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Am J Physiol Heart Circ Physiol 288: H302-H309, 2005. First published September 23, 2004; doi:10.1152/ajpheart.00661.2004
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Cyclooxygenase- and lipoxygenase-dependent relaxation to arachidonic acid in rabbit small mesenteric arteries

David X. Zhang, Kathryn M. Gauthier, Yuttana Chawengsub, Blythe B. Holmes, and William B. Campbell

Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 6 July 2004 ; accepted in final form 15 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We recently reported that the lipoxygenase product 11,12,15-trihydroxyeicosatrienoic acid (THETA) mediates arachidonic acid (AA)-induced relaxation in the rabbit aorta. This study was designed to determine whether this lipoxygenase metabolite is involved in relaxation responses to AA in rabbit small mesenteric arteries. AA (10–9–10–4 M) produced potent relaxations in isolated phenylephrine-preconstricted arteries, with a maximal relaxation of 99 ± 0.5% and EC50 of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 µM), NS-398 (10 µM, selective for COX-2), and SC-560 (100 nM, selective for COX-1) caused a marked rightward shift of concentration responses to AA. With the use of immunohistochemical analysis, both COX-1 and COX-2 were detected in endothelium and smooth muscle of small mesenteric arteries. Indomethacin-resistant relaxations were further reduced by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 µM), nordihydroguaiaretic acid (NDGA; 1 µM), and ebselen (1 µM). HPLC analysis showed that [14C]AA was metabolized by mesenteric arteries to PGI2, PGE2, THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-hydroxyeicosatetraenoic acid (15-HETE). The production of PGI2 and PGE2 was blocked by indomethacin, and the production of THETAs, HEETAs, and 15-HETE was inhibited by CDC and NDGA. Column fractions corresponding to THETAs were further purified, analyzed by gas chromatography/mass spectrometry, and identified as 11,12,15- and 11,14,15-THETA. PGI2, PGE2, and purified THETA fractions relaxed mesenteric arteries precontracted with phenylephrine. The AA- and THETA-induced relaxations were blocked by high K+ (60 mM). These findings provide functional and biochemical evidence that AA-induced relaxation in rabbit small mesenteric arteries is mediated through both COX and lipoxygenase pathways.

endothelium-derived factors; trihydroxyeicosatrienoic acid; prostaglandin E2; prostaglandin I2; endothelium-derived hyperpolarizing factor

ACETYLCHOLINE, BRADYKININ, AND OTHER VASODILATORS elicit endothelium-dependent relaxation by release of a number of soluble factors from the vascular endothelium (8, 17, 22, 23). These relaxing factors include nitric oxide (NO), prostacyclin, and a group of compounds termed EDHFs. Although the chemical identity of EDHF remains controversial, its action has been demonstrated in a number of vascular beds, where a significant portion of endothelium-dependent relaxation persists in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors such as N-nitro-L-arginine (L-NNA) and indomethacin. This endothelium-dependent, non-NO, and non-prostacyclin relaxation is associated with smooth muscle hyperpolarization and is sensitive to K+ channel blockers such as apamin and charybdotoxin (7, 9, 14, 16, 24). The mediators of EDHF vary with species, vascular bed, and vascular size. In bovine, porcine, and canine coronary arteries, the cytochrome P-450 (CYP) metabolites of arachidonic acid (AA), epoxyeicosatrienoic acids (EETs), act as EDHFs (4, 15, 18, 30), whereas K+ may represent EDHF in rat hepatic arteries (11). In rabbit small mesenteric arteries, acetylcholine-induced relaxations are also highly resistant to L-NNA and indomethacin but are sensitive to high K+, thus indicating the activity of EDHF (16). Although evidence suggests that EDHF may not be CYP metabolites of AA in this vasculature, the identity of the mediator(s) remains elusive (16).

Recently, we reported that the rabbit aortic endothelium metabolizes AA by 15-lipoxygenase to produce 11,12,15-trihydroxyeicosatrienoic acids (THETAs), and this lipoxygenase metabolite serves as an EDHF to mediate non-NO and non-prostacyclin relaxations to AA and acetylcholine (5, 19). However, little is known about the role of lipoxygenase metabolites in relaxation responses of resistance arteries. The present study examined the effects of lipoxygenase inhibitors on AA-induced relaxations in isolated rabbit small mesenteric arteries. In addition, lipoxygenase metabolites of AA were isolated and characterized for their vasodilator activity. Because relaxation responses to AA were inhibited by indomethacin, the role of COX pathway in relaxation responses was also explored.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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The experimental protocol was approved by the Animal Care Committee of the Medical College of Wisconsin, and procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996).

