.05); in contrast, plasma tumor necrosis factor-α and monocyte chemoattractant protein-1 levels increased. Ang II-treated animals had higher levels of superoxide anion and inducible nitric oxide synthase and increased urinary protein and plasma creatinine levels. Lentiviral transduction with the VECAD-HO-1 construct attenuated the increase in blood pressure (p < 0.05), improved vascular relaxation, increased plasma adiponectin, and prevented the elevation in urinary protein and plasma creatinine in Ang II-treated rats. Endothelial-specific expression of HO-1 also reduced oxidative stress and levels of inflammatory cytokines resulting in increased expression of the anti-apoptotic proteins phosphorylated AKT, phosphorylated AMP-activated protein kinase, peNOS, and eNOS. Collectively, these findings demonstrate that endothelial-specific increases in HO-1 expression attenuate Ang II hypertension and the associated vascular dysfunction that is associated with increases in adiponectin and peNOS and reductions in oxidative stress and levels of inflammatory cytokines.

The beneficial effects of coronary angioplasty are limited by the proliferation and migration of vascular smooth muscle cells leading to restenosis. We hypothesized that increased activity of phosphodiesterase (PDE) after angioplasty in response to growth factors such as platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF), leads to reduced cAMP levels, which, in turn, may contribute to vascular smooth muscle cell proliferation. In rats subjected to angioplasty, aortic expression and activity of PDE3/PDE4 were increased within 24 h and associated with reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP-dependent protein kinase A (PKA). Inhibition of PDE3 increased VASP phosphorylation in aortic rings from rats subjected to angioplasty, whereas inhibition of PDE4 or stimulation of adenylate cyclase with isoproterenol was without effect; however, combined inhibition of PDE3 and PDE4 produced a synergistic effect on VASP phosphorylation. In cultured vascular smooth muscle cells, exposure to PDGF-BB resulted in increased expression of PDE3, which was prevented by an inhibitor of PI3 kinase but not by inhibitors of the MAP kinase signaling pathway. In contrast, FGF increased the expression of PDE4 in vascular smooth muscle cells but did not influence expression of PDE3. This study shows that angioplasty results in increased expression/activity of PDE, possibly arising from stimulation by PDGF-BB and FGF, and decreased cAMP levels, which may promote restenosis. These results provide a rational explanation for the beneficial effects of PDE inhibitors.

We confirmed that release of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated perfused kidney of diabetic rats is greatly reduced compared with age-matched control rats. The present studies were undertaken to examine potential mechanisms for the deficit in renal 20-HETE in rats with streptozotocin-induced diabetes of 3-4 wk duration. A role for oxidative stress was excluded, inasmuch as treatment of diabetic rats with tempol, an SOD mimetic, for 4 wk did not affect the renal release of 20-HETE. Similarly, chronic inhibition of nitric oxide formation with nitro-l-arginine methyl ester or aldose reductase with zopolrestat failed to alter the release of 20-HETE from the diabetic rat kidney. Inasmuch as 20-HETE may be metabolized by cyclooxygenase (COX), the expression/activity of which is increased in diabetes, we included indomethacin in the perfusate of the isolated kidney to inhibit COX but found no effect on 20-HETE release. Diabetic rats were treated for 3 wk with fenofibrate to increase expression of cytochrome P-450 (CYP4A) in an attempt to find an intervention that would restore release of 20-HETE from the diabetic rat kidney. However, fenofibrate reduced 20-HETE release in diabetic and control rat kidneys but increased expression of CYP4A. Only insulin treatment of diabetic rats for 2 wk to reverse the hyperglycemia and maintain blood glucose levels at <200 mg/dl reversed the renal deficit in 20-HETE. We conclude that oxidative stress, increased aldose reductase activity, or increased COX activity does not contribute to the renal deficit of 20-HETE in diabetes, which may be directly related to insulin deficiency.

Renal cyclooxygenase (COX)-2 expression is increased in the diabetic rat and has been linked to increased glomerular filtration rate (GFR) and renal injury. Our studies indicate that oxidative stress in the form of peroxynitrite (ONOO) may be the stimulus for induction of COX-2. In this study, we addressed the effects of a peroxisome proliferator-activated receptor alpha agonist on renal COX-2 expression as fibrates exert renal protective effects. Forty-eight hours after the induction of diabetes with streptozotocin in male Wistar rats, fenofibrate treatment (100 mg/kg/day) was started, and the effects were compared with untreated diabetic rats and treated and untreated age-matched control rats (n = 5 per group). After 12 to 14 weeks of treatment, the right kidney was perfused to determine prostaglandin release in response to arachidonic acid (AA), and the left kidney was used to examine the expression of COX-2 and nitrotyrosine, an index of ONOO formation. Release of prostaglandin (PG) E(2) in response to AA was enhanced in the diabetic rat kidney compared with control (4.8 +/- 0.7 versus 1.9 +/- 0.7 ng/min) and reduced by fenofibrate to 0.6 +/- 0.2 ng/min. A similar pattern was obtained for AA-stimulated release of 6-ketoPGF(1alpha). The effects of fenofibrate were associated with reduced renal expression of COX-2 and nitrotyrosine in diabetic rats. We used creatinine clearance as an index of GFR, which was increased in the diabetic rat, 3.09 +/- 0.4 versus 1.15 +/- 0.1 ml/min for control, and reduced by fenofibrate treatment to 1.87 +/- 0.3 ml/min. These results show that fenofibrate treatment of diabetic rats decreases renal COX-2 expression, possibly by reducing nitrosative stress, and is associated with a reduction of the enhanced GFR.

It is known that cAMP and cGMP are important for vasorelaxation, and cyclic nucleotide phosphodiesterases (PDEs) regulate their levels. Balloon angioplasty (BAL) is associated with reduced cAMP and cGMP levels, and inhibition of PDE-3 reduces restenosis. In this study, we found that BAL increased PDE-3 activity, which affected vasoreactivity of rat aortic rings 24-h post-BAL; these were compared with intact (INT) and ex vivo endothelium-denuded rings (RUB) from sham rats. In BAL and RUB rings, vasorelaxant responses to ACh were abolished. The EC(50) for phenylephrine (PE) was 1.8-fold less in RUB than in INT or BAL, whereas the maximal contractile effect of PE was greater in BAL than in INT or RUB. PDE-3 inhibitors reduced the maximal response to PE by >65% in BAL compared with 10-30% in INT and RUB; the reduction of the maximal response to U-46619 was 37% in BAL compared with 8% in INT with no reduction in RUB. PDE-4 inhibitors reduced PE-induced tone by <30% in an endothelium-dependent manner. Vasorelaxant responses to agonists that utilize cAMP were greatly impaired in BAL and RUB rings, and inhibition of PDE-3 enhanced the vasorelaxant responses in BAL or RUB. Inhibition of PDE-4 increased vasorelaxant responses to isoproterenol (ISO) to a much lesser degree. Thus PDE-3 and PDE-4 inhibitors exhibited differential effects on PE-induced tone and vasorelaxant responses to ISO. Inhibition of PDE-3 also produced a greater increase in cAMP in BAL than INT or RUB rings. These results suggest that increased PDE-3 activity after BAL may promote a vasospastic state and that the reduction in cAMP may, possibly, influence vessel remodeling.

