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【摘要】
Objective- Aortic 15-lipoxygenase (15-LO) metabolizes arachidonic acid (AA) to 15-hydroperoxyeicosatetraenoic acid, which is then converted to the vasodilators 15-hydroxy-11,12-epoxyeicosatrienoic acid and 11,12,15-trihydroxyeicosatrienoic acid. These metabolites contribute to endothelium-dependent relaxations of rabbit aorta to AA and acetylcholine. We investigated the identity of rabbit aortic 15-LO and studied its importance in the regulation of vascular tone.
Methods and Results- RT-PCR using 12-lipoxygenase/15-LO specific primers resulted in a 572-bp product with a sequence identical to 15-LO-I from rabbit aorta. A RT-PCR/restriction digest strategy excluded expression of 12-lipoxygenase. Immunoblotting revealed 15-LO-I expression in rabbit endothelial and smooth muscle cells. Aortic homogenates and cytosolic fractions metabolize AA to 15(S)-hydroxyeicosatetraenoic acid and linoleic acid to 13(S)-hydroxyoctadecadienoic acid. This activity was blocked by LO inhibitors. The kinetic characteristics (Michaelis constant of aortic 15-LO is 2.2±0.3 µmol/L for AA and 23.5±3.3 µmol/L for linoleic acid) of aortic 15-LO were similar to those of the purified 15-LO-I. An antisense oligonucleotide inhibited 15-LO-I expression in rabbit aorta. Indomethacin and nitro- L -arginine-resistant relaxations to acetylcholine were inhibited by 15-LO-I antisense oligonucleotide but not by the scrambled oligonucleotide.
Conclusions- 15-LO-I is expressed in rabbit aortic endothelium and is important in endothelium-dependent regulation of vascular tone.
15-LO-I is expressed in rabbit aorta. 15-LO regulates vasodilatory eicosanoid synthesis and vascular tone.
【关键词】 endothelium arachidonic acid lipoxygenase endotheliumderived hyperpolarizing factor
Introduction
Endothelial cells (ECs) release factors that control vascular tone in response to acetylcholine, bradykinin, and other hormones, as well as by shear stress. 1 Some of these vasoactive factors are metabolites of arachidonic acid (AA). In rabbit aortic ECs, AA is metabolized through cyclooxygenase, lipoxygenase (LO), and cytochrome P 450 pathways into bioactive eicosanoids. 2-4 Rabbit aorta relaxes to acetylcholine and AA in the presence of indomethacin and L-nitro-arginine (L-NA), which block cyclooxygenase and nitric oxide synthase, respectively. 2,5 These relaxation responses are blocked by LO inhibitors, such as nordihydroguaiaretic acid (NDGA), cinnamyl-3,4-dihydroxy-a-cyanocinnamate (CDC), and ebselen. 4-6 AA is oxidized via the 15-LO pathway to 15(S)-hydroperoxyeicosatetraenoic acid, which is additionally converted to hydroxy-epoxyeicosatrienoic acids (HEETAs) and trihydroxyeicosatrienoic acids (THETAs). Both the HEETA and 11,12,15-THETA relax the rabbit aorta. 7 These studies implicate the aortic 15-LO pathway in the regulation of vascular tone. Thus, the rabbit aortic 15-LO, which initiates the synthesis of these vasodilatory eicosanoids, represents an important regulatory site of vascular eicosanoid metabolism.