Wire myograph. Small mesenteric arteries corresponding to a second- or third-order branch from superior mesentery arteries (200–300 µm) were isolated from 4-wk-old, male New Zealand White rabbit (New Franken Research Rabbits; New Franken, WI) and placed in physiological saline solution (PSS). The tissue was carefully cleaned of adhering fat and connective tissue and cut into rings (1.5 mm long). Arterial segments were threaded on two stainless steel wires (40 µm diameter) and mounted on a four-chamber wire myograph (model 610M, Danish Myo Technology A/S) as we described previously (33). Briefly, arteries were set to an initial luminal diameter at which passive tension was first measurable and equilibrated at 37°C for 30 min in PSS bubbled with 95% O2-5% CO2. Arteries were then gradually stretched during an additional 30-min equilibration period to a resting tension of 1 mN, which was the optimal preload for active tension development as determined by the length-tension curve method (20). Thereafter, the preparation was stimulated two times with KCl (60 mM) plus phenylephrine (10 µM) for 3–5 min at 10-min intervals. Arteries were then allowed to equilibrate for another 30 min before the initiation of experimental protocols.

To examine relaxation responses, a submaximal concentration of phenylephrine (1 µM) was added to the bath to precontract the arteries to 50–75% of maximal phenylephrine contraction. After the contraction reached steady state, cumulative concentration-response curves to AA (10–9–10–4 M), PGI2 (10–10–10–6 M), PGE2 (10–11–10–6 M), 15(S)-hydroxyeicosatetraeonic acid (HETE; 10–10–10–6 M), or sodium nitroprusside (10–8–10–5 M) were determined. To examine the possible role of COX and lipoxygenase metabolites in relaxation responses to AA, arteries were pretreated for 15–30 min with the COX inhibitor indomethacin (10 µM), the selective COX-1 inhibitor SC-560 (100 nM, IC50: 9 nM and 6.3 µM for human recombinant COX-1 and -2, respectively) (32), the selective COX-2 inhibitor NS-398 (10 µM, IC50: 1.77 µM and 75 µM for human recombinant COX-2 and -1, respectively) (2), or the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 µM), nordihydroguaiaretic acid (NDGA; 1 µM), or ebselen (1 µM) (5). Similar studies were performed in vessels pretreated with the hydroperoxide isomerase inhibitor miconazole (2 µM) (28, 29). Relaxations were examined as paired rings or before and after the application of inhibitors. To examine the contribution of smooth muscle hyperpolarization to relaxation responses, arteries were preconstricted with KCl (60 mM), and the concentration responses to a vasodilator were then determined. Experiments were performed on arteries with intact endothelium and in the presence of the endothelial NOS inhibitor L-NNA (30 µM). The endothelium was considered intact if acetylcholine (1 µM) caused >80% relaxation of arteries precontracted with phenylephrine.

Metabolism of [14C]AA. Small mesenteric arteries isolated from two to four animals were pooled, cut into 3-mm-long segments, and incubated for 15 min at 37°C in HEPES buffer. Vessels were preincubated with vehicle, indomethacin (10 µM), or indomethacin plus NDGA (1 µM) for 10 min, followed by the sequential addition of [14C]AA (0.05 µCi, 100 nM) for 10 min and A23187 [GenBank] (10 µM) for another 10 min. Reactions were stopped by adding ethanol to a final concentration of 15%. HEPES buffer was removed, acidified (pH < 3.5) with glacial acetic acid, and extracted on ODS solid-phase extraction columns, as previously described (27, 28). The extracts were evaporated to dryness under a stream of nitrogen gas and stored at –40°C until analysis by HPLC.

