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Am J Physiol Heart Circ Physiol 293: H1553-H1563, 2007. First published May 25, 2007; doi:10.1152/ajpheart.00151.2007
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Influence of sex, high-fat diet, and exercise training on potassium currents of swine coronary smooth muscle

Yan Yang,2,5 Allan W. Jones,2,4,5 Tom R. Thomas,3 and Leona J. Rubin1,4,6

1Departments of Biomedical Sciences, 2Medical Pharmacology and Physiology, 3Nutrition Science, 4Dalton Cardiovascular Research Center, 5Center for Diabetes and Cardiovascular Health, and 6Center for Gender Physiology and Environmental Adaptations. University of Missouri, Columbia, Missouri

Submitted 6 February 2007 ; accepted in final form 24 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Potassium channels in vascular smooth muscle (VSM) control vasodilation and are potential regulatory targets. This study evaluated effects of sex differences, exercise training (EX), and high-fat diet (HF) on K+ currents (IK) of coronary VSM cells. Yucatan male and female swine were assigned to either sedentary confinement (SED), 16 wk of EX, 20 wk of HF, or 20 wk of HF with 16 wk of EX (HF-EX). VSM cells of normal-diet SED animals exhibited three components of IK: 4-aminopyridine-sensitive IK(KV), TEA-sensitive IK(BK), and 4-aminopyridine + TEA-insensitive IK. Females exhibited significantly higher basal IK than males in the same group. EX increased basal IK in males and females. HF reduced IK in males and females and nullified effects of EX. Endothelin-1 increased IK significantly in males but not in females. In the presence of endothelin-1, 1) IK(KV) was similar in SED males and females and EX increased IK(KV) to a greater extent in males than in females and 2) IK(BK) was greater in SED females than in males and EX increased IK(BK) to a greater extent in males, resulting in IK(BK) similar to EX females. Importantly, HF nullified effects of EX on IK(KV) and IK(BK). These data indicate that basal IK of SED female swine is inherently greater than that shown in SED males and that males require EX to achieve comparable levels of IK. Importantly, HF reduced IK in males and females and nullified effects of EX, suggesting HF abrogates beneficial effects of EX on coronary smooth muscle.

gender; potassium channel; hyperlipidemia

MULTIPLE RISK FACTORS INCLUDING sex and hyperlipidemia are associated with human coronary artery disease (CAD). For instance, increased risk associated with sex differences has been shown, with the incidence of CAD in men exceeding that in premenopausal women of similar age (57). The pig has often been used as a model for human vascular disease. In one study that emphasized sex effects, the relaxation of endothelial-denuded porcine coronary rings to natriuretic peptide was greater in females than in males, and this response was attenuated by K+ channel blockers (1). Sex differences in adenosine-induced relaxation of porcine arterioles also involved K+ channels such as voltage-dependent K+ channels (21). Sex differences have also been reported in the sensitivity of porcine coronary arteries to endothelin-induced constriction (26), which appears to result from differences in K+ channel activity. Evidence supports the hypothesis that coronary vasculature of female pigs may be more resistant to contractile agents because of increased K+ channel activity (21).

Hyperlipidemia resulting from a high-fat diet (HF) is a primary independent risk factor for CAD (57) and is associated with altered ion channel function of coronary microcirculation. Voltage-dependent K+ currents (IK) contributed less to adenosine relaxations in arterioles from hyperlipidemic swine than in arterioldes from animals fed a normal diet (22, 38). Hyperlipidemia was also associated with decreased voltage-dependent IK and adenosine sensitivity in arteries from swine coronary circulation (17). The small body of available evidence indicates that vascular K+ channels are a target for dysfunction induced by HF.

In contrast to the risk of hypercholesterolemia, exercise training (EX) appears to be beneficial and decreases the incidence of CAD, possibly by improving vascular endothelial cell function (47) and, as reviewed by Gielen and Hambrecht (19), by improving vascular function and myocardial perfusion in general. Miniature swine subjected to EX exhibited an increased coronary artery capacity that included an increase in blood flow capacity and capillary exchange (30). Some of the improved vascular function in swine could be attributed to altered vascular smooth muscle (VSM) function. For example, coronary arteries from EX male swine exhibited less contractile activity to endothelin than those from swine subjected to sedentary confinement (SED) (4). At least some of the altered tone associated with EX may result from increased K+ channel contribution (5, 26). Thus IK are targets for hyperlipidemia and EX and are sex dependent. To date, there have been no systematic studies of IK from swine models of both sexes treated with EX and HF. We report here an integrative study that addresses these factors. The approach of this study was first to compare IK in male and female smooth muscle cells from pig coronary arteries and then to assess whether specific K+ channel subtypes are modulated by EX and HF. This work tests the hypothesis that IK is greater in females than in males, thereby providing a greater level of protection to females from vasospasm. Furthermore, we predicted EX would increase IK and reduce the adverse effects of hyperlipidemia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal models and EX protocol. This study used sexually mature male (n = 28) and female (n = 30) Yucatan miniature swine of similar age (8–10 mo) and weight (25–40 kg). The animal research protocol was approved and conducted in compliance with the University of Missouri Animal Care and Use Committee guidelines. Miniature swine were randomly divided into eight groups. There were four female and four male subgroups, including normal diet + SED, normal diet + EX, HF + SED (HF-SED), and HF + EX (HF-EX). The HF composition, EX protocols, treadmill performance test, and assay for hypercholesterolemia were described previously (53). Pigs were anesthetized with ketamine (30 mg/kg) and pentobarbital sodium (35 mg/kg), injected with heparin (1,000 U/kg), and euthanized by a left thoracotomy.

