Several studies have shown that an increased dietary intake of cholesterol results in hypercholesterolemia and eventually atherosclerosis (1,2). Different animal models have been developed to investigate the atherosclerotic process, with one consistent feature of the disease being an enhanced reactivity of the vessels to various vasoactive agents (3-6). As well, the synthesis of endothelium-derived relaxing factor (EDRF) appears to be impaired in atherosclerotic vessels (3,7-9).
Little is known about the vascular changes that occur in animals fed a high-cholesterol diet for a period of time that result in hypercholesterolemia, but not atherosclerosis. We have previously shown that feeding 1-month-old New Zealand White rabbits a 2% cholesterol diet for 2 weeks results in hypercholesterolemia without morphologic changes and a depressed aortic contractile response to norepinephrine (10). These studies differ from studies in both rabbit carotid artery and monkey aorta, which reported an enhanced response to norepinephrine (7,11). The responses to serotonin were unaltered in our study (10) but enhanced in a number of other studies (4,6). A distinct difference in our studies compared with those by other investigators is the period for cholesterol feeding. Our experiments were performed before the development of any gross evidence of atherosclerosis.
The synthesis of arachidonic acid also has been shown to be altered in atherosclerosis (12-15). Specifically, aortic synthesis of prostacyclin (PGI2) is decreased, whereas the platelet synthesis of thromboxane A2 (TXA2) is increased (13,14). We recently reported the ability of aortas from cholesterol-fed, but not normal, rabbits to synthesize epoxyeicosatrienoic acids (EETs), cytochrome P450 epoxygenase metabolites of arachidonic acid (12). We hypothesized that the early changes in vascular reactivity are mediated by an arachidonic acid metabolite. The aim of our study was to better characterize vasoactive responses in cholesterol-fed rabbit aorta and to investigate the role of arachidonic acid metabolites in these responses.
MATERIALS AND METHODS
For a period of 2 weeks, two groups of 1-month-old male New Zealand White rabbits received either control rabbit diet or control diet supplemented with 2% cholesterol. The cholesterol diet was prepared by adding 200 g of cholesterol to 1 L of corn oil and mixing with 10 kg of commercial rabbit chow (Tekland). Rabbits had free access to both food and water. There was a significant increase in plasma cholesterol concentrations in animals fed the 2% cholesterol diet compared with the animals fed the control diet (1,506 ± 123 vs. 87 ± 6 mg/dl) (12). No differences in weight gain were measured between the two groups. After 2 weeks of feeding the 2% cholesterol diet, no visible fatty streaks or plaques were noted in the aortas obtained from the cholesterol-fed rabbits.
Normal and cholesterol-fed rabbits were killed with pentobarbital (120 mg/kg), and the thoracic aorta was removed and placed in Kreb's bicarbonate buffer (118 mM NaCl, 3.3 mM CaCl2, 24 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4 and 11 mM glucose). The tissue was carefully cleaned of adhering fat and connective tissue and cut into rings (3 mm thick), taking care not to damage the endothelium. In some vessels, the endothelium was purposefully removed by gently rubbing the intimal surface with a cotton-tipped swab (16). Aortic rings were suspended in 15-ml tissue baths containing Krebs-bicarbonate buffer maintained at 37°C and continuously bubbled with 95% O2-5% CO2. Isometric tension was adjusted to its length-tension maximum of 2 g, and vessels were allowed to equilibrate for 1 h. Reproducible contractions were produced by increasing the KCl concentration of the baths to 40 mM. After the vessels reached peak contraction, tissue baths were rinsed, and vessels allowed to return to resting tension. After the aortic rings had reproducible stable responses to KCl, cumulative concentration-response curves to norepinephrine (10-9-10-6M) were obtained. The responses to norepinephrine were repeated in the presence of various inhibitors including the specific nitric oxide synthase inhibitor, nitro-L-arginine (LNA; 3 × 10-5M), the cyclooxygenase inhibitor, indomethacin (10-5M), the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA; 5 × 10-5M), the cytochrome P450 epoxygenase inhibitor, metyrapone (10-4M), and the specific TXA2/prostaglandin (PG) H2-receptor antagonist, SQ 29548 (10-7M). The inhibitors were present for 10 min before the addition of norepinephrine. Additional studies examined the effect of the specific α1-adrenergic receptor agonist, phenylephrine (10-9-10-6M) and the specific α2-adrenergic receptor agonist, xylazine (10-9-10-6M) in normal and cholesterol-fed rabbit aortas. Vascular responses in aortic rings from both normal and cholesterol-fed rabbits were always measured in parallel. In time-control studies, it was found that multiple concentration-response curves to norepinephrine in the absence of added inhibitors remained identical throughout the experiment.