Renal cyclooxygenase (COX)-2 expression and arachidonic acid-stimulated prostaglandin release are increased in streptozotocin-diabetic rats and are reduced by tempol treatment, indicating a role for superoxide. Generation of nitric oxide (NO) and its product with superoxide, peroxynitrite, is also increased in diabetes and can induce COX-2. To investigate a role of NO, rats were treated with L-nitroarginine methyl ester (L-NAME; 100 mg/kg/day) to inhibit NO synthase (NOS) for 14-18 days. In isolated perfused kidneys from diabetic rats, prostaglandin release and vasoconstrictor responses to arachidonic acid were increased and renal cortical expression of COX-2 was 2-fold that of control rats. Treatment of diabetic rats with L-NAME reduced arachidonic acid-stimulated release of prostaglandins and the expression of COX-2. L-NAME increased vasoconstrictor responses to AA in diabetic and non-diabetic rats but abolished the differences between the two. These results, coupled with those using tempol, suggest that NO or its product with superoxide may contribute to the induction of renal COX-2 in the diabetic rat.

Renal cyclooxygenase (COX)-2 expression is increased in the streptozotocin (STZ)-diabetic rat and is associated with enhanced renal prostaglandin release in response to arachidonic acid (AA). Endoperoxide-mediated vasoconstrictor responses to AA were also enhanced in the diabetic rat kidney. Because oxidative stress is increased in diabetes and has been shown to induce COX-2, we assessed its contribution to prostaglandin release by treating diabetic rats with tempol (120 mg/kg/day) for 28 days. Release of AA-stimulated prostaglandins PGE(2) and 6-ketoPGF(1alpha) from the isolated perfused kidney was used as an index of COX activity, and Western analysis was used to determine COX-2 protein expression. In untreated diabetic rats, the release of prostaglandins in response to AA was markedly enhanced; the increase in release of both 6-ketoPGF(1alpha) and PGE(2) after AA was twice that in control rats. Renal cortical COX-2 expression in diabetic rats was 3-fold that of control rats. Tempol treatment reduced the AA-stimulated release of prostaglandins to levels seen in control rats; this was associated with reduced expression of COX-2 protein to levels not different from that in control rats. However, the enhanced vasoconstrictor response to AA in diabetic rats was unaffected by tempol treatment but abolished by inhibition of COX-1 with SC58560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole]. The addition of tempol to the perfusate of kidneys from diabetic and control rats had only a slight effect on prostaglandin release. We conclude that oxidative stress is an integral component of the mechanism involved in the induction of renal COX-2 in diabetes.

Increased extracellular K+ is reported to cause endothelium-independent vasodilation and K+ has been proposed as an endothelium-derived hyperpolarizing factor. However, the endothelium is endowed with K+ channels that may also be responsive to increased K+. We examined the vasodilator effect of bolus administration of 20, 40 and 60 micromol KCl in the rat isolated kidney in which perfusion pressure was elevated with phenylephrine. KCl produced dose-dependent vasodilator responses that were virtually abolished by removal of the endothelium which also abolished the vasodilator effect of bradykinin without affecting that to nitroprusside. The vasodilator effect of KCl was unaffected by inhibition of cyclooxygenase, nitric oxide synthase or cytochrome P450 but reduced by inhibition of K+ channels with tetraethylammonium (TEA). Barium chloride reduced the vasodilator effects of KCl but charybdotoxin/apamin was without effect. These results indicate that KCl results in endothelium-dependent vasodilation that is independent of nitric oxide (NO), prostaglandins and cytochrome P450 but dependent on activation of endothelial K+ channels.

Epoxyeicosatrienoic acids (EETs) are candidate endothelium-derived hyperpolarizing factors that demonstrate a wide range of biological effects. The presence of both cis- and trans-EETs in rat plasma was identified with HPLC-electrospray ionization tandem mass spectrometry in this study. The total EETs in plasma are 38.2 ng/ml with cis-EETs representing 21.4 +/- 0.4 ng/ml and trans-EETs 16.8 +/- 0.4 ng/ml. EETs in RBCs were estimated to be 20.2 ng/10(9) RBCs, which corresponds to 200 ng in RBCs contained in 1 ml blood. RBC incubation with 10 mM tert-butyl hydroperoxide resulted in 4.4-fold increase of total cis-EETs (from 9.2 to 40.2 ng/10(9) RBCs) and 5.5-fold increase of total trans-EETs (from 11.0 to 60.8 ng/10(9) RBCs). EETs were released (2 ng/ml) from RBCs after incubation at 37 degrees C for 10 min even after being washed 3 times, indicating that RBCs are reservoirs of plasma EETs. The identification of cis- and trans-EETs in RBCs and in plasma as well as their release from RBCs suggest a vasoregulatory role of RBCs in view of their potent vasoactivity.

A novel eicosanoid, 5,6-trans-epoxy-8Z,11Z,14Z-eicosatrienoic acid (5,6-trans-EET), was identified in rat red blood cells. Characterization of 5,6-trans-EET in the sn-2 position of the phospholipids was accomplished by hydrolysis with phospholipase A(2) followed by gas chromatography/mass spectrometry as well as electrospray ionization-tandem mass spectrometry analyses. The electron ionization spectrum of 5,6-erythro-dihydroxyeicosatrienoic acid (5,6-erythro-DHET), converted from 5,6-trans-EET in the samples, matches that of the authentic standard. Hydrogenation of the extracted 5,6-erythro-DHET with platinum(IV) oxide/hydrogen resulted in an increase of the molecular mass by 6 daltons and the same retention time shift as an authentic standard in gas chromatography, suggesting the existence of three olefins as well as the 5,6-erythro-dihydroxyl structure in the metabolite. Match of retention times by chromatography indicated identity of the stereochemistry of the red blood cell 5,6-erythro-DHET vis à vis the synthetic standard. High pressure liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of the phospholipase A(2)-hydrolyzed lipid extracts from red blood cells revealed match of the mass spectrum and retention time of the compound with the authentic 5,6-trans-EET standard, providing direct evidence of the existence of 5,6-trans-EET in red blood cells. The presence of other trans-EETs was also demonstrated. The ability of both 5,6-trans-EET and its product 5,6-erythro-DHET to relax preconstricted renal interlobar arteries was significantly greater than that of 5,6-cis-EET. In contrast, 5,6-cis-EET and 5,6-trans-EET were equipotent in their capacity to inhibit collagen-induced rat platelet aggregation, whereas 5,6-erythro-DHET was without effect. We propose that the red blood cells serve as a reservoir for epoxides which on release may act in a vasoregulatory capacity.