Isolation of coronary smooth muscle cells. Smooth muscle cells were isolated according to the method of Quayle et al. (46) with modifications. Briefly, immediately after the thoracotomy, the heart was removed and placed in ice-cold Krebs solution containing (in mM) 131.5 NaCl, 5.0 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 11.2 glucose, 13.5 NaHCO3, and 0.025 EDTA (pH 7.3). The proximal right coronary artery was dissected from the heart and placed in iced low-Ca2+ (0.5 mM CaCl2) MEM (Sigma-Aldrich, St. Louis, MO) buffered with HEPES (20 mM) adjusted to pH 7.3 with NaOH. The adventitial layer was removed, and a vessel segment (10–15 mm) was slit open longitudinally and pinned adventitial side down in a 1.5-oz. vial containing a rubber base (Sylgard, Dupont). The endothelium was removed by gently abrading the luminal surface with moist filter paper. The vessel segment was incubated in low-Ca2+ MEM containing 1.5 mg/ml papain (Sigma) and 1 mg/ml DTT (Sigma) in a shaking water bath for 35 min at 35°C. This first digestion solution containing residual endothelial cells was discarded. Fresh low-Ca2+ MEM was added, which contained 1.5 mg/ml collagenase (type IV, Sigma) and 1 mg/ml hyaluronidase (Sigma). Segments were incubated another 20 min (35°C), and the solution was discarded. Segments were then washed one or two times with enzyme-free low-Ca2+ MEM and gently triturated with a wide-bore pipette to separate smooth muscle cells. On average, 90% of these cells were spindle shaped, relaxed, and exhibited morphology similar to that reported by DeFeo and Morgan (13) for relaxed myocytes. Yield of relaxed, spindle-shaped cells was less from all HF animals (30–50%). Cells were stored in low-Ca2+ MEM at 4°C and used within 6 h of isolation.

Whole cell recording of IK. IK results were measured by conventional whole cell patch-clamp technique. An aliquot of cell suspension was placed in a recording chamber (0.5 ml) mounted on the stage of an inverted microscope (Diaphot 200, Nikon), and cells were allowed to settle to the bottom of the dish for ~5–10 min before continuous perfusion was initiated. The perfusion solution contained (in mM) 138 NaCl, 5.4 KCl, 1.8 CaCl2, 0.1 CdCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4, adjusted with NaOH. CdCl2 was added to block Ca2+ currents (20). Patch pipettes were prepared from 0.2-mm-thick glass capillary tubing (Fisherbrand, 1.2 mm ID) and fire-polished to a final resistance of 2–4 M{Omega}. Pipettes were filled with pipette solution containing (in mM) 80 potassium aspartate, 50 KCl, 20 NaCl, 1 MgCl2, 3 MgATP, 10 EGTA, and 10 HEPES, pH adjusted to 7.2 with KOH. EGTA was included to buffer intracellular Ca2+. MgATP was included to inhibit ATP-sensitive K+ channels and provide substrate for energy-dependent processes. The liquid junction potential was +7 mV measured and +9 mV calculated. Calculations were done with the Junction Potential Calculator available from Axon Instruments. Liquid junction potential was not subtracted from current measures but was similar for all groups. Thus all measures, including zero current potentials, are shifted approximately +8 mV. Zero current potentials were calculated from the average cell current for each animal under each condition by interpolation using the following formula: y = ya + [(x – xa)(yb – ya)]/(xb – xa), where y represents voltage corresponding to xa as negative current and xb as positive current on either side of the zero current potential.

Whole cell IK was measured with an EPC 9 patch-clamp amplifier (HEKA), and the signal was filtered at 2.5 kHz using an eight-pole low-pass Bessel filter (Frequency Devices). The signal was digitized at 5 kHz with the ITC-16 interface and analyzed by Pulse + Pulsefit 8.30, Igor Pro, and Sigmaplot 9.0 computer programs. The series resistance (<10 M{Omega}) was compensated to minimize the duration of the capacitive surge. Cell membrane capacitance was measured using the internal circuit for capacitance-current compensation after formation of the whole cell configuration. Subtraction of leak currents was not performed. Whole cell currents were normalized to cell capacitance and expressed as picoampere per picofarad (pA/pF). All experiments were conducted at room temperature (22–25°C). Endothelin-1 (ET-1)-sensitive currents ({Delta}IK) were determined by subtracting basal IK from IK in the presence of ET-1. The 4-aminopyridine (4-AP; 1 mM)-sensitive current [{Delta}IK(KV)] is the current in the absence of 4-AP minus the current in the presence of 4-AP. The TEA (1 mM)-sensitive current [{Delta}IK(BK)] is the current in the absence of TEA minus the current in the presence of TEA. Only spindle-shaped, relaxed cells were examined to eliminate potential confounding effects of differing cell populations based on morphological criteria (44).

