INTRODUCTION

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THE BALANCE BETWEEN endothelium-derived nitric oxide (NO) and endothelin-1 (ET-1) may contribute to altered vasomotor function in pathophysiological states, including hypercholesterolemia and atherosclerosis (11). For example, in experimental hypercholesterolemia, circulating levels of ET-1 increase, whereas circulating levels of plasma oxidized products of NO (NOx) decrease (2, 3). At the level of the coronary arteries, endothelium-dependent relaxations are reduced, as is immunoreactivity for endothelial NO synthase (NOS; eNOS) (2, 13). These observations, taken together with reductions in contractions to infusion of the arginine analog N^G-monomethyl-L-arginine, provide indirect evidence that activity of NO synthase (NOS) decreases with hypercholesterolemia (13). Chronic inhibition of hypercholesterolemic pigs with antagonists for ET_A receptors or combined inhibition of ET_A plus ET_B receptors increases circulating levels of NO and restores endothelium-dependent relaxations and immunostaining for NOS in coronary arteries of pigs (2, 3). Whether endothelin-receptor antagonists affect changes in NOS at the transcriptional or posttranscriptional level is unclear. Therefore, experiments were designed to extend observational studies of changes in NOx and NOS immunostaining by directly determining changes in mRNA for endothelial NO and enzyme activity in the setting of chronic hypercholesterolemia and endothelin-receptor blockade. It was hypothesized that chronic endothelin-receptor blockade in hypercholesterolemia would increase activity of eNOS.

	METHODS

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Animals. All study procedures using animals were reviewed and approved by the Mayo Foundation Institutional Animal Care and Use Committee and were designed in accordance with the National Institutes of Health Guidelines. Female juvenile domestic crossbred pigs (23-35 kg) were placed on an atherogenic diet of 2% cholesterol and 15% lard by weight (TD-93296; Harlan Teklad, Madison, WI; Table 1) for 12 wk (11). The mean nitrate concentration is 8.1 parts/million (range 0.1-22), and nitrite concentration is 1.8 parts/million (range 0.1-6.9). Animals were assigned to one of three groups. They either did not receive any medications (control group), or were treated with oral ABT-627 (Abbott Laboratories, Abbott Park, IL), an ET_A receptor antagonist, on a weight-adjusted scale to maintain a dose of 4 mg · kg¹ · day¹, or were treated with oral RO-48-5696 (Hoffman-LaRoche, Basel, Switzerland), a combined ET_A plus ET_B-receptor antagonist, on a weight-adjusted scale every 3 wk to maintain the dose at 3 mg · kg¹ · day¹ (2, 3, 16, 19). After 12 wk of treatment, hearts and aorta were removed for study. Responses of the coronary arteries from these animals were studied in separate experiments (2, 3). Endothelial cells were scraped from the aorta of each animal and prepared either for measurement of activity of eNOS or mRNA for eNOS. Because of technical problems, aortic endothelial cells were obtained from only three of seven control animals (2, 3).

Plasma assays. Plasma was collected at baseline and after 12 wk of treatment. Total cholesterol, high-density-lipoprotein (HDL), low-density-lipoprotein (LDL), triglyceride levels, plasma ET-1, and NOx were measured as previously described (2, 3).

