INTRODUCTION

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MULTIPLE RISK FACTORS are associated with human coronary artery disease (CAD) (10). One factor, gender, has been known for many years, with the incidence of CAD in men exceeding that in premenopausal women of similar age (18), and, recently, a sedentary lifestyle has been identified as a risk factor (7). Few studies, however, have evaluated the effects of both gender and exercise (Ex) on vascular responses. One study showed that the increased blood flow conductance in calf muscles associated with exercise training occurred equally in young men and women (23).

Isolated arteries from animal models have been used to study gender differences independent of acutely acting neural and endocrine factors. For instance, aorta (denuded of endothelium) from male rats exhibited a two- to fourfold increase in sensitivity to serotonin, phenylephrine (PE), norepinephrine (NE), and PGF₂ compared with that of females (16, 19, 22). Similarly, hepatic arteries (endothelium intact) from male rabbits exhibited a twofold increase in sensitivity to NE (3). On the other hand, no gender differences were observed in the sensitivity of coronary arteries (denuded of endothelium) to endothelin-1 (ET-1) in either pigs or rabbits (1, 13). It has been proposed that estrogens reduce the risk of vascular disease by their influence on endothelial functions (12, 27). However, direct effects on vascular smooth muscle have been reported recently that are independent of endothelial release of nitric oxide (5). The relaxation response to estrogen was closely related to increased K-channel activity and reduced Ca entry.

Ex is associated with significant coronary adaptations that involve both smooth muscle and the endothelium (17). The sensitivity of proximal coronary arteries to K depolarization, ET-1, PGF₂, and NE was unaffected by Ex in female pigs, however (25, 26). In contrast to pigs, Ex reduced the sensitivity of male rabbit aorta to NE (4) and can negatively influence excitation-contraction coupling as indicated by a reduced release of cellular Ca (29). It is not known whether this negative effect occurs at the level of second-messenger production or interaction between the sarcoplasmic reticulum and membrane channels.

We designed this study to test the hypotheses that Ex reduces the sensitivity of coronary smooth muscle to ET-1, with the adaptation being greater in males. It was reasoned that estrogen levels in sedentary (Sed) females convey a protective effect (perhaps by activating K channels, which reduce the sensitivity to agonists), making them less responsive to the effects of Ex. We were concerned that previous Ex studies on porcine coronary arteries were limited to large conduit vessels from females (25, 26). The generality of these findings was tested by the inclusion of first-order branches of males, a group that is at higher risk for CAD in humans.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Tissues. The hearts were obtained from anesthetized male and female miniature swine (Charles River). The left circumflex arteries (1.6- to 2.2-mm OD) were significantly larger than the branches (P < 0.001; 0.7-1.5 mm OD, measured by a calibrated reticle in the dissection microscope) of all the major coronary arteries. All arteries were trimmed of loose connective tissues in a dissection solution at room temperature, and, for contractile studies, rings were denuded of endothelium by rotating them over the sharp edge of a scissor. This procedure reduced the bradykinin (1 µM)-induced relaxation to 10 ± 2 (SD) % of an ET-1 contracture, whereas rings with intact endothelium relaxed 89 ± 17%. No differences were observed in maximal force development to K depolarization [2.7 ± 0.2 vs. 2.5 ± 0.2 (SE) G; n = 11, with and without endothelium]. Rings were mounted on a force transducer (Grass) with an attached micrometer drive and underwent a series of four stretches (1.4 times the resting length) to achieve a stable and reproducible resting length. This was followed by incubation at 35°C in a physiological salt solution (PSS) gassed with air. The rings were tested with a high-K solution (K = 80 mM, substituted for Na) to establish a reference contracture. Only one concentration-response curve was done on each ring, and siliconized cups were used to hold solutions that contained the cumulative additions of ET-1. Arteries used for phosphatidic acid (PA) assay were mounted on wires to facilitate handling. They were exposed to N^G-nitro-L-arginine methyl ester (30 µM) and indomethacin (3 µM) to inhibit the production of endothelial dilators and bovine serum albumin (0.02% wt/vol) to maintain ET-1 in solution during the final incubation.