Chemicals. ET-1 was purchased from American Peptide (Sunnyvale, CA) and prepared as a 0.1 mM stock solution in 10 mM acetic acid and stored in the refrigerator. ET-1 final concentration in the bath was 10 nM. 4-AP, TEA, and iberiotoxin (IBTX) were purchased from Sigma-Aldrich, and stocks were prepared in distilled water and stored frozen. Final concentrations were 1 mM, 1 mM, and 100 nM, respectively. Selected cells were exposed to diluted vehicle alone (acetic acid), which had no effect on bath pH or IK.

Data analysis. Statistical differences in the current-voltage relationships between groups of pigs were compared by two-way repeated-measures ANOVA followed by Tukey's post hoc test for multiple comparisons. Differences in lipid levels were tested by two-way ANOVA followed by Holm-Sidak post hoc for multiple comparisons. Efficacy of EX was determined by paired Student's t-test (endurance time) and one-way ANOVA [heart weight-to-body weight ratio (HW/BW) and citrate synthase]. P values of <0.05 were considered significant. Data are reported as means ± SE, and n refers to the number of animals. Data of IK were collected from 8–10 cells from each animal.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hypercholesterolemia. HF-fed male and female swine exhibited increased serum total cholesterol (TC). Similar to previous reports with this model, EX had no effect on either TC or triglyceride levels (53); therefore, SED and EX group data for males and SED and EX group data for females have been combined. As shown in Fig. 1, TC significantly increased after 4 wk of HF and remained increased for an additional 16 wk of feeding. TC levels of SED female pigs were significantly greater than levels for male pigs at both 4 and 20 wk of HF. Females also exhibited a trend toward greater basal triglycerides and exhibited a significant elevation in triglycerides at 4 wk compared with male pigs, which had no elevation of triglycerides. HF pigs also experienced a greater increase in body weight than animals on a normal diet. The average body weight for SED male pigs increased 11% over the 20-wk study (34.3 ± 2.4 to 38.0 ± 2.2 kg), whereas body weight for HF-fed SED male pigs increased 19% (34.8 ± 2.3 to 41.5 ± 1.7 kg). This hypercholesterolemia model is presently extensively used by others in our research group (3, 5, 22).


Figure 1
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  		Fig. 1. Serum triglyceride and total cholesterol of male and female swine on normal diet and high-fat diet (HF). Values (mean ± SE) were obtained from pigs before (basal) and after 4 and 20 wk of HF. Values were not different between animals subjected to sedentary confinement (SED) and exercise training (EX) for either male or female pigs; therefore, data were combined. *Females had significantly higher lipid levels than males of the same treatment group (P < 0.05). #Significant difference from basal of the same sex (P < 0.05).

 
Efficacy of EX. The efficacy of the training program was judged by the following three markers: exercise endurance time, HW/BW, and skeletal muscle citrate synthase activity. As shown in Table 1, exercise endurance time was increased significantly after 16 wk of training for both normal diet-EX and HF-EX animals, and there were no differences related to sex. Likewise, HW/BW results were significantly increased in EX and HF-EX male and female groups compared with SED and HF-SED animals. In EX male pigs, HW/BW was increased 20% in normal diet and 19% in HF animals. In EX female pigs, HW/BW was increased 29% in normal diet and 37% in HF animals. Therefore, HF did not eliminate the effect of EX on HW/BW for males and females. The oxidative capacity of skeletal muscle was also significantly increased in both EX male and female pigs independent of diet. There was no statistical difference related to sex. From the above three endpoints, we conclude that EX caused adaptive changes in the pig model independent of sex or diet.


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  		Table 1. Efficacy of exercise training in Yucatan swine

 
Coronary smooth muscle cell properties. No apparent difference was observed in morphology between normal diet and HF VSM cells. Cell length ranged from 100 to 150 µm and width from 5 to 8 µm. Measures of cell capacitance (pF) for each group were not statistically different [23.0 ± 1.7 pF in SED (27 cells), 21.3 ± 1.8 pF in EX (22 cells), 22.6 ± 1.7 pF in HF-SED (24 cells), 23.3 ± 1.3 pF in HF-EX (23 cells)]. The average for cell capacitance was 22.9 ± 0.8 pF in males and 22.6 ± 0.5 pF in females. Thus we observed no difference in cell capacitance between cells taken from normal diet and HF groups. All cells used in our study remained relaxed during the perfusion procedure.

