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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Basic Clin Pharmacol Toxicol. 2009 Dec 7;106(5):378–388. doi: 10.1111/j.1742-7843.2009.00501.x

A Synthetic Analogue of 20-HETE, 5,14-HEDGE, Reverses Endotoxin-Induced Hypotension via Increased 20-HETE Levels Associated with Decreased iNOS Protein Expression and Vasodilator Prostanoid Production in Rats

Tuba Cuez 1, Belma Korkmaz 1, C Kemal Buharalioglu 1, Seyhan Sahan-Firat 1, John Falck 2, Kafait U Malik 3, Bahar Tunctan 1
PMCID: PMC2882512  NIHMSID: NIHMS158967  PMID: 20002062

Abstract

Nitric oxide (NO) produced by inducible NO synthase (iNOS) is responsible for endotoxin-induced hypotension and vascular hyporeactivity and plays a major contributory role in the multiorgan failure. Endotoxic shock is also associated with an increase in vasodilator prostanoids as well as a decrease in endothelial NO synthase (eNOS) and cytochrome P450 4A protein expression, and production of a vasoconstrictor arachidonic acid product, 20-hydroxyeicosatetraenoic acid (20-HETE). The aim of this study was to investigate the effects of a synthetic analogue of 20-HETE, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), on the endotoxin-induced changes in eNOS, iNOS and heat shock protein 90 (hsp90) expression as well as 20-HETE and vasodilator prostanoid (6-keto-PGF1α and PGE2) production. Endotoxin-induced fall in blood pressure and rise in heart rate were associated with an increase in iNOS protein expression and a decrease in eNOS protein expression in heart, thoracic aorta, kidney and superior mesenteric artery. Endotoxin did not change hsp90 protein expression in the tissues. Endotoxin-induced changes in eNOS and iNOS protein expression were associated with increased 6-keto-PGF1α and PGE2 levels and a decrease in 20-HETE levels, in the serum and kidney. These effects of endotoxin on the iNOS protein expression and 6-keto-PGF1α, PGE2 and 20-HETE levels were prevented by 5,14-HEDGE. Furthermore, a competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, prevented the effects of 5,14-HEDGE on the endotoxin-induced changes in systemic and renal levels of these prostanoids and 20-HETE. These data are consistent with the view that an increase in systemic and renal 20-HETE levels associated with a decrease in iNOS protein expression and vasodilator prostanoid production contributes to the effect of 5,14-HEDGE to prevent the hypotension during rat endotoxemia.

The expression of inducible nitric oxide (NO) synthase (iNOS) is enhanced in many tissues in response to mediators released by endotoxin [1, 2]. This leads to increased generation of NO, which contributes to fall in blood pressure, vascular hyporeactivity, multiple organ failure and high mortality rate that are associated with septic shock [2–5]. Systemic blockade of iNOS opposes the fall in blood pressure in endotoxic shock [2, 3, 5]. This is not only due to withdrawal of vasodilator effects of NO, but also is associated with increased activity of vasoconstrictor arachidonic acid products including 20-hydroxyeicosatetraenoic acid (20-HETE) [2, 6]. In contrast to iNOS, a potential role of the constitutive endothelial cell isoform of NOS (eNOS) in the pathophysiology of endotoxic shock has recently gained controversy due to findings that indicated eNOS as a pro-inflammatory candidate in inflammatory disease conditions [7]. Recent studies not only demonstrate the importance of eNOS for the up-regulation of pro-inflammatory protein expression, but also indicate the autoregulation of NOS expression by NO, since iNOS-derived NO is known to inhibit the expression and activity of eNOS [8–14]. Collectively, these data give rise to the hypothesis that eNOS plays a key role in the protein expression of iNOS and the pathogenesis of endotoxic shock.

20-HETE, an eicosanoid synthesized from arachidonic acid primarily by cytochrome P450 (CYP) isoforms of the 4A and 4F classes in the vasculature, is one of the primary eicosanoids produced in the microcirculation [6, 15]. 20-HETE participates in the regulation of vascular tone by blocking the large conductance calcium-activated potassium channels and by a direct effect on L-type calcium channels in several vascular beds, including renal, aortic, mesenteric and coronary arteries [6, 15]. It has been reported that 20-HETE-induced endothelium-dependent constriction is abolished by inhibition of cyclooxygenase (COX) with indomethacin and by an endoperoxide/thromboxane receptor antagonist, SQ-29548 [16–19]. It has also been demonstrated that prostaglandin analogues of 20-HETE, 20-OH-PGG2 and 20-OH-PGH2, produced by COX in vascular endothelial cells mediate the vasoconstrictor effects of 20-HETE [18, 19]. As opposed to its vasoconstrictor effect, 20-HETE also produces vasodilation in renal, coronary, pulmonary and basiler arteries [20–22]. These vasodilatory responses to 20-HETE have been attributed to NO release, conversion of 20-HETE to 20-OH-PGE2 and 20-OH-PGF2α by COX, and increased formation of PGE2 and prostacyclin (PGI2) [18, 20–23]. Our previous studies with COX inhibitors demonstrated that prostanoids produced during endotoxemia increase iNOS protein expression and NO synthesis, and decrease CYP 4A1 protein expression and CYP 4A activity. These results suggested that dual inhibition of iNOS and COX restores blood pressure presumably due to increased production of 20-HETE derived from CYP 4A in endotoxemic rats [24, 25].