Training protocols. The procedures have been described and evaluated in detail (25, 26). Sexually mature male and female miniature swine of similar age (8-10 mo) and weight (25-35 kg) were randomly assigned to Ex or Sed groups. The swine were fed laboratory minipig diet (PMI Feeds) in the amount of 2.5% body weight daily. The Ex group initially ran on the treadmill at 3 miles/h (mph), 0% grade, for 20-30 min and at 5 mph for 15 min. The speed and duration were progressively increased, yielding a typical session at 12 wk of 1) 5-min run at 2.5 mph, 2) 15 min at 5-8 mph (depending on the particular pig's ability), 3) 60 min at 4-5 mph, and 4) 5 min at 2 mph. The pigs received positive reinforcement by being fed at the end of the sessions. The estrous cycle (22 days) was not significantly altered by the Ex protocol. The plasma 17-estradiol measured by radioimmunoassay (Animal Sciences Research Center, Univ. of Missouri, Columbia, MO) were also similar (3.3 ± 1.0 vs. 2.7 ± 0.4 ng/ml for Sed females vs. Ex females). The training protocol lasted 16-20 wk, and at term the Ex and Sed swine were of similar age (13-16 mo).

Treadmill performance test. The test was similar to that applied previously, which consisted of four stages (25, 26). The pigs ran at 3.1 mph, 0% grade, for 5 min during stage 1 followed by 3.1 mph, 10% grade, for 10 min at stage 2. The pigs then ran for 10 min at 4.3 mph, 10% grade, at stage 3 followed by 6 mph, 10% grade, until exhausted. Heart rates were monitored as well as the time for each performance stage. The efficacy of training was evaluated by comparing the heart weight-to-body weight ratios (g/kg), the running time to exhaustion for the treadmill test, and the skeletal muscle oxidative capacity between Sed and Ex. At time of death, samples from the long head of the triceps brachii muscle were frozen in liquid N₂ and stored at 70°C until assayed. Citrate synthase activity in whole-muscle homogenates (µmol · min¹ · g wet wt¹) was measured spectrophotometrically by the method of Srere (28).

Reagents and solutions. Normal PSS had the following composition (in mM): 138 NaCl, 5 KCl, 1.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 10 Na HEPES (pH 7.4), and 11.2 glucose. The dissection and the PO₄-free solutions were the same except the former contained low Ca (0.2 mM), and PO₄ was omitted from the latter. ET-1 was purchased from Peninsula Laboratory and was diluted into 10 mM acetic acid.

PA assay. Arteries were labeled with ³²PO₄ (50 µCi/ml in PO₄-free PSS) for 2 h at 35°C. The segments were placed in nonlabeled solutions 10 min before exposure to ET-1. After 10 min in ET-1, the arteries were freeze clamped to stop the response and to disrupt the tissue to facilitate extraction. They were placed immediately into 3 ml chloroform-methanol (1:2 vol/vol) for PA extraction and processing (14). The lipid phase was dried, resuspended in 30 µl chloroform, and spotted onto a Whatman LKGD plate (Whatman Laboratories). The plate was developed by using a TLC solvent system of chloroform-pyridine-88% formic acid (10:6:1 vol/vol). PA separated with a retardation factor of 0.5, which was identified by standards (Sigma Chemical). ³²P was measured by means of a storage phosphor screen exposed to the TLC plate for 2 h. The screen was scanned on a Molecular Dynamics phosphor imager and was analyzed by using Molecular Dynamics software. The PA values are expressed as a percentage of total ³²P counts per lane. The coefficient of variation for the method, estimated in Sed and Ex female pigs, was 16 and 20% of the mean at high ET-1 concentrations (1 × 10⁸ and 1 × 10⁷ M).