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.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450-derived constrictor eicosanoid produced by the preglomerular vasculature where it contributes to regulation of tone. Removal of the tonic inhibitory influence of nitric oxide (NO) has been reported to increase renal 20-HETE release. Because inhibition of NO synthesis enhances responses to vasoconstrictor agents, we examined a contribution for increased 20-HETE generation. In the rat kidney perfused with Krebs' buffer, responses to U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF2alpha), a thromboxane A2 mimetic, were compared before and after 50 microM L-nitroarginine (L-NA) to inhibit NO synthase. L-NA raised perfusion pressure (PP) from 79 +/- 3 to 190 +/- 7 mm Hg and enhanced constrictor responsiveness to U46619. U46619 (10, 30, 100, and 300 ng) increased PP by 7 +/- 1, 17 +/- 2, 50 +/- 7, and 67 +/- 7 mm Hg, respectively, before L-NA and 15 +/- 1, 37 +/- 7, 68 +/- 10, and 85 +/- 11 mm Hg, respectively, after L-NA, which did not increase 20-HETE efflux from the kidney. Nonetheless, an inhibitor of omega-hydroxylase, dibromododecencyl methylsulfonimide (DDMS), which reduced 20-HETE release, normalized the enhanced responsiveness to U46619. When PP was elevated with phenylephrine, vasoconstrictor responses to U46619 were similarly enhanced, an effect that was also prevented by DDMS. DDMS and an antagonist of 20-HETE, 20-HEDE [20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid], also reduced vasoconstrictor responses to U46619 in the absence of elevation of PP. Because 20-HETE inhibits K+ channels, we examined the effects of K+ channel inhibitors on vasoconstrictor responses and showed that both tetraethylammonium (TEA) and charybdotoxin enhanced renal vasoconstrictor responses to U46619. However, the inhibitory effects of 20-HEDE on vasoconstrictor responses remained after treatment with TEA. These results support a role for 20-HETE vasoconstrictor responses but suggest an action independent of K+ channels.

In the rat isolated perfused kidney, arachidonic acid elicits cyclooxygenase-dependent vasoconstriction through activation of PGH2/TxA2 receptors; responses are enhanced in kidneys from diabetic rats. This study examined the roles of cyclooxygenase-1/cyclooxygenase-2 in the enhanced renal vasoconstrictor effect of arachidonic acid in streptozotocin-diabetic rats. Release of 20-HETE was also determined, as this eicosanoid has been reported to elicit cyclooxygenase-dependent vasoconstriction. We confirmed that vasoconstrictor responses to arachidonic acid were enhanced in the diabetic rat kidney associated with a 2-fold-greater increase in the release of 6-ketoPGF1alpha, which was used as an index of cyclooxygenase activity. One and three micrograms of arachidonic acid increased perfusion pressure by 85+/-37 and 186+/-6 mm Hg, respectively, in diabetic rat kidneys compared with 3+/-1 and 17+/-8 mm Hg, respectively, in control rat kidneys. Inhibition of both cyclooxygenase isoforms with indomethacin (10 micromol/L) abolished the vasoconstrictor response to arachidonic acid in both diabetic and control rat kidneys, whereas inhibition of cyclooxygenase-2 with nimesulide (5 micromol/L) reduced perfusion pressure responses to 1 and 3 microg arachidonic acid only in the diabetic rat kidney to 15+/-8 and 108+/-26 mm Hg, respectively, consistent with a 3-fold increase in the renal cortical expression of cyclooxygenase-2. 20-HETE release from the diabetic rat kidney was reduced almost 6-fold and was not increased in response to arachidonic acid. These results demonstrate that the renal vasoconstrictor effect of arachidonic acid is solely dependent on cyclooxygenase activity, with no evidence for a contribution from 20-HETE; in the diabetic rat, cyclooxygenase-2 activity contributes to the renal vasoconstrictor effect of arachidonic acid.

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.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid that elicit vasodilation via activation of K(+) channels. They have been implicated as endothelium-derived hyperpolarizing factors (EDHFs), mediating the effect of some endothelium-dependent vasodilator agents such as bradykinin in some vascular tissues. We reasoned that an agent that increases the availability of free arachidonic acid should also elicit cytochrome P450-dependent vasodilation that is associated with increased release of EETs and attenuated by agents that inhibit the synthesis or action of EETs. Thus, we used thimerosal as an inhibitor of reacylation of arachidonic acid and determined the contribution of prostaglandins, nitric oxide, and EETs to the vasodilator effect in the isolated, perfused, preconstricted kidney of the rat. Thimerosal elicited vasodilator responses that were unaffected by inhibition of cyclooxygenase with indomethacin but were reduced by the further inhibition of nitric oxide synthesis. The vasodilator activity that remained after inhibition of cyclooxygenase and nitric oxide synthase was reduced by inhibition of K(+) channels with tetraethylammonium and was associated with increased release of EETs measured by gas chromatography-mass spectroscopy following hydrolysis to the corresponding diols. Inhibition of cytochrome P450 with miconazole or epoxygenase with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexamide reduced the nitric oxide- and prostaglandin-independent vasodilator effect of thimerosal and attenuated the increase in the release of EETs. We conclude that thimerosal causes vasodilation of the isolated perfused kidney via nitric oxide-dependent and -independent mechanisms. The nitric oxide-independent component of the response involves activation of K(+) channels and is likely mediated by EETs, possibly acting as EDHFs.