Characteristics of basal, whole-cell IK. Depolarization pulses were applied to each cell from a holding potential of –80 stepped to +80 mV in increments of 10 mV with durations of 500 ms. Voltage-dependent outward currents exhibited similar characteristics in VSM cells from both males and females, and traces from a SED male are shown in Fig. 2. The currents displayed little or no inactivation during the 500-ms voltage step and could be eliminated by replacing bath KCl with CsCl and adding TEA to the pipette solution, (4 cells, data not shown). IK could be partially inhibited by a voltage-dependent K+ channel blocker, 4-AP (Fig. 2, B and D), and by a relatively nonselective K+ channel blocker, TEA (Fig. 2, C and D), which further distinguished three components of IK as shown in Fig. 2E. One component that was inhibited by 4-AP (1 mM) was activated near –30 mV and tended to peak at +30 to +50 mV. This component had characteristics similar to those attributed to the delayed rectifier current and the voltage-dependent IK (KV) family of K+ channels (43). The second component was inhibited by TEA (1 mM) and activated at potentials positive to 0 mV. This current was maximally activated in our protocol at +80 mV. This current also exhibited large current fluctuations (Fig. 2, A and B) that are characteristic of the large-conductance Ca2+-activated IK, which also are activated by voltage (BK currents) (43). A third component was resistant to a combined 4-AP and TEA blockade, and we termed this a leak component. This component showed a linear relation to increasing voltage (Fig. 2, E and F).


Figure 2
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  		Fig. 2. Representative traces of coronary smooth muscle outward K+ currents (IK) from normal diet SED male pig. Currents are shown under basal conditions (A), in the presence of a KV blocker [4-aminopyridine (4-AP), 1 mM; B], in the presence of 1 mM TEA (C), and in the presence of both blockers (D). E: current-voltage (I-V) curves obtained from A–D. F: difference currents obtained from B–D (subtracted from A).

 
IBTX (100 nM), a selective BK current blocker, also inhibited IK (Fig. 3) with characteristics similar to TEA blockade at 1 mM. Both IBTX and TEA blocked large current fluctuations (Fig. 3, B and C), consistent with BK channel activity. The data are consistent with the proposal that at least three components of IK can be identified in coronary VSM cells.


Figure 3
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  		Fig. 3. Representative traces of coronary smooth muscle outward IK from normal diet SED female pig. Currents shown are under basal conditions (A), in the presence of the BK channel blocker iberiotoxin (IBTX, 100 nM; B), and in the presence of TEA (1 mM; C). D: I-V curves obtained from A–C. E: difference currents obtained from B and C (subtracted from A).

 
Effect of sex, EX, and diet on basal IK. Averaged basal current-voltage curves from all male and female swine groups appear in Fig. 4, A and B. IK was greatest for EX males and females on normal diets but was not significantly greater than results for the SED group within the same sex groups. However, EX was significantly greater than HF groups for both males and females, with the effect of EX being absent in the HF groups. EX also had a significant effect on the zero current potential of males but not females (Fig. 4, C and D). The calculated zero current potential of EX males was –40 ± 4 mV and was significantly greater than SED males (–34 ± 1 mV) or HF-EX males (–28 ± 2 mV). Female swine had significantly (P < 0.05) higher basal IK results in all groups than males of the same groups (Table 2). Finally, IK results for HF groups were consistently lower than the corresponding normal diet groups.


Figure 4
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  		Fig. 4. Influence of sex, EX, and HF on basal IK-voltage relationship. IK results for male groups (A) were consistently less than results shown in the respective female groups (B), and results for all HF groups were reduced. C and D: data from A and B, respectively, that are graphed to demonstrate zero current potential for each group. Calculated zero current potential for EX males ({circ}) was significantly different from all other groups. Data are means ± SE for n = 6–8 animals. *Significant difference in the curves (P < 0.05).

 

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  		Table 2. Total IK in coronary vascular smooth muscle cells of male and female swine

 
Effect of ET-1 on IK. Previous studies on coronary arteries showed that contractile responses to ET-1 were significantly reduced in EX male swine and that enhanced K+ channel activity might be involved in limiting contraction (26). Because the contractile studies were conducted in the presence of ET-1, we chose to determine the TEA and 4-AP effects under similar conditions. First, we measured the IK-voltage relationships for the various groups in the presence of 10 nM ET-1 (Fig. 5). The ET-1-sensitive currents ({Delta}IK, Fig. 5, E and F) exhibited a significant sex effect, with the male groups exhibiting a significant increase and the females showing little or no response to ET-1. The effect of ET-1 on IK was greatest in EX males and blunted in both HF groups. ET-1 also shifted the zero current potential to more negative values in EX males (Fig. 5, C and D). This value was –46 ± 3 mV and was significantly more negative than results for any other group, male or female.