It has been reported that NO inhibits renal CYP ω-hydroxylase activity and the production of 20-HETE [26–28]. Conversely, recent studies have indicated that 20-HETE affects both the release and actions of eNOS-derived NO [29, 30]. It has been shown that increased production of 20-HETE in the vasculature is associated with endothelial dysfunction and increased vascular tone which contributes to the development of hypertension in animal models [31]. At least 3 different pathways have been suggested to play a role in these responses including increased vascular expression of subunits of reduced nicotinamide-adenine dinucleotide phosphate oxidase by 20-HETE, leading to production of superoxide [32], decreased association of eNOS with heat shock protein 90 (hsp90) by 20-HETE, leading to diminished formation of NO and increased formation of superoxide [29, 33, 34], and increased formation of superoxide directly by 20-HETE in endothelial cells [34]. We have previously demonstrated that the fall in mean arterial pressure and increase in heart rate in endotoxemic rats is also associated with a decrease in the expression of CYP 4A1/A3 protein and CYP 4A activity in the kidney and increased levels of nitrite in serum, kidney, heart, thoracic aorta and superior mesenteric artery [24, 35–38]. Furthermore, administration of a synthetic analogue of 20-HETE, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), prevented hypotension and vascular hyporeactivity associated with the changes in systemic and tissue NO production in rats treated with endotoxin [39]. These findings led us to hypothesize that 20-HETE regulates eNOS, iNOS and hsp90 protein expression in cardiac, vascular and renal tissues during endotoxemia. To test this hypothesis, we have investigated the effects of a stable 20-HETE analogue 5,14-HEDGE, on endotoxin-induced changes in eNOS, iNOS and hsp90 protein expression as well as 20-HETE and vasodilator prostanoid production. Preliminary results have been presented in an abstract form [40, 41].

Materials and Methods

Endotoxic shock model

Experiments were performed on male Wistar rats (n = 59) (Research Center of Experimental Animals, Mersin University, Mersin, Turkey) weighing 250 to 300 g that were fed a standard chow. The rats were housed in an animal facility with a 12-hr light: dark cycle. All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Ethics Committee of Mersin University School of Medicine. Endotoxic shock was induced as previously described by Tunctan et al. [35]. Briefly, conscious rats received a 10 mg/kg (i.p.) (sublethal dose) injection of endotoxin (Escherichia coli lipopolysaccharide, O111:B4; Sigma Chemical Co., St. Louis, USA) or an equivalent volume of saline (4 ml/kg, i.p.) at time 0. Mean arterial pressure and heart rate were measured using a tail-cuff device (MAY 9610 Indirect Blood Pressure Recorder System, Commat Ltd., Ankara, Turkey) during a control period at time 0 and 1, 2, 3 and 4 hr later. Separate groups of endotoxin-treated rats were given a synthetic analogue of 20-HETE, 5,14-HEDGE (30 mg/kg, s.c.) [39], a competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE; WIT002) (30 mg/kg, s.c.) [39] 1 hr after injection of saline or endotoxin. 5,14-HEDGE and 20-HEDE were synthesized in Department of Biochemistry University of Texas Southwestern Medical Center, Dallas, Texas, USA. The rats were euthanized 4 hr after the administration of endotoxin, and a blood sample, kidney, heart, thoracic aorta and superior mesenteric artery were collected from all animals. Sera were obtained from blood samples by centrifugation at 14,000 × g for 15 min. at 4°C and stored at −20°C until analyzed for the measurement of 20-HETE, 6-keto-PGF1α and PGE2 levels. The tissues were homogenized in 1 ml of an ice-cold 20 mM HEPES buffer (pH 7.5) containing 20 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 2 mM EDTA, 20 mM sodium fluoride, 10 mM benzamidine 10, 1 mM dithiothreitol, 20 mM leupeptin and 10 mM aprotinin [35]. Cell debris was removed by centrifugation at 14,000 × g for 10 min. at 4°C followed by sonication for 15 sec. on ice with 50 µl ice-cold Tris (50 mmol/l, pH 8.0) and KCl (0.5 M). The samples were centrifuged at 14,000 × g for 15 min. at 4°C and then supernatants were removed and stored at −20°C until analyzed for the measurement of α-actin, eNOS, iNOS and hsp90 protein levels. The total protein amount was determined by Coomassie blue method using bovine serum albumin (BSA) as standard [35].