In humans, there are 2 15-LO isozymes (15-LO-I and 15-LO-II) originating from 2 different genes. 8,9 15-LO-I was cloned from rabbit reticulocytes 10 and human leukocytes and oxygenates AA to 15(S)-hydroperoxyeicosatetraenoic acid (90%) and 12(S)-hydroperoxyeicosatetraenoic acid (10%). 8 It is expressed in various cell types, such as reticulocytes, airway epithelial cells, and eosinophilic granulocytes. 11 It uses as substrates AA, linoleic acid (LA), 12,13 or 2-arachidonoylglycerol (2-AG), 14 as well as phospholipids or cholesterol esters containing AA or LA. 15,16 15-LO-I is predominately localized in the cytosol; however, a variable proportion of the enzyme may be membrane bound. 17 15-LO-I has been implicated in many physiological and pathological events. Distinct from 15-LO-I, 15-LO-II selectively metabolizes free fatty acids and prefers AA to LA as a substrate. 9,18 The cDNA sequences for human 15-LO-I and 15-LO-II share a low degree of identity (36%). These distinct enzymatic properties, as well as the tissue- and cell-specific expression patterns, suggest different biological functions of the 15-LO isoforms. Rabbits express 15-LO-I and a 12-LO in the same tissue. Both enzymes share a 99% amino acid homology with 4 amino acid exchanges. One of these exchanges (Phe353Leu) is responsible for the different positional specificities for oxygenation of the 2 enzymes. 19
Rabbit aorta and cultured ECs convert AA to 15-HPETE and 15-HETE. 5,7,20 However, the cellular expression patterns of the enzyme(s) responsible for the enzymatic properties of aortic 15-LO are still not clear. Also, because LO inhibitors also block 5-LO, 12-LO, 15-LO, and possibly other pathways of AA metabolism, 21 a more specific approach is needed to study 15-LO function. Here, we report the expression of 15-LO-I in rabbit aorta and exclude significant expression of 12-LO. We also demonstrated clear similarities in the basic kinetic characteristics of 15-LO from aortic cytosol and purified rabbit reticulocyte 15-LO-I. These data support our conclusion that the 15-LO activity of rabbit aorta is attributable to 15-LO-I. Moreover, using antisense oligonucleotides, we suppressed the expression of 15-LO-I in isolated rabbit aorta and decreased the relaxation responses to acetylcholine. These studies provide evidence for the important role of 15-LO-I in regulation vascular tone in the rabbit aorta.
Methods
Tissue Preparation
One-week-old New Zealand white rabbits were euthanized by carbon dioxide inhalation. Aortas were dissected and cleaned of adhering connective tissue and fat in cold HEPES buffer [in mmol/L: 10 HEPES, 150 NaCl, 5 KCl, 1 EDTA, 1 MgCl 2, and 6 glucose (pH 7.4)]. The tissue was homogenized in 25% sucrose buffer with 1 mmol/L EDTA and was then sonicated on ice. The homogenate was centrifuged for 20 minutes at 8500 x g to remove tissue debris. The supernatant was collected and centrifuged for 1 hour at 100 000 x g to separate the cytosolic (supernatant) and membrane fractions (pellet). The pellet was resuspended in 25% sucrose buffer. Protein concentrations were determined by the Bio-Rad method. Rabbit aortic ECs and smooth muscle cells were prepared and cultured as described previously. 3 Human aortic ECs were purchased from Cell Applications.
Western Immunoblotting Assay
Protein lysates (30 µg) were loaded in each lane and separated by SDS gel electrophoresis as described previously. 17,22 After being transferred to nitrocellulose membranes, membranes were exposed to the guinea pig antirabbit 15-LO-I antibody (1:1000 overnight at 4°C and then rinsed repeatedly). Membranes were then incubated with 1:2000 goat antiguinea pig IgG (horseradish peroxidase-conjugated, Jackson Immunoresearch) for 1 hour at room temperature and then rinsed. Immunoreactive bands were identified using the chemiluminescence detection.
RT-PCR
Total RNA was prepared from rabbit aortic tissue by using Trizol total RNA isolation reagent (Life Technologies) as described previously. 23 PCR reactions were performed using PCR Supermix kit (Life Technologies). The 50-µL PCR mixture consisted of primers [0.2 µmol/L, High Fidelity buffer (1x), 2'-deoxynucleoside 5'-triphosphate (1 µmol/L), MgSO 4 (1 mmol/L), and Platinum Taq (GIBCO)]. The forward primer was made against 1 to 20 nucleotide (nt) of rabbit reticulocyte 15-LO complete mRNA sequence: 5'- ACAAGGCGTGCAACGACC CT-3', and reverse primer, against 592 to 621 nt, 5'-CCAGGGAGCAGAACATTGAGTC CTT-3'. The program for the thermocycler was 94°C for 30 s, 58°C for 1 minute, and 72°C for 1.5 minutes, repeated 30 times followed by final extension at 72°C for 7 minutes. PCR products were separated by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The PCR band was isolated from the gel, excised, and subcloned into TOPO2.1 TA cloning vector (Invitrogen) and sequenced.
