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J Appl Physiol 94: 234-244, 2003. First published September 13, 2002; doi:10.1152/japplphysiol.00246.2002
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Vol. 94, Issue 1, 234-244, January 2003

Short-term training enhances endothelium-dependent dilation of coronary arteries, not arterioles

M. Harold Laughlin1, Leona J. Rubin1, James W. E. Rush2, E. M. Price1, William G. Schrage1, and Christopher R. Woodman1

1 Departments of Veterinary Biomedical Sciences and Physiology and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211; and 2 Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our objective was to test the hypothesis that short-term exercise training (STR) of pigs increases endothelium-dependent dilation (EDD) of coronary arteries but not coronary arterioles. Female Yucatan miniature swine ran on a treadmill for 1 h, at 3.5 mph, twice daily for 7 days (STR; n = 28). Skeletal muscle citrate synthase activity was increased in STR compared with sedentary controls (Sed; n = 26). Vasoreactivity was evaluated in isolated segments of conduit arteries (1-2 mm ID, 3-4 mm length) mounted on myographs and in arterioles (50-100 µm ID) isolated and cannulated with micropipettes with intraluminal pressure set at 60 cmH2O. EDD was assessed by examining responses to increasing concentrations of bradykinin (BK) (conduit arteries 10-12-10-6 M and arterioles 10-13-10-6 M). There were no differences in maximal EDD or BK sensitivity of coronary arterioles from Sed and STR hearts. In contrast, sensitivity of conduit arteries (precontracted with PGF2alpha ) to BK was increased significantly (P < 0.05) in STR (EC50, 2.33 ± 0.62 nM, n = 12) compared with Sed animals (EC50, 3.88 ± 0.62 nM, n = 13). Immunoblot analysis revealed that coronary arteries from STR and Sed animals had similar levels of endothelial nitric oxide synthase (eNOS). In contrast, eNOS protein was increased in STR aortic endothelial cells. Neither protein nor mRNA levels of eNOS were different in coronary arterioles from STR compared with Sed animals. STR did not alter expression of superoxide dismutase (SOD-1) protein in any artery examined. We conclude that pigs exhibit increases in EDD of conduit arteries, but not in coronary arterioles, at the onset of exercise training. These adaptations in pigs do not appear to be mediated by alterations in eNOS or SOD-1 expression.

nitric oxide; endothelial nitric oxide synthase; superoxide dismutase; bradykinin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ENDOTHELIUM IS THE PRIMARY sensory tissue of the arterial wall as it detects chemical substances within the blood and physical forces imparted to blood vessel walls (i.e., shear stress and vessel distention). In response to these signals, the endothelium releases substances that modulate vascular tone and blood vessel structure (3, 6, 23). Endothelial function has been reported to be enhanced by chronic exercise training in the aorta and in arteries that perfuse cardiac and skeletal muscle (5, 7, 15, 19, 26, 32, 34, 35). Evidence indicates that increased endothelium-dependent dilation is produced at least partially as a result of increased release of, or effectiveness of, nitric oxide (NO) produced by endothelial cell NO synthase (eNOS). However, in the coronary circulation, there are conflicting reports as some indicate that exercise training does not result in increased endothelium-dependent dilation (28), whereas others report that endothelium-dependent dilation is enhanced after exercise training (26, 34).

Wang and colleagues (34) reported that exercise training dogs 2 h/day for 7-10 days resulted in enhanced endothelium-dependent dilation in conduit coronary arteries. They reported increased flow-induced dilation (reactive dilation of the circumflex artery after a brief coronary occlusion), increased ACh-induced dilation, and increased ACh-induced NO production in conduit coronary arteries from these dogs (32, 34). These changes in the conduit coronary arteries were not as apparent in the coronary microcirculation because exercise hyperemia, reactive hyperemic blood flows, and ACh-induced increases in coronary blood flow were similar in sedentary dogs and in dogs trained for 7-10 days (34). However, microvessels isolated from the hearts of the short-term exercise trained dogs exhibited increased ACh-induced NO production (32). Finally, Sessa et al. (32) report that eNOS-mRNA was significantly increased in aortic endothelial cells (AECs) of these dogs trained for 7-10 days. Thus Wang et al. and Sessa et al. report that endothelium-mediated dilation was enhanced in the large-conduit coronary arteries but not in the coronary resistance vasculature (arterioles) of dogs trained intensely for 7-10 days. In contrast, we find a different pattern of coronary artery adaptation in adult miniature swine exercise trained for 16-20 wk, which exhibit no change in endothelium-mediated relaxation (28) or in eNOS protein content of conduit coronary arteries (22), whereas coronary arterioles exhibit enhanced endothelium-mediated dilation (26), increased eNOS-mRNA (35), and eNOS protein content (22). It is possible that species differences between pigs and dogs produce the difference between the results of our laboratory's training studies (22, 26, 28, 35) and those of Wang et al. and Sessa et al. However, there are other reports of no change in endothelium-mediated dilation of conduit coronary arteries of exercise-trained dogs (30) and rats (29), suggesting that conflicting results between our pigs and the dogs of Wang et al. and Sessa et al. are not the result of species differences. Instead, the hypothesis of the study reported herein is that these conflicting observations result from the fact that measurements were made at two different stages (time points during a progressive process) of adaptation stimulated by exercise training.