Immunoblotting

Immunoblotting for α-actin, eNOS, iNOS and hsp90 proteins were performed according to the method as described previously [37]. Briefly, tissue homogenates (100 µg of protein) were subjected to a 10% SDS-polyacrylamide gel electrophoresis and then proteins were transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (mmol/l: Tris-HCl 25 [pH 7.4], NaCl 137, KCl 27 and 0.05% Tween 20) and incubated overnight with anti-eNOS monoclonal antibody (Calbiochem, San Diego, CA, USA), (1:500 in 5% BSA), anti-iNOS monoclonal antibody (BD Transduction Laboratories, San Jose, CA, USA) (1:500 in 5% BSA) or anti-hsp90 monoclonal antibody (Calbiochem, San Diego, CA, USA) (1:500 in 5% BSA) followed by incubation with a sheep anti-mouse immunoglobulin secondary antibody conjugated with a horse radish peroxidase (Amersham Life Sciences, Cleveland, OH, USA) (1:1,000 in 0.1% BSA) for 1 hr. The blots were developed with enhanced chemiluminescence (ECL) (ECL Plus Western Blotting Detection Reagents) (Amersham Life Sciences, Cleveland, OH, USA) according to the manufacturer's instructions. Immunoreactive proteins were visualized using a gel imaging system (EC3-CHEMI HR imaging system) (Ultra-Violet Products, UVP, UK). Densitometric analysis was performed with NIH image software (ImageJ 1.29). The same membrane was used to determine α-actin expression using a monoclonal antibody against α-smooth muscle actin (Sigma Chemical Co., St. Louis, USA) (1:500 for heart, thoracic aorta and superior mesenteric artery, and 1:1,000 for kidney in 5% BSA) and the content of the latter was used to normalize the expression of eNOS, iNOS and hsp90 proteins in each sample.

Measurement of systemic and renal eicosanoid levels

Serum and tissue 20-HETE, 6-keto-PGF1α and PGE2 concentrations were measured as indexes for CYP 4A and COX activity by ELISA according to the manufacturer's instructions in the 20-HETE (Detroit R&D, Inc., Detroit, MI, USA), and 6-keto-PGF1α and PGE2 (Cayman Chemical Co., Ann Arbor, MI, USA) assay kits, respectively.

Statistical analysis

All data were expressed as means ± SEM. Data were analysed by one-way ANOVA followed by Student-Newman-Keuls test for multiple comparisons, Kruskal-Wallis test followed by Dunns test for multiple comparisons and Student's t or Mann-Whitney U tests when appropriate. A P value < 0.05 was considered to be statistically significant.

Results

Effect of 5,14-HEDGE on the endotoxin-induced decrease in mean arterial pressure and increase in heart rate

We have previously demonstrated that mean arterial pressure fell by 31 mmHg and heart rate rose by 90 bpm in rats treated with endotoxin (P < 0.05) (table 1) [39]. 5,14-HEDGE completely prevented the fall in mean arterial pressure and the increase in heart rate in rats given endotoxin (P < 0.05). A competitive antagonist of the vasoconstrictor effects of 20-HETE, 20-HEDE prevented the ability of 5,14-HEDGE to oppose the effects of endotoxin on mean arterial pressure and heart rate (P < 0.05). 5,14-HEDGE and 20-HEDE had no effect on mean arterial pressure or heart rate when given to rats treated with vehicle (P > 0.05).

Table 1.

Time course of the effects of N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE) and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) on mean arterial presure (MAP) and heart rate (HR) following administration of saline (vehicle) or endotoxin (ET) to rats.