Quantitation of mRNA for NOS. RT-PCR was performed on RNA extracted from thoracic aortic endothelial cells. Endothelial cells were scraped from the luminal surface of aortas and then stored in 1 ml of RNA STAT-60 (TEL-TEST "B"). Total RNA was then extracted with 0.2 ml chloroform and precipitated with 0.5 ml isopropanol. The supernatant was removed, and the RNA pellet was washed with 1 ml of 75% ethanol, air dried, and reconstituted in diethyl pyrocarbonate-treated water. RNA concentration was determined by measuring absorbance at 260 nm in a spectrophotometer (Beckman DU 640, Fullerton, CA). DNase treatment of 1 µg of total RNA was carried out with 1 µl of DNase buffer (200 mM Tris · HCl at pH 8.4, 500 mM KCl, 20 mM MgCl₂) and 2 µl of amplification-grade DNase I (Life Technologies) for 15 min at room temperature. DNase I was then inactivated by heating to 65°C after the addition of 1 µl of 25 mM EDTA. First-strand cDNA synthesis was next performed (Superscript Preamplification System, Life Technologies) by sequential reactions after the addition of 1 µl of oligo(dT)_12-18 primers to hybridize to 3' poly(A) tails on mRNA (70°C for 10 min) and then 7 µl of reaction mixture (2 µl 10× PCR buffer, 2 µl 25 mM MgCl₂, 1 µl dNTP mix, and 2 µl 0.1 M dithiothreitol at 42°C for 5 min) and 1 µl of Superscript II RT (42°C for 50 min). Reaction was terminated by heating to 70°C for 15 min followed by incubation with RNase H for 20 min at 37°C. Target cDNA was next amplified by PCR: 38 cycles of denaturation (94°C for 45 s), annealing (60°C for 45 s), and polymerization (72°C for 60 s). The primers used detected eNOS (transfected and endogenous): 5' primer (TCA ACC AGT ACT ACA GCT CC) and 3' primer (GTG GTT GCA GAT GTA GGT GA). A 251-bp product was visualized on 2% agarose gel electrophoresis.

Activity of NOS. NOS activity was determined by measuring the conversion of L-[³H]arginine to L-[³H]citrulline by methods originally described by Myatt et al. (18) and modified by Miller and Barber (17). In brief, homogenates of aortic endothelial cells were prepared and eluted through 10-DG desalting columns. To quantitate eNOS activity, duplicate reactions were carried out in the presence of calcium (total activity), in the absence of calcium plus EGTA (calcium-independent activity), and in the absence of calcium plus EGTA in the presence of N^G-monomethyl-L-arginine (nonspecific activity). Reactions were started by adding 150 µl of cofactor mix. The reaction was incubated on a shaker at 37°C for 1 h and terminated by the addition of ice-cold stop buffer. Separation of L-[³H]arginine from L-[³H]citrulline was accomplished by using affinity column containing AG 50S-X8 Na⁺ form 200- to 400-mesh resin (Bio-Rad Laboratories, Hercules, CA). Calcium-dependent activity (eNOS) equals total activity after correcting for nonspecific activity.

Statistical analysis. All values are expressed as means ± SE. Analysis of variance followed by correction for repeated measures (Bonferroni correction) was used to analyze data with a Gaussian distribution. A Kruskal-Wallis test followed by pairwise comparisons of distributions using a Mann-Whitney U-test was used to analyze the quantified PCR results that had a non-Gaussian distribution. A P value of <0.05 was considered significant.

	RESULTS

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Animals and blood chemistry. All pigs gained weight during the 12 wk of high-cholesterol diet [77.3 ± 1.6 kg at baseline (n = 17), to 149.0 ± 5.8 kg in control animals (n = 3), 117.1 ± 2.8 kg in the ET_A-antagonist-treated group (n = 8), and 121.7 ± 3.6 kg in the ET_A plus ET_B-treated group (n = 6)]. Total cholesterol, HDL, and LDL increased significantly in all groups. Increases in total cholesterol, LDL, and HDL were not affected by endothelin-receptor antagonists (Table 2).

Quantitation of mRNA for eNOS. Quantitative RT-PCR performed on RNA extracted from the aortic endothelial cells showed no significant differences in the mRNA levels for eNOS among groups [control: 0.0250 ± 0.0125 amol/µl (n = 3); ET_A blocked: 0.0424 ± 0.017 amol/µl (n = 8); ET_A plus ET_B blocked: 0.03708 ± 0.0197 amol/µl (n = 6); Fig. 1; P value for the exact version of the Kruskal-Wallis test was 0.9420].