Figure 5
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  		Fig. 5. Effects of endothelin-1 (ET-1) on IK-voltage relationships of sex, EX, and HF groups. Data are plotted as in Fig. 4. Total IK results in the presence of ET-1 (10 nM) are presented in A for male groups and in B for female groups. Currents near the reversal potential are shown as expanded graphs in C (male) and D (females). Calculated zero current potential for ET-1-treated EX males ({circ}) was significantly different from all other groups, including EX male in the absence of ET-1 (data not shown). E and F: ET-1-sensitive currents [{Delta}IK determined by subtracting basal IK (data from Fig. 4) from IK in the presence of ET-1 (A and B)]. {Delta}IK was calculated for each cell and averaged to represent n = 1 animal. {Delta}IK results for all male groups (E) were consistently greater than results for respective female groups (F), which showed small to no {Delta}IK in the presence of ET-1. Data are means ± SE for n = 6–8 animals. *Significant difference between curves (P < 0.05).

 
Effects of sex, EX, and diet on 4-AP- and TEA-sensitive IK. 4-AP- and TEA-sensitive {Delta}IK were measured in the presence of ET-1 and are shown in Fig. 6. All male and female groups exhibited significant 4-AP-sensitive (KV channel activity) currents [{Delta}IK(KV)], as illustrated in Fig. 6, A and B. {Delta}IK(KV) was activated at around –30 mV and peaked between +30 and +50 mV, consistent with KV channel behavior. The males exhibited greater differences between groups than the females. Of note, EX males on normal diet had significantly greater {Delta}IK(KV) than the corresponding SED groups, whereas EX had only a small effect on {Delta}IK(KV) in females. HF reduced greatly or nullified the effect of EX on {Delta}IK(KV) in female and male swine and also consistently reduced the {Delta}IK(KV) in SED males.


Figure 6
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  		Fig. 6. Effects of sex, EX, and HF on 4-AP- and TEA-sensitive IK in the presence of ET-1. Data are plotted as in Fig. 5, and data from Fig. 5 (A and B) representing basal currents in the absence of blocker were used to calculate difference currents. A (male) and B (female) show 4-AP (1 mM)-sensitive currents [{Delta}IK(KV), current in the absence of 4-AP minus the current in the presence of 4-AP]. C (male) and D (female) show TEA (1 mM)-sensitive currents [{Delta}IK(BK), current in the absence of TEA minus the current in the presence of TEA]. Values are means ± SE for n = 6–8 animals. *Significant differences between curves (P < 0.05).

 
All male and female groups exhibited significant TEA-sensitive (mostly BK channel activity) currents [{Delta}IK(BK)], as illustrated in Fig. 6, C and D. {Delta}IK(BK) was activated at around –10 to –20 mV and continued to rise with increasing voltages, consistent with BK channel behavior. The female groups exhibited greater {Delta}IK(BK) at higher voltage (>60 mV) than the corresponding male groups, with the exception of that shown in normal diet EX animals; this effect was about twofold greater in males than in females. Similar to the effects on {Delta}IK(KV), HF diet nullified the effect of EX on {Delta}IK(BK) in female and male swine and also consistently reduced the {Delta}IK(BK) in SED females and males.

Because IK results of females differed from males as shown in Fig. 5, 4-AP- and TEA-sensitive IK components are presented as percentage of total current in Table 3. Comparisons are made at +30 mV for 4-AP-sensitive currents because they peaked near that voltage. Comparisons were also made at +70 mV for TEA-sensitive currents where the contribution of KV activity was relatively small. SED and EX males on a normal diet exhibited a greater percentage of 4-AP-sensitive currents than the corresponding females. The contribution of IK(KV) was reduced in HF. On the other hand, at +70 mV, EX females on a normal diet had a significantly greater percentage of TEA-sensitive currents than SED females. A similar trend was noted in EX males, although the values did not reach significance. This difference was absent in HF males and females.


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  		Table 3. Normalized inhibitor sensitive IK in the presence of endothelin-1

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study resulted in several important findings concerning the relation of sex, exercise, and HF to K+ channel function in VSM. The basal IK of females exceeded that of males in the same treatment group, and EX tended to increase basal IK in both sexes given a normal diet. Of special importance, HF, which caused hypercholesterolemia, reduced basal IK and nullified the effects of EX. We used established K+ channel blockers to determine the channel types that contribute to the sex and exercise effects. In the presence of ET-1, the EX male and female animals on a normal diet exhibited increased IK(KV) and IK(BK), with the males having the greater response. HF nullified the effect of exercise on both channel types in both sexes. With the assumption that the pig is a reliable model for human cardiovascular disease, we have presented evidence that HF can nullify the beneficial effects of exercise on vascular function.

