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Jun 16, 2005 - Submitted 16 March 2005; accepted in final form 17 June 2005. Woodman, Christopher R. ..... J Appl Physiol • VOL 99 • OCTOBER 2005 • www.jap.org by 10.220.33.4 on ..... Med Sci Sports Exerc 27: 1135–1144,. 1995. 10.
J Appl Physiol 99: 1412–1421, 2005. First published June 16, 2005; doi:10.1152/japplphysiol.00293.2005.

Endurance exercise training improves endothelium-dependent relaxation in brachial arteries from hypercholesterolemic male pigs Christopher R. Woodman, Mark A. Thompson, James R. Turk, and M. Harold Laughlin Department of Biomedical Sciences, University of Missouri, Columbia, Missouri Submitted 16 March 2005; accepted in final form 17 June 2005

(16), and/or greater reliance on NO to mediate endotheliumdependent vasodilator responses (13). On the basis of experimental evidence that hypercholesterolemia-induced endothelial dysfunction is due in part to decreased bioavailability of NO, previous studies were conducted to test the hypothesis that endurance exercise training attenuates or reverses the detrimental effects of hypercholesterolemia on EDR in coronary arteries by enhancing the production and stability of NO (22, 27). Results from these studies revealed that EDR is improved in coronary arteries of hypercholesterolemic male and female pigs by enhancing the release and/or bioactivity of NO (22, 27). Interestingly, results from a recent study using hypercholesterolemic female pigs revealed that endurance exercise training improved EDR in brachial (Br) arteries by enhancing a vasodilator pathway independent of NO, possibly endotheliumderived hyperpolarizing factor (EDHF) (28). Therefore, the purpose of the present study was to test the hypothesis that there is no sex effect on these processes, i.e., that endurance exercise training also attenuates or reverses the detrimental effects of hypercholesterolemia in Br arteries of adult male pigs by enhancing NO- and/or EDHF-mediated, EDR. METHODS

Experimental Animals

HYPERCHOLESTEROLEMIA IS REPORTED to induce endothelial dysfunction in coronary and peripheral arteries (1- 3, 6 – 8, 17, 18, 25). This dysfunction is characterized by impaired endothelium-dependent vasodilator responses and is often associated with a disruption of the nitric oxide synthase (NOS) pathway (5, 14) resulting in reduced bioavailability of nitric oxide (NO; Ref. 23). It is well documented that endurance exercise training can improve endothelium-dependent relaxation (EDR) in some, but not all, vascular beds (9 –13, 15, 19, 20, 24). In arteries where exercise training has been shown to improve EDR, experimental evidence indicates that enhanced endothelial function is frequently associated with increased expression of endothelial NOS (eNOS; Refs. 10, 16, 26), enhanced production of NO

Before this study was initiated, approval was received from the Animal Care and Use Committee at the University of Missouri. The experimental animals were adult male Yucatan miniature swine (n ⫽ 46) that were purchased from a commercial breeder (Sinclair Research Farm, Columbia, MO). The pigs were 8 –12 mo of age and weighed 25– 40 kg. All of the pigs were housed in the animal care facility in the Department of Biomedical Sciences in a room maintained at 20 –23°C with a 12:12-h light-dark cycle. Some of the pigs (n ⫽ 24) were provided a normal-fat (NF) diet (Purina Lab Mini-pig Chow) in which 8% of daily caloric intake was derived from fat. The remaining pigs (n ⫽ 22) were provided a high-fat (HF) diet consisting of pig chow supplemented with cholesterol (2.0%), coconut oil (17.1%), corn oil (2.3%), and sodium cholate (0.7%), such that 46% of daily caloric intake was derived from fat (4). Four weeks after the diet was initiated, pigs were exercise trained (Ex) or remained sedentary (Sed) for 16 –20 wk. During this time period, pigs continued to consume the HF or NF diet. The resulting experimental design consisted of four groups of pigs: 1) NF-Sed (n ⫽ 12), 2) NF-Ex (n ⫽ 12), 3) HF-Sed (n ⫽ 11), and 4) HF-Ex (n ⫽ 11). Plasma lipid and coronary artery function data from the pigs used in the present study have been reported elsewhere (21, 22). The results indicated that the HF diet significantly elevated plasma levels of cholesterol, triglycerides, and LDL cholesterol (21). In addition, EDR was impaired in coronary arteries of HF-Sed pigs (22).

Address for reprint requests and other correspondence: C. R. Woodman, Dept. of Biomedical Sciences, W108 Veterinary Medicine, 1600 E. Rollins Rd., Univ. of Missouri, Columbia, MO 65211 (e-mail: [email protected]).