Available evidence is consistent with the concept that endothelium-mediated control of conduit coronary artery diameter is enhanced early in the exercise-adaptive process (7-10 days) but returns toward normal later in the training period, when structural adaptations produce increased conduit artery diameter (11) and a decrease in coronary shear stress (compared with shear stress during the first training bout) in these arteries during exercise training bouts (20). If our hypothesis is correct, then pigs trained for 7 days with a program similar to that used by Wang et al. (34) and Sessa et al. (32) should exhibit enhanced endothelium-dependent relaxation in conduit coronary arteries, but not in coronary arterioles. Therefore, in the study reported here we tested this hypothesis by training pigs with a training program modeled after the one used by Wang et al. and Sessa et al. If the hypothesis is correct, conduit coronary arteries from these pigs should exhibit enhanced bradykinin (BK)-induced relaxation, whereas arterioles should not show enhanced responses. Second, when results of our vasomotor function experiments supported this hypothesis, we also determined whether 7 days of intense exercise training altered expression of eNOS in conduit arteries and coronary arterioles of the size used for BK-induced dilation experiments. Because it is well established that increased content of superoxide dismutase (SOD-1) can increase the bioactivity of NO produced by eNOS and because SOD-1 expression is increased in coronary arterioles from fully trained pigs (31), we also examined SOD-1 expression in conduit arteries and coronary arterioles. It is important to emphasize that expression of eNOS and SOD-1 was examined in coronary arteries in these experiments because previous results indicated that fully trained pigs exhibit increased expression of these enzymes and because the training-induced increases in endothelial function reported by Wang and colleagues and Sessa and colleagues appear to involve increased expression of eNOS. Here we report results revealing that a short-term training program increases BK-induced dilation in conduit arteries, but not in coronary arterioles. This short-term training program increased eNOS levels in aortic endothelium but did not appear to alter expression of eNOS or SOD-1 in coronary arteries. These results, combined with current literature, indicate that the exercise training-induced adaptations of coronary arterial endothelium are highly dependent on artery size (conduit artery vs. arteriole) (22) and the time point studied (1 wk or months) (32, 34) during the adaptive process stimulated by exercise training.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental Animals

All animals used in this study were housed and maintained in accordance with standards set forth by the American Association for Laboratory Animal Care and the University of Missouri Institutional Animal Care and Use Committee. Adult female miniature swine weighing 25-40 kg were obtained from the breeder (Charles River) and trained to walk on the treadmill for 3 min at 3 mph for 3 days. The pigs were randomly divided into groups of short-term exercise trained (STR) pigs and sedentary control (Sed) pigs. The STR pigs completed the treadmill training program outlined below and modeled after that used by Wang et al. (34) and Sessa et al. (32). Sed pigs were confined to their pens during the training period. Female pigs were selected for this study because the previous studies of fully trained pigs used female pigs (26, 28).

Training Program

STR was accomplished with a motorized treadmill (Quinton). STR pigs were exposed to twice-daily bouts of exercise (1 h, at 3.5 mph, no incline) once in the morning and once in the afternoon, for 7 consecutive days. Exercise performance was rewarded by feeding at the end of the second training bout each day (13, 25). Sed pigs were fed daily in their cages at the same time as their paired STR pigs. Heart weights and body weights were recorded at the end of the training or sedentary confinement program. Skeletal muscle samples were taken from the middle of the triceps brachii muscles, frozen, and stored at -70°C until processed. Citrate synthase activity was measured in these samples by using the spectrophotometric assay described by Srere (33).

Conduit Coronary Artery Reactivity

Conduit coronary artery preparation. After completion of exercise training or sedentary confinement, the pigs were sedated with ketamine (30 mg/kg) and anesthetized with pentobarbital sodium (Fort Dodge; 35 mg/kg). Hearts were removed and placed in iced (4°C) Krebs bicarbonate buffer solution during vessel isolation. Segments of conduit coronary artery of ~2.0 mm outer diameter (OD) were carefully dissected from the myocardium, trimmed of fat and connective tissue, and cut into rings of 3.5-4 mm long. Vessel samples were taken from similar sites in hearts from Sed and STR pigs. OD, inside diameter (ID), and length of each vessel ring were measured with a Filar calibrated micrometer eye piece. For studies of rings devoid of endothelium, the endothelium was removed by gentle rubbing of the luminal surface over the edge of a forceps. Adequate denudation was tested by examining responses to maximal BK (30 µM). A vessel was considered denuded if BK produced <5% relaxation of a 30 µM PGF2alpha constriction.

Length-tension relationship. Vascular rings were mounted on two stainless steel wires passed through the vessel lumen. One wire was attached to a force transducer (Grass FT03), and the other to a micrometer microdrive (Stoelting) to allow the vessel to be stretched by known increments. Each vessel apparatus was placed in an individual 20-ml tissue bath containing Krebs bicarbonate buffer equilibrated at 37°C with 95% O2-5% CO2. Isometric tension was continuously recorded and collected by the Dataq computerized data-acquisition system. Rings were individually stretched to the maximum of the length-developed tension relationship (Lmax) by repeated test exposures to KCl (50 mM) at increasing vessel diameters. All subsequent pharmacological responsiveness studies were performed with arteries at Lmax. The arteries were allowed 30-min stabilization at Lmax before further study.

Experimental design for arteries. After completion of the length-tension experiment, the arteries were challenged by two test exposures to 80 mM KCl and one test exposure to 100 µM of norepinephrine to test smooth-muscle responsiveness. Endothelium-mediated vasorelaxation was evaluated by adding cumulatively increasing concentrations of BK (10-12-10-6 M) to the organ bath and measuring changes in force. Arteries were contracted with 30 µM PGF2alpha , and relaxation produced with BK was examined to reflect endothelium-mediated relaxation. Arteries were rinsed, reequilibrated for 1-1.5 h, and then contracted with 30 µM PGF2alpha , and relaxation produced by adenosine (Ado) was examined. In one subset of pigs, responses were examined in both intact and denuded arteries to separate endothelium-dependent Ado-induced relaxation from endothelium-independent Ado-induced relaxation.

Solutions and drugs used in artery experiments. Krebs solution contained 131.5 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.5 mM CaCl2, 11.2 mM glucose, and 13.5 mM NaHCO3. All solutions contained propranolol (3 µM) and 0.025 mM ethylenediamine-tetraacetic acid. Solutions were aerated with 95% O2-5% CO2 (pH 7.4) and maintained at 37°C. All other drugs and chemicals were purchased from Sigma Chemical, St. Louis, MO.