Animal model. The pig has long been considered an excellent model of humans for studies involving the coronary circulation and heart function. The Yucatan miniature pig has been used by many groups to study the impact of exercise and HF on vascular lipids (16, 48) because the pig lipoprotein metabolism can be related to that of humans (6). Unlike humans, however, the male pig has been considered hyperestrogenized because of high levels of circulating estrogen-like compounds (10). To address this issue, Laughlin et al. (31) utilized a sensitive estrogen bioassay and showed that the bioactive estrogen levels of male pigs were 35 pg/ml and were not dissimilar to human males. Therefore, the male porcine model of hypercholesterolemia is still employed and has been characterized (53). Less is known regarding the female model of hypercholesterolemia, although the female pig has been used extensively for studies of EX. In our study, a significant effect of sex was observed on blood cholesterol levels, with female pigs exhibiting significantly higher plasma levels than males and females also exhibiting increases in both triglycerides and TC (Fig. 1), whereas only TC was increased in the males. These data are consistent with previous reports demonstrating increased TC and triglyceride in pigs (27, 36, 45) and suggest that the hypercholesterolemia model is useful for both males and females. The efficacy of EX is acceptable from the data shown in Table 1 based on the typical three markers of EX.

Basal IK. K+ channels, particularly KV and BK, represent a primary ion-conducting pathway of VSM that plays an essential role in the regulation of resting membrane potential and therefore vascular tone and blood flow (43). Thus it is reasonable to propose that adaptations in IK would occur with EX or HF. Our data support adaptations of K+ channel activity and that these adaptations differed based on sex, EX, and diet. Under basal conditions (in the absence of ET-1), coronary smooth muscle of females expressed functionally greater IK than males, independent of diet or EX status (Table 2). This may explain the observation that coronary arteries from females exhibited reduced contractile sensitivity to ET-1 compared with male swine (26). In the presence of high concentrations, TEA caused the females to exhibit ET-1 sensitivity similar to the males (26). This sex effect that we previously reported appears to be based on differences in K+ conductance, with the females exhibiting greater conductance to K+ and reduced responses to agents associated with depolarization and contraction.

EX tended to increase IK (in the absence of ET-1) in both females and males, with the increase being proportionately greater in males (Fig. 4). EX also induced a significant shift in the zero current potential to more negative values in males but not in females, suggesting greater channel availability in EX males. A shift to more negative values may be associated with increased coupling of BK with beta1-regulatory subunits (11). The beta1-subunit is also an estrogenic target (15), possibly underlying sex differences in BK activity. It is unclear whether adaptations in subunit expression or coupling underlie EX or sex differences in the present study, particularly because only the zero current potential shifted in EX males, and this shift, even in the presence of ET-1, could be blocked by either 4-AP or TEA. Furthermore, we saw no change in the overall voltage sensitivity within male groups (data not shown). Consistent with EX effects on overall IK, EX had a greater effect in reducing the sensitivity of male coronary arteries to ET-1-mediated constriction (26). We had hypothesized that the females were protected from constriction by having a high basal IK, whereas males had a greater capacity to respond to EX with increased IK and subsequently reduced sensitivity to agonists. We tested this proposal here with direct measures of IK in the presence of ET-1, to duplicate conditions used for contraction in our previous study. We found IK to be higher in females and less responsive to the effects of EX. These observations provide a firmer base for our previous proposal.

In the absence of ET-1, HF had only a small effect in reducing basal IK in SED females and males (Fig. 4). However, the increase in IK induced by EX was nullified by HF, with the effect being greater in males (Fig. 4). These data are contrary to our initial premise that EX would provide a dominant beneficial influence over HF on IK. Bowles et al. (5) and Mokelke et al. (41, 42) studied IK in smooth muscle cells from conduit coronary arteries that were taken from pigs similar to those used in our study. Bowles et al. measured total IK in SED and EX female pigs and found no difference in VSM with EX. These findings are similar to our female group (Fig. 4B). They did not study the effects of HF, however. Mokelke et al. (42) did study these effects and found that smooth muscle cells from HF coronaries had greater IK than controls and diabetic male pigs given a normal diet. This differs from our observations in which HF was consistently associated with little change in IK (Fig. 4A). They also observed that EX reduced IK in the HF diabetic groups. Unfortunately, no measures were made in EX groups without diabetes, as done in the present study. Despite the differences in groups, it is puzzling why EX was associated with increased IK in our studies, whereas EX was associated with a decrease in the studies on diabetes (41, 42). Major differences, however, were present in the techniques employed to measure cellular current. Mokelke et al. used a perforated-patch technique that allowed cellular Ca2+ to vary depending on the experimental conditions. For instance, the caffeine-induced increases in IK were reduced by EX compared with the corresponding diabetic-HF group, which were interpreted to result from differences in Ca2+ coupling and/or Ca2+ release.