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.

nitric oxide; prostacyclin; endothelium-derived hyperpolarizing factor; endothelial nitric oxide synthase; vascular smooth muscle

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Woodman, Christopher R., Mark A. Thompson, James R. Turk, and M. Harold Laughlin. Endurance exercise training improves endothelium-dependent relaxation in brachial arteries from hypercholesterolemic male pigs. J Appl Physiol 99: 1412–1421, 2005. First published June 16, 2005; doi:10.1152/japplphysiol.00293.2005.— We tested the hypothesis that exercise (Ex) training attenuates hypercholesterolemia-induced impairment of endothelium-dependent relaxation (EDR) in brachial (Br) arteries of adult male pigs by enhancing nitric oxide (NO)-mediated EDR. Adult male pigs were fed a normalfat (NF) or high-fat/cholesterol (HF) diet for 20 wk. Four weeks after the diet was initiated, pigs were trained or remained sedentary (Sed) for 16 wk, yielding four groups: 1) NF-Sed, 2) NF-Ex, 3) HF-Sed, and 4) HF-Ex. EDR of Br artery rings was assessed in vitro with acetylcholine (ACh) and bradykinin (BK). ACh- and BK-induced relaxation was not impaired by HF; however, relaxation responses were enhanced by Ex in NF and HF arteries. To determine the mechanism(s) by which Ex improved EDR, ACh- and BK-induced relaxation was assessed in the presence of NG-nitro-L-arginine methyl ester (LNAME; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), or L-NAME ⫹ Indo. ACh- and BK-induced relaxation was inhibited by L-NAME, and L-NAME ⫹ Indo, in all groups of arteries. Indo did not inhibit ACh-induced relaxation in any group but did inhibit BK-induced relaxation in HF-Ex arteries. In the presence of L-NAME or L-NAME ⫹ Indo, ACh- and BK-induced relaxation in HF-Ex arteries remained greater than in HF-Sed arteries. However, in the presence of Indo, ACh-induced relaxation in HF-Ex arteries was no longer greater than in HF-Sed arteries. These results indicate that EDR is not impaired by hypercholesterolemia in Br arteries from adult male pigs; however, Ex improves EDR in HF Br arteries by enhancing production of endothelium-derived hyperpolarizing factor and/or prostacyclin.

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Training Program All pigs were familiarized with running on a motorized treadmill and randomly assigned to Ex or Sed groups for 16-wk. The Ex group completed a 16-wk endurance exercise training program as described previously (13, 21, 28). Pigs assigned to the Sed group were restricted to their enclosures (2 ⫻ 4-m pens) and did not exercise. The training program resulted in significant increases in run time to exhaustion, heart weight-to-body weight ratio, and citrate synthase activity measured in the deltoid muscle. These data have been published previously (21). In Vitro Assessment of Vasorelaxation

Krebs bicarbonate buffer solution contained (in mM) 131.5 NaCl, 5.0 KCL, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 11.2 glucose, 20.8 NaHCO3, 0.003 propranolol, and 0.025 EDTA. Solutions were aerated with 95% O2-5% CO2 (pH 7.4) and maintained at 37°C. All drugs and chemicals were purchased from Sigma Chemical. Statistical Analysis All values are means ⫾ SE. Between-group differences in arterial ring characteristics, maximal relaxation, and half-maximal effective dose (ED50) values were determined using one-way ANOVA. Means of the ED50 values are presented as the negative log of the molar concentration. Concentration-response curves were analyzed by twoway ANOVA with repeated measures on one factor (dose). When a significant F value was obtained, post hoc analyses were performed using Duncan’s multiple-range test. Statistical significance was set at the P ⱕ 0.05 probability level. RESULTS

Quantification of eNOS, Superoxide Dismutase-1, and Caveolin-1 Expression

Vascular Ring Characteristics

Immunohistochemistry. Samples of Br artery were dissected and immersed in 10% formalin for a minimum of 24 h. Three-millimeterlong rings were processed routinely to paraffin embedment. Fivemicrometer-thick sections were cut with an automated microtome (Microm), floated onto positively charged slides (Fischer), and deparaffinized. The slides were steamed in citrate buffer at pH 6.0 (Dako target retrieval solution S1699) for 30 min to achieve antigen retrieval and then cooled for 30 min. The slides were stained manually with sequential Tris buffer, and water wash steps were performed after each protocol step. Sections were incubated with avidin biotin twostep blocking solution (Vector SP-2001) to inhibit background staining and in 3% hydrogen peroxide to inhibit endogenous peroxidase. Nonserum protein block (Dako X909) was applied to inhibit nonspecific protein binding. The primary antibodies utilized were mouse monoclonal anti-eNOS (BD Transduction Laboratories), rabbit polyclonal anti-superoxide dismutase-1 (SOD-1; Stressgen Biotechnology), and rabbit polyclonal anti-caveolin-1 (Cav-1; Santa Cruz LabJ Appl Physiol • VOL

Solutions and Drugs

Br artery ring characteristics are presented in Table 1. One-way ANOVA revealed that outer diameter, inner diame-

Table 1. Characteristics of brachial arteries Variable

NF-Sed (n ⫽ 12)

NF-Ex (n ⫽ 12)

HF-Sed (n ⫽ 11)

HF-Ex (n ⫽ 11)

Outer diameter, mm Inner diameter, mm Wall thickness, mm Axial length, mm

2.39⫾0.06 1.23⫾0.11 0.58⫾0.04 2.90⫾0.09

2.43⫾0.09 1.17⫾0.11 0.63⫾0.03 2.64⫾0.09

2.24⫾0.05 1.08⫾0.06 0.58⫾0.02 2.84⫾0.09

2.23⫾0.16 1.09⫾0.10 0.57⫾0.04 2.54⫾0.15

Values are means ⫾ SE; n, no. of pigs. NF-Sed, normal-fat sedentary; NF-Ex, normal-fat exercise trained; HF-Sed, high-fat sedentary; HF-Ex, highfat exercise trained. All data were analyzed by one-way ANOVA. ANOVA revealed no significant between-group differences.