Separation and identification of AA metabolites. Extracted samples were chromatographed on a reversed-phase HPLC (Nucleosil-C18 column, 5 µm, 4.6 x 250 mm) using three difference solvent systems. COX metabolites were resolved using solvent system I in which solvent A contained 0.025 M phosphoric acid in distilled water and solvent B was acetonitrile (3). The sample was redissolved and injected in 200 µl of acetonitrile-water-phosphoric acid (31:69:0.01). The program consisted of a 40-min isocratic phase with 31% sovent B in solvent A, followed by a 20-min linear gradient to 100% solvent B and 20-min isocratic phase with 100% solvent B. Flow rate was 1 ml/min. Column effluent was collected in 0.5-ml fractions by a fraction collector and analyzed for radioactivity by liquid scintillation spectrometry. Lipoxygenase metabolites were resolved using solvent system II in which solvent A was water and solvent B was acetonitrile containing 0.1% glacial acetic acid (5). The sample was redissolved and injected in 200 µl of acetronitrile-water-acetic acid (50:50:0.01). The program was a 40-min linear gradient from 50% solvent B in solvent A to 100% solvent B with a flow rate of 1 ml/min. Column effluent was collected in 0.2-ml fractions and analyzed for radioactivity. For some experiments, the fractions corresponding to THETAs (fractions 27–35 of solvent system II, 5–7.5 min) were collected, acidified, and extracted with a 50:50 cyclohexane-ethylacetate mixture. The solvent was removed, and the extract was rechromatographed on a reverse-phase HPLC with solvent system III. This solvent system consisted of solvent A (water containing 0.1% glacial acidic acid) and solvent B (acetonitrile). The program was a 5-min isocratic phase with 35% solvent B in solvent A, followed by a 35-min linear gradient to 85% solvent B. The flow rate was 1 ml/min. The column eluate was collected in 0.2-ml aliquots. The pooled fractions containing THETAs from solvent system III (fractions 87–93, 17.5–18.5 min) were evaporated to dryness under nitrogen, derivatized, and analyzed by gas chromatography-mass spectrometry (GC-MS) as described previously (28).

Immunohistochemical analysis of COX. Briefly, small mesenteric arteries were fixed in ethanol and embedded in 1% agarose, followed by the preparation of 7-µm cryostat sections. The tissue sections were permeabilized with 0.2% Triton in 10 mM phosphate-buffered saline (Sigma) and blocked with 1% normal goat serum for 30 min. The sections were then incubated with a monoclonal antibody against COX-1 or -2 (1:500 dilution for 1 h, Oxford Biochemical Research) or a monoclonal antibody against PECAM-1 (1:500 dilution for 1 h, gift from Dr. P. J. Newman). A separate arterial section was incubated with blocking solution as a negative control. After being washed, the sections were stained with Texas red-labeled goat anti-mouse IgG (1:1,000 dilution for 1 h, Molecular Probes), and fluorescence images were obtained using an epifluorescence microscope (Nikon E600) coupled with SPOT software (Diagnostic Instruments) at x200 magnification.

Data analysis. Relaxation responses are expressed as the percent relaxation relative to phenylephrine or KCl precontraction, with 100% relaxation representing basal tension. Where appropriate, the concentration of the drug required to produce 50% of the maximal response (EC50) was calculated from the concentration-response curves by fitting data to a logistic sigmoid equation using the GraphPad Prism program (GraphPad; San Diego, CA). Data are presented as means ± SE. Significance of differences between mean values was evaluated by Student's t-test or ANOVA followed by the Student-Newman-Keuls multiple-comparison test. P < 0.05 was considered statistically significant.

Drugs and solutions. Phenylephrine, AA (sodium salt), sodium nitroprusside, L-NNA, indomethacin, NDGA, ebselen, and miconazole were purchased from Sigma, CDC was obtained from Bio-Mol Research laboratories, SC-560 and NS-398 were from EMD Biosciences, and PGI2, PGE2 and 15(S)-HETE were from Cayman Chemical. [14C-U]AA (920 mCi/mmol) was obtained from New England Nuclear. All solvents were HPLC grade and purchased from Burdick and Jackson. We used PSS of the following composition (in mM): 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.17 MgSO4, 24 NaHCO3, 1.18 KH2PO4, 0.026 EDTA, and 5.5 glucose. The HEPES solution consisted of the following composition (in mM): 150 NaCl, 5.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 10 HEPES, and 5.5 glucose, pH 7.4.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In rabbit small mesenteric arteries contracted with phenylephrine, AA (10–9–10–4 M) produced concentration-dependent relaxations, with a maximal relaxation of 99 ± 0.5% and EC50 of 50 nM. Pretreatment of arteries with the COX inhibitor indomethacin (10 µM) caused a significant rightward shift of the concentration-response curve to AA (maximal relaxation of 92 ± 0.2% and EC50 of 10 µM; Fig. 1A). The selective COX-2 inhibitor NS-398 (10 µM) and the selective COX-1 inhibitor SC-560 (100 nM) also caused a marked rightward shift of concentration responses to AA (maximal relaxations and EC50s of 97 ± 0.6 and 97 ± 0.4% and 7.4 and 1.6 µM, respectively; Fig. 1, B and C). The combination of SC-560 with NS-398 further inhibited AA-induced relaxations compared with SC-560 alone (maximal relaxation of 97 ± 0.3% and EC50 of 2.4 µM; Fig. 1C).