Background: Activation of endothelial K+ channels and the subsequent increase in intracellular Ca2+, may be an important step in the release of relaxant factors in response to endothelium-dependent vasodilator agents. However, the type of K+ channel involved in hyperpolarization of the endothelium and the subsequent release of relaxing factors remains to be defined., Material and Methods: Rat aortic rings precontracted with U46619 were used to address the effects of inhibitors of K+ channels on the vasorelaxant response to acetylcholine (Ach). As responses to Ach were mediated solely by endothelium-derived NO and responses to NO derived from nitroprusside were unaffected by inhibition K+ channels, any effect of K+ channel inhibitors could be attributed to actions on endothelial K+ channels to modify NO release., Results: Tetraethylammonium (TEA) and elevated K+ attenuated the relaxant effect of Ach, indicating a role for K+ channels in NO release. The Ca2+-activated K+ channel inhibitors, apamin, charybdotoxin and iberiotoxin as well as glibenclamide and BaCl2, inhibitors of ATP-sensitive K+ channels and inwardly rectifying K+ channels, respectively, did not affect the response to Ach. However, a combination of apamin and charybdotoxin, but not apamin and iberiotoxin, attenuated the vasorelaxant response to Ach., Conclusions: The results of this study indicate that NO release in response to Ach involves activation of an endothelial K+ channel that is inhibited by a combination of apamin and charybdotoxin.

The properties of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids, vasoactivity and modulation of ion transport and mediation/modulation of the effects of vasoactive hormones, such as angiotensin II and endothelin, underscore their importance to renal vascular mechanisms and electrolyte excretion. 20-Hydroxyeicosatetraenoic acid is an integral component of renal autoregulation and tubuloglomerular feedback as well as cerebral autoregulation, eliciting vasoconstriction by the inhibition of potassium channels. Nitric oxide inhibits 20-hydroxyeicosatetraenoic acid formation, the removal of which contributes to the vasodilator effect of nitric oxide. In contrast, epoxyeicosatrienoic acids are generally vasodilatory by activating potassium channels and have been proposed as endothelium-derived hyperpolarizing factors. 20-Hydroxyeicosatetraenoic acid modulates ion transport in key nephron segments by influencing the activities of sodium--potassium-ATPase and the sodium--potassium--chloride co-transporter; however, the primacy of the various arachidonate oxygenases that generate products affecting these activities changes with age. The range and diversity of activity of 20-hydroxyeicosatetraenoic acid is influenced by its metabolism by cyclooxygenase to products affecting vasomotion and salt/water excretion. 20-Hydroxyeicosatetraenoic acid is the principal renal eicosanoid that interacts with several hormonal systems that are central to blood pressure regulation. This article reviews the most recent studies that address 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids in vascular and renal tubular function and hypertension.

In the present study, we examined the effects of peroxynitrite on reperfusion injury using a rat model of hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia, followed by 4 h of reperfusion. Groups A and B rats were sham-operated controls that received vehicle or peroxynitrite; groups C and D rats were subjected to HI/R and received peroxynitrite or vehicle, respectively. A dose of 2 micromol/kg body wt of peroxynitrite, diluted in saline (pH 9.0, 4 degrees C), was administered as a bolus through a portal vein catheter at 0, 60, and 120 min after reperfusion. Results showed that superoxide generation in the ischemic lobes of the liver and plasma alanine aminotransferase (ALT) activity of group C were decreased by 43% and 45%, respectively, compared with group D. Leukocyte accumulations in the ischemic lobes of liver and circulating leukocytes were decreased by 40% and 27%, respectively, in group C vs. D. The ratios of mRNA of P-selectin and intercellular adhesion molecule-1 (ICAM-1) to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA extracted from the ischemic lobes of the liver of group C were decreased compared with group D. There were no differences between the groups A and B in terms of plasma ALT activity, circulating leukocytes, superoxide generation, and leukocyte infiltration in the ischemic lobes of the liver. Moreover, hemodynamic parameters (i.e., mean arterial blood pressure, cardiac index, stroke index, and systemic vascular resistance) were not significantly different among groups B, C, and D. These results suggest that administration of peroxynitrite via the portal vein only has a local effect. Exogenous peroxynitrite at physiological concentrations attenuates leukocyte-endothelial interaction and reduces leukocyte infiltration. The mechanism of the reduction of leukocyte infiltration into ischemic lobes of the liver appears because of decreased expression of mRNA of P-selectin and ICAM-1. The net effect of administration of peroxynitrite may be to reduce adhesion molecule-mediated, leukocyte-dependent reperfusion injury.

The vascular effects of arachidonic acid (AA) were addressed in the rat perfused heart in terms of metabolic pathways and effector mechanisms. Under basal perfusion pressure, AA elicited dilator responses. However, in hearts treated with nitroarginine to eliminate nitric oxide and to elevate perfusion pressure, the predominant effect of AA was vasoconstriction which was converted to a vasodilator effect by inhibition of cyclooxygenase or antagonism of TP receptors. The vasodilator effect of AA in nitroarginine- and indomethacin-treated hearts was greatly attenuated by clotrimazole, an inhibitor of cytochrome P450, and by inhibition of K(+) channels with tetraethylammonium; in the absence of indomethacin, clotrimazole enhanced the vasoconstrictor effect of AA. When endothelin was used to constrict the coronary vasculature, AA also produced cyclooxygenase-dependent vasoconstriction. In hearts constricted with the endoperoxide analogue, U46619, only endothelium-dependent vasodilator effects of AA were observed that were reduced by indomethacin or clotrimazole. These results indicate that the coronary vasoconstrictor effect of AA which is expressed with elevated tone, results from its conversion by cyclooxygenase to a product(s) that activates TP receptors. The vasodilator effect exhibits two endothelium-dependent components, one mediated by cyclooxygenase products and the other by a cytochrome P450-derived product that activates K(+) channels.

Renal function is perturbed by inhibition of nitric oxide synthase (NOS). To probe the basis of this effect, we characterized the effects of nitric oxide (NO), a known suppressor of cytochrome P450 (CYP) enzymes, on metabolism of arachidonic acid (AA), the expression of omega-hydroxylase, and the efflux of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated kidney. The capacity to convert [(14)C]AA to HETEs and epoxides (EETs) was greater in cortical microsomes than in medullary microsomes. Sodium nitroprusside (10-100 microM), an NO donor, inhibited renal microsomal conversion of [(14)C]AA to HETEs and EETs in a dose-dependent manner. 8-bromo cGMP (100 microM), the cell-permeable analogue of cGMP, did not affect conversion of [(14)C]AA. Inhibition of NOS with N(omega)-nitro-L-arginine-methyl ester (L-NAME) significantly increased conversion of [(14)C]AA to HETE and greatly increased the expression of omega-hydroxylase protein, but this treatment had only a modest effect on epoxygenase activity. L-NAME induced a 4-fold increase in renal efflux of 20-HETE, as did L-nitroarginine. Oral treatment with 2% sodium chloride (NaCl) for 7 days increased renal epoxygenase activity, both in the cortex and the medulla. In contrast, cortical omega-hydroxylase activity was reduced by treatment with 2% NaCl. Coadministration of L-NAME and 2% NaCl decreased conversion of [(14)C]AA to HETEs without affecting epoxygenase activity. Thus, inhibition of NOS increased omega-hydroxylase activity, CYP4A expression, and renal efflux of 20-HETE, whereas 2% NaCl stimulated epoxygenase activity.