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Vascular ring preparation. Segments of the Br artery were removed and trimmed of connective tissue and fat. Vessel segments were taken from the same sites in all pigs. A Filar-calibrated micrometer eyepiece was used to measure axial length, inner diameter, and outer diameter of each Br ring. Vasomotor reactivity was examined with the rings stretched to the length that produced maximal active tension, as described previously (27, 28). Relaxation responses of arterial rings. Procedures used to assess vasoactive responses of Br rings have been published previously in detail (28). Before dose-response curves were initiated, all arterial rings were preconstricted with prostaglandin F2␣ (PGF2␣; 30 ␮M). To determine whether hypercholesterolemia produced generalized or selective endothelial dysfunction, EDR was assessed using acetylcholine (ACh; 10⫺10 to 10⫺4 M) and bradykinin (BK; 10⫺11 to 10⫺6 M). Endothelium-independent relaxation was assessed with sodium nitroprusside (SNP; 10⫺10 to 10⫺4 M). A total of 4 Br rings were studied in parallel from each pig. In arterial ring 1, vasorelaxation to agonist alone was measured by adding cumulatively increasing doses of the selected drug to the organ bath while measuring changes in force. In arterial ring 2, the role of NO in vasoactive responses was assessed in the presence of NG-nitro-L-arginine methyl ester (L-NAME; 300 ␮M) to block NOS. In arterial ring 3, the importance of prostacyclin (PGI2) in vasoactive responses was assessed in the presence of indomethacin (Indo; 5 ␮M) to block cyclooxygenase (COX). In arterial ring 4, double blockade with L-NAME ⫹ Indo was used to assess the importance of NOS- and COX-independent mechanisms of relaxation. The experimental protocol was designed such that ACh was always the first agonist administered, followed by BK and SNP. At the end of each dose-response protocol, bicarbonate buffer solution was replaced to wash out the drug, and the arterial segments were allowed 1 h to stabilize before initiation of the next protocol.

oratories). All primary antibodies were diluted 1:800 and incubated with the tissue sections overnight at 4°C. After appropriate washing steps were completed, the sections were incubated with biotinylated anti-mouse or -rabbit link secondary antibody in PBS containing 15 mM sodium azide and peroxidase-labeled streptavidin (Dako LSAB⫹ kit, peroxidase, K0690). Diaminobenzidine (Dako) applied for 5 min allowed visualization of primary antibody staining. Sections were counterstained with Mayer’s hematoxylin stain for 1 min, dehydrated, and coverslipped. For negative controls, histological sections were prepared as described above, but incubation in primary antibody was excluded from the protocol. Sections were examined and photographed using an Olympus BX40 photomicroscope. Immunoblot analysis. Relative differences in eNOS, SOD-1, and Cav-1 protein content in Br artery rings were assessed using immunoblot analysis as described previously in detail (28). eNOS protein content was evaluated with a monoclonal antibody (1: 1,600; Transduction Laboratories). SOD-1 protein content was assessed with a polyclonal antibody (1:1,600; Stressgen). Cav-1 protein content was determined with a monoclonal antibody (1: 250, Transduction Laboratories). Immunoblots were evaluated by densitometry by using NIH Image software (National Institutes of Health, Bethesda, MD). Data were standardized such that the mean value of the NF-Sed arteries was set to 1.0, while NF-Ex, HF-Sed, and HF-Ex data were expressed as fold increase relative to the NF-Sed arteries.

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Fig. 1. Acetylcholine (ACh)-induced relaxation in brachial artery rings. Values are means ⫾ SE; n ⫽ 11–12 pigs per group. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)induced tension. NF, normal fat; HF, high fat; Sed, sedentary; Ex, exercise trained. Significantly different from 1NF-Sed and 3HF-Sed, P ⱕ 0.05.

ter, wall thickness, and axial length were similar in all groups of Br arteries. ACh Responses ACh elicited a concentration-dependent relaxation of Br rings from all groups of pigs (Fig. 1). Relaxation to ACh was not impaired by HF but was improved by exercise, such that ACh-induced relaxation was significantly greater in NF-Ex and HF-Ex arteries than in NF-Sed and HF-Sed arteries (Fig. 1, Table 2). In the presence of L-NAME, or L-NAME ⫹ Indo, ACh-induced relaxation was attenuated in all groups of Br arteries (Figs. 2 and 3); however, relaxation Table 2. Maximal relaxation and ED50 values for ACh-induced relaxation of brachial arteries from normal-fat- and high-fat-fed pigs Parameter Control Maximal relaxation, % ED50, ⫺log M L-NAME Maximal relaxation, % ED50, ⫺log M Indo Maximal relaxation, % ED50, ⫺log M L-NAME ⫹ Indo Maximal relaxation, % ED50, ⫺log M