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  		Fig. 1. Effect of cyclooxygenase (COX) inhibitors on arachidonic acid (AA)-induced relaxation of rabbit small mesenteric arteries. Arteries were pretreated with indomethacin (10 µM; A), the selective COX-2 inhibitor NS-398 (10 µM; B), or the selective COX-1 inhibitor SC-560 (100 nM) alone or in combination with NS-398 (C). Arteries were contracted with phenylephrine (1 µM). Values are means ± SE; n = 6–10. *P < 0.05 vs. control.

 
The expression of COX was determined using immunohistochemical analysis. As shown in Fig. 2, A and B, both COX-1 and -2 were detected in small mesenteric arteries, although the signal for COX-2 was stronger than that of COX-1. The fluorescence staining was mainly in the endothelium with less staining in smooth muscle cells. In the absence of the primary antibody, no significant fluorescence was observed (Fig. 2D). As a positive control, the staining of PECAM-1 (an endothelial marker) was localized to the endothelium, indicating that the endothelium of the preparation was intact (Fig. 2C).



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  		Fig. 2. Immunohistochemical analysis of COX in rabbit small mesenteric arteries. A and B: representative artery sections labeled with a monoclonal antibody against COX-1 and -2, respectively. C: control arteries labeled with anti-PECAM-1. D: negative control with only the secondary antibody. Magnification: x200.

 
We next measured the COX metabolites of AA in mesenteric arteries. Isolated arteries were incubated with [14C]AA in the absence and presence of indomethacin (10 µM). The metabolites were then extracted and analyzed by reverse-phase HPLC and liquid scintillation spectrometry. Mesenteric arteries produced metabolites of AA that comigrated with 6-keto-PGF1{alpha} (a stable metabolite of PGI2) and PGE2 standards. The production of these two metabolites was blocked by indomethacin (10 µM; Fig. 3).



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  		Fig. 3. Effect of indomethacin on metabolism of [14C]AA by rabbit small mesenteric arteries. Arteries were treated with vehicle (A) or indomethacin (10 µM; B). Metabolites were extracted and resolved by reverse-phase HPLC using solvent system I (see MATERIALS AND METHODS). Migration times of known standards are noted above each chromatogram. CPM, counts per minute; THETAs, trihydroxyeicosatrienoic acids; HETEs, hydroxyeicosatetraeonic acids.

 
To provide further evidence that PGI2 and PGE2 mediate indomethacin-sensitive relaxations induced by AA, we examined the vascular response to exogenous PGI2 and PGE2. As shown in Fig. 4, PGI2 and PGE2 elicited potent concentration-related relaxations of small mesenteric arteries contracted with phenylephrine. Maximal relaxations and EC50s for PGI2 and PGE2 were 100 ± 0 and 96 ± 0.6% and 1.8 and 11 nM, respectively. The lipoxygenase metabolite 15(S)-HETE induced a small contraction at 10–6 M (19 ± 1%) and had no effect at lower concentrations. The small mesenteric arteries also relaxed to sodium nitroprusside. These relaxations were not affected by indomethacin (10 µM) or the lipoxygenase inhibitor CDC (1 µM).



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  		Fig. 4. Relaxation responses to PGI2 (A), PGE2 (B), 15-HETE (C), and sodium nitroprusside (D) in rabbit small mesenteric arteries. For sodium nitroprusside responses, arteries were pretreated with indomethacin (10 µM) or the lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 µM). Arteries were contracted with phenylephrine (1 µM). Values are means ± SEM; n = 6–14.