View larger version (27K): [in this window] [in a new window]   Fig. 1.   A: representative ethidium bromide-stained 1.6% agarose gel used to visualize comparison of target and MIMIC bands for quantification of endothelial nitric oxide synthase (eNOS) target DNA derived from mRNA of aortic endothelial cells from cholesterol-fed pigs. mRNA concentration (amol/µl) was determined by visual comparison of the target (eNOS) and MIMIC bands. Lane 1, 100-bp standard; lane 2, eNOS plasmid; lane 3, water control; lanes 4-7, titrates of a constant amount of the experimental target DNA against serial dilutions (10-fold) of the PCR MIMIC (nonhomologous internal standards). Note, 2-fold dilutions were used to actually quantitate the target, i.e., to quantify cDNA band 5. B: summary of quantification of eNOS mRNA in aortic endothelial cells of cholesterol-fed pigs using internal nonhomologous standards. ET, endothelin. Results are presented as individual data points. No significant differences were observed in eNOS mRNA levels among groups (P value for the exact version of the Kruskal-Wallis test was 0.9420).

NOS activity. Citrulline accumulation from total and calcium-dependent NOS enzyme was greater in cells from cholesterol-fed pigs treated with ET_A plus ET_B antagonists compared with those treated with ET_A-receptor antagonists alone (Fig. 2). Calcium-independent NOS enzyme accumulation was similar in all three groups [control: 144.8 ± 78.2 pmol · mg protein¹ · h¹ (n = 3); ET_A blocked: 116.3 ± 87.1 pmol · mg protein¹ · h¹ (n = 8); ET_A plus ET_B blocked: 127.6 ± 50.6 pmol · mg protein¹ · h¹ (n = 6)].

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Nitric oxide synthase enzyme activity in aortic endothelial cells from cholesterol-fed pigs (L-[3H]citrulline in pmol · mg protein1 · h1). Nitric oxide synthase enzyme activity was determined by measuring the conversion of L-[3H]arginine to L-[3H]citrulline using modification of methods previously described (17). Values are means ± SE; n, no. of pigs. Total (A) and calcium-dependent (B) nitric oxide synthase activity were significantly higher in aortic endothelial cells of pigs treated with ETA plus ETB antagonists compared with those from pigs treated with a selective ETA antagonist. No significant differences were observed in calcium-independent nitric oxide synthase activity among the different treatment groups (values in text). * Statistical difference from control, P < 0.05.

	DISCUSSION

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Previous studies have demonstrated that high-cholesterol feeding of pigs increases plasma cholesterol and ET-1 with concomitant decreases in plasma NOx and endothelium-dependent relaxations in coronary arteries (2, 3, 13). Furthermore, chronic treatment of hypercholesterolemic pigs with endothelin-receptor antagonists improves endothelium-dependent relaxations of coronary arteries and attenuates decreases in plasma NOx (2, 13). These observations suggest indirectly that endothelin-receptor antagonism increases activity of NOS (2, 3). Results of the present study extend these observations to provide direct evidence that combined chronic antagonism of ET_A and ET_B receptors results in increased eNOS enzyme activity (as defined by citrulline accumulation at a single time point) in pigs with hypercholesterolemia. Increases in eNOS activity would account in part for increases in plasma NOx in animals with combined endothelin-receptor antagonism and improved endothelium-dependent relaxation (3). It is important to note that increases in NOS activity were due to an increase in calcium-dependent activity and not the calcium-independent activity typically defining activity of inducible NOS. This probably represents, in part, posttranscriptional regulation of the enzyme, as mRNA for eNOS was similar between the tissue from animals receiving ET_A and ET_A plus ET_B antagonists. However, NOS activity was significantly greater in the group receiving ET_A plus ET_B antagonist compared with the group receiving only ET_A-receptor antagonists. These data should not be interpreted to mean that endothelin-receptor antagonists would not influence transcription for eNOS, as only a single time point was studied. Indeed, 12 wk of treatment may represent a "steady-state" condition and may not be indicative of changes in message at earlier time points after treatment.