Considerable data exist to suggest Ca2+ regulatory mechanisms are altered by diabetic dyslipidemia and EX. The case for hyperlipidemia alone is less clear. Ca2+ levels, as measured by fura 2, are elevated in coronary VSM from diabetic dyslipidemic swine but unchanged from control in VSM from hyperlipidemic swine (58). Similarly, Ca2+ efflux, sarcoendoplasmic reticulum Ca2+-ATPase activity, and Ca2+ channel activity were unaltered by hyperlipidemia alone (23, 58). Exercise, in either normal diet animals (4, 52) or diabetic dyslipidemic animals, significantly modifies cellular Ca2+ handling (58). Given the impact of changes in Ca2+ on IK either directly (BK channels) or indirectly through modulation of signaling pathways, we adopted an approach that would reduce the confounding effects of Ca2+ changes by employing a dialyzed cell technique in which Ca2+ was buffered and Ca2+ influx was inhibited (20).

ET-1. We further evaluated the effects of ET-1 on IK and pharmacologically dissected IK in the presence of ET-1 to determine whether the effects of HF as well as sex and EX were selective for one class of K+ channel. Even though ET-1 is a potent vasoconstrictor, it also affects IK (24, 49). Multiple cellular mechanisms underlie ET-1 constriction of VSM and depend on the vascular bed (17, 49) culture conditions (29, 40) and receptor subtype expression (49, 56). ET-1 modulates multiple smooth muscle Ca2+ channels, including voltage-dependent Ca2+ channels (33), voltage-independent receptor-operated Ca2+ channels (18, 55), store-operated Ca2+ channels (2), and nonselective cation channels (7, 28), as well as channels dependent on changes in cellular Ca2+ [i.e., Ca2+-dependent Cl– channels (49) and Ca2+-dependent K+ channels (25)]. However, ET-1 may also modulate voltage-dependent K+ channels (35) independent of changes in Ca2+. In the present study, we restricted changes in cellular Ca2+ by including EGTA in the pipette and blocking voltage-dependent and voltage-independent Ca2+ entry with Cd2+ (14). These data suggest that the effect of ET-1 on IK was independent of Ca2+ changes, possibly involving other signaling pathways. Endothelin increased phosphorylation of tyrosine in VSM cells (34) and appears to regulate smooth muscle proliferation through activation of p38 and/or ERK pathways among others (9, 50, 51). Tyrosine kinase activity in turn may also regulate cellular Ca2+ (37) and other cellular targets.

Surprisingly, VSM cells treated with ET-1 in the present study had enhanced {Delta}IK only in the male groups (Fig. 5, E and F). Most studies from other laboratories used only male animals and would miss this important effect of sex. The reasons for this difference are unclear but could reflect sex differences in ET receptor expression or signaling pathways. Miller et al. (39) reported no effect of sex on ET receptor expression in VSM but greater contractility in females, which they attributed to differences in Ca2+ regulation. ET receptor coupling to VSM Ca2+ levels was modulated by diet in male swine that exhibited reduced ETA receptor Ca2+ coupling due to enhanced ETB-receptor inhibition (34). Thus adaptations in ET-receptor activity can be significant. The ET-1-induced {Delta}IK for males was greatest in the EX group on a normal diet. Similarly, the EX effect was nullified by HF, which is consistent with increased contractile responsiveness to ET-1 in HF and reduced sensitivity to relaxants (e.g., adenosine), which act via activation of IK (17).

Altered vascular reactivity induced by an atherogenic diet is well recognized, although the underlying mechanisms are less clear. It was reported that L-type Ca2+ current in VSM cells from the coronary macrocirculation was inhibited in a hypercholesterolemic pig model (3). Functional experiments suggested hypercholesterolemia abolished IK(KV) contribution to adenosine-mediated relaxation in porcine coronary arterioles (22). Comparisons of normal-diet SED and HF-SED in the present study indicate that HF reduced the current-voltage relationship in males and females by ~10%. These differences are less than the ~50% reduction in IK reported for portal vein myocytes from rabbits fed an atherogenic HF diet (12). The membrane cholesterol content was increased by HF in the rabbit model (8), which led to the proposal that K+ channel properties are modulated downward by the altered membrane lipid environment (8, 12). Our findings are consistent with this conclusion, although there may be species and/or sex differences in the size of the response. It should be noted, however, that lipid levels of females were consistently greater than those in males, although the effect of diet on IK appeared more pronounced in males.

KV and BK currents. The determination of 4-AP [IK(KV)]- and TEA [IK(BK)]-sensitive currents was done in the presence of ET-1. Although measuring the properties of these currents under basal conditions (as done in most published studies) is important, we wished to reproduce conditions under which vascular contractile reactivity was assessed. We previously assayed the sensitivity of coronary arteries to constrictors and to dilators by means of ET-1-induced contractions (17, 26). We especially wished to determine which IK were associated with the sex and EX effects on coronary contractile sensitivity to ET-1 (26). The SED males exhibited a greater sensitivity to ET-1 than did the SED females (26). In the present study, {Delta}IK(KV) in SED males and females are similar (peak IK was ~6 pA/pF; Fig. 6, A and B). However, {Delta}IK(BK) was consistently smaller in the SED males than in females (e.g., 16 vs. 21 pA/pF at +80 mV; Fig. 6, C and D). Therefore, it appears that the sex difference in contractile sensitivity to ET-1 had more of a contribution from differences in BK channel activity in SED males and females. The higher IK(BK) in the presence of ET-1 could have an inhibitory effect on contractures in females. We propose that such an effect would provide SED females added protection against vasospasm often associated with CAD.