Time after ET injection (h)
0 1 2 3 4
Vehicle
    MAP 123.40±1.52
(n = 21)		 126.70±1.47
(n = 21)		 127.70±1.62
(n = 15)		 127.00±1.33
(n = 14)		 124.20±1.77
(n = 21)
    HR 345.10±5.44
(n = 14)		 321.50±6.98
(n = 12)		 351.00±8.29
(n = 15)		 333.90±9.49
(n = 14)		 336.40±7.20
(n = 21)
ET
    MAP 125.10±1.43
(n = 20)		 94.20±1.76af
(n = 20)		 92.88±1.83af
(n = 17)		 92.65±1.48af
(n = 17)		 93.50±2.03af
(n = 16)
    HR 372.50±8.25
(n = 20)		 407.10±7.51af
(n = 19)		 404.20±10.43af
(n = 19)		 412.10±14.02af
(n = 18)		 426.30±8.95af
(n = 20)
5,14-HEDGE
    MAP 124.30±3.09
(n = 12)		 124.90±3.57
(n = 11)		 120.60±6.62
(n = 10)		 120.30±3.78
(n = 12)		 121.30±3.92
(n = 12)
    HR 341.20±7.81
(n = 12)		 321.80±6.38
(n = 10)		 343.30±8.03
(n = 12)		 349.60±8.15
(n = 12)		 352.80±11.53
(n = 12)
ET+5,14-HEDGE
    MAP 129.20±1.55
(n= 21)		 98.10±1.98acf
(n= 21)		 124.00±3.51bg
(n= 21)		 128.60±2.62bg
(n= 21)		 126.80±2.63bg
(n= 21)
    HR 346.40±4.95
(n = 21)		 384.20±5.73acf
(n = 19)		 357.60±5.16bg
(n = 18)		 357.40±4.63bg
(n = 16)		 365.20±6.29bg
(n = 9)
20-HEDE
    MAP 125.00±1.00
(n = 6)		 126.20±0.79
(n = 6)		 124.30±1.02
(n = 6)		 123.70±0.62
(n = 6)		 125.20±0.91
(n = 6)
    HR 304.80±6.72
(n = 5)		 316.00±5.21
(n = 6)		 345.70±8.36
(n = 6)		 328.30±4.88
(n = 6)		 346.50±6.68
(n = 6)
ET+5,14-HEDGE+20-HEDE
    MAP 127.20±1.58
(n = 6)		 93.50±1.09acef
(n = 6)		 101.80±0.60acdefg
(n = 6)		 101.70±0.42acdehi
(n = 6)		 102.30±0.56acdehi
(n = 6)
    HR 334.80±11.40
(n = 4)		 361.80±13.42acef
(n = 6)		 406.30±3.69acdefg
(n = 6)		 413.20±9.01acdefg
(n = 6)		 434.00±9.60acdefg
(n = 6)
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5,14-HEDGE (30 mg/kg, s.c.) or 20-HEDE (30 mg/kg, s.c.) was given at 1 hr after vehicle (4 ml/kg, i.p.) or ET (10 mg/kg, i.p.) injection. Data are expressed as means ± S.E.M. Number in parentheses indicate the number of animals studied per group.

a

indicates a significant difference from the corresponding value seen in rats treated with saline (P < 0.05).

b

indicates a significant difference from the corresponding value seen in the rats treated with vehicle and ET (P < 0.05).

c

indicates a significant difference from the corresponding value seen in the rats treated with vehicle and 5,14-HEDGE (P < 0.05).

d

indicates a significant difference from the corresponding value seen in the rats treated with ET and 5,14-HEDGE (P < 0.05).

e

indicates a significant difference from the corresponding value seen in the rats treated with vehicle and 20-HEDE (P < 0.05).

f

indicates a significant difference from the time 0 value within a group (P < 0.05).

g

indicates a significant difference from the time 1 value in each group (P < 0.05).

Effect of 5,14-HEDGE on the endotoxin-induced decrease in 20-HETE levels

Endotoxin caused a decrease in 20-HETE levels in serum and kidney of rats (P < 0.05) (fig. 1). A synthetic analogue of 20-HETE, 5,14-HEDGE, prevented the decrease in systemic and tissue 20-HETE levels in endotoxin-treated rats (P < 0.05) (fig. 1). A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-HEDE, reversed the effects of 5,14-HEDGE on 20-HETE levels in endotoxemic rats (P < 0.05) (fig. 1). On the other hand, 20-HEDE also caused a decrease in systemic and renal 20-HETE levels of control rats (P < 0.05) (fig. 1).

Fig. 1.

Fig. 1

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The effects of N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE) and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) on changes in systemic and renal 20-hydroxyeicosatetraenoic acid (20-HETE) levels measured 4 hr after saline (vehicle) (4 ml/kg, i.p.) or endotoxin (ET) (10 mg/kg, i.p.) injection to Wistar rats. 5,14-HEDGE (30 mg/kg, s.c.) or 20-HEDE (30 mg/kg, s.c.) was given at 1 hr after ET injection. Data are expressed as means ± S.E.M. of 5–14 animals. a indicates a significant difference from the corresponding value seen in rats treated with saline (P < 0.05). b indicates a significant difference from the corresponding value seen in the rats treated with vehicle and ET (P < 0.05). c indicates a significant difference from the corresponding value seen in the rats treated with vehicle and 5,14-HEDGE (P < 0.05). d indicates a significant difference from the corresponding value seen in the rats treated with ET and 5,14-HEDGE (P < 0.05). e indicates a significant difference from the corresponding value seen in the rats treated with vehicle and 20-HEDE (P < 0.05).