RT-PCR/Restriction Cleavage Strategy
Total RNA (1 µg) was reverse transcribed at 37°C for 170 minutes in 45 µL 28 mmol/L Tris-HCl buffer (pH 8.3) containing 1.7 mmol/L MgCl 2, 42 mmol/L KCl, 5.5 mmol/L DTT, 0.277 mmol/L of 2'-deoxynucleoside 5'-triphosphate, 100 µg/mL BSA, 11 ng/µL of oligo d(T)18 primer, and 200 units of reverse transcription. The PCR sample (total volume of 25 µL) consisted of a 10 mmol/L Tris-HCl buffer (pH 9.0) containing 3 µL of reverse transcription sample, 4 mmol/L MgSO 4, 100 nmol/L forward (5' TGG CTG CCC CGC TGG TCA TG 3') and reverse (5' CCT GGC GCG GAC GTT GAT CTC 3') primers, 120 µmol/L 2'-deoxynucleoside 5'-triphosphate, and 1 unit of Pyra exo(-) DNA polymerase. The PCR program consisted of 2 minutes at 94°C, 45 s at 95°C, 30 s at 60°C, and 2 minutes at 68°C. After 34 cycles of amplification, a postconditioning phase (10 minutes at 70°C) was carried out, and the storage was at 4°C. PCR products were digested at 60°C for 3 hours in 10 µL of 10 mmol/L Tris-HCl buffer (pH 7.9) containing 50 mmol/L NaCl, 10 mmol/L MgCl 2, 1 mmol/L DTT, 100 µg/mL BSA, and 2 units of Bst NI. Digestion products were resolved by electrophoresis in a 2% agarose gel.
15-LO Enzymatic Activity
Cytosolic proteins in HEPES buffer (0.2 mg/2 mL) were incubated with LA or AA (10 -3 to 10 -5 mol/L) at 23°C for various times. Also, cytosolic proteins were incubated in HEPES buffer with 10 -7 to 10 -4 mol/L of AA or LA at 23°C for 20 minutes. The reaction was terminated with glacial acetic acid (40 µL) and 95% ethanol (0.3 mL), 8-HETE (100 ng) was added, and lipids were extracted by solid phase extraction. 22 13-(S)-hydroxyoctadecadienoic acid (13-HODE), 8-HETE, and 15-HETE were resolved and measured by reverse-phase (RP) high-performance liquid chromatography (HPLC) on a Phenomenex Kromasil C18 column (2.0 x 250 mm) with a Hewlett-Packard 1090 liquid chromatograph. The flow rate was 0.2 mL/min. Solvent A was deionized water with 0.1% glacial acetic acid, and solvent B was acetonitrile with 0.1% glacial acetic acid. The program consisted of a linear gradient from 55% to 60% B over 10 minutes, 10 minutes at 60% B, a linear gradient from 60% to 75% B over 15 minutes, and a linear gradient of 75% to 100% over 5 minutes. Absorbance at 235 nm was monitored with a diode array UV detector and was recorded and analyzed with Chemstation software. The amount of 15-HETE or 13-HODE was measured by comparing the peak area to the internal standard 8-HETE.
Liquid Chromatography-Electrospray Ionization-Mass Spectrometric Analysis
Samples were analyzed on Agilent 1100 LC/MSD, as described previously. 24 Metabolites were separated by RP-HPLC as for 15-LO activity. The detection was made in the negative ion mode for the major ions for 15-HETE (319 m/z) and 13-HODE (295 m/z), respectively.
Inhibition of Aortic 15-LO-I Expression by Antisense Oligonucleotides
Aortas from 1-week-old rabbits were excised, cleaned, and cut into 1-mm rings. Two rings were placed in each cell culture well with 0.5 mL of Krebs solution. The rings were transfected with either scrambled (5'-ggtccttctccaataacgtgg-3') or antisense (5'-gctcatcaacctggaagtcag-3') phosphorothiolated oligonucleotides. Oligofectamine (Invitrogen, 4 µL) was diluted in 11 µL of Opti-MEM (Qiagen). After 10 minutes at room temperature, the oligofectamine mixture was added to the scrambled or antisense oligonucleotides (35 µmol/L) diluted in Opti-MEM to a final volume of 100 µL. The mixture was left for 20 minutes at room temperature. Rings were placed in Krebs solution (325 µL), 100 µL of the mixture overlaid onto the rings, and incubated for 12 hours at 30°C. The tissue was used immediately for Western immunoblotting or vascular reactivity studies.