The synthesis of TXA2 and PGI2 were compared in cholesterol-fed and normal rabbit aortas. Segments of aortas were obtained from both normal and cholesterol-fed rabbits and incubated for 30 min at 37°C in N-2-hydroxyethylpiperazie-N′-2-ethanesulfonic acid (HEPES) buffer containing norepinephrine (10-6M). The synthesis of TXB2, the stable metabolite of TXA2, and 6-keto PGF1α, the stable metabolite of PGI2, was measured in the buffer by specific radioimmunoassays by using the method of Campbell and Ojeda (17). The antibody for TXB2 and 6-keto PGF1α was produced in rabbits. The sensitivity of the assay is 1 pg/0.3 ml for TXB2 and 5 pg/0.3 ml for 6-keto PGF1α. The cross-reactivity of the antisera with known arachidonic acid (AA) metabolites is <0.1%.
Identification of arachidonic acid metabolites
To identify the arachidonic acid metabolites produced by cholesterol-fed rabbit aortas, intact aortic segments from cholesterol-fed rabbits (500 mg of wet weight) were incubated with 0.5 μCi [3H]arachidonic acid dissolved in HEPES buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 6 mM glucose, pH 7.4) containing indomethacin (10-5M) for 4 h at 37°C. In one experiment, the endothelial layer was removed before the prelabeling with arachidonic acid. Under these labeling conditions, 90% of the added radioactivity was lost from the labeling buffer and assumed to be incorporated in the tissue or cellular lipids (18). After the prelabeling period, vessels were washed 4 times with HEPES buffer containing 1% of fatty acid-free bovine serum albumin. The washed vessels were incubated in fresh protein-free HEPES buffer containing indomethacin (10-5M) in the presence or absence of norepinephrine (10-6M) for 15 min at 37°C. Vessels were homogenized in HEPES buffer, and radioactive metabolites were extracted with ethyl acetate. The extracted metabolites were evaporated to dryness under a stream of nitrogen and stored at -40°C until analysis by high-pressure liquid chromatography (HPLC). The arachidonic acid metabolites were resolved by reverse-phase (Nucleosil-C18 column, 5 μm, 4.6×250 mm) HPLC by using the following solvent system. Solvent A was water, and solvent B was acetonitrile containing 0.1% glacial acetic acid. The program was a 40-min linear gradient from 50% solvent B in A to 100% solvent B. Radioactivity of the column eluate was collected in 0.2-ml aliquots and measured by liquid scintillation spectrometry.
Data are presented as the mean + SEM. Statistical evaluation was made by using a one-way analysis of variance followed by the Sidak multiple comparison test when significant differences were found. A p value of <0.05 was considered significant. The concentration of drug producing 50% of the maximal response (EC50) was determined by using a computer-assisted (PRISM) nonlinear regression analysis.
There was no significant difference in the maximal contractions caused by KCl (40 mM) in vascular rings from normal and cholesterol-fed rabbits (2.6 ± 0.2 vs. 2.4 ± 0.2 g; normal vs. cholesterol-fed; NS). Removal of the endothelium also did not change the maximal contractions to KCl in either group of rabbits (2.3 ± 0.2 vs. 2.3 ± 0.2 g; normal vs. cholesterol-fed; NS). The effect of KCl in denuded vessels was identival to that in a previous report (16). Constraction to the various agents is reported as a percentage of the maximal contraction elicited by KCl.