 
To define the role of lipoxygenase metabolites in indomethacin-resistant relaxations induced by AA, vascular responses were determined in arteries in the presence of three chemically unrelated lipoxygenase inhibitors plus indomethacin (10 µM). Treatment of arteries with CDC (1 µM), NDGA (1 µM), and ebselen (1 µM) caused a similar inhibition of AA-induced relaxations. The relaxations at 10–4 M AA were reduced to 36 ± 6%, 54 ± 8%, and 67 ± 7%, respectively (Fig. 5, A–C). Vehicle alone had no significant effect on AA-induced relaxations (Fig. 5B). The relaxations to AA were eliminated in arteries contracted with high K+ (60 mM; Fig. 5D).



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  		Fig. 5. Effect of lipoxygenase inhibitors, hydroperoxide isomerase inhibitor, and high K+ on indomethacin-resistant AA relaxations of rabbit small mesenteric arteries. Arteries were pretreated with the lipoxygenase inhibitors CDC (1 µM; A), nordihydroguaiaretic acid (NDGA; 1 µM) or vehicle (B), or ebselen (1 µM; C) in the presence of indomethacin (10 µM). For some experiments, arteries were pretreated with the hydroperoxide isomerase inhibitor miconazole (2 µM) alone or in combination with CDC (1 µM) in the presence of indomethacin (10 µM) (A). Arteries were contracted with phenylephrine (1 µM; A–C) or high K+ (60 mM; D). Values are means ± SE; n = 7–9. *P < 0.05 vs. control.

 
In the rabbit aorta, we previously showed that the hydroperoxide isomerase is also involved in biosynthesis of THETAs (28). To examine the role of this enzyme in the THETA-mediated component of relaxations to AA in small mesenteric arteries, arteries were pretreated with indomethacin (10 µM) and miconazole (2 µM), a hydroperoxide isomerase inhibitor. As shown in Fig. 5A, miconazole also inhibited indomethacin-resistant relaxations to AA. Miconazole plus CDC (1 µM) further reduced the relaxations.

Campbell et al. (5) previously reported that the rabbit aorta metabolizes AA to 11,12,15-THETA by 15-lipoxygenase and 11,12,15-THETA mediates the relaxation response to AA. Using a similar approach, we incubated mesenteric arteries with indomethacin and [14C]AA and examined the lipoxygenase metabolites by reverse-phase HPLC. Three main metabolites detected in the incubation solution comigrated with the THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-HETE (Fig. 6A). The production of these metabolites was inhibited by NDGA (1 µM; Fig. 6B). Similar inhibition was observed with CDC (1 µM; data not shown).



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  		Fig. 6. Effect of the lipoxygenase inhibitor NDGA on metabolism of [14C]AA by rabbit small mesenteric arteries. Arteries were treated with vehicle (A) or NDGA (1 µM) plus indomethacin (10 µM) (B). Metabolites were extracted and resolved by reverse-phase HPLC using solvent system II (see MATERIALS AND METHODS). Migration times of known standards are noted above each chromatogram. HEETAs, hydroxyepoxyeicosatrienoic acids.

 
The column fractions from solvent sytem II (fractions 27–35, 5–7.5 min) containing THETAs were collected and further purified by reverse-phase HPLC using solvent system III (Fig. 7A). This gave a single radioactive peak. The THETA fractions collected from solvent system III (fractions 87–93, 17.5–18.5 min) were tested for their ability to relax small mesenteric arteries precontracted with phenylephrine or high K+. As shown in Fig. 7, B and C, the THETA fractions elicited a marked relaxation in arteries contracted with phenylephrine, whereas they had no effect in arteries contracted with 60 mM KCl.



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  		Fig. 7. Effect of the THETA fraction on isometric tension in rabbit small mesenteric arteries. A: separation of the THETA fraction (fractions 27–35 from solvent system II, see solid bar in Fig. 6A) by reverse-phase HPLC using solvent system III (see MATERIALS AND METHODS). THETA fractions collected from solvent system III (fractions 87–93; solid bar) were dried under nitrogen, redissolved in ethanol, and added to the precontracted vessel. Arteries were contracted with either phenylephrine (1 µM) or 60 mM K+. B: original traces. C: summarized data. Values are means ± SE; n = 2–3. *P < 0.05 vs. vehicle.