Reactivity of Coronary Arterioles

Preparation of coronary arterioles. With the heart maintained in cold physiological saline solution (PSS) (4°C), a portion of the left ventricular (LV) wall was isolated and placed in a dissection chamber containing cold PSS. Coronary arterioles 50-100 µm in intraluminal diameter and ~1 mm in length were isolated from the surrounding tissue 0.5-3.0 mm below the epicardial surface with the aid of a dissecting microscope. Arterioles were placed in a Plexiglas chamber containing PSS-albumin solution equilibrated with room air at ambient temperature. Each end of the vessel was cannulated with a glass micropipette (~50-µm diameter and filled with filtered PSS-albumin) and secured with 11-0 ophthalmic suture. The vessel was stretched to the length measured before removal from the myocardium. The glass micropipettes were connected to independent reservoirs, and intraluminal pressure was set at 60 cmH2O, with zero intraluminal flow, by raising the reservoirs 60 cm above the vessel chamber. Pressures were measured through side arms of the two reservoirs with low-volume displacement transducers (AD Instruments, model BP100 transducer). If the arteries would not maintain pressure (due to leaks), they were removed from the chamber and discarded. The cannulated vessel was viewed through an inverted microscope (Nikon Diaphot TMD) with ×20-40 lens and numerical aperture of 0.4 (ELWD). The microscope was coupled to a video camera (Panasonic WV 1500x) and TV monitor (Panasonic TR930B). An image of the vessel was displayed on the TV monitor, and intraluminal diameter measurements were made continuously by using a video tracking device (Texas A&M) as described previously (16, 26). The tracking system was calibrated with a stage micrometer showing 10-µm divisions. The resolution of the system allowed measurement of changes in vessel diameter as small as 2 µm. The tracking device produced a direct-current signal, which was recorded on a computer data-acquisition system (MacLab).

The PSS used in these experiments consisted of (in mM) 145 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS buffer. The PSS pH was adjusted to 7.4 and filtered through 0.22 microfilters (Fisher Scientific, Pittsburgh, PA). All drugs were obtained from Sigma Chemical (St. Louis, MO), unless otherwise specified. Drugs were dissolved in PSS-albumin and administered to the bath surrounding the artery. PSS-albumin contained 10 mg/ml bovine serum albumin (US Biochem Bovine; fraction V: 98-99% albumin) as described previously (16, 21, 26).

Experimental procedure for arterioles. Arterioles were allowed to equilibrate at an intraluminal pressure of 60 cmH2O for 1 h during which time the temperature of the chamber was raised to, and maintained at, 37 ± 1°C with a circulating water bath. During the 1-h equilibration period, the PSS-albumin was replaced three times with fresh PSS-albumin (37°C).

Sensitivity to BK was evaluated on arterioles that exhibited spontaneous tone. In arterioles that did not develop at least 35% spontaneous tone, endothelin-1 (ET-1) was administered to stimulate this amount of tone. (Sixteen of 19 Sed arterioles and 14 of 23 STR arterioles required ET-1. There was no difference between the average dose of ET-1 needed between the two groups: 0.51 ± 0.1 nM for the Sed arterioles and 0.41 ± 0.1 nM for the STR arterioles.)

Concentration-response curves were obtained by cumulative additions of small aliquots of concentrated stock solution directly into the bath; drug concentrations (BK: 10-13-10-6M) were increased after the response to the preceding dose was maximal.

At the end of each experiment, each artery was exposed to 100 µM sodium nitroprusside (SNP) in PSS-albumin solution to determine maximal diameter at 60-cmH2O intraluminal pressure. Diameters were normalized to this measurement as described previously (16, 26). This pressure was selected because in vivo microvascular pressure measurements obtained by Chilian et al. (2) in the cat beating ventricle indicate that intravascular pressure in vessels of this size is ~60 cmH2O.

eNOS and SOD-1 Protein Levels of Arteries and Arterioles

Protein content of coronary arteries. Pigs were anesthetized, and hearts were removed and maintained at 0-4°C during vessel isolation, as described previously (22, 26-28). Segments of conduit coronary artery and coronary arterioles were carefully dissected from LV myocardium, fat, and connective tissue. Diameters and lengths were measured, and the samples were placed in microcentrifuge tubes and frozen.

Standard immunoblot analysis for conduit coronary arteries. Relative levels of eNOS and SOD-1 protein were measured with standard immunoblot analysis. Lanes were loaded with equal amounts of protein (30 µg total protein), electrophoresed on 7.5% SDS-PAGE gels, and transferred onto nitrocellulose or polyvinylidene difluoride (PVDF) membranes. Each gel was loaded with an equal number of Sed and STR samples to facilitate comparison of bands between groups. Estimates of the amount of protein in the immunoreactive bands were determined by using scanning densitometry applying the NIH Image software (National Institutes of Health, Bethesda, MD). Proteins were detected by enhanced chemiluminescence (ECL, Amersham) and evaluated by densitometry (NIH Image). After data had been collected for eNOS and SOD protein content, each blot was stained with the Sypro Ruby Protein Blot Stain (Molecular Probes, Eugene, OR) to determine whether the blot exhibited adequate and uniform transfer of protein from gel to blot. The amount of protein in each band of interest was determined with STORM fluorescence imager, and the signals were evaluated by densitometry with NIH Image. Loading was standardized to an arbitrarily selected internal standard band in the size range of eNOS. This standard was used to correct for the effects of loading and transfer differences for the eNOS and SOD-1 signal on the luminogram. For each blot, data were also normalized by expressing all values relative to the average Sed value for the blot.

Immunoblot analysis of eNOS and SOD-1 protein in arterioles. Arterioles were isolated from myocardium (LV free wall) by dissecting along the length of 300-µm ID arteries to the smallest branches. We used two approaches to examine protein expression in the same-sized coronary arterioles used for the BK-induced dilation experiments: a single arteriole immunoblot technique and a modified standard immunoblot analysis with a pooled sample of five arterioles of similar diameter (50-100 µm) and length isolated from each pig.