Norepinephrine evoked concentration-dependent contractions in aortic rings from both normal and cholesterol-fed rabbits (Fig. 1A). However, the maximal response to norepinephrine was significantly less in cholesterol-fed rabbits (126 ± 2% vs. 100 ± 2%; normal vs. cholesterol-fed; p < 0.01). Cholesterol feeding elicited an ≈26% reduction in norepinephrine-induced contractions in cholesterol-fed rabbits in comparison with normal rabbits. The EC50 was not different in the cholesterol-fed rabbits compared with normal rabbits (Table 1). In constrast, when aortic rings without endothelium were obtained from normal and cholesterol-fed rabbits and treated with increasing concentrations of norepinephrine, no significant difference in norepinephrine-induced contractions was observed (maximal response, 155 ± 5% vs. 150 ± 5%; normal vs. cholesterol-fed; NS; Fig. 1B). These results indicated that removal of the endothelium enhanced norepinephrine-induced contractions in both normal and cholesterol-fed rabbit aortas. Pretreatment of intact aortas from normal and cholesterol-fed rabbits with a specific inhibitor of the endothelium-derived factor, nitric oxide, LNA, did not affect norepinephrine-induced contractions in normal and cholesterol-fed rabbits (Fig. 1C). In the presence of LNA, norepinephrine-induced contractions were reduced in cholesterol-fed rabbits compared with normal rabbits (maximal response, 139 ± 6% vs. 108 ± 4%; normal vs. cholesterol-fed; p < 0.01).
The next experiment investigated the α-receptor subtype mediating norepinephrine-induced contractions in the normal and cholesterol-fed rabbits. The selective α1-agonist, phenylephrine, elicited concentration-related contractions that were significantly greater in normal rabbits than in cholesterol-fed rabbit aortas (maximal response, 133 ± 6% vs. 106 ± 2%; normal vs. cholesterol-fed; p < 0.01; Fig. 2). When aortic rings from normal and cholesterol-fed rabbits were treated with the seletive α2-agonist, xylazine, no contractions were measured (data not shown). In addition, if vessels were precontracted with the TXA2-mimetic, U 46619 (10-7M) and increasing concentrations of xylazine added, there was no vasorelaxant effect (data not shown). Pretreatment with the β-adrenergic antagonist, propranolol (10-6M), had no effect on contractile responses to norepinephrine (data not shown).
Pretreatment of rings with the cyclooxygenase inhibitor, indomethacin (10-5M) produced a slight decrease in norepinephrine-induced contractions in both normal and cholesterol-fed rabbit aortas (Fig. 3A). Because cyclooxygenase metabolites of arachidonic acid have been shown to produce vasoconstriction through TXA2/PGH2 receptors (19), we investigated norepinephrine-induced contractions in aortas pretreated with the TXA2/PGH2-receptor antagonist, SQ 29548. SQ 29548 (10-7M) had no effect on norepinephrine-induced contractions in control or indomethacin-treated vessels (data not shown) obtained from either normal or cholesterol-fed rabbits. Aortic segments from normal and cholesterol-fed rabbits were incubated with norepinephrine, and the release of TXB2 and 6-keto PGF1α was measured. Results are shown in Table 2 and indicated that norepinephrine did not increase the production production of TXB2 and 6-keto PGF1α in either group of rabbits. Additionally, there were no differences in the basal or stimulated release of TXB2 or 6-keto PGF1α in normal or cholesterol-fed aortas.
The lipoxygenase inhibitor, NDGA, elicited a small increase in norepinephrine-induced contractions in cholesterol-fed rabbits (Fig. 3B, bottom) but not in the normal rabbits (Fig. 3B, top). The ability of NDGA to enhance contractions was seen only at the higher concentrations of norepinephrine; however, the effect was not statistically significant. Pretreatment of vessels with an inhibitor of cytochrome P450 epoxygenase, metyrapone, increased norepinephrine-induced contractions in the cholesterol-fed rabbits (Fig. 4, bottom). Metyrapone had no effect on norepinephrine-induced contractions in normal rabbit aortas (Fig. 4, top). Table 1 summarizes the EC50 levels and maximal contractile responses for the various drug treatments in the normal and cholesterol-fed rabbit aortas.