Isometric Tension in Aortic Rings
Aortic rings were mounted in a 4-chamber wire myograph (Danish MyoTechnology A/S). 3 The rings were equilibrated in Krebs solution (in mmol/L, 119 NaCl, 4.7 KCl, 2.5 CaCl 2, 1.17 MgSO 4, 25 NaHCO 3, 1.18 KH 2 PO 4, 0.027 EDTA, and 5.5 glucose) bubbled with 95% O 2 /5% CO 2 at 37°C for 30 minutes under 0.5 g of resting tension (the length-tension maximum). After pretreatment for 15 minutes with L-NA (30 µmol/L) and indomethacin (10 µmol/L), submaximal concentration of serotonin (0.1 to 1 µmol/L) was added to precontract the arteries to 50% to 75% of the maximal KCl contraction. Cumulative concentration-response curves were determined for acetylcholine (10 -9 to 10 -5 mol/L).
Statistical Analysis
Data are expressed as mean±SEM. Significance was evaluated by Student t test or ANOVA followed by the Student-Newman-Kuel?s multiple comparison test.
Results
Identification of Aortic 15-LO
RT-PCR was performed with total aortic RNA using primers made against rabbit reticulocyte 15-LO. 10,23 A PCR product of the expected size (572 bp) was obtained ( Figure 1 A). A product at the same size was detected in human aortic ECs. The sequence of the rabbit aortic PCR product was identical to 15-LO-I cDNA ( Figure 1 C). Immunoblotting using a specific antibody 17 against rabbit 15-LO-I showed a single 75-kDa band from rabbit aorta, rabbit aortic ECs, rabbit aortic smooth muscle cells, and human aortic ECs. This band has the same molecular weight as 15-LO-I ( Figure 1 B). Immunohistochemistry was performed on a histological section of the rabbit aorta. An antibody against PECAM-1 was used to identify ECs (data not shown). Vascular smooth muscle cells were labeled with anti- -actin antibody (data not shown). 15-LO-I immunofluorescence was strongly associated with vascular endothelium (Figure Ia and Ib, available online at http:atvb.ahajournals.org). Smooth muscle cells located close to the endothelial layer were also stained by the 15-LO antibody. No immunoreactivity was observed when the primary antibody was omitted or when antigen-absorbed antiserum was applied (data not shown). Another antirabbit 15-LO-I antiserum (Cayman Chemical) gave similar results (data not shown). These data indicate that transcription and translation of the 15-LO-I gene occurs primarily in rabbit aortic ECs.
Figure 1. Identification of aortic 15-LO. (A) RT-PCR assay of 15-LO-I expression in rabbit aortic tissue (RA) and human aortic ECs (HAOEC). Control is without reverse transcriptase. (B) Western immunoblotting using an anti-15-LO-I antibody in RA, RA-EC, RA-SMC, and HAOEC. (C) Alignment of the sequence of aortic PCR product with rabbit 15-LO cDNA and 12-LO cDNA.
A RT-PCR/restriction digest strategy was developed to quantify the relative expression of 15-LO and 12-LO. For this purpose, we amplified a 325-bp fragment that contains the functionally most important amino acid difference between the 15-LO-I and the 12-LO ( Figure 2 ). The annealing sequence of the PCR primers was identical for the 2 LO isoforms so that both mRNA species were amplified to the same extent. This PCR product contained a single BstN1 restriction site when the 15-LO-I mRNA was amplified ( Figure 2 A). In contrast, the restriction site was absent when the 12-LO mRNA constituted the template ( Figure 2 B). Thus, when the PCR product was digested with BstN1, it was possible to obtain the relative expression of the LO-isoforms ( Figure 2 C). The PCR product obtained from the control 15-LO plasmid (positive control) was completely digested under these experimental conditions as was the PCR product obtained from aortic mRNA extracts. In contrast, no restriction cleavage was seen when a control plasmid of the 12-LO was used as PCR template (negative control). These data indicate the expression of the 15-LO-I but not 12-LO in rabbit aorta.