Posttranscriptional regulation of eNOS could include changes in intracellular regulation of calcium by phosphoinosital as occurs in hypertension (24), autocrine regulatory systems associated with production or release of other endothelium-derived factors such as prostaglandin or adrenomedullin (15), or changes in oxidative stress (3, 9).

It is unlikely that changes in circulating NOx represented changes in dietary intake of nitrate and nitrites. All pigs received the same diet, and pigs with the highest plasma NOx (those treated with ET_A plus ET_B antagonist) actually weighed less than the control pigs, which would suggest less dietary intake of nitrates and nitrites.

A shortcoming of this study is that NOS activity was determined in aortic rather than coronary endothelial cells. Coronary arteries from these animals were used for functional studies (2, 3). Heterogeneity in distribution of NO and ET-1 throughout the vasculature is well recognized. Although NOS activity and mRNA were not measured in coronary arterial endothelial cells, it should be pointed out that changes in circulating NOx and ET-1 probably represent mean production and secretion from several vascular beds. Increases in NOS activity in aortic endothelial cells of animals treated with the ET_A plus ET_B antagonists are consistent with increases in systemic concentrations of NOx and increases in functional expression of endothelium-dependent responses observed in the coronary arteries (2, 3). Therefore, chronic endothelin-receptor antagonism is likely to affect endothelin receptors and NOS activity throughout the vasculature.

ET_A receptors are located on vascular smooth muscle cells, whereas ET_B receptors are located on both endothelial and vascular smooth muscle cells (12, 21, 23). Stimulation of ET_B receptors has been associated with release of NO from the endothelium in experimental animals and humans (4, 5, 8, 10, 20, 22). Therefore, it is unclear how antagonism of ET_B receptors would act to maintain NOx in the setting of hypercholesterolemia. In hypercholesterolemia, there is an enhanced vasoconstrictor response to the selective ET_B agonist sarafotoxin in the coronary microcirculation (14). In support of this observation, there was a trend, albeit statistically insignificant, for increases in both ET_B-receptor affinity and number in conduit coronary arteries with hypercholesterolemia (14). Although the receptor-binding assay did not differentiate receptors on the smooth muscle or endothelial cells, data suggest that there are changes in ET_B receptors in hypercholesterolemia. Clearly, additional experiments are needed to better define regulation of endothelin-receptor subtypes, their distribution, affinity for both agonists and antagonists, and intracellular signaling pathways in hypercholesterolemia.

Antagonism of ET_A plus ET_B receptors but not ET_A receptors alone also increased plasma ET-1. This result is consistent with previous studies demonstrating a negative feedback between the stimulation of ET_B receptors and the half-life of exogenously administered ET-1 (6). The increase in ET-1 concentrations in ET_A plus ET_B-blocked animals suggests that ET_B-receptor binding may be an important mechanism in clearance of endogenous ET-1. The relationship between clearance of ET-1 and regulation of NOS remains to be defined. Chronic administration of exogenous ET-1 has been shown to increase NO-dependent reactivity of resistance vessels in rats (7).

In summary, in hypercholesterolemia, plasma ET-1 increases, whereas plasma NOx decreases. Chronic antagonism of ET_A and ET_B receptors attenuates the decrease in plasma NOx associated with hypercholesterolemia. The greatest restoration in plasma NOx was observed during simultaneous blockade of both ET_A plus ET_B receptors, a treatment that further elevated circulating concentrations of ET-1. After 12 wk of treatment, mRNA for eNOS was similar between animals treated with the ET_A-receptor antagonist alone and those treated with ET_A plus ET_B antagonist. However, because NOS activity increased only with ET_A plus ET_B antagonist, this suggests that ET_B receptors are associated with posttranscriptional regulation of NOS in hypercholesterolemia.