Effect of 5,14-HEDGE on the endotoxin-induced increase in 6-keto-PGF1α and PGE2 levels

Endotoxin caused an increase in 6-keto-PGF1α, a stable metabolite of PGI2, and PGE2 levels in serum (fig. 2A) and kidney (fig. 2B) of endotoxin-treated rats (P < 0.05). 5,14-HEDGE prevented the increase in 6-keto-PGF1α and PGE2 levels in serum (fig. 2A) and kidney (fig. 2B) of endotoxemic rats (P < 0.05). 20-HEDE opposed the effect of 5,14-HEDGE on prostanoid levels in endotoxin-treated rats (P < 0.05) (fig. 2). However, 5,14-HEDGE and 20-HEDE decreased systemic (fig. 2A) and tissue (fig. 2B) basal prostanoid levels in control rats (P < 0.05).

Fig. 2.

Fig. 2

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The effects of N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE) and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) on changes in (A) systemic and (B) renal prostanoid levels measured 4 hr after saline (vehicle) (4 ml/kg, i.p.) or endotoxin (ET) (10 mg/kg, i.p.) injection to Wistar rats. 5,14-HEDGE (30 mg/kg, s.c.) or 20-HEDE (30 mg/kg, s.c.) was given at 1 hr after ET injection. Data are expressed as means ± S.E.M. of 3–4 animals. a indicates a significant difference from the corresponding value seen in rats treated with saline (P < 0.05). b indicates a significant difference from the corresponding value seen in the rats treated with vehicle and ET (P < 0.05). c indicates a significant difference from the corresponding value seen in the rats treated with vehicle and 5,14-HEDGE (P < 0.05). d indicates a significant difference from the corresponding value seen in the rats treated with ET and 5,14-HEDGE (P < 0.05). e indicates a significant difference from the corresponding value seen in the rats treated with vehicle and 20-HEDE (P < 0.05).

Effect of 5,14-HEDGE on the endotoxin-induced decrease in eNOS protein expression

Endotoxin decreased eNOS protein levels in kidney (fig. 3A), heart (fig. 3B), thoracic aorta (fig. 3C) and superior mesenteric artery of rats (fig. 3D) (P < 0.05). Neither 5,14-HEDGE nor 20-HEDE had any effect on the decrease in the eNOS protein expression in the tissues of rats treated with vehicle or endotoxin (P > 0.05) (fig. 3).

Fig. 3.

Fig. 3

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The effects of N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE) and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) on changes in endothelial nitric oxide synthase (eNOS) protein expression in (A) kidney, (B) heart, (C) thoracic aorta and (D) superior mesenteric artery 4 hr after saline (vehicle) (4 ml/kg, i.p.) or endotoxin (ET) (10 mg/kg, i.p.) injection to Wistar rats. 5,14-HEDGE (30 mg/kg, s.c.) or 20-HEDE (30 mg/kg, s.c.) was given at 1 hr after ET injection. Data are expressed as means ± S.E.M. of 6 animals.

Effect of 5,14-HEDGE on the endotoxin-induced increase in iNOS protein expression

In contrast to eNOS protein expression, endotoxin increased iNOS protein levels in kidney (fig. 4A), heart (fig. 4B), thoracic aorta (fig. 4C) and superior mesenteric artery of rats (fig. 4D) (P < 0.05). 5,14-HEDGE prevented the increase in iNOS protein expression in the tissues of endotoxemic rats (P < 0.05) (fig. 4). 20-HEDE did not prevent the effect of 5,14-HEDGE on iNOS protein expression in endotoxin-treated rats (P > 0.05) (fig. 4).

Fig. 4.

Fig. 4

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The effects of N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE) and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) on changes in inducible nitric oxide synthase (iNOS) protein expression in (A) kidney, (B) heart, (C) thoracic aorta and (D) superior mesenteric artery 4 hr after saline (vehicle) (4 ml/kg, i.p.) or endotoxin (ET) (10 mg/kg, i.p.) injection to Wistar rats. 5,14-HEDGE (30 mg/kg, s.c.) or 20-HEDE (30 mg/kg, s.c.) was given at 1 hr after ET injection. Data are expressed as means ± S.E.M. of 6 animals. a indicates a significant difference from the corresponding value seen in rats treated with saline (P < 0.05). b indicates a significant difference from the corresponding value seen in the rats treated with vehicle and ET (P < 0.05).