NF-Sed (n ⫽ 12)

NF-Ex (n ⫽ 12)

HF-Sed (n ⫽ 11)

HF-Ex (n ⫽ 11)

74.4⫾2.5 86.6⫾3.4 78.0⫾7.1 ⫺7.77⫾0.07 ⫺8.21⫾0.09* ⫺7.61⫾0.07†

85.6⫾4.7 ⫺7.93⫾0.07†‡

48.8⫾6.3 63.8⫾5.6 ⫺7.37⫾0.09 ⫺7.72⫾0.10

40.4⫾5.1 ⫺7.22⫾0.08†

57.9⫾7.4 ⫺7.94⫾0.27*‡

65.5⫾5.0 82.2⫾3.5 ⫺7.68⫾0.07 ⫺8.03⫾0.10

64.0⫾5.2 ⫺7.38⫾0.16

71.2⫾5.5 ⫺7.71⫾0.37

49.5⫾6.4 60.5⫾7.0 ⫺7.20⫾0.17 ⫺7.55⫾0.09

37.8⫾6.4 54.5⫾6.2 ⫺6.04⫾0.74*†§ ⫺7.50⫾0.17

Values are means ⫾ SE; n, no. of pigs. ED50, half-maximal effective dose; ACh, acetylcholine; L-NAME, N G-nitro-L-arginine methylester; Indo, indomethacin. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)-induced tension. Significantly different from *NFSed, †NF-Ex, ‡HF-Sed, §HF-Ex: P ⱕ 0.05.

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Fig. 2. ACh-induced relaxation in brachial artery rings in the absence or presence of N G-nitro-L-arginine methyl ester (L-NAME; 0.3 mM) to inhibit nitric oxide synthase. A: NF pigs. B: HF pigs. Values are means ⫾ SE; n ⫽ 11–12 pigs per group. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)-induced tension. Significantly different from 1NF-Sed, 2NF-Ex, 3NF-Sed ⫹ L-NAME, 5HF-Sed, 6HF-Ex, and 7 HF-Sed ⫹ L-NAME: P ⱕ 0.05.

in the Ex arteries remained significantly greater than in Sed arteries (Figs. 2 and 3). Indo did not inhibit ACh-induced relaxation in NF arteries; thus, in the presence of Indo, ACh-induced relaxation in NF-Ex arteries remained greater than in NF-Sed arteries (Fig. 4A). In HF arteries, Indo did not significantly inhibit ACh-induced relaxation; however, in the presence of Indo, ACh-induced relaxation in HF-Ex arteries was no longer greater than in HF-Sed arteries (Fig. 4B).

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SNP Responses SNP elicited a concentration-dependent relaxation in Br rings from all groups of pigs (Fig. 9). Direct smooth muscle relaxation induced by SNP was similar in NF-Sed, NF-Ex, HF-Sed, and HF-Ex arteries. Immunohistochemistry Immunohistochemistry revealed staining for eNOS, SOD-1, and Cav-1 in Br arteries from all groups of pigs (Figs. 10 –12). eNOS was confined to the endothelium of Br arteries in all groups of arteries (Fig. 10). SOD-1 and Cav-1 stained both endothelium and smooth muscle of the tunica media (Figs. 11 and 12). In the absence of primary antibody

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Fig. 3. ACh-induced relaxation in brachial artery rings in the absence or presence of L-NAME (0.3 mM) and indomethacin (Indo; 5.0 ␮M) (both) to inhibit nitric oxide synthase and cyclooxygenase. A: NF pigs. B: HF pigs. Values are means ⫾ SE; n ⫽ 11–12 pigs per group. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)induced tension. Significantly different from 1NF-Sed, 2NF-Ex, 3NF-Sed ⫹ both, 5HF-Sed, 6HF-Ex, 7HF-Sed ⫹ both: P ⱕ 0.05.

BK Responses BK elicited a concentration-dependent relaxation of Br rings from all groups of pigs (Fig. 5). Relaxation to BK was not impaired by the HF diet, but it was improved by exercise in HF arteries, such that relaxation was greater in HF-Ex than in HF-Sed (Fig. 5, Table 3). In the presence of L-NAME, or L-NAME ⫹ Indo, BK-induced relaxation was inhibited in all groups of arteries (Figs. 6 and 7); however, relaxation in HF-Ex remained significantly greater than in HF-Sed arteries (Figs. 6 and 7). In the presence of Indo, BK-induced relaxation was not significantly inhibited in NF-Sed, NF-Ex, or HF-Sed arteries (Fig. 8, A and B). Indo inhibited BK-induced relaxation in HF-Ex arteries (Fig. 8B); however, in the presence of Indo, relaxation remained greater in HF-Ex than in HF-Sed arteries (Fig. 8B). J Appl Physiol • VOL

Fig. 4. ACh-induced relaxation in brachial rings in the absence or presence of Indo (5 ␮M) to inhibit cyclooxygenase. A: NF pigs. B: HF pigs. Values are means ⫾ SE; n ⫽ 11–12 pigs per group. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)-induced tension. Significantly different from 1NF-Sed, 2NF-Ex, 3NF-Sed ⫹ Indo, and 6HF-Ex: P ⱕ 0.05.