Single arteriole immunoblots. Arteriolar diameters and lengths were matched for equal numbers of STR and Sed arterioles, to control for total protein (22). Frozen arterioles were solubilized in 20-µl Laemmli buffer (18): 62.5 mM Tris, pH 6.8, 6 M urea, 160 mM DTT, 2% SDS, 0.001% bromophenol blue, boiled for 2 min, and vortexed vigorously. Cell lysates were subjected to SDS-PAGE under reducing conditions, and proteins were transferred to PVDF membrane (Hybond-ECL, Amersham). The membrane was blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline-Tween (20 mM Tris · HCl, 137 mM NaCl, and 0.1% Tween 20). Blots were incubated overnight at room temperature with primary antibody against eNOS (1:1,600; Transduction Laboratories) and a monoclonal antibody against GAPDH (1:10,000, Chemicon) followed by incubation for 1 h with secondary antibody (1:2,500; horseradish peroxidase-conjugated anti-mouse). Specific eNOS and GAPDH proteins were detected by ECL (Amersham) and evaluated by densitometry (NIH Image). For comparison between samples, eNOS protein was expressed as arbitrary densitometric units on blots with paired STR and Sed samples.

Immunoblot procedures for pooled arterioles with equal diameters. eNOS and SOD-1 protein content of arterioles was determined by loading equal amounts of total arteriole protein (5-µg protein) from equal numbers of Sed and STR pigs on the same gel, allowing comparisons among arterioles of similar size on the same gel (22). Because there is not sufficient protein in a single arteriole to allow measurement of protein content and have sufficient sample to run on an SDS gel, it was necessary to pool samples of five arterioles. Total protein was measured with NanoOrange Protein Quantitation kits (Molecular Probes), which allows measurement of total protein in small samples. Samples were then processed as described above for single-arteriole immunoblots. Blots were incubated overnight (25°C) with primary antibody against eNOS (1:1,600; Transduction Laboratories) followed by incubation for 1 h with secondary antibody (1:2,500; horseradish peroxidase-conjugated anti-mouse). Subsequently, blots were again incubated overnight with primary antibody against SOD-1 (1:2,500; Stressgen) followed by incubation for 1 h with secondary antibody (1:2,500; horseradish peroxidase-conjugated anti-rabbit). Proteins were detected by ECL (Amersham) and evaluated by densitometry (NIH Image). After data had been collected for eNOS and SOD protein content, each blot was stained with the Sypro Ruby Protein Blot Stain (Molecular Probes) to determine whether the blot exhibited adequate and uniform transfer of protein from gel to blot. The amount of protein in each band of interest was determined from the fluorescence with a STORM 850 (Amersham Biotech, Piscataway, NJ) and NIH Image. Loading was standardized to an arbitrarily selected internal standard band in the size range of eNOS. This standard was used to correct for the effects of loading and transfer differences for the eNOS and SOD-1 signal on the luminogram. For each blot, data were also normalized by expressing all values relative to the average Sed value for the blot.

Isolation of AEC protein. A 15-cm segment of excised thoracic aorta was isolated starting ~3 cm distal to the end of the arch of the aorta. This segment of aorta was cleaned of adhering tissue while submersed in a dish of Krebs buffer (4°C), transferred to a fresh dish of buffer, and cut longitudinally to expose the luminal surface. The tissue was then transferred to a clean glass plate with the luminal surface up. Endothelial cells were harvested by applying a 2-ml aliquot of TRI Reagent (Molecular Research Center) to the luminal surface and, 30 s later, scraping this surface with the edge of a glass microscope slide. The cell lysate was collected into a tube and immediately frozen in liquid nitrogen. Proteins were isolated from the TRI Reagent extracts according to the manufacturer's instructions. Isolated and washed protein pellets were dissolved in a protein solubilization buffer consisting of 50 mM Tris · HCl, pH 7.4, 6 M urea, and 2% SDS. Protein concentration was determined by using the bicinchoninic acid assay (Pierce). Before electrophoresis, aliquots of these samples were supplemented with 150 mM DTT and boiled for 1 min. Isolated aortic endothelial cell preparations were tested for vascular smooth-muscle cell contamination by performing immunoblots for smooth-muscle alpha -actin (primary antibody 1:2,500; Chemicon), which revealed no detectable contamination.

AEC immunoblots. Samples containing 30-µg protein were loaded onto gels and electrophoresed and electroblotted to PVDF membranes. Proteins were detected by using primary antibodies specific for SOD-1 (1:2,500; Stressgen) and eNOS (1:3,500; Transduction Laboratories). Secondary antibodies were conjugated with horseradish peroxidase. All antibody and blocking solutions contained 5% nonfat milk and 0.1% Tween in Tris-buffered saline. Immunoblot signals were generated via chemiluminescence (ECL, Amersham) and captured on X-ray film (Amersham). Scanning densitometry (NIH Image) was used to quantify immunoblot signals. For each blot, data were normalized by expressing all values relative to the average Sed value for the blot.