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

A cytochrome P450-derived metabolite of arachidonic acid, namely an epoxyeicosatrienoic acid (EET), has many of the properties of a hyperpolarizing factor that mediates endothelium-dependent, nitric oxide-independent vasodilation. As there are four EET regioisomers, we used pharmacological criteria, based on previous observations with bradykinin (BK), to evaluate which, if any, of the EETs could be considered a potential mediator of vasodilator responses to BK in the rat isolated heart treated with indomethacin and nitroarginine to eliminate prostaglandin and nitric oxide components of the response. Nifedipine, used as a probe for dilator mechanisms dependent on closure of voltage-dependent Ca++ channels, almost abolished the vasodilator effect of cromakalim and attenuated those of BK and 5,6 EET. The vasodilator effects of the other EETs were not reduced and were excluded from consideration as mediators of BK-induced vasodilation. The vasodilator effect of 5,6 EET, as with that of BK, was markedly reduced by charybdotoxin but not iberiotoxin, suggesting the contribution of a similar type K+ channel to the vascular response to both agents. As expected for a putative endothelium- and cytochrome P450-derived mediator, the coronary vasodilator effect of 5,6 EET was not affected by either removal of the endothelium or inhibition of cytochrome P450 with clotrimazole, interventions that virtually abolished the vasodilator activity of BK. Thus, of the four EET regioisomers, 5,6 EET is the most likely mediator of the vasodilator effect of BK in the isolated heart under these experimental conditions.

Angiotensin II (AII) is a potent vasoconstrictor which, at physiological plasma concentrations, produces antinatriuresis, whereas high intrarenal concentrations cause natriuresis and diuresis. We examined the effects of a selective AT1 receptor antagonist, losartan, and a nonselective AT receptor antagonist, Sar1Thr8AII, on the response to infusion of AII in the isolated rat kidney perfused at constant pressure with a recirculating modified Krebs-Henseleit buffer. AII increased renal vascular resistance (RVR), glomerular filtration rate (GFR) and urinary volume (UV) and sodium excretion (UNaV) without changing the fractional excretion of water or electrolytes. Thus, changes in GFR can account for the natriuresis/diuresis. Both AII receptor antagonists prevented the increase in RVR. However, losartan was without effect on angiotensin-induced increases in GFR, UV or UNaV, whereas Sar1Thr8 AII also prevented the increases in GFR, UV and UNaV. The angiotensin receptor mediating the increase in GFR can be dissociated from that mediating the increase in RVR, providing functional evidence of angiotensin receptor subtypes in the rat kidney.

The mediator of nitric oxide-(NO) independent vasodilation attributed to endothelium-derived hyperpolarizing factor remains unidentified although there is evidence for a cytochrome P450-derived eicosanoid. Anandamide, the ethanolamide of arachidonic acid and an endogenous ligand for cannabinoid receptors, was proposed as an endothelium-derived hyperpolarizing factor-mediating mesenteric vasodilation to acetylcholine and the hypotensive effect of bradykinin. Using pharmacological interventions that attenuate responses to bradykinin, we examined the possibility of anandamide as a mediator of the NO-independent vasodilator effect of bradykinin in the rat perfused heart by determining responses to anandamide and arachidonic acid. Hearts were treated with indomethacin to exclude prostaglandins and nitroarginine to inhibit NO synthesis and elevate perfusion pressure. The cannabinoid receptor antagonist, SR 141716A (2 microM), reduced dose-dependent vasodilator responses to anandamide (1-10 microgram) but was without effect on responses to AA (1-10 microgram), bradykinin (10-1000 ng) or cromakalim (1-10 microgram). Inhibition of voltage-dependent Ca++ channels with nifedipine (5 nM) attenuated vasodilation to anandamide and arachidonic acid whereas inhibition of Ca++-activated K+ channels with charybdotoxin (10 nM) reduced responses to arachidonic acid but had no effect on vasodilation induced by anandamide. Inhibition of cytochrome P450 with clotrimazole (1 microM) greatly reduced vasodilator responses to bradykinin with less effect on those to anandamide. Finally, the time course of the coronary vasodilator responses to anandamide and bradykinin were dissimilar. These results argue against a role of anandamide in the vasodilator effect of bradykinin in the rat heart.

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-lactone and 5,6-DHT with a t1/2 approximately 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.

The role of K+ channels in the nitric oxide-independent renal vasodilator effect of acetylcholine (Ach) was examined to address the hypothesis that the mechanism underlying this response was different from that of bradykinin, because an earlier study indicated the possibility of different mediators. We used the rat isolated, perfused kidney that was constricted with phenylephrine and treated with nitroarginine and indomethacin to inhibit nitric oxide synthase and cyclooxygenase, respectively. The nonspecific K+ channel inhibitors, procaine and tetraethylammonium (TEA), reduced vasodilator responses to Ach and cromakalim, but not those to nitroprusside. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced vasodilator responses to cromakalim but did not affect those to Ach or nitroprusside. Charybdotoxin, an inhibitor of Ca(++)-activated K+ channels, reduced vasodilator responses to Ach without affecting those to cromakalim or nitroprusside. Iberiotoxin and apamin, inhibitors of large- and small-conductance Ca(++)-activated K+ channels, respectively, did not reduce vasodilation induced by Ach, cromakalim or nitroprusside. The inhibitor of cytochrome P450, clotrimazole, reduced the renal vasodilator effects of Ach and bradykinin but not those of nitroprusside or SCA 40, an agonist for Ca(++)-activated K+ channels. These results suggest that in the rat kidney, Ach, like bradykinin, utilizes a charybdotoxin-sensitive Ca(++)-activated K+ channel of intermediate conductance to elicit vasodilation and that this effect may be dependent on cytochrome P450 activity.