EX was associated with reduced ET-1 sensitivity in males to the level found in females (26). Females remained unchanged by EX in that study. As shown in Fig. 5, A and B, in the presence of ET-1, the total IK in EX males was similar to that in EX females on normal diets. ET-1 also further shifted the zero current potential to more negative levels in EX males. Measures of selective currents in Fig. 6 shed some light on the types of channels that are involved. EX had little effect on IK(KV) in females (~1 pA/pF at peak current), whereas a significant increase occurred in EX males (3 pA/pF at peak current). BK channels are also involved, with significant EX-associated increases in IK for males (10 pA/pF) that exceeded those for females (6 pA/pF). We propose that such an effect of EX could provide EX males protection against coronary vasospasm. This protection appears to be present in SED females without an added benefit from EX.

HF reduced IK(KV) by one-third in SED males (~2 pA/pF). Our group (17) reported earlier that HF caused ET-1 contractures in coronary arteries from males to be more resistant to adenosine relaxation during metabolic inhibition than that shown in males on a normal diet. Such an effect was mimicked by the treatment of arteries from normal diet males with 4-AP but not with glibenclamide (an inhibitor of ATP-sensitive K+ channels). It was concluded that HF had an inhibitory effect on IK(KV) in SED males, which would place them at greater risk during an ischemic episode. The evaluation of HF effects associated with sex, EX, and BK channels was not part of this earlier study, however. We addressed these issues in the present study.

In the presence of ET-1, HF had no significant effect on IK(KV) in SED females (Fig. 6B); thus a sex difference is noted that would cause HF to differentially place males at risk. HF had a small but consistent effect in reducing IK(BK) from both SED males and females (Fig. 6, C and D), with the HF females still exhibiting greater IK(BK) than males. These sex-related effects of HF on IK(BK) appear to be relatively small in the SED groups. This did not appear to be the case for EX, however. In the presence of ET-1, HF nullified the effect of EX on IK(KV) in males (Fig. 6A). As noted above, EX affected females to a lesser extent (+1 pA/pF), which was also nullified by HF (–1 pA/PF). HF also nullified the EX effect on IK(BK) in both sexes by reducing the currents ~10 pA/pF (Fig. 6, C and D). The apparently beneficial effects of EX on male IK(KV) and IK(BK), as well as IK(BK) in females, were prevented by HF diet. It has been shown that the hyperlipidemia caused by HF was not reduced by EX in males and females (53, 53). Hence, benefits of EX are not realized during hyperlipidemia and the associated membrane changes in VSM (8, 12). However, it should be noted that the diets involve extremely high lipid intake. Even so, these findings emphasize the importance of incorporating a moderate to low-fat diet into an exercise program, if it is to promote cardiovascular health.

In summary, systematic comparison of whole cell IK in smooth muscle cells from coronary arteries demonstrated the following major findings: 1) basal IK of females was greater than that of males for the same treatment group; 2) EX tended to increase basal IK in both males and females on a normal diet, although the effect was greatest in males; 3) a diet high in fat caused hypercholesterolemia, reduced the basal IK, and nullified the affects of EX in males and females; 4) ET-1 increased IK in all male groups, whereas females were unchanged; 5) IK(KV) results measured in the presence of ET-1 were similar in SED males and females on a normal diet; 6) exercise increased IK(KV) to a greater extent in males than in females and HF nullified this effect; 7) IK(BK) results measured in the presence of ET-1 were greater in females than in males on a normal diet; and 8) exercise increased IK(BK) to a greater extent in males than in females and HF nullified this effect. Considering that BK and KV channels represent the primary control mechanism regulating membrane potential and that loss or reduction in either BK or KV activity results in membrane depolarization and constriction (32, 54), these data suggest that female swine, compared with males, experience greater protection from coronary vasospasm or hypertensive episodes by exhibiting higher basal IK. Males on an EX program can become similar to the females, in terms of absolute IK, and thus may have increased cardioprotection compared with SED males. HF nullifies the effect of exercise on IK in both sexes, suggesting membrane potential control by IK would be less effective at resisting an increase in vascular tone. Thus the full benefit of EX modulation of IK activity requires incorporation of a moderate-fat diet into the exercise program.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-15852 and HL-52490 and the National Aeronautics and Space Administration.


   ACKNOWLEDGMENTS
 
We thank Drs. Douglas Bowles and Virginia Huxley for comments on an earlier version of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. J. Rubin, Dept. of Biomedical Science, E102 Veterinary Medicine, Univ. of Missouri-Columbia, Columbia, MO 65211 (e-mail: RubinL{at}missouri.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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