Measurement of eNOS mRNA in Single Arterioles

Relative differences in eNOS mRNA expression in coronary arterioles (50-100 µm ID) were assessed as described previously (35). In brief, single arterioles were homogenized in a lysis buffer by vortexing the arteriole vigorously for 60 s, four to five times over a 15-min period of time. Poly-A RNA was isolated from crude lysate with paramagnetic oligo(dT) beads [Dynabeads oligo(dT)25, Dynal], and first-strand cDNA synthesis was performed in a 20-µl volume (Superscript Preamplification System, Gibco-BRL Life Technologies). Five microliters of cDNA were used in a PCR by using previously published primers and cycling conditions for eNOS and GAPDH (35). Each PCR was spiked with 10 µCi of [alpha -32P]dCTP (3,000 Ci/mmol). GAPDH and eNOS were coamplified in the same PCR. The PCR amplified products were electrophoresed on 1.5% agarose gel, and the eNOS and GAPDH bands were excised, placed in separate scintillation vials, and counted for 1 min with a Packard 1600CA liquid scintillation counter. The eNOS-to-GAPDH ratio was calculated for each arteriole. GAPDH was used as an internal standard because it is a constitutively expressed enzyme whose mRNA level is not affected by exercise training (32, 35). Consequently, expressing eNOS relative to GAPDH allows for relative comparisons between coronary arterioles isolated from Sed and STR pigs.

Data analysis. For conduit arteries, responses to constrictors are expressed in grams of developed tension, determined as the tension developed above the resting tension. Responses to vasorelaxing agents are expressed as relative relaxation from precontracted levels. For calculation of EC50, the concentration-relaxation responses for each ring were fitted by regression analysis to a four-parameter sigmoidal equation by using the SigmaPlot equation's library and software (SPSS). EC50 is normally distributed according to a log rather than arithmetic scale; therefore, log values were used to make statistical comparisons. Statistical differences in the log of the EC50 values were determined by using a Student's t-test. For clarity, EC50 values are graphed arithmetically. Statistical differences in relaxation curves were determined by a two-way repeated-measures ANOVA, and significant differences were determined by using least squares means post hoc test.

Vasomotor responses of arterioles are presented as absolute diameters and as normalized diameters. The diameter measured at 60 cmH2O intraluminal pressure in the presence of 100 µM SNP was defined as the reference diameter and assigned a value of 1.0. All normalized diameter measurements are expressed relative to this diameter, as described previously (17, 21). Responses were compared between STR and Sed groups by ANOVA for repeated measures. Diameters measured at each dose were compared between STR and Sed groups with ANOVA for repeated measures by using SuperANOVA software. No post hoc test was employed because ANOVA did not indicate that differences existed between groups. Significance of differences between mean values for protein contents, treadmill performance times, citrate synthase activity, and heart-to-body weight ratios were determined by the unpaired Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Efficacy of Exercise Training

Adaptation produced by the training program was evident in that citrate synthase activity of the long head of the triceps brachii muscle was significantly greater for STR pigs than Sed values (STR citrate synthase activity was 19% greater than that for Sed pigs). This confirms the shift in skeletal muscle oxidative capacity reported previously in pigs trained for 7 days (13, 25). Average heart weight was 153 ± 4 g for the Sed pigs (n = 26) and 171 ± 5 g for the STR group (n = 28) (P < 0.05). The heart weight-to-body weight ratio for the Sed pigs was also significantly less than the corresponding STR value (Sed = 4.3 ± 0.1 g/kg, STR = 4.9 ± 0.1 g/kg; P < 0.05).

Conduit Coronary Arteries

Characteristics of conduit arteries. Conduit coronary arteries isolated from Sed and STR pigs had similar dimensions for OD, luminal diameter, and wall thickness at rest (Table 1). Per experimental design, arterial segments isolated from Sed and STR pigs also had similar axial lengths.

                              
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  		Table 1.   Conduit artery physical parameters

Arteries were progressively stretched by increments of 5-20% of the initial resting diameter. Contractile responses to 50 mM KCl were determined at each length until additional increases in resting length increased maximal active tension by <5%. Arteries from Sed and STR pigs were of the same resting ID and OD and were stretched to the same relative length at Lmax (Table 1). In addition, resting tension at Lmax was not different in vessels from Sed (6.78 ± 0.84 g) and STR (6.52 ± 0.77 g) pigs.

Contractile responses. Maximal contractile tensions stimulated by 80 mM KCl were not different in vessel segments from Sed and STR animals [Sed (n = 11): 10.44 ± 1.13 g; STR (n = 13): 11.48 ± 0.89 g]. Similarly, the developed tension to maximal concentrations of norepinephrine (100 µM) (Sed: 2.80 ± 0.5 g; STR: 2.87 ± 0.5 g) or PGF2alpha (30 µM) (Sed: 10.91 ± 0.8 g; STR: 11.79 ± 1.2 g) were not different in vessel segments from Sed and STR.

Relaxation responses. Cumulative addition of BK to the bath produced concentration-dependent decreases in the PGF2alpha -induced precontraction (Fig. 1) in coronary arteries from both Sed and STR pigs. Arteries from STR pigs exhibited greater relaxation in the dose range of 3 × 10-10 M to 3 × 10-9 M BK than did coronary arteries from Sed pigs. This difference in sensitivity was reflected in the significantly greater EC50 value for Sed compared with STR (log M: Sed -8.49 ± 0.08; STR -8.78 ± 0.1). After endothelium removal, BK-induced vasodilation was abolished (3.2 ± 1% vasodilation in response to 30 µM BK).


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  		Fig. 1.   Cumulative addition of bradykinin-induced relaxation of PGF2alpha -induced precontraction in conduit coronary arteries isolated from sedentary (Sed) and short-term exercise-trained (STR) pigs. Values are means ± SE. P = 0.03 is the probability that the Sed and STR curves are different as determined with 2-way repeated-measures ANOVA. * Arteries from STR pigs exhibited greater relaxation in the dose range of 3 × 10-10 M to 3 × 10-9 M bradykinin than did coronary arteries from Sed pigs (P < 0.05). This difference in sensitivity is also reflected in the significantly greater EC50 value (P < 0.05) for Sed compared with STR pigs (inset).