Data analyses. The contractile responses were normalized in terms of maximal response to ET-1. The excitatory concentration causing a 50% response (EC₅₀) was determined for each ring by linear interpolation between the log concentrations that produced responses just below and above 50%. Because the EC₅₀ values were normally distributed by a log rather than arithmetic scale, log values were used to make statistical comparisons. These were transformed to arithmetic means for presentation in the text. Student's t-test was used to test for differences between group means where one treatment was being evaluated. A two-way ANOVA and Tukey's post hoc test were used for multiple comparisons between treatments, e.g., Ex and gender. P < 0.05 was taken to be significant. Data are presented as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Three adaptations to Ex were evident in the pigs used for this study. As shown in Table 1, the heart weight-to-body weight ratios were increased 1.20- to 1.26-fold by Ex with no significant gender differences. Ex also increased the oxidative capacity of skeletal muscle (1.35-fold) without significant gender differences. The endurance time for the performance tests was increased 1.33- to 1.41-fold by Ex with no significant gender differences during Ex. These results indicate no significant gender effect on the efficacy of training, similar to findings in humans (23).

The maximal tension responses to K depolarization in branches from Sed males (2.4 ± 0.2 g) was not significantly different from the Ex groups used for ET-1 contractile responses (Fig. 1). Similarly, the ratio of maximum ET-1 response to K depolarization did not differ between groups (1.12 ± 0.03 and 1.13 ± 0.07 in Sed and Ex males vs. 1.17 ± 0.05 and 1.15 ± 0.03 in females, respectively). Although we found no evidence for a significant difference in the maximal contractile force generated by arteries from any of the groups, we still normalized the concentration responses to ET-1 as a percentage of the maximal response to facilitate the computation of EC₅₀. The ET-1 contractile response of the coronary branches (Fig. 1) was significantly shifted to the right in the Ex males, whereas Ex had no significant effect on the responses of the females. The EC₅₀ was significantly changed by Ex in males (0.24 ± 0.08 log unit, ~2-fold; P < 0.05; n = 8), whereas no change took place in females. Similar responses were observed in the left circumflex arteries, with Ex inducing a shift of 0.35 ± 0.15 log unit, (P < 0.05; n = 8) in males and only 0.02 ± 0.08 (not significant; n = 15) in females.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Contractile responses to endothelin-1 by branches of left circumflex artery from miniature pigs. Values are means ± SE; n = 19 for both sedentary (Sed) and exercise-trained (Ex) females (A), n = 8 for Sed males and n = 7 for Ex males (B). Values of log excitatory concentration causing a 50% response (EC50) for Sed and Ex females are 9.15 ± 0.07 and 9.13 ± 0.08, respectively, and are 9.37 ± 0.06 and 9.13 ± 0.06 for Sed and Ex males, respectively. Brackets denote concentration. Note that Ex males exhibited a significant decrease in response to 3 × 1010 and 1 × 109 M ET-1 and a shift of the curve to the right (* P <0.05 repeated-measures 2-way ANOVA). No significant differences were observed for Ex in the females.

Changes in K-channel activity are regarded to have important modulatory effects in response to agonists with estrogenic effects recently reported (5, 24). We measured the sensitivity of branches to ET-1 in the presence of high concentrations of a K-channel inhibitor, tetraethylammonium (TEA), to provide a general test of the hypothesis that K channels play an inhibitory role in the sensitivity to ET-1. As shown in Fig. 2, TEA significantly changed the EC₅₀ in Ex males (1.25-fold from baseline) and Ex females (2.2-fold from baseline) to lower concentrations with no significant effect in the Sed groups. The females (combined Sed and Ex) exhibited a greater response to TEA (0.26 ± 0.08, n = 14) than did males (0.03 ± 0.04; n = 15; P < 0.01, ANOVA).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Change () in contractile EC50 for ET-1 in coronary branches associated with incubation in tetraethylammonium (TEA; 30-50 mM). Control values were subtracted from those determined in presence of TEA and plotted as log. Values are means ± SE; n = 8 for Sed male, n = 7 for Ex male, and n = 7 in each female group. Note that TEA reduced EC50 significantly from baseline (log = 0) in both Ex males and Ex females (* P < 0.05) but not in Sed pigs.