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against eNOS, SOD-1, or Cav-1, no immunoreactivity was detected. Immunoblot Analysis The effect of hypercholesterolemia and exercise training on eNOS, SOD-1, and Cav-1 protein content in Br rings is shown in Fig. 13. eNOS, SOD-1, and Cav-1 protein expression was not significantly altered by diet or exercise. DISCUSSION

The purpose of the present study was to test the hypothesis that hypercholesterolemia impairs EDR in Br arteries from adult male pigs. In addition, we hypothesized that exercise training would attenuate or reverse the detrimental effects of HF by enhancing NO- and/or EDHF-mediated, EDR. Results Table 3. Maximal relaxation and ED50 values for BKinduced relaxation of brachial arteries from normal-fat- and high-fat-fed pigs Parameter Control Maximal relaxation, % EC50, ⫺log M L-NAME Maximal relaxation, % EC50, ⫺log M Indo Max relaxation, % EC50, ⫺log M L-NAME ⫹ Indo Maximal relaxation, % EC50, ⫺log M

NF-Sed (n ⫽ 12)

NF-Ex (n ⫽ 12)

HF-Sed (n ⫽ 11)

HF-Ex (n ⫽ 11)

81.0⫾4.7 ⫺8.92⫾0.07

84.8⫾3.4 ⫺8.99⫾0.14

78.8⫾3.6 ⫺8.73⫾0.09

86.7⫾4.4 ⫺9.23⫾0.14‡

58.9⫾8.7 ⫺8.39⫾0.07

63.8⫾4.9 ⫺8.58⫾0.07

43.7⫾5.6 ⫺7.81⫾0.28†§

63.6⫾6.6 ⫺8.71⫾0.27

71.1⫾6.2 ⫺8.70⫾0.06

78.2⫾5.3 ⫺8.86⫾0.09

67.4⫾4.9 ⫺8.91⫾0.33

74.9⫾5.5 ⫺9.12⫾0.12

61.6⫾4.8 ⫺8.29⫾0.07

60.2⫾5.3 ⫺8.21⫾0.30

50.3⫾6.4 ⫺7.53⫾0.43*§

62.3⫾3.6 ⫺8.62⫾0.10

Values are means ⫾ SE; n, no. of pigs. BK, bradykinin. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣-induced tension (30 ␮M). Significantly different from *NF-Sed, †NF-Ex, ‡HF-Sed, §HF-Ex: P ⱕ 0.05.

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Fig. 6. BK-induced relaxation in brachial rings in the absence or presence of L-NAME (0.3 mM) to inhibit nitric oxide synthase. A: NF pigs. B: HF pigs. Values are means ⫾ SE; n ⫽ 11–12 pigs per group. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)induced tension. Significantly different from 1NF-Sed, 2NF-Ex, 5HF-Sed, 6 HF-Ex, and 7HF-Sed ⫹ NAME: P ⱕ 0.05.

indicate that, contrary to previous studies on female pigs (28), EDR was not impaired in Br arteries from HF male pigs. Consequently, these results reveal a sex difference in the adaptive response of Br arteries to conditions of hypercholesterolemia. Importantly, endothelial function was improved by exercise training in Br arteries from the male pigs used in the present study. Thus male pigs received a potentially important benefit from the endurance exercise training program, despite the lack of HF-induced endothelial dysfunction. The beneficial effect of exercise on ACh- and BK-induced relaxation of HF arteries persisted in the presence of L-NAME and L-NAME ⫹ Indo; however, in the presence of Indo, ACh-induced relaxation of HF-Ex arteries was no longer greater than in HF-Sed arteries. These results indicate that exercise training improves EDR in HF Br arteries from male pigs by enhancing production of EDHF and/or PGI2.

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Fig. 5. Bradykinin (BK)-induced relaxation in brachial artery rings. Values are means ⫾ SE; n ⫽ 11–12 pigs per group. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)-induced tension. Significantly different from 3HF-Sed, P ⱕ 0.05.

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the HF diet. Interestingly, the absence of endothelial dysfunction in Br arteries of HF male pigs occurred, despite significantly elevated plasma levels of cholesterol, triglyceride, and LDL cholesterol (21). In addition, EDR was impaired in coronary arteries isolated from these pigs (22). Thus, in adult male pigs, hypercholesterolemia induces endothelial dysfunction in coronary but not Br arteries, whereas in female pigs, hypercholesterolemia elicits endothelial dysfunction in coronary and Br arteries (27, 28). Influence of Exercise Training on EDR A primary objective of this study was to determine whether endurance exercise training attenuates or reverses the effects of hypercholesterolemia on EDR responses in male porcine Br arteries. Previous studies demonstrated that endurance exercise

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Fig. 7. BK-induced relaxation in brachial rings in the absence or presence of L-NAME (0.3 mM) and Indo (5.0 ␮M) to inhibit nitric oxide synthase and cyclooxygenase. A: NF pigs. B: HF pigs. Values are means ⫾ SE; n ⫽ 11–12 pigs per group. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)-induced tension. Significantly different from 1NF-Sed, 2NF-Ex, 5HF-Sed, 6HF-Ex, and 7HF-Sed ⫹ both: P ⱕ 0.05.