Cumulative addition of Ado to the bath produced concentration-dependent decreases in the PGF2alpha -induced precontraction (Fig. 2) in coronary arteries from both Sed and STR pigs. ANOVA indicates that the curves are different between Sed and STR in intact arteries, with arteries from STR pigs exhibiting significantly greater relaxation in the dose range of 1 × 10-6 M to 1 × 10-5 M Ado than coronary arteries from Sed pigs (Fig. 2A). The EC50 values were not significantly different (P = 0.079) between Sed and STR arteries (Sed = 13.4 ± 2.1 µM; STR = 10.1 ± 2.4 µM; log M: Sed -4.95 ± 0.07; STR -5.24 ± 0.1). After endothelium removal, there was no longer a difference between responses of Sed and STR coronary arteries to Ado (Fig. 2B).


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  		Fig. 2.   Cumulative addition of adenosine-induced relaxation of PGF2alpha -induced precontraction in conduit coronary arteries isolated from Sed and STR pigs with (A) and without (B) endothelium. Values are means ± SE. A: responses of intact coronary arteries to adenosine. n = No. of animals. P = 0.04 is the probability that the Sed and STR curves are different as determined with 2-way repeated-measures ANOVA. * Arteries from STR pigs exhibited greater relaxation in the dose range of 1 × 10-6 M to 1 × 10-5 M adenosine than did coronary arteries from Sed pigs (P < 0.05). The EC50 values were not significantly different (P = 0.079) (Sed = 13.4 ± 2.1 µM; STR = 10.1 ± 2.4 µM). B: responses of denuded coronary arteries to adenosine. In this subset of animals, the responses of the intact arteries were similar to those illustrated in A. There is no difference between responses of Sed and STR arteries after removal of the endothelium (EC50 values: Sed = 9.2 ± 2.3 µM; STR = 11.0 ± 3.4 µM).

Reactivity of Coronary Arterioles

Characteristics of isolated coronary arterioles. Mean intraluminal diameter measured at 60 cmH2O in the presence of 100 µM SNP was similar in arterioles isolated from STR (111 ± 6 µm, n = 11) and Sed (111 ± 6 µm, n = 9) pigs. During the initial equilibration period at 60 cmH2O, vessels from STR and Sed pigs developed similar tone.

Endothelium-mediated vasodilator responses. BK produced concentration-related increases in relative diameter (Fig. 3) in coronary arterioles from Sed and STR pigs. There were no differences between the responses of arterioles from Sed and STR pigs. A-23187 was used to examine endothelium-mediated dilation that is not mediated by receptor and/or second-messenger systems. The results indicate that A-23187 produces similar amounts of vasodilation in arterioles from both groups (Fig. 4).


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  		Fig. 3.   Cumulative addition of bradykinin-induced dilation of coronary arterioles isolated from Sed (111 ± 6 µm, n = 9) and STR (111 ± 6 µm, n = 11) pigs. Luminal diameters were normalized to the passive diameter measured with intraluminal pressure of 60 cmH2O in the presence of nitroprusside (10-4 M). Values are means ± SE. Arterioles were equilibrated at intraluminal pressures of 60 cmH2O as described in the text. There were no statistically significant differences between Sed and STR values.



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  		Fig. 4.   A-23187-induced dilation of coronary arterioles isolated from Sed and STR pigs. Luminal diameters were normalized to the passive diameter measured with intraluminal pressure of 60 cmH2O in the presence of nitroprusside (10-4 M). Values are means ± SE. Arterioles were equilibrated at intraluminal pressures of 60 cmH2O as described in the text. There were no statistically significant differences between Sed and STR values.

Expression of eNOS and SOD-1 in Coronary Arteries and Arterioles

Figure 5 presents results of immunoblot analysis of eNOS content in conduit coronary arteries [large coronary (Lg Cor)] and for coronary arterioles (pooled; 50-100 µm). Because there were no differences in the results from the different conduit arteries, the results are pooled as large coronary arteries in Fig. 5 for clarity. As described above in MATERIALS AND METHODS, the conduit artery data in Fig. 5 are expressed relative to the mean Sed value for each blot. We also analyzed the NIH image optical density data, and these raw optical density data corrected for differences in protein transfer as determined from Sypro fluorescence. Results indicate that there was no difference between STR or Sed optical density data (P = 0.29), transfer-corrected data (P = 0.34), or the data expressed relative to the average Sed values as presented in Fig. 5 (P = 0.25). The sample blot in Fig. 5 illustrates data for pooled arterioles, and the data in the bar graph indicate that there were no differences between Sed and STR (P = 0.45). Results in Fig. 5 indicate that STR has little or no effect on eNOS protein levels in conduit coronary arteries or coronary arterioles. Figure 6 presents results from eNOS and GAPDH immunoblots for single coronary arterioles. These results also indicate that STR did not alter eNOS or GAPDH content of coronary arterioles. Similarly, SOD-1 protein expression appeared similar in coronary arteries of STR and Sed (Fig. 7). Finally, we measured eNOS and SOD-1 protein content in AECs from three Sed and three STR pigs. Results indicate that eNOS protein content is increased ~50% in AECs isolated from STR pigs, but SOD-1 protein content was similar in cells from Sed and STR (Fig. 8).


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  		Fig. 5.   Effects of short-term exercise training on endothelial nitric oxide synthase (eNOS) expression in conduit coronary arteries and coronary arterioles. A: eNOS content results from immunoblots prepared from pooled arteriolar samples (5 arterioles, 50- to 100-µm internal diameter, and 1-2 mm in length) as described in the text. HEL, human endothelial cell lysates. Each lane represents 5 µg of arteriolar protein from arterioles isolated from 3 different Sed pigs and 5 different STR pigs. B: bar graphs presenting average eNOS protein content of conduit coronary arteries [large coronary (Lg Cor)] from 17 STR and 11 Sed pigs and from arterioles of equal diameter (5 arterioles pooled into 1 sample). Values are means ± SE (normalized by expressing all values relative to the average Sed value for each blot). There were no statistically significant differences between Sed and STR values.