ET-1 has been shown to activate phospholipase (PL) activity in vascular smooth muscle (21). PA is produced either directly from phospholipid by PLD or indirectly by phosphorylation of diacylglycerol produced by PLC (14). We measured changes in [³²P]PA as an indicator of increased phospholipase activity in branches exposed to ET-1 (Fig. 3). ET-1 increased [³²P]PA in both males and females, with similar EC₅₀ values. The magnitude of the responses, however, were reduced by Ex in both males and females. No gender differences were observed in the Sed and Ex groups.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Changes in [32P]phosphatidic acid (PA) caused by ET-1 in coronary branches from miniature pigs. Values are means ± SE; n = 4-7 for Sed females, n = 4-7 Sed females, n = 5-9 for Ex females (A); n = 10-13 for Sed males, n= 4-8 for Ex males (B). Log EC50 values for Sed and Ex females are 8.72 ± 0.05 and 8.75 ± 0.09, respectively, and for Sed and Ex males are 8.91 ± 0.08 and 8.83 ± 0.08, respectively. Note that Ex females exhibited a significant decrease in response to 1 × 108 and 1 × 107M ET-1 (* P < 0.05, ANOVA). Both Ex females (P < 0.02, ANOVA) and Ex males (P < 0.05, ANOVA) exhibited a significant decrease in response over the range of 1 × 1010 to 1 × 107 M.

	DISCUSSION

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Our study of gender and Ex showed Ex to have a greater effect on males in reducing the contractile sensitivity to ET-1 without changing the maximal contractile force. Branches from Ex of both males and females showed a significant increase in ET-1 sensitivity to nonselective blockade of K-channels, whereas the Sed did not. A gender difference was observed, with the females exhibiting a greater response to TEA than males. Receptor-PL signaling was also affected by Ex with a reduced magnitude in the [³²P]PA response; however, no significant gender effects were observed.

Gender. Studies in rats and rabbits have shown increased sensitivity of male arteries to serotonin, NE, PE, and PGF₂ (3, 16, 19, 22). No significant difference in the maximal response of males and females was reported, consistent with our observations. Our measures of sensitivity were in agreement with those made in pig and rabbit coronary arteries where major differences were not observed (1, 13). It is relevant that a systematic study of ET-1-receptor binding in pig coronary arteries showed no significant differences in maximum binding or affinity between males and females (1).

Exercise training. Our results indicated that Ex significantly reduced the sensitivity of coronary arteries to ET-1 in male pigs. The shift was present in two vascular sites and could be expressed throughout the coronary bed. Evidence that ET-1 can contribute to angina and myocardial infarction has been reviewed with the conclusion that ET-1-receptor antagonists would be effective therapy (8). The antagonists would have a similar effect to Ex by shifting the ET-1 concentration-response curves to the right. A positive outcome of our study is that males exhibit an Ex response that reduces ET-1 sensitivity and perhaps risk to CAD.

Conclusions. The contractile sensitivity of male coronary arteries to ET-1 is reduced by Ex but is not altered in females with similar efficacy of Ex. Females (combined Sed and Ex) exhibited a greater shift than did males in ET-1 contractile sensitivity (EC₅₀) when K-channels were blocked with TEA. TEA significantly reduced the EC₅₀ in both Ex males and females, whereas Sed males and females were not significantly affected. Ex reduced the ET-1-stimulated production of [³²P]PA in both males and females. Further study is required to delineate why Ex had a significant effect on ET-1 contractile sensitivity only in females, whereas Ex significantly affected contractile responses to TEA and ET-1 stimulation of [³²P]PA production in both males and females. A potential gender difference in K-channel function may contribute to this discrepancy.