Effect of 5,14-HEDGE on the hsp90 protein expression

Endotoxin did not alter the expression level of hsp90 in kidney (Fig. 5A), heart (fig. 5B), thoracic aorta (fig. 5C) and superior mesenteric artery of rats (fig. 5D) (P > 0.05). Neither 5,14-HEDGE nor 20-HEDE had any effect on the basal hsp90 protein expression in the tissues of rats treated with vehicle or endotoxin (P > 0.05) (fig. 5).

Fig. 5.

Fig. 5

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The effects of N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE) and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) on changes in heat shock protein 90 (hsp90) protein expression in (A) kidney, (B) heart, (C) thoracic aorta and (D) superior mesenteric artery 4 hr after saline (vehicle) (4 ml/kg, i.p.) or endotoxin (ET) (10 mg/kg, i.p.) injection to Wistar rats. 5,14-HEDGE (30 mg/kg, s.c.) or 20-HEDE (30 mg/kg, s.c.) was given at 1 hr after ET injection. Data are expressed as means ± S.E.M. of 6 animals.

Discussion

This is the first study to provide an evidence that, 5,14-HEDGE, a synthetic analogue of 20-HETE, prevents the endotoxin-induced increase in iNOS protein expression, but not decrease in eNOS protein level, in cardiac, vascular and renal tissues of rats and that inhibition of iNOS protein expression and vasodilator prostanoid production by 5,14-HEDGE restores mean arterial pressure and heart rate by increasing systemic and renal 20-HETE levels. Moreover, the hsp90 protein expression level in the cardiovascular tissues was not altered in endotoxemic rats and by 5,14-HEDGE.

There are several reports suggesting a direct link between COX, NOS and CYP 4A enzymes [6, 15, 42, 43]. For example, arachidonic acid and its metabolites generated by COX isoforms have been shown to interfere with NO biosynthesis [42, 43]. NO has also been demonstrated to activate COX enzymes, an event leading to overt production of prostanoids [42, 43]. Moreover, it has been reported that NO inhibits renal CYP ω-hydroxylase activity and the production of 20-HETE [6, 15]. More recently, it has been demonstrated that 20-HETE inhibits both the release and actions of eNOS-derived NO in vitro and in vivo [29, 30]. Therefore, these data suggest that increased production of COX-derived prostanoids and NO might contribute to the endotoxin-induced decrease in 20-HETE levels during endotoxemia in rats.

Our previous findings with a selective iNOS inhibitor, 1,3-PBIT, suggest that the endotoxemia-induced increase in iNOS-derived NO production suppresses renal CYP 4A protein expression and activity [37]. By using a non-selective COX inhibitor, indomethacin, we have also demonstrated that prostanoids produced during endotoxemia increase iNOS protein expression and NO synthesis, and decrease CYP 4A1 protein expression and CYP 4A activity [24]. Moreover, dual inhibition of iNOS or COX by indomethacin restored renal CYP 4A protein level and CYP 4A activity and mean arterial pressure in endotoxin-treated rats, suggesting that the effects of non-selective inhibition of COX might be due to increased production of arachidonic acid metabolites derived via CYP 4A. Our recent studies with a selective COX-2 inhibitor, NS-398, also suggest that decreased production of vasodilator prostanoids, PGI2 and PGE2, and NO as well as increased levels of a vasoconstrictor eicosanoid, 20-HETE, contribute to the effect of selective COX-2 inhibitor to prevent the endotoxin-induced decrease in mean arterial pressure and increase in heart rate in endotoxemic rats [25]. In the present study, a synthetic analogue of 20-HETE, 5,14-HEDGE, prevented the endotoxin-induced increase in the production of vasodilator prostanoids, PGI2, measured as its stable metabolite 6-keto-PGF1α, and PGE2, and the decrease in the levels of the vasoconstrictor eicosanoid, 20-HETE, in endotoxin-treated rats. A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-HEDE, prevented the effects of 5,14-HEDGE. Based on the results from previous studies [24, 37, 39] and our present findings, it is likely that a decrease in vasodilator prostanoid production by COX-2 might contribute to the effects of 5,14-HEDGE in preventing the decrease in blood pressure and vascular hyporeactivity in endotoxemic rats. In contrast to the previous findings with 20-HETE [20–22], our results also suggest that 5,14-HEDGE is not to be converted to vasodilator prostanoids or increased prostanoid production in the rat model of endotoxemia because the glycine substitution renders 5,14-HEDGE less susceptible to β-oxidation and the absence of the double bonds across the 8,9- and 11,12-carbons prevent its degradation by COX to arachidonic acid products [44]. Although we did not investigate the effect of 5,14-HEDGE on COX-2 at transcriptional level in the present study, it is possible that 5,14-HEDGE might directly or indirectly, via 20-HETE, NO, peroxynitrite and/or transcription factors such as nuclear factor-κB (NF-κB) [42, 43], inhibit COX-2 mRNA expression leading to a decrease in COX-2 protein expression and prostanoid production in endotoxemic rats. In the present study, 5,14-HEDGE and 20-HEDE also caused a decrease in PGI2 and PGE2 levels in serum and kidney of control rats. A possible mechanism by which 20-HEDE decreases systemic and renal PGI2 and PGE2 levels in control rats could be through inhibiting the effects of endogenously produced 20-HETE on prostanoid production. It is also possible that both 5,14-HEDGE and 20-HEDE might inhibit COX-1 activity leading to decreased prostanoid levels. However, additional experiments need to be conducted to demonstrate the validity of the proposed hypothesis.