 
The purified THETAs from solvent system III were also derivatized to the methyl and TMS esters and analyzed by positive ion chemical ionization GC-MS. Figure 8A illustrates a typical reconstructed ion chromatogram of the THETA fractions. Figure 8, B and C, shows the mass spectra of peaks B and C. The two peaks had similar mass spectra with major ions (m/z) of the following: 585 (M++1), 569 (M-15, loss of CH3), 405 [M-179, loss of (CH3)3SiOH and (CH3)3SiO+], 301 (M-283), 283 [M-301, (CH3)3SiO-CH-(CH2- CH=CH)2-(CH2)3-COOCH3], and 173 [M-411, (CH3)3SiO-(CH2)4-CH3]. However, the two peaks differed in the intensity of the 283 m/z ions indicating the favored cleavage between the 11,12 (peak B)- and 14,15 (peak C)-vicinal diols, respectively. Therefore, these mass spectra are consistent with 11,12,15- and 11,14,15-THETA.



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  		Fig. 8. Identification of THETAs isolated from rabbit small mesenteric arteries by gas chromatography-mass spectroscopy (GC-MS). Arteries were incubated with [14C]AA (0.05 µCi, 100 nM) and indomethacin (10 µM). Metabolites were extracted and resolved by sequential reverse-phase HPLC using solvent systems II and III (see MATERIALS AND METHODS). The fractions containing THETAs from solvent system III (fractions 87–93) were collected, derivatized, and analyzed by GC/MS. A: reconstituted ion chromatogram. B and C: positive ion chemical ionization mass spectra of the methyl and TMS esters of peaks B and C in A.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Vascular cells metabolize AA through several enzymatic pathways, including COX, lipoxygenase, and CYP pathways. The present study examined mediators of AA-induced relaxations in rabbit small mesenteric arteries. We found that AA was a potent vasodilator of small mesenteric arteries. The relaxations were markedly shifted to the right by the COX inhibitors, whereas the remaining relaxations were further reduced by lipoxygenase inhibitors. The small mesenteric arteries metabolized AA to PGI2 and PGE2 and to THETAs, HEETAs and 15-HETE, and the production of these metabolites was inhibited by COX and lipoxygenase inhibitors, respectively. Furthermore, exogenous PGI2, PGE2, and purified THETA fractions relaxed small mesenteric arteries. Together, these results suggest that AA relaxes rabbit small mesenteric arteries through COX- and lipoxygenase-dependent mechanisms.

COX metabolizes AA to a number of vasoactive products, with PGI2 and PGE2 being two major products with potent vasodilator activity (23). Our findings that PGI2 and PGE2 mediate AA-induced relaxations of rabbit small mesenteric arteries are consistent with their vasodilatory properties. To date, two isoforms of COX have been identified: COX-1, which is constitutively expressed, and COX-2, which is an inducible form. In rabbit small mesenteric arteries, AA-induced relaxations were markedly reduced by both COX-1 and -2 inhibitors. In addition, small mesenteric arteries were found to express both isozymes as indicated by immunohistochemical analysis. These findings confirm that both isoforms of COX are present, functional, and involved in relaxation responses to AA. The expression of COX-2 in normal mesenteric arteries was unexpected, given that this isoform is predominantly expressed in disease states such as inflammation and atherosclerosis (1, 6, 10, 31). However, a previous study (12) has reported that both COX-1 and -2 are constitutively expressed in the rat lung vasculature and that COX-2 plays a predominant role in prostanoid-related regulation of pulmonary vascular tone. Further studies are needed to determine the precise role of vascular COX-2 under physiological conditions.

Two lipoxygenase isoforms have been described in endothelial cells: 12- and 15-lipoxygenase, corresponding to the carbon where the oxygenation reaction occurs (13, 28). These lipoxygenases convert AA to hydroperoxyeicosatetraenoic acids (HPETEs), which are unstable and rapidly reduced by cellular peroxidases to HETEs or by a heme-containing hydroperoxide isomerase to HEETAs (25). Hydrolysis of the epoxy group of the HEETA by an epoxide hydrolase results in the formation of THETAs (28). Some of these metabolites have been reported to have vasodilatory effects. For example, 11,12,15-THETA produced by 15-lipoxygenase hyperpolarizes rabbit aortic smooth muscle and induces concentration-dependent relaxation of preconstricted aortas, whereas 11,14,15-THETA has no vasoactive effect (5, 19). In the present study, we found that lipoxygenase inhibitors reduced indomethacin-resistant relaxations to AA and the production of THETAs in rabbit small mesenteric arteries. Additionally, the THETAs relaxed the preconstricted mesenteric arteries. GC-MS analysis indicated that the THETA fractions contained 11,12,15- THETA. Therefore, these findings clearly demonstrate that lipoxygenase-derived THETAs are involved in relaxation responses of AA in small resistance arteries. The relaxation induced by THETAs was blocked by high extracellular K+, indicating that K+ channel-mediated hyperpolarization is involved. The possible activation of K+ channels in mediating relaxations induced by the THETAs implies that THETAs act as EDHF. The identity of the active regional isomer or stereoisomer of THETAs is currently unknown and will require the synthesis and evaluation of the vasomotor activity of the possible isomers.