There is now overwhelming evidence for factors, other than nitric oxide (NO), that mediate endothelium-dependent vasodilation by hyperpolarizing the underlying smooth muscle via activation of Ca2+-activated K+ channels. Although the identity of endothelium-derived hyperpolarizing factor (EDHF) remains to be established, cytochrome P450 (CYP)-dependent metabolites of arachidonic acid (AA), namely, the epoxides, fulfill several of the criteria required for consideration as putative mediators of endothelium-dependent hyperpolarization. They are produced by the endothelium, released in response to vasoactive hormones, and elicit vasorelaxation via stimulation of Ca2+-activated K+ channels. Our studies in the rat indicate that, of the epoxides, 5,6-epoxyeicosatrienoic acid (5,6-EET) is the most likely mediator of NO-independent, but CYP-dependent coronary vasodilation in response to bradykinin. Studies in the rat kidney, however, support the existence of additional EDHFs as acetylcholine also exhibits NO-independent vasodilation that is unaffected by CYP inhibitors in concentrations that attenuate responses to bradykinin. In some blood vessels, NO may tonically suppress the expression of CYP-dependent EDHF. In the event of impaired NO synthesis, therefore, a CYP-dependent vasodilator mechanism may serve as a backup to a primary NO-dependent mechanism, although they may act in concert. In other vessels, particularly microvessels, an EDHF may constitute the major vasodilator mechanism for hormones and other physiological stimuli. EDHFs appear to be important regulators of vascular tone; alterations in this system can be demonstrated in hypertension and diabetes, conditions associated with altered endothelium-dependent vasodilator responsiveness.

Depression of GFR and antinatriuresis in response to high chloride has been linked to a cyclooxygenase (COX)-dependent mechanism involving thromboxane A2 (TxA2) and prostaglandin endoperoxide (PGH2), because inhibition of COX prevented the fall in GFR and antinatriuresis produced by hyperchloremia. However, hyperchloremia did not increase, but unexpectedly decreased, renal prostaglandin and TxA2 efflux (Yin et al., 1995). To resolve questions regarding the role of eicosanoids in mediating the renal functional effects of high chloride (117 mM), by stimulating either TxA2 synthesis or TxA2/PGH2 receptors, we compared the ability of indomethacin to block high-chloride effects in the rat isolated kidney with that of BMS 180291 and SQ 29548, antagonists of the TxA2/PGH2 receptor. These antagonists differ in terms of their selectivity and their capacity to inhibit isoforms of the TxA2/PGH2 receptor. Indomethacin and SQ 29548 had identical actions, preventing the decrease of GFR and antinatriuresis evoked by hyperchloremia, e.g., sodium excretion rate in the SQ 29548 and indomethacin groups increased to 7.2 +/- 1.3 and 7.1 +/- 1.2 microEq/min, respectively, compared with 2.6 +/- 0.7 microEq/min in the control group. In contrast, neither BMS 180291 nor the TxA2 synthase inhibitors, OKY 046 and CGS 13080, modified the negative effects of high chloride on GFR or sodium excretion. These results argue against either TxA2 or PGH2 acting as mediator of the effects of high chloride on renal function and suggest a product of COX activity such as a 20-HETE analog of prostaglandin endoperoxide. Evidence to support this proposal was obtained: 1) Hyperchloremia increased 20-HETE release from the rat kidney by 2-fold when compared with low-chloride conditions of renal perfusion. 2) The renal vasoconstrictor action of 20-HETE was shown to be dependent on COX activity and to be antagonized by blockade of the TxA2/PGH2 receptor.

The coronary vasodilator effect of bradykinin (BK) in the rat is independent of NO but dependent on activation of phospholipases with involvement of cytochrome P450 mono-oxygenase (P450) and stimulation of Ca++-activated K+ channels, implicating an unidentified hyperpolarizing factor generated via P450 metabolism of arachidonic acid (AA). Because P450 activity also generates free radicals, such as superoxide, which can lead to the formation of hydrogen peroxide and hydroxyl radicals, which are vasoactive, we addressed the contribution of superoxide to the vasodilator effect of BK in the rat heart. Using rat renal microsomes as a source of P450, we verified that P450-dependent metabolism of AA generated superoxide, as detected by chemiluminescence with lucigenin. The signal was almost abolished by inhibition of P450 with clotrimazole and the superoxide scavenger 4,5-dihydroxy-1,3-benzene sulfonic acid. However, base-line superoxide formation, detected by chemiluminescence, in cardiac slices and perfused hearts was unchanged in response to BK or AA. Furthermore, in perfused hearts treated with nitroarginine and indomethacin to eliminate NO and prostaglandins and elevate perfusion pressure, dose-dependent vasodilator responses to BK were unaffected by superoxide dismutase plus catalase, a combination that abolished dilator responses to hydrogen peroxide. Similarly, the superoxide scavengers 4,5-dihydroxy-1,3-benzene sulfonic acid and 4-hydroxy-2,2,6,6-tetramethylpiperidine-noxyl were without effect on vasodilator responses to BK. Thus, the coronary vasodilator action of BK is independent of superoxide or its derivatives, which can be excluded as hyperpolarizing factors mediating NO-independent vasodilation in the rat.

The cytochrome P450-dependent component of the coronary vasodilator action of bradykinin which requires activation of K+ channels was examined in terms of the contribution of phospholipases in the rat Langendorff heart preparation. This component was isolated by inhibition of nitric oxide synthase with nitroarginine and cyclooxygenase with indomethacin, neither of which affects the coronary vasodilator action of bradykinin. However, nitroarginine elevated coronary perfusion pressure from approximately 40 to 130 mm Hg. The phospholipase C inhibitor, U73122 {1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione}, reduced coronary vasodilator responses to bradykinin by greater than 80%. U73122 also diminished the coronary vasodilator action of cromakalim which activates ATP-sensitive K+ channels. The maleimide moiety of U73122 that has the capacity to affect K+ channels inhibited cromakalim-induced coronary vasodilation, but did not affect that to bradykinin. Inhibition of diacylglycerol lipase with RHC 80267 {1,6-bis-(cyclohexyloximinocarbonylamino)-hexane} was without an overall effect on coronary vasodilator responses to bradykinin. The cytosolic phospholipase A2 inhibitor, AACOCF3 {arachidonyl trifluoromethyl ketone¿} decreased responses to bradykinin by up to 90% whereas inhibitors of the secretory form of phospholipase A2 oleyloxyethyl phosphorylcholine and ONO-RS-082 {2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid} were less effective than either AACOCF3 or U73122. The phospholipase inhibitors demonstrated selectivity as they did not affect the coronary vasodilator responses to nitroprusside. We obtained additional evidence for the antiphospholipase activity of the inhibitors by demonstrating their capacity to suppress bradykinin-stimulated increases in the release of prostacyclin, measured as 6-keto prostaglandin F1 alpha. The phospholipase inhibitors did not affect cyclooxygenase activity as the ability of arachidonic acid to stimulate prostaglandin formation was unimpaired. These results indicate that the coronary vasodilator action of bradykinin is linked to the activities of both phospholipase C and A2.