A chaperone molecule, hsp90, has been identified as a signalling molecule in the activation of all the isoforms of NOS [45]. Hsp90 associates with NOS and facilitates its phosphorylation, and this in turn increases NO production from the enzyme. Vo et al. [13] demonstrated that maximal vascular iNOS expression and its function are achieved via the up-regulation and increased association of eNOS with hsp90, and that in the absence of functional eNOS, the vascular effects of iNOS are delayed during rodent endotoxemia. Yoshida and Xia [46] also reported that hsp90 is an important post-translational modulator of iNOS in iNOS-transfected cells. On the other hand, there are several studies reporting that endotoxin up-regulates iNOS protein, but does not alter basal hsp90 expression in in vitro [47] and in vivo studies [48]. Recently, Cheng et al. [29] have shown that 20-HETE impairs NO production in vitro and its function in vivo by inhibiting association of eNOS with hsp90. We have previously demonstrated that 5,14-HEDGE did not prevent the endotoxin-induced decrease in endothelium-dependent relaxations induced by acetylcholine in thoracic aorta and superior mesenteric artery, although it reversed the effects of endotoxin on vascular hyporeactivity to norepinephrine and over-production of nitrite in the tissues [39]. Moreover, 20-HEDE also had no effect on the endothelial dysfunction in the endotoxemic rats treated with 5,14-HEDGE. In the present study, endotoxin-induced fall in systemic and renal 20-HETE levels was accompanied by increased iNOS protein levels and decreased eNOS protein expression without alterations in hsp90 expression in the kidney, heart, thoracic aorta and superior mesenteric artery of endotoxemic rats. These effects of endotoxin, except for eNOS protein expression, were prevented by 5,14-HEDGE. Based on these data and our previous findings [35–39], it is likely that iNOS-derived NO inhibits eNOS protein expression in cardiac, vascular and renal tissues of endotoxemic rats. On the other hand, the ineffectiveness of 5,14-HEDGE in preventing the decrease in eNOS protein expression does not completely support this hypothesis. However, the negative feedback of iNOS on eNOS expression could be explained by several mechanisms. For example, down-regulation of eNOS at post-transcriptional level associated with up-regulation of iNOS mNA by endotoxin [49] may contribute to decrease in eNOS protein levels. Several kinases such as, Rho-kinase, p38 mitogen-activated protein kinase, 5′-adenosine monophosphate-activated protein kinase, protein kinase A and protein kinase G as well as impaired phosphotidylinositol 3-kinase/Akt pathway have been shown to modulate eNOS expression and activity [50, 51]. Therefore, further studies are required to clarify mechanisms of the endotoxin-induced decrease in eNOS protein expression. More importantly, these results indicate that an increase in the production of 20-HETE contributes to the effect of 5,14-HEDGE to prevent the increase in iNOS protein expression during rat endotoxemia. Our results also demonstrate that 20-HEDE does not prevent the effect of 5,14-HEDGE on increased iNOS protein expression in endotoxemic rats. 20-HEDE is an inactive analogue of 20-HETE and it competitevely blocks vasoconstrictor effects of 20-HETE in arteries and prevents the rise in intracellular calcium induced by 20-HETE [44, 52]. Since NO synthesis by iNOS is calcium-dependent, therefore, it is possible that 20-HEDE reverses the effect of 5,14-HEDGE on iNOS activity, but not its protein expression. Although we did not investigate the effect of 5,14-HEDGE on iNOS at transcriptional level in the present study, it is possible that 5,14-HEDGE might directly or indirectly, via 20-HETE, PGI2, PGE2, peroxynitrite and/or transcription factors (such as NF-κB) [42, 43], inhibits iNOS mRNA expression leading to a decrease in iNOS protein expression and NO production in endotoxemic rats. Further characterization of the molecular mechanisms of 5,14-HEDGE on eNOS and iNOS protein expression and association of these enzymes with hsp90 will provide the framework for extension of this work in understanding the role of 20-HETE and NO on the decrease in blood pressure during endotoxemia.