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  		Fig. 6.   Immunoblots for eNOS (A) and GAPDH (B) from single arterioles (50- to 100-µm internal diameter). Each lane represents material from a single coronary arteriole. Three Sed and STR samples shown were matched for diameter and length. Bar graphs present average immunoreactive eNOS (A) and GAPDH (B) protein levels of electrophoretically separated protein from coronary arterioles isolated from Sed and STR pigs. Protein content was quantified by scanning densitometry for arterioles from 3 Sed and 3 STR hearts. Values are means ± SE for relative densitometric units. There were no statistically significant differences between Sed and STR values.



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  		Fig. 7.   A: SOD-1 content results from standard immunoblots prepared from pooled arteriolar samples (5 arterioles 50- to 100-µm internal diameter, and 1-2 mm in length) as described in the text. Each lane represents 5-µg equal amounts of arteriolar protein from arterioles isolated from 3 different Sed pigs and 5 different STR pigs. B: bar graphs present average SOD-1 protein content. Data are conduit coronary arteries (Lg Cor) from 17 STR and 11 Sed pigs and from pooled arterioles of equal diameter as described in MATERIALS AND METHODS. Values are means ± SE (normalized by expressing all values relative to the average Sed value for each blot). There were no statistically significant differences between Sed and STR values.



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  		Fig. 8.   A: Results of immunoblots for eNOS and SOD-1 content of aortic endothelial cells isolated from 3 Sed and 3 STR pigs. Duplicates of blots prepared from the same 3 Sed and 3 STR aortas are shown. Each lane represents 30 µg of endothelial cell protein. B: bar graphs present average eNOS and SOD-1 protein content quantified by scanning densitometry. Values are means ± SE. * STR eNOS protein content is significantly greater than Sed (P < 0.05).

Expression of eNOS mRNA in coronary arterioles. Figure 9 presents results from RT-PCR analysis of eNOS-mRNA expression in single coronary arterioles. The bar graphs in Fig. 9A present average results, where data from multiple arterioles in each pig are pooled so that results from each pig equal one observation. In Fig. 9B, bar graphs reflect average results for all arterioles, where each arteriole equals one observation. Although these results suggest that 7 days of exercise training produced a modest increase in eNOS-mRNA, this modest change is not statistically significant (P = 0.07).


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  		Fig. 9.   RT-PCR analysis of STR effect on eNOS mRNA expression in single coronary arterioles. Inset: sample agarose gel visualized with ethidium bromide staining. MW, 123-base pair molecular marker: lanes 1-3 are single arterioles from 3 different Sed pigs, and lanes 4-6 are single arterioles from 3 different STR pigs. A: comparison of eNOS-to-GAPDH mRNA ratio in single coronary arterioles from Sed and STR pigs presented as means ± SE for 6 Sed and 8 STR pigs. B: comparison of eNOS-to-GAPDH mRNA ratio in coronary arterioles from Sed and STR pigs presented as means ± SE for 8 Sed and 15 STR arterioles. dagger  STR value significantly greater than Sed with P = 0.07.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The findings of this study demonstrate that short-term (7 days) exercise training of pigs induces increased BK-induced, endothelium-dependent relaxation in conduit coronary arteries but not coronary arterioles. Importantly, these results support our hypothesis and indicate that, in response to a short-term (7 days), high-intensity (2 h/day) exercise training program, porcine coronary artery endothelium exhibits adaptations that are similar to adaptations reported in coronary arteries of dogs trained with a short-term program (7-10 days) (32, 34). Also consistent with Sessa et al. (32), who reported increased eNOS message in aortic endothelium of short-term exercise-trained dogs, we find that aortic endothelium from STR pigs contained 50% more eNOS protein than endothelium from Sed pigs. There was no significant difference between eNOS protein levels in conduit coronary arteries of STR and Sed pigs, and STR did not alter eNOS protein content or eNOS-mRNA in coronary arterioles. Finally, STR did not alter SOD-1 protein expression in conduit arteries or in coronary arterioles.

Effects of Training on Conduit Coronary Arteries

Wang and colleagues (34) reported that dog conduit coronary arteries exhibited enhanced endothelium-dependent dilation after a short-term exercise training program. Their evidence for enhanced endothelium-dependent dilation of the circumflex coronary artery included increased flow-induced dilation (stimulated both by exercise and reactive hyperemia) and increased ACh-induced dilation (34). Our results indicate that a short-term training program in adult miniature swine, modeled after that used by these investigators (32, 34), also produces increased (BK-induced) endothelium-dependent relaxation in conduit coronary arteries. Ado-induced relaxation was also enhanced in the STR conduit arteries due to increased endothelium-dependent relaxation as reflected in the fact that there were no differences between responses of STR and Sed arteries after denudation (Fig. 2B).

These results, combined with those in the literature, support the concept that endothelium-mediated control of conduit coronary artery diameter is enhanced early in the exercise-adaptive process (7-10 days) but returns toward normal later in the training period. Thus studies that have examined endothelium-dependent dilation after a short-term exercise training program report enhanced dilation in conduit coronary arteries (Fig. 1) (34). Furthermore, studies that have examined endothelium-dependent dilation in fully trained dogs (30), rats (29), and pigs (28) report that endothelium-dependent dilation of conduit coronary arteries is unchanged from sedentary values, after chronic exercise training. It is possible that the endothelium of the conduit arteries returns to a normal phenotype in a fully trained subject because of structural adaptations of these arteries, i.e., increased conduit artery diameter, produced by chronic exercise training (11). Such increases in coronary artery diameter could result in a normalization of coronary shear stress during exercise (or at least attenuate the increase in shear stress during exercise) and perhaps, thereby, signal a return to "normal" endothelial phenotype (20, 25).