Influence of Hypercholesterolemia on EDR Hypercholesterolemia-induced endothelial dysfunction has been reported previously in coronary and Br arteries of adult female pigs (27, 28). In addition, hypercholesterolemia impairs endothelial function in coronary arteries from adult male pigs (22). In each of these studies, the endothelial dysfunction associated with hypercholesterolemia was attributed in part to a reduction in NO bioavailability. In the present study, we examined the effects of hypercholesterolemia on EDR in Br arteries from adult male swine. Contrary to results obtained from HF female pigs (28), endothelial function was not impaired in Br arteries from hypercholesterolemic male pigs (Figs. 1 and 5). Nor were the relative contributions of known endothelium-derived dilator substances (NO, PGI2, EDHF) accounting for EDR altered by J Appl Physiol • VOL

Fig. 8. BK-induced relaxation in brachial rings in the absence or presence of Indo (5 ␮M) to inhibit cyclooxygenase. A: NF pigs. B: HF pigs. Values are means ⫾ SE; n ⫽ 11–12 pigs per group. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)induced tension. Significantly different from 5HF-Sed, 6HF-Ex, and 7HFSed ⫹ Indo: P ⱕ 0.05.

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training prevents or reverses the detrimental effects of hypercholesterolemia on EDR of coronary and Br arteries from female pigs by enhancing NO- and EDHF-mediated relaxation, respectively (27, 28). Therefore, we hypothesized that exercise training would attenuate or reverse the detrimental effects of hypercholesterolemia in Br arteries from male pigs by enhancing NO- and/or EDHF-mediated, EDR. Results from the present study revealed that exercise improved EDR in Br arteries from HF male pigs. The improvement in EDR in HF arteries was characterized by increased sensitivity to BK and ACh. Indeed, BK-induced relaxation was enhanced in HF arteries at the lowest dose of BK administered. Enhanced sensitivity of HF arteries to BK and ACh, in the absence of change in maximal relaxation, suggests the possibility that exercise altered receptor-second-messenger coupling in these arteries. Thus exercise enhanced endothe-

Fig. 10. Immunohistochemistry on porcine brachial artery rings. Cross section of brachial arteries was stained for endothelial nitric oxide synthase (eNOS). Arrows show intense staining in endothelial cells.

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Fig. 9. Sodium nitroprusside (SNP)-induced relaxation in brachial artery rings. Values are means ⫾ SE; n ⫽ 11–12 pigs per group. Percent relaxation was calculated as percent reduction in force from prostaglandin F2␣ (30 ␮M)induced tension. ANOVA revealed no significant between-group differences.

lial function in HF Br arteries, despite the lack of HFinduced endothelial dysfunction. Interestingly, in NF arteries exercise enhanced ACh-induced EDR, whereas BKinduced relaxation was not altered. The mechanism accounting for the differential effect of exercise on EDR responses in NF arteries is not known. To determine whether exercise improved endothelial function of Br arteries by enhancing bioavailability of NO, AChand BK-induced relaxation was assessed in the presence of L-NAME to inhibit NOS. Although results clearly indicate that NO contributed to EDR in male Br arteries, the finding that ACh- and BK-induced relaxation in HF-Ex remained greater than in HF-Sed in the presence of L-NAME indicates that the beneficial effect of exercise was not mediated by increased production of NO. These results are similar to previous results in adult female pigs, indicating that exercise training-induced improvements in endothelial function of Br arteries are mediated by a vasodilator other than NO (28). To further evaluate the effects of exercise on the NO pathway, immunohistochemical and immunoblot analyses for eNOS, SOD-1 and Cav-1 were performed on Br artery rings. In NF and HF arteries, eNOS expression was restricted to the endothelium, whereas SOD-1 and Cav-1 were expressed in endothelium and smooth muscle cells of the tunica media (Figs. 10 –12). Importantly, eNOS, SOD-1, and Cav-1 protein content was not significantly altered by exercise training (Fig. 13). Thus these results are consistent with the interpretation that the beneficial effect of exercise was not mediated by enhanced production or stability of NO. To determine whether exercise improved endothelial function of Br arteries by enhancing bioavailability of PGI2, relaxation responses were assessed in the presence of Indo to inhibit COX. In the presence of Indo, ACh-induced relaxation was no longer greater than in HF-Sed arteries (Fig. 4B). These results suggest that enhanced EDR in Br

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Fig. 11. Immunohistochemistry on porcine brachial artery rings. Cross section of Br arteries was stained for superoxide dismutase-1 (SOD-1). Arrows show intense staining in endothelial cells. Arrowheads show moderate staining in smooth muscle cells.

indicating that the beneficial effect of exercise on Br arteries is mediated by a vasodilator pathway independent of NOS and COX (possibly EDHF). To assess the effects of hypercholesterolemia and exercise training on vascular smooth muscle function, relaxation responses to SNP were assessed in Br artery rings (Fig. 9). SNP-induced relaxation was similar in all groups of arteries, indicating that the ability of vascular smooth muscle to relax to NO was not impaired by hypercholesterolemia or improved by exercise training. These results are in agreement with results from female pigs, indicating that improved relaxation responses in Br artery rings are not due to

Fig. 12. Immunohistochemistry on porcine brachial artery rings. Cross section of Br arteries was stained for caveolin-1 (Cav-1). Arrows show intense staining in endothelial cells. Arrowheads show moderate staining in smooth muscle cells.