The final series of experiments examined the effect of norepinephrine on the metabolism of [3H]arachidonic acid in aortas from cholesterol-fed rabbits. A representative chromatogram is shown in Fig. 5. Cholesterol-fed rabbit aortas synthesized radioactive products that comigrated with the PGs, dihydroxyeicosatrienoic acids (DHETs) and dihydroxyeicosatetranoic acids (DiHETEs), the hydroxyeicosatetranoic acids (HETEs), and the EETs. In the presence of norepinephrine, there was an increase in the production of metabolites migrating with the DHETs-DiHETEs, HETEs, and EETs. The radioactive fractions corresponding to the DiHETEs-DHETs, HETEs, and EETs contained 6,370 ± 410, 4,928 ± 995, and 900 ± 78 cpm, respectively, for the untreated aortas, and 8,297 ± 992, 7,187 ± 493, and 1,546 ± 23 cpm, respectively, for the norepinephrine-treated aortas. In a separate experiment in denuded aortas, there was no production of DHETs-DiHETEs and EETs either under basal conditions or in the presence of norepinephrine. Minor radioactive peaks were observed that comigrated with the HETEs, but the synthesis of these products was not enhanced by norepinephrine (data not shown).
This study indicated that norepinephrine-induced contractions are reduced in aortic tissue obtained from cholesterol-fed rabbits compared with rabbits maintained on normal rabbit chow. Several clinical and experimental studies have suggested that atherosclerosis alters vascular reactivity (3-9). However, although some studies have shown norepinephrine contractions are less (20), other studies have reported enhanced vasoconstriction to norepinephrine in atherosclerotic tissue (7,11). Several hypothesis explain the different responses of atherosclerotic vessels to α-adrenergic receptor agonists including such factors as whether atherosclerotic lesions are present, the size of the artery studied, and the subtype of the α-receptor mediating the response (21). Our study examined norepinephrine-induced contractions in a large-diameter vessel, the thoracic aorta. Experiments were performed after a period of cholesterol feeding in which serum cholesterol concentration was elevated in the rabbits, but there was no gross evidence of lesions or plaque development in the aorta. Finally, in these studies, the α-adrenergic receptor subtype mediating the response in the aorta to norepinephrine was found to be α1. This was based on the following observations: (a) the specific α1-adrenergic agonist, phenylephrine, produced decreased contractions in cholesterol-fed compared with normal rabbits, (b) the specific α2-adrenergic agonist, xylazine, failed to contract aortas from either normal or cholesterol-fed rabbits, and (c) the β-adrenergic antagonist, propranolol, did not alter contractile responses to norepinephrine.
Removal of the endothelium enhanced norepinephrine-induced contractions in both normal and cholesterol-fed rabbit aortas (without affecting the maximal contractile response to KCl), suggesting that an endothelium-derived factor released by norepinephrine modulates vascular tone. A number of vasodilators are produced by vascular endothelial cells including EDRF or nitric oxide. There is evidence that α2-receptors are located on endothelial cells, and these receptors mediate the release of EDRF or nitric oxide (22). Alternatively, recent studies suggested that α1-receptors located on vascular endothelial cells also release EDRF in response to norepinephrine (23). Therefore we examined the role of nitric oxide in norepinephrine-induced contractions in both normal and cholesterol-fed rabbit aortas. Inhibition of nitric oxide with LNA did not increase norepinephrine-induced contractions in either normal or cholesterol-fed rabbit aortas. Thus our results suggest that an endothelium-derived vasodilator other than nitric oxide is released by norepinephrine and mediates the reduced contractile responses observed in hypercholesterolemia. Although we did not characterize the α-adrenergic receptor subtype mediating the endothelial-mediated release of the factor, our data suggest that the factor released by norepinephrine mediates relaxation by an α1-subtype and not an α2-subtype because the specific α2-adrenergic agonist, xylazine, failed to elicit relaxations in normal or cholesterol-fed rabbit aortas.
We first investigated the role of cyclooxygenase arachidonic acid metabolites in the vascular responses to norepinephrine. Stimulation of sympathetic nerves in blood vessels causes an increase in PG formation (24). In rabbit aorta with intact endothelium, the principal arachidonic acid metabolite formed by the cyclooxygenase pathway is PGI2(12,25). Whereas PGI2 is a potent vasodilator in most vascular tissue, the rabbit aorta is relatively insensitive to the relaxant effects of PGI2(26). Alternatively, we previously showed that the cyclooxygenase metabolite, TXA2, mediates endothelium-dependent contractions of rabbit pulmonary artery (19). Our study showed that preptreatment with indomethacin, a cyclooxygenase inhibitor, decreased norepinephrine-induced contractions in both normal and cholesterol-fed rabbits. These data suggest that TXA2 may be released by norepinephrine and contribute to vascular tone. However, if TXA2 receptors were blocked with a specific TXA2/TGH2-receptor antagonist, SQ 29548, there was no effect on norepinephrine-induced contractions in either normal or cholesterol-fed aortas. Additional evidence showed that release of TXB2 or 6-keto PGF1α was not different between normal and cholesterol-fed rabbit aortas under either basal or norepinephrine-stimulated conditions.