It appears that similar forces are in operation in signaling vascular adaptations of endothelium in other conduit arteries of large mammals (13, 20, 25). It is of interest that Johnson et al. (13) reported increased endothelium-dependent relaxation of pulmonary arteries and increased eNOS protein content of pulmonary arteries of pigs trained with this training program (13). Thus, in this model of exercise training, evidence indicates increased expression of eNOS in conduit arteries, from several different anatomic locations, with increased blood flow during exercise. Equally interesting is the finding that these conduit arteries do not retain this adaptation after the pigs become fully exercise trained (12, 22, 24, 28). Perhaps this is further evidence that these conduit arteries remodel to larger diameters during chronic exercise training, thus blunting the effects of exercise on shear stress in these arteries.

Mechanisms Responsible for the Effects of Training on Conduit Coronary Arteries

The increased BK-induced relaxation responses in conduit coronary arteries from STR pigs could be the result of several mechanisms, including the following: 1) increased eNOS protein expression; 2) increased bioactivity of NO; 3) increased eNOS activity without altered eNOS content; 4) increased activity of endothelium-mediated dilator pathways other than eNOS; or 5) increased responsiveness of vascular smooth muscle to endothelium-derived dilators. Our experimental design tested the first two of these possibilities, and we expected that conduit coronary arteries from STR pigs would exhibit increased eNOS and/or SOD-1 protein content. The results presented in Figs. 5 and 7 indicate that STR did not increase eNOS or SOD-1 content in the conduit coronary arteries.

Our expectation of increased eNOS and/or SOD-1 was based on a number of observations in the literature concerning the effects of training on eNOS expression and endothelium-dependent dilator responses in conduit arteries (13, 20, 25) and coronary arterioles (22, 26, 35). Furthermore, Sessa et al. (32) reported that short-term exercise training of dogs resulted in increased expression of eNOS mRNA in AECs, suggesting that a similar adaptation may occur in the coronary arteries. Consistent with these results, STR pigs also exhibited increased eNOS protein content in AECs (Fig. 8), whereas neither eNOS nor SOD-1 protein content was different in coronary arteries from STR pigs. If the mechanisms for producing increased endothelium-dependent dilation in conduit arteries in STR dogs and pigs are the same, then the fact that Sessa et al. observed increased ACh-induced NO release from coronary arteries isolated from STR dogs suggests that NOS activity in the arteries was increased. Our experimental design does not allow conclusions about whether increased eNOS activity without altered eNOS content, or increased activity of endothelium-mediated dilator pathways other than eNOS, is responsible for the increased BK-induced relaxation responses measured in the coronary arteries. Lastly, it is possible that BK-induced dilation was enhanced in conduit coronary arteries from the STR pigs because of increased responsiveness of vascular smooth muscle to endothelium-derived dilators. Our results argue against this possibility because the responses of the coronary arteries to KCl, norepinephrine, PGF2alpha , and Ado (in arteries without endothelium) were not altered by 7 days of exercise training. Thus we believe that our results suggest that only endothelium-mediated responses were modified by training.

It is important and interesting that the time course of vascular adaptations in conduit arteries appears different in rodent models after the onset of exercise training. Aortas and pulmonary arteries from fully trained rats appear to have increased endothelial function and increased eNOS protein content (5, 14) as do those of rabbits (1). Furthermore, it appears that enhanced endothelium-dependent dilation and increased eNOS expression are not present in rats until 4 wk of exercise training (4). It seems possible that the relative changes in hemodynamics produced during exercise are different in the conduit arteries of rats vs. those of larger mammals producing a modified time course of vascular adaptation.

Effects of Training on Coronary Arterioles

Wang et al. (34) reported that endothelium-dependent dilation of the coronary resistance arteries and arterioles (as reflected in coronary blood flow during exercise, coronary blood flow during reactive hyperemia, and coronary blood flow during ACh infusion into the coronary circulation) was not different in the short-term trained dogs. Their results suggest that the responsiveness of the coronary resistance arteries and arterioles to flow and shear stress and ACh were not altered by the 7-10 days of training. On the other hand, in similarly trained dogs, Sessa et al. (32) reported that isolated myocardial microvessels exhibited increased ACh-induced nitrite production, suggesting that the coronary resistance arteries and/or arterioles had increased eNOS activity. It is not apparent why the isolated microvessel preparations from the short-term trained dogs produced greater ACh-induced nitrite production, but the intact coronary circulation did not exhibit enhanced ACh-induced decreases in coronary vascular resistance (32, 34). It is possible, indeed likely, that results from the isolated microvessel preparation represent results from microvessels other than the coronary arterioles, which control vascular resistance (i.e., capillaries, veins, and perhaps lymphatics). If so, the increased nitrite production may not be primarily focused in the coronary resistance arteries (or arterioles) that determine coronary vascular resistance. Alternatively, the results of Wang and colleagues and Sessa and colleagues may suggest that adaptations have been initiated in the coronary microcirculatory resistance arteries but are not fully developed. If this is so, these results are consistent with the results of the present study in which we also find no change in endothelium-dependent dilation of coronary arterioles (Figs. 3 and 4) and no change in eNOS protein content (Figs. 5 and 6), but our data suggest that eNOS-mRNA is increased in the arterioles (Fig. 9). The results from dogs and pigs are consistent with the possibility that adaptations are in the initial stages in these arterioles. An increase in eNOS mRNA, stimulated by 7 days of increased coronary blood flow (during two 1-h exercise training bouts), is credible as it is known that increased intraluminal flow can stimulate increased eNOS-mRNA expression in porcine coronary arterioles (36). Also, consistent with an increased eNOS message in response to training, eNOS message is increased (35) and eNOS protein content is increased in coronary arterioles isolated from fully trained pigs (trained for 16-20 wk) (22). Finally, in fully trained pigs, endothelium-dependent dilation of coronary arterioles is increased (26). Hence available information indicates that short-term training enhances endothelium-dependent dilation in conduit arteries, whereas adaptations in vascular endothelium appear to be focused in the arteriolar resistance arteries in fully trained animals (22, 26, 28, 30, 35). It is important to emphasize that there is evidence that the distribution of exercise training-induced endothelial adaptations in the coronary arterial