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arteries from HF-Ex pigs may be due in part to increased production of PGI2. To evaluate the potential role of EDHF in exerciseinduced improvements in EDR of Br arteries (28), we also examined ACh- and BK-induced relaxation in the presence of L-NAME ⫹ Indo. In the presence of double blockade, residual relaxation to ACh or BK can be attributed to a vasodilator molecule other than NO or PGI2. Results indicated that ACh- and BK-induced relaxation remained significantly greater in Ex arteries than in Sed arteries in the presence of both enzyme inhibitors (Figs. 3 and 7). These results are in accord with data obtained from female pigs (28),

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ACKNOWLEDGMENTS The authors gratefully acknowledge the expert technical assistance of Pam Thorne, Denise Holiman, Jennifer Casati, and Tammy Strawn. GRANTS This work was supported by National Heart, Lung, and Blood Institute Grants HL-52490 and HL-36088 (to M. H. Laughlin) and National Institute on Aging Grant AG-00988 (to C. R. Woodman). REFERENCES

Fig. 13. Immunoblot analysis for eNOS (A), SOD-1 (B), and Cav-1 (C) protein expression in porcine brachial arteries. Values are means ⫾ SE; n ⫽ 8 pigs per group. ANOVA revealed no significant between-group differences.

vascular smooth muscle adaptation to the exercise-training program (28). Sex Perspectives A primary objective of this study was to determine whether hypercholesterolemia-induced endothelial dysfunction, and exercise training-induced improvements in EDR, reported previously in Br arteries from hypercholesterolemic female pigs (28), also occurs in Br arteries from male pigs. Results indicated that neither EDR nor the mechanisms accounting for EDR were altered in Br arteries from hypercholesterolemic male pigs (Figs. 1 and 5). By comparison, hypercholesterolemia attenuates EDR in Br arteries from female pigs fed the same HF diet by impairing production and/or release of NO and PGI2 (28). Thus the primary effects of hypercholesterolemia on endothelial function of Br arteries are influenced by J Appl Physiol • VOL

1. Best PJM, Lerman LO, Romero JC, Richardson D, Holmes DR, and Lerman A. Coronary endothelial function is preserved with chronic endothelin receptor antagonism in experimental hypercholesterolemia in vitro. Arterioscler Thromb Vasc Biol 19: 2769 –2775, 1999. 2. Casino PR, Kilcoyne CM, Quyyumi AA, Hoeg JM, and Panza JA. The role of nitric oxide in endothelium-dependent vasodilation of hypercholesterolemic patients. Circulation 88: 2541–2547, 1993. 3. Cohen RA, Zitnay KM, Haudenschild CC, and Cunningham LD. Loss of selective endothelial cell vasoactive functions caused by hypercholesterolemia in pig coronary arteries. Circ Res 63: 903–910, 1988. 4. Dixon JL, Stoops JD, Parker JL, Laughlin MH, Weisman GA, and Sturek M. Dyslipidemia and vascular dysfunction in diabetic pigs fed an atherogenic diet. Arterioscler Thromb Vasc Biol 19: 2981–2992, 1999. 5. Dzau, VJ. Pathobiology of atherosclerosis and plaque complications. Am Heart J 128: 1300 –1304, 1994. 6. Komori K, Shimokawa H, and Vanhoutte PM. Hypercholesterolemia impairs endothelium-dependent relaxations to aggregating platelets in porcine iliac arteries. J Vasc Surg 10: 318 –325, 1989. 7. Kuo L, Davis MJ, Cannon MS, and Chilian WM. Pathophysiological consequences of atherosclerosis extend into the coronary microcirculation. Restoration of endothelium-dependent responses by L-arginine. Circ Res 70: 465– 476, 1992. 8. Lamping KG, Nuno DW, Chappell DA, and Faraci FM. Agonistspecific impairment of coronary vascular function in genetically altered, hyperlipidemic mice. Am J Physiol Regul Integr Comp Physiol 276: R1023–R1029, 1999. 9. Laughlin MH. Endothelium-mediated control of coronary vascular tone after chronic exercise training. Med Sci Sports Exerc 27: 1135–1144, 1995. 10. Laughlin MH, Pollock JS, Amann JF, Hollis ML, Woodman CR, and Price EM. Training induces nonuniform increases in eNOS content along the coronary arterial tree. J Appl Physiol 90: 501–510, 2001.