Figure 2. Identification of aortic 15-LO by RT-PCR/restriction digestion. A 326-bp segments from aortic 15-LO, 15-LO plasmid, and 12-LO plasmid were amplified by PCR. A single BstN1 restriction site was used to distinguish 15-LO-I (A) from 12-LO (B). In C, RT-PCR products before (lane A) and after BstN1 digestion (lane B).
Aortic 15-LO Activity
Rabbit aortic homogenates and cytosol converted AA and LA into 15-HETE and 13-HODE, respectively. AA was converted by aortic cytosol to a major metabolite comigrating with 15-HETE standard on HPLC ( Figure 3 A). Liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometric (MS) analysis of this metabolite showed a major ion of 319 m/z (M-1), confirming it as 15-HETE ( Figure 3 C). Small amounts of 12-HETE were also formed. LA was metabolized to a metabolite comigrating with 13-HODE. A small amount of 9-HODE was also detected ( Figure 3 B). LC-ESI-MS analysis indicated a major ion of 295 m/z (M-1) confirming biosynthesis of 13-HODE ( Figure 3 D). The 15-LO inhibitors, CDC and NDGA, inhibited the synthesis of 15-HETE from AA ( Figure 3 E) and 13-HODE from LA ( Figure 3 F).
Figure 3. Identification of AA (A) and LA (B) metabolites of rabbit aortic cytosol. AA or LA (10 -5 mol/L) metabolites were analyzed by RP-HPLC (A and B). Mass spectra of the 15-LO products, 15-HETE (C) and 13-HODE (D). CDC and NDGA, LO inhibitors, inhibited the metabolism of AA (E) and LA (F) by aortic cytosol.
When aortic cytosol was incubated for various times with AA or LA (10 -5 mol/L), the 15-HETE and 13-HODE production increased linearly over 20 minutes ( Figure 4A and 4 B). The synthesis of 15-HETE and 13-HODE depended on the concentration of AA and LA, respectively, and Michaelis-Menten kinetics were observed ( Figure 4C and 4 D). We determined Michaelis constant ( K m ) values of 2.2±0.3 and 23.5±3.3 µmol/L for AA and LA, respectively. The maximum velocity for AA oxygenation was lower than that for LA. K m values of 6.0±2.2 (AA) and 80.2±22.2 µmol/L (LA) were determined for the purified rabbit reticulocyte 15-LO-I ( Figure 4E and 4 F). For the purified enzyme, maximum velocity for LA was higher than that for AA. Thus, the kinetic characteristics of the rabbit aortic 15-LO were similar to those of the purified 15-LO-I.
Figure 4. Kinetic analysis of AA and LA metabolism by rabbit aortic cytosol and purified rabbit 15-LO-I. Rate of 15-HETE (A) and 13-HODE (B) produced from AA and LA (10 -5 mol/L), respectively. Effect of concentration of AA on 15-HETE synthesis or LA on 13-HODE synthesis by aortic cytosol (C and D) and purified rabbit reticulocyte 15-LO (E and F). Each point represents the mean±SEM (n=3).
When AA and LA were added together, LA metabolism to 13-HODE was selectively quantified. AA decreased the synthesis of 13-HODE from LA (50 µmol/L) by 30% and 80% at 1 and 10 µmol/L, respectively ( Figure 5 A). In contrast, LA inhibited the synthesis of 15-HETE from AA less. LA decreased 15-HETE from AA (20 µmol/L) by 17% and 51% at 1 and 10 µmol/L, respectively ( Figure 5 B). Interestingly, the K m for AA and LA were only slightly altered by the addition of the other substrate, which may imply a mixed competitive/noncompetitive mode of action. AA strongly decreased LA metabolism (IC 50 =0.8 µmol/L; Figure 5 C). In contrast, LA was a weaker inhibitor of AA metabolism (IC 50 =6.9 µmol/L; Figure 5 D). When aortic cytosol was incubated with 14 C-AA, the major metabolites comigrated with 15-HETE, HEETA, and THETA (Figure II, available online at http://atvb.ahajournals.org). 14 C-15-HETE production was slightly decreased by the addition of 10 µmol/L LA but not with lower concentration. Unlabeled AA inhibited 14 C-15-HETE synthesis in a concentration-related manner. 14 C-13-HODE was the major metabolite from 14 C-LA. Its production was inhibited by LA and AA with a reduction by 61% with 1 mmol/L AA. These data suggest that AA is preferred as a substrate over LA by the rabbit aortic 15-LO-I.