Objective: The success of thrombolytic therapy in acute stroke relies on timely reperfusion. The current study examines the efficacy of antiplatelet agents as adjuvants for thrombolytic therapy., Methods: Using an established rabbit model of clot embolization and a randomized blinded design, rabbits (n = 8 in each group) were orally pretreated daily for 5 days with adjuvant aspirin (1 mg/kg of body weight or 20 mg/kg), ticlopidine (100 mg/kg), or vehicle (sodium carbonate). On the 6th day, tissue plasminogen activator (6.3 mg/kg administered intravenously over 2 h), was initiated 1 hour after embolization., Results: In all groups, cerebral blood flow (CBF) was reduced to < 10 ml/100 g/min immediately after clot embolization. After the initiation of tissue plasminogen activator (t-PA), there was significant restoration of CBF in the control (t-PA only) and ticlopidine groups (P < 0.05) only. Restoration of CBF generally correlated with brain infarct size (percent hemisphere, mean +/- standard error of the mean), which was 18.0 +/- 7.0 in the t-PA only group versus 11.0 +/- 3.3, 26.5 +/- 5.8, and 21.5 +/- 3.4 in the ticlopidine, low-dose aspirin, and high-dose aspirin groups, respectively (ticlopidine versus aspirin, P < 0.05). Clot lysis was identical in the control and ticlopidine groups, with 6 of 8 animals demonstrating complete clot lysis. Aspirin antagonized clot lysis in a dose-related manner, with low-and high-dose aspirin groups noting clot lysis in four of eight and two of eight animals, respectively., Conclusions: Pretreatment with ticlopidine significantly reduced brain infarct size when compared with aspirin treatment (P < 0.05). Moreover, whereas ticlopidine treatment did not affect clot lysis or CBF relative to t-PA alone, aspirin therapy resulted in antagonism of clot lysis and was associated with a more modest restoration of blood flow. This study provides a background for a more comprehensive understanding of the balance of thrombogenicity and thrombolysis and may assist in the development of novel therapies to expedite cerebrovascular patency and reduce ischemic and reperfusion-mediated neuronal injury.

1. NO- and prostaglandin-independent, endothelium-dependent vasodilator responses to bradykinin are attributed to release of a hyperpolarizing factor. Therefore, the contribution of K+ channels to the renal vasodilator effect of bradykinin was examined in rat perfused kidneys that were preconstricted with phenylephrine and treated with NG-nitro-L-arginine (L-NOARG) and indomethacin to inhibit NO and prostaglandin synthesis. 2. The non-specific K+ channel inhibitors, TEA and TBA reduced vasodilator responses to bradykinin and cromakalim but not those to nitroprusside. 3. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, blocked the vasodilator response to cromakalim without affecting responses to bradykinin. 4. Charybdotoxin, a selective inhibitor of Ca(2+)-activated K+ channels, greatly attenuated vasodilator responses to bradykinin without affecting those to cromakalim or nitroprusside. 5. Iberiotoxin and leiurotoxin, inhibitors of large and small conductance Ca(2+)-activated K+ channels, respectively, were without effect on vasodilator responses to bradykinin, cromakalim or nitroprusside. 6. These results implicate K+ channels, specifically Ca(2+)-activated K+ channels of intermediate conductance, in the renal vasodilator effect of bradykinin and, thereby, support a role for a hyperpolarizing factor.

5,6-Epoxyeicosatrienoic acid (5,6-EET), a cytochrome P450-dependent arachidonate product, is a substrate for cyclooxygenase (COX) and, in some vascular preparations, elicits COX-dependent vasodilation. In the blood perfused rat kidney, 5,6-EET causes COX-dependent renal vasoconstriction, whereas in the rat isolated kidney perfused with a physiological buffer, 5,6-EET produces dose-dependent vasodilation that is unaffected by indomethacin. We examined the possible contribution of platelet COX to the vasoconstrictor action of 5,6-EET. Incubation of labeled 5,6-EET with rat washed platelets yields additional products that elute between 14 to 17 min on high-performance liquid chromatography (HPLC) and cause constriction of the perfused kidney. Indomethacin decreased the formation of these products and reduced the vasoconstrictor capacity of the corresponding HPLC fractions. Thus, platelet COX can metabolize 5,6-EET to vasoconstrictor products that may contribute to the in vivo vasoconstrictor effect of this eicosanoid.

Three major enzyme systems-cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases (CYP)-generate biological mediators from arachidonic acid (AA). A distinct profile of arachidonate products characterizes each renal tubular segment and each section of the renal vascular tree from arteries to microvessels to veins. The most recent discoveries on renal mechanisms subserved by arachidonate metabolites relate to CYP pathways of AA metabolism. These arachidonate products have prominent effects on blood vessels and ion transport, including modulation and mediation of the actions of vasoactive hormones. The diverse properties of these AA metabolites and the wide distribution of the CYP system make them prime candidates for participation in regulatory mechanisms that affect the circulation and transporting epithelia. CYP-AA products are as important to renal and circulatory control as are nitric oxide and prostaglandins., (Copyright © 1996. Published by Elsevier Inc.)

1. Vasodilator responses to bradykinin (BK) in the rat heart are reported to be independent of NO and cyclo-oxygenase/lipoxygenase products of arachidonic acid (AA). 2. We verified that inhibition of NO synthase with L-nitroarginine (50 microM) and cyclo-oxygenase with indomethacin (2.8 microM) were without effect on vasodilator responses to BK (10-1000 ng) in the Langendorff rat heart preparation. 3. L-Nitroarginine elevated perfusion pressure, signifying a crucial role of NO in the maintenance of basal vasculature tone. 4. In hearts treated with L-nitroarginine to eliminate NO and elevate perfusion pressure, vasodilator responses were reduced by inhibitors of cytochrome P450 (P450), clotrimazole (1 microM) and 7-ethoxyresorufin (1 microM). 17-Octadecynoic acid (17-ODYA 2 microM), a mechanism based inhibitor of P450-dependent metabolism of fatty acids, also reduced vasodilator responses to BK. 5. These results confirm that NO and prostaglandins do not mediate vasodilator responses to BK in the rat heart but suggest a major role for a P450-dependent mechanism via AA metabolism.