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sex. It is important to note, however, that hypercholesterolemic male pigs received an important benefit from exercise, despite the lack of HF-induced endothelial dysfunction. Specifically, ACh- and BK-induced relaxation was improved by exercise training. The improvement in relaxation in HF arteries persisted in the presence of L-NAME, indicating that exercise improved EDR in HF Br arteries by enhancing production of a vasodilator other than NO. In addition, the finding that enhanced EDR in HF Br arteries persisted in the presence of L-NAME ⫹ Indo indicates that the beneficial effect of exercise was mediated in part by enhanced production of EDHF. These data are in accord with results obtained from hypercholesterolemic female pigs, indicating that exercise improves EDR of Br arteries by enhancing EDHF-mediated relaxation. Thus, whereas there are sex differences in adaptation of Br arteries to hypercholesterolemia, adaptations induced by exercise training are similar in Br arteries from male and female pigs. In conclusion, the results of this study indicate that EDR of Br arteries from male pigs was not impaired by 20 wk of hypercholesterolemia. Nor was the mechanism mediating EDR altered by the HF diet. Endurance exercise training improved endothelial function in HF Br arteries by enhancing production of EDHF and/or PGI2.

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20. Sun D, Huang A, Koller A, and Kaley G. Adaptation of flow-induced dilation of arterioles to daily exercise. Microvasc Res 56: 54 – 61, 1998. 21. Thomas TR, Pellechia J, Rector RS, Sun GY, Sturek MS, and Laughlin MH. Exercise training does not reduce hyperlipidemia in pigs fed a high fat diet. Metabolism 51: 1587–1595, 2002. 22. Thompson MA, Henderson KK, Woodman CR, Turk JR, Rush JWE, Price EM, and Laughlin MH. Exercise preserves endothelium-dependent relaxation in coronary arteries of hypercholesterolemic male pigs. J Appl Physiol 96: 1114 –1126, 2004. 23. Vita JA and Keaney JF. Endothelial function: a barometer for cardiovascular risk? Circulation 106: 640 – 642, 2002. 24. Wang J, Wolin MS, and Hintze TH. Chronic exercise enhances endothelium-mediated dilation of epicardial coronary artery in conscious dogs. Circ Res 73: 829 – 838, 1993. 25. Wilson SH, Simari RD, Best PJM, Peterson TE, Lerman LO, Aviram M, Nath KA, Holmes DR, and Lerman A. Simvastatin preserves coronary endothelial function in hypercholesterolemia in the absence of lipid lowering. Arterioscler Thromb Vasc Biol 21: 122–128, 2001. 26. Woodman CR, Muller JM, Laughlin MH, and Price EM. Induction of nitric oxide synthase mRNA in coronary resistance arteries isolated from exercise-trained pigs. Am J Physiol Heart Circ Physiol 273: H2575– H2579, 1997. 27. Woodman CR, Turk JR, Rush JWE, and Laughlin MH. Exercise attenuates the effects of hypercholesterolemia on endothelium-dependent relaxation in coronary arteries from adult female pigs. J Appl Physiol 96: 1105–1113, 2004. 28. Woodman CR, Turk JR, Williams DP, and Laughlin MH. Exercise training preserves endothelium-dependent relaxation in brachial arteries from hyperlipidemic pigs. J Appl Physiol 94: 2017–2026, 2003.

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11. Laughlin MH, Schrage WG, McAllister RM, Garverick HA, and Jones AW. Interaction of gender and exercise training: vasomotor reactivity of porcine skeletal muscle arteries. J Appl Physiol 90: 216 –227, 2001. 12. McAllister RM and Laughlin MH. Short-term exercise training alters responses of porcine femoral and brachial arteries. J Appl Physiol 82: 1438 –1444, 1997. 13. Muller JM, Meyers PR, and Laughlin MH. Vasodilator responses of coronary resistance arteries of exercise-trained pigs. Circulation 89: 2308 –2314, 1994. 14. Nava E, Noll G, and Luscher TF. Nitric oxide in cardiovascular diseases. Ann Med 27: 343–351, 1995. 15. Parker JL, Oltman CL, Muller JM, Meyers PR, Adams HR, and Laughlin MH. Effects of exercise training on regulation of tone in coronary arteries and arterioles. Med Sci Sports Exerc 26: 1252–1261, 1994. 16. Sessa WC, Pritchard K, Seyedi N, Wang J, and Hintze TH. Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res 74: 349 –353, 1994. 17. Shimokawa H and Vanhoutte PM. Impaired endothelium-dependent relaxation to aggregating platelets and related vasoactive substances in porcine coronary arteries in hypercholesterolemia and atherosclerosis. Circ Res 64: 900 –914, 1989. 18. Shimokawa H and Vanhoutte PM. Hypercholesterolemia causes generalized impairment of endothelium-dependent relaxation to aggregating platelets in porcine arteries. J Am Coll Cardiol 13: 1402–1408, 1989. 19. Sun D, Huang A, Koller A, and Kaley G. Short-term daily exercise activity enhances endothelial NO synthesis in skeletal muscle arterioles of rats. J Appl Physiol 76: 2241–2247, 1994.

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