Figure 5. Competition between AA and LA for metabolism by rabbit aortic 15-LO. (A) Metabolism of LA in the presence of 10 ( ), 1( ), and 0 µmmol/L AA ( ). (B) AA metabolism is measured in the presence of 10 ( ), 1 ( ) and 0 µmol/L LA ( ). (C) Inhibition of 13-HODE production from 10 ( ) and 1 µmol/L LA ( ) by various concentrations of AA. (D) Inhibition of 15-HETE production from 10 ( ) and 1 µmol/L AA ( ) with various concentrations of LA.
Inhibition of 15-LO-I Expression on Isometric Tension
Determination of the nucleotide sequence of aortic 15-LO cDNA enables us to suppress 15-LO-I expression by antisense oligonucleotide targeting of the Phe353Leu region of the enzyme. The antisense oligonucleotide strongly decreased 15-LO after 12 hours, yet the scrambled oligonucleotide did not affect 15-LO ( Figure 6 A). Because 11,12,15-THETA mediates acetylcholine-induced relaxations in the rabbit aorta, 5,7 we compared the endothelium-dependent relaxation to acetylcholine in antisense and scrambled oligonucleotide-treated aortas. In aortic rings pretreated with indomethacin (10 µmol/L) and L-NA (30 or 300 µmol/L), increasing concentrations of acetylcholine induced relaxation. The control and scrambled oligonucleotide-treated group showed a significant relaxation response to 10 -7 mol/L acetylcholine ( Figure 6 B). Maximal relaxations at 10 -5 mol/L were 28% for the control and 23% for the scrambled oligonucleotide-treated group. In contrast, aortas treated with 15-LO-I antisense oligonucleotide had a significantly reduced relaxation response to acetylcholine, with only 10% relaxation at 10 -5 mol/L. These data support the importance of 15-LO-I and its metabolites in endothelium-dependent relaxation.
Figure 6. Effect of inhibition of aortic 15-LO-I expression on relaxations to acetylcholine. Aortic rings treated with vehicle (control), scrambled oligonucleotide, or 15-LO-I antisense oligonucleotide. (A) Western immunoblot of aortic rings. (B) Relaxations responses to acetylcholine were determined in the 3 groups of aortic rings treated with L-NA and indomethacin. Each value represents the mean±SEM for n=9 to 13. * P <0.05; ** P <0.01 control versus antisense.
Discussion
The 15-LO pathway in rabbit aorta produces vasoactive lipid metabolites that mediate vascular relaxation in response to AA and acetylcholine. 4,5,7,25 Incubation of rabbit aortic rings in vitro with interleukin 13 upregulates aortic 15-LO activity. 22 This treatment enhances vascular relaxations to AA indicating the importance of 15-LO in regulating vascular tone. 15-LO metabolites also relax canine arteries 26 and rat aorta. 27 However, the 12-LO metabolite 12(S)-HETE mediates the relaxations to AA in porcine coronary and rat mesenteric arteries. 28,29
We wanted to determine the specific LO isoform expressed in the vasculature. Three lines of experimental evidence indicate that 15-LO-I but not 12-LO is expressed: (1) the sequence of the PCR product obtained with 12/15-LO-specific primers matched 15-LO-I; (2) activity assays using aortic cytosol revealed 15-HETE as the major metabolite of AA; and (3) our RT-PCR/restriction digestion strategy excluded the expression of 12-LO.