Because blockade of the cyclooxygenase pathway with indomethacin can cause shunting of arachidonic acid to lipoxygenase or cytochrome P450 epoxygenase pathways, we next examined the role of these metabolites in the response to norepinephrine. Our results indicated that blockade of the lipoxygenase pathway with NDGA had no effect on norepinephrine-induced contractions in normal aortas and caused only a small increase in contractions in the cholesterol-fed rabbits. Inhibition of the epoxygenase pathway with metyrapone elicited a much greater increase in norepinephrine-induced contractions in cholesterol-fed rabbit aortas, but the cytochrome P450 epoxygenase inhibitor had no effect on contractions in normal rabbit aortas. These results suggest that lipoxygenase or epoxygenase metabolites of arachidonic acid or both may contribute to the reduced norepinephrine-induced contractions observed in hypercholesterolemia. Although limited, some studies have investigated the lipoxygenase metabolism of arachidonic acid in atherosclerosis (12,27,28). In Watanabe Heritable Hyperlipidemic rabbits, a genetic model of atherosclerosis, the aortic synthesis of the lipoxygenase metabolites is impaired (15). Others have shown that 15-lipoxygenase activity is increased in the aorta of hypercholesterolemic rabbits (27), whereas cholesterol-rich macrophages have an increased production of 12-lipoxygenase metabolites (28). An oxygenation reaction catalyzed by specific lipoxygenase enzymes converts arachidonic acid to hydroperoxide derivative (HPETEs), which possess a variety of biologic actions (29). The HPETEs are short-acting compounds that are further metabolized to HETEs, hepoxilins, lipoxins, and leukotrienes. Biologic actions of the HPETEs and, to a lesser degree, the HETEs include vasorelaxation (30), vasoconstriction (31), inhibition of PGI2 synthetase (32), and chemotaxis (33).
Much less is known about the role of the epoxygenase metabolites of arachidonic acid in atherosclerosis. We reported that aortic tissue obtained from cholesterol-fed but not normal rabbits produced 14,15-,11,12-,8,9- and 5,6-EET (12). The production of these products was dependent on an intact endothelial layer (12). By using cultured human vein endothelial cells, Pritchard et al. (34) found a similar enhancement of EET production by low-density lipoprotein-cholesterol. Various EETs have been reported to produce changes in vascular tone; Proctor et al. (35) reported the ability of the EETs to elicit vasodilatation of intestinal blood flow, and Carroll et al. (36) showed the ability of 5,6-EET to relax rat tail artery. The four EET isomers can also produce endothelium-independent relaxation of isolated canine coronary artery and rabbit aorta vessels (12,37). It is not known what effect norepinephrine has on the metabolism of arachidonic acid by the lipoxygenase and cytochrome P450 epoxygenase pathways. Therefore, the effect of norepinephrine was investigated in our study by analyzing the metabolism of [3H]arachildonic acid in aortic tissue obtained from cholesterol-fed rabbits. Results indicated that norepinephrine increased the production of radiolabeled metabolites that comigrated with the DHETs-DiHETEs, HETEs, and EETs only in vessels with an intact endothelial layer. Based on these results, coupled with the studies that described the ability of these metabolites to produced vasodilation of vascular tissue, this study demonstrated that (a) an endothelium-derived metabolite of arachidonic acid regulates vascular tone, (b) this metabolite may be a lipoxygenase or cytochrome P450 product or both, and (c) the activity or synthesis of the factor is enhanced by hypercholesterolemia.
Acknowledgment: We thank Donna Kotulock for her invaluable technical assistance and Gretchen Barg for her excellent secretarial assistance. Support was provided by National Heart, Lung, and Blood Institute Grant HL-37981.
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