As discussed previously, a heme-containing hydroperoxide isomerase is also involved in the biosynthesis of THETAs. Blockade of this enzyme inhibits AA-induced relaxations in rabbit aorta (5, 28). In addition, the CYP epoxygenase, possibly CYP2J2, may function as the hydroperoxide isomerase in this vascular bed (29). In the present study, we also found that miconazole, a hydroperoxide isomerase inhibitor, reduced relaxation responses to AA in small mesenteric arteries. This provides further support for the potential role of hydroperoxide isomerase in AA-induced responses.

The rabbit mesenteric arteries are more sensitive to AA than is the aorta. The relaxations to AA have an approximate ED50 of 5 x 10–8 M in the mesenteric arteries and 5 x 10–5 M in the rabbit aorta as well as the indomethacin-resistance component of relaxations to AA (5, 28). This indicates that the contribution of lipoxygenase metabolite to the regulation of vascular tone may increase as vascular diameter decreases.

Recently, Miller et al. (21) have shown that rat small mesenteric arteries metabolize AA to 12-HETE through 12-lipoxygenase and that exogenous 12-HETE induced a concentration-dependent relaxation in this arterial bed. However, in rabbit small mesenteric arteries as well as aortas (5), the main radioactive HETEs comigrated with 15-HETE standard, indicating that 15-lipoxygenase is the major isoform in these arteries. In addition, 15-HETE has no vasodilator activity in rabbit mesenteric arteries as well as in rabbit aortas (26). The reasons for these discrepancies are not clear but may represent species variability.

In addition to COX and lipoxygenase pathways, endothelial cells can metabolize AA through CYP enzymes. These enzymes convert AA to four regional isomers of EETs, which can be quickly hydrolyzed by cellular epoxide hydrolase to dihydroxyeicosatrienoic acids (DHETs). Both groups of metabolites are vasoactive and act as EDHFs to induce smooth muscle hyperpolarization and relaxation in coronary arteries (4, 15, 18, 30). However, in rabbit small mesenteric arteries, there was no production of CYP metabolites when the arteries were incubated with AA, suggesting no role for CYP metabolites in these arteries. Similar findings have been reported in other arteries of rabbit such as aortas (5). These results are in accordance with a recent study (16) that concluded that acetylcholine-induced relaxation is also not mediated by the CYP metabolites in rabbit mesenteric arteries. However, it is possible that CYP metabolites are involved in the regulation of vascular function under other conditions. Indeed, we have previously reported that the EET synthesis via CYP epoxygenase is enhanced in cholesterol-fed rabbit aortas, indicating a potential for altered AA metabolism via epoxygenase pathway in pathophysiological states such as hypercholesterolemia (26).

In summary, the present study is the first to examine the mediators of relaxation responses to AA in rabbit small mesenteric arteries. The data from our functional and biochemical studies suggest that AA-induced relaxation is mediated through both COX and lipoxygenase pathways. Both PGI2 and PGE2 are produced by the mesenteric arteries and cause relaxation. Importantly, we have also shown that THETAs, the 15-lipoxygenase metabolites, are produced and mediate relaxation of these arteries. It remains of interest to determine whether these metabolites also contribute to relaxations of rabbit small mesenteric arteries to other dilator agonists such as acetylcholine.


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 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-37981. D. X. Zhang is a postdoctoral fellow of the American Heart Association, Greater Midwest Affiliate, and a recipient of the Jenkins Cardiovascular Research Fellowship.


   ACKNOWLEDGMENTS
 
The authors thank Gretchen Barg for secretarial assistance and Xin Tang for help with the immunohistochemistry studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. B. Campbell, Dept. of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: wbcamp{at}mcw.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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