1. The role of K+ channels in the nitric oxide (NO)-independent coronary vasodilator effect of bradykinin was examined in the Langendorff heart preparation in which nitroarginine was used to inhibit NO synthesis and elevate perfusion pressure; cyclo-oxygenase was inhibited with indomethacin. 2. The K+ channel inhibitors, tetraethylammonium, procaine and charybdotoxin, but not glibenclamide, further increased perfusion pressure suggesting a role for K+ channels, other than ATP-sensitive K+ channels, in the regulation of coronary vascular tone under the experimental conditions adopted here. 3. The non-specific K+ channel inhibitors, tetraethylammonium and procaine, reduced vasodilator responses to bradykinin and cromakalim but not those to nitroprusside in the perfused heart treated with nitroarginine and indomethacin. 4. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced vasodilator responses to cromakalim but did not affect those to bradykinin or nitroprusside. 5. Charybdotoxin, an antagonist of Ca(2+)-activated K+ channels, inhibited responses to bradykinin but did not affect those to cromakalim or nitroprusside. 6. Nifedipine inhibited vasodilator responses to bradykinin and cromakalim without affecting those to nitroprusside. 7. Inhibition of cytochrome P450 with clotrimazole reduced responses to bradykinin but did not modify those to cromakalim or nitroprusside. 8. These results suggest that bradykinin utilizes a Ca(2+)-activated K+ channel to produce vasodilatation in the rat heart.

Bradykinin stimulates phospholipases to release arachidonic acid (AA) which can be metabolized by cyclooxygenase, lipoxygenase and cytochrome P450 (P450) to yield vasoactive products that may contribute to the effect of the peptide. In the rat kidney, pharmacological evidence suggests that a substantial component of the vasodilator response is dependent on P450-AA metabolism. In the heart, the vasodilator response to bradykinin is independent of NO and prostaglandins but reduced by inhibitors of P450, including 17-ODYA, an inhibitor of fatty acid metabolism, also suggesting a role of P450-AA. Moreover, the renal and coronary vasodilator responses to bradykinin are associated with release of P450-AA products measured by gas chromatography-mass spectrometry (GC-MS). The coronary vasodilator response to bradykinin is also dependent on activation of K+ channels linking P450-AA and hyperpolarization. Formation of vasodilator eicosanoids derived via the P450 pathway may make important contributions to the control of vascular tone, local blood flow and, thereby, blood pressure.

Based on the use of inhibitors, cytochrome P450 has been implicated in endothelium-dependent relaxant responses via metabolism of arachidonic acid (AA). However, the contribution of cytochrome P450 and its AA metabolites to the regulation of vascular tone has not been established due, in part, to questions of specificity of cytochrome P450 inhibitors which have not been extensively characterized in terms of their vascular effects. Consequently, we addressed the effects of several inhibitors on vasorelaxant responses of phenylephrine-contracted, rabbit, aortic rings to agents that utilize different transduction mechanisms to determine any actions unrelated to inhibition of cytochrome P450 and/or AA metabolism. Octadecynoic acid (2.5 and 5 microM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, and eicosatetrayenoic acid (10 and 20 microM), an inhibitor of AA metabolism, were without effect on vasorelaxant responses to acetylcholine, sodium nitroprusside, isoproterenol and diazoxide. 7-Ethoxyresorufin (2-10 microM), a substrate for cytochrome P450, and clotrimazole (2.5-10 microM) which binds to the heme moiety of the enzyme, concentration-dependently reduced responses to acetylcholine but not the other agonists indicating an effect on nitric oxide synthesis although neither affected the conversion of L-arginine to L-citrulline by endothelial cells. SKF 525A (50-200 microM), the prototypical inhibitor of cytochrome P450, which is metabolized to an inhibitory intermediate, also reduced responses to acetylcholine and, in addition, impaired the vasorelaxant activities of isoproterenol and diazoxide.

1. Inhibition of nitric oxide generation with Nw-nitro-L-arginine (nitroarginine) reduced vasodilator responses to bradykinin and acetylcholine and enhanced those to nitroprusside in the rat isolated perfused kidney, preconstricted with phenylephrine. 2. Inhibition of cyclo-oxygenase with indomethacin, decreased the vasodilator responses to bradykinin by approximately 25% without affecting those to acetylcholine or nitroprusside. 3. BW755c, a dual inhibitor of cyclo-oxygenase and lipoxygenase, reduced renal vasodilator responses to bradykinin, comparable to the effect of indomethacin suggesting an effect related to inhibition of cyclo-oxygenase rather than lipoxygenase. 4. ETYA, an inhibitor of all arachidonic acid metabolic pathways, markedly reduced vasodilator responses to bradykinin but was without effect on the renal vasodilatation induced by acetylcholine or nitroprusside. 5. Clotrimazole and 7-ethoxyresorufin, inhibitors of cytochrome P450, greatly attenuated vasodilator responses to bradykinin without affecting those to acetylcholine or nitroprusside. 6. These data suggest that the renal vasodilator response to bradykinin is subserved by arachidonic acid metabolites as well as nitric oxide, the former accounting for up to 70% of the vasodilator effect of bradykinin.

Streptozotocin-induced diabetes resulted in diminished vasodilator responses to bradykinin in the preconstricted isolated perfused kidney of the rat which were associated with decreased renal phospholipase A2 activity and reduced release of PGE2 into the renal venous effluent.

Our previous studies indicated a role for cytochrome P-450-dependent enzymes in generating the mediators of the vasodilator effect of arachidonic acid (AA) in the preconstricted indomethacin-treated perfused kidney of the rat. We report that in vivo induction of cytochrome P-450 enzymes with 3-methylcholanthrene-beta-naphthoflavone or dexamethasone enhanced the renal vasodilator effect of AA in this experimental preparation. Conversely, depletion of cytochrome P-450 enzymes with stannous chloride or cobalt chloride diminished the vasodilator response to AA. Injection of AA resulted in the release of relaxant material into the renal effluent detected by superfusion of rabbit aortic rings. Inhibition of cytochrome P-450 with 7-ethoxyresorufin reduced the release of vasorelaxant material. Metabolism of labeled AA by the kidney revealed four peaks of radioactivity that were recovered from the renal effluent. The heights of these peaks were reduced by 7-ethoxyresorufin. These results provide further evidence for cytochrome P-450-dependent metabolism of AA to one or more vasodilator products by the rat kidney.