Using in situ hybridization, 12/15-LO is expressed in aorta from atherosclerotic rabbits. 30 In addition, we tested for the presence of other 15-LO isoforms. However, we detected neither 12-LO mRNA nor transcripts similar to human 15-LO-II. To determine whether 15-LO-I mRNA is translated into protein in aortic tissue, immunoblotting was performed using 2 specific 15-LO-I antibodies. Both antibodies recognized a single 75-kDa band, the molecular weight of 15-LO-I. 17 15-LO-I mRNA, but not protein expression, was reported in human umbilical ECs treated with interleukin 4. 31 We detected 15-LO-I mRNA and protein expression from human aortic ECs. Using immunohistochemistry, 15-LO-I was expressed in both rabbit aortic endothelium and smooth muscle cells adjacent to the endothelium, which agrees with the localization of 15-LO mRNA. 30 It is interesting that 15-LO is present in smooth muscle cells close to the endothelium but not more distal layers. It is possible that an endothelial factor may induce 15-LO expression in adjacent smooth muscle cells.
The 15-LO-I in rabbit aorta is enzymatically active. Exogenous AA is converted by rabbit aortic cytosol mainly to 15-HETE and LA to 13-HODE. The rabbit 15-LO is a cytosolic enzyme, which translocates to intracellular membranes when the calcium concentration is increased to 1 to 10 µmol/L. 17 We also found that aortic 15-LO is located primarily in the cytosol when the calcium concentration is <1 mmol/L (data not shown).
The kinetic characteristics for aortic 15-LO are similar to purified rabbit reticulocyte 15-LO. For both enzyme preparations, K m values were in the lower micromole per liter range. 13,14,32,33 15-LO-I, both aortic cytosol and purified enzyme, exhibits a higher affinity for AA than for LA. These data are consistent with other findings, suggesting that AA is a better substrate of mammalian 15-LO than LA. 32,34 Endothelial 15-LO metabolizes AA to 15-HPETE, which is additionally metabolized to vasoactive THETA and HEETA. 7 A higher affinity of aortic 15-LO-I for AA is consistent with an important role for 15-LO and its AA metabolites in endothelial function.
LO inhibitors, such as CDC, ebselen, and NDGA, block acetylcholine-induced relaxations in aortic rings pretreated with L-NA and indomethacin. 5,7 However, these LO inhibitors can also block the activity of 5-, 12-, and 15-LO. 21 Thus, studies with these inhibitors may not establish the role of 15-LO in regulating vascular tone. Obtaining the nucleotide sequence of aortic 15-LO allowed us to design antisense oligonucleotides to specifically reduce the expression of 15-LO-I. We suppressed 15-LO-I expression in isolated aortic rings while preserving vascular smooth muscle and endothelial function. Interestingly, vessels treated with an antisense oligonucleotide were less sensitive to acetylcholine stimulation, and the maximum relaxation to acetylcholine was reduced. This strongly supports a role for 15-LO-I in regulating vascular tone.
In summary, our data indicate that rabbit aortic 15-LO activity is attributable to expression of the 15-LO-I in vascular ECs and a subset of smooth muscle cells. This enzyme is responsible for the formation of the vasodilators 15-H-11,12-EETA and 11,12,15-THETA. 3,5,7 These 15-LO products mediate AA-induced relaxations and function as endothelium-derived hyperpolarizing factors. 3,5,7 Consistent with this conclusion, reducing 15-LO expression with an antisense oligonucleotide decreases relaxation to acetylcholine. These studies indicate that 15-LO-I contributes to regulation of vascular tone in the rabbit aorta.
Acknowledgments
These studies were supported in part by grants from the National Heart, Lung, and Blood Institute (HL-37981), Deutsche Forschungsgemeinschaft (Ku961/8-2), and the European Commission (FP6-LSHM-CT-2004-DD50333). We thank Gretchen Barg for secretarial assistance and Drs. Sandra Pfister and Kathryn Gauthier for their review of the manuscript and helpful discussions.
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作者单位：Department of Pharmacology and Toxicology (X.T., B.B.H., K.N., C.J.H., W.B.C.), Medical College of Wisconsin, Milwaukee; and the Institute of Biochemistry (H.K.), University Medicine Berlin-Charité, Berlin, Germany.