The mechanism by which 5,14-HEDGE increases the 20-HETE levels in endotoxemic rats could be through inhibiting prostanoid and NO production that is known to reduce 20-HETE synthesis [24–27, 37]. Another possible mechanism could be conversion of 5,14-HEDGE to 20-HETE either enzymatically or non-ezymatically leading to increase in systemic and tissue levels of 20-HETE in endotoxemic rats. Because 5,14-HEDGE is structurally similar to 20-HETE, it may also compete for the normal degradation pathways to prevent its catabolism and allow 20-HETE levels to increase. It has been reported that 20-HETE is stored in the kidney and vascular endothelial cells through incorporation into phospholipids [53, 54]. Therefore, it is possible that 5,14-HEDGE might also cause the release of incorporated 20-HETE leading to increased 20-HETE levels. Further studies on the characterization of the effect of 5,14-HEDGE on the increase in 20-HETE levels will provide the framework for extension of this work in understanding the mechanism of action of 5,14-HEDGE during endotoxemia.

In conclusion, the present study indicates that a fall in the production of 20-HETE, a vasoconstrictor arachidonic acid metabolite, and an increase in the levels of vasodilator prostanoids, PGI2 and PGE2, is associated with increased iNOS protein expression and decreased eNOS protein levels in rats treated with endotoxin (fig. 6). Our findings also demonstrate that tissue hsp90 protein expression level does not change in endotoxemic rats. Overall, these findings provide cogent evidence that an increase in systemic and renal 20-HETE levels associated with a decrease in iNOS protein expression and vasodilator prostanoid production contributes to the effect of 5,14-HEDGE to prevent the hypotension during rat endotoxemia. Although we did not investigate the effect of 5,14-HEDGE on COX-2 protein expression in the present study, our results suggest that an increase in 20-HETE levels by 5,14-HEDGE causes dual inhibition of iNOS and COX-2 protein expression and activity in endotoxemic rats. Impairment of cardiovascular and renal function is critically involved in the pathophysiological sequale in septic shock finally resulting in multiorgan failure and death; restoration of these impaired functions should improve therapeutic benefit. In light of the important role of 20-HETE, prostanoids and NO in endotoxin-induced hypotension and vascular hyporeactivity, the interaction of CYP 4A, COX and NOS pathways should be considered when developing new strategies for drug development in the treatment of endotoxic shock. More importantly, further studies with arachidonic acid metabolites generated via CPY 4A including stable analogues of 20-HETE in models of endotoxemia could provide a novel approach to treat hypotension in septic shock.

Fig. 6.

Fig. 6

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Schematic diagram showing the involvement of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), COX-2 and cytochrome P450 4A (CYP 4A) in endotoxin-induced vascular hyporeactivity and hypotension based on the results of the present study and our previous findings (24, 25, 35–39). Endotoxin induces iNOS and cyclooxygenase-2 (COX-2) protein expression associated with increased production of nitric oxide (NO) and vasodilator prostanoids (PGI2 and PGE2), while the protein expression of heat shock protein 90 (hsp90), an allosteric activator of NOS enzymes, remains unchanged. Conversely, endotoxin causes a decrease in eNOS and CYP 4A protein expression, and 20-hydroxyeicosatetraenoic acid (20-HETE) production. Endotoxin-induced changes in protein expression and/or activity of these enzymes lead to a decrease in vascular reactivity and blood pressure. A synthetic 20-HETE analogue, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,4-HEDGE), prevents the effects of endotoxin on the increase in iNOS protein expression and activity, and prostanoid formation as well as the decrease in 20-HETE levels, and thus, restores vascular reactivity and blood pressure during endotoxemia. It should be noted that, a competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, prevents the effects of 5,14-HEDGE on the endotoxin-induced changes in vascular reactivity and blood pressure as well as NO, PGI2, PGE2 and 20-HETE levels. Finally, it can be concluded that increased production of iNOS-derived NO and COX-2-derived prostanoids might be responsible for the decrease in 20-HETE levels in endotoxemic rats; thus an increase in 20-HETE levels by 5,14-HEDGE might cause dual inhibition of iNOS and COX-2 protein expression and/or activity. (Inline graphic): stimulation; (Inline graphic) inhibition.

Acknowledgements

This work was supported by the Research Foundation of Mersin University (Project Code No: BAP ECZF EMB (BT) 2006-3 and BAP SBE EMB (TC) 2008-6 DR), Novartis Turkey, USPHS NIH Grant HLBI-19134-34, NIH Grant GM31278 and the Robert A. Welch Foundation. We greatly acknowledge Dr. Mehmet Sami Serin for all of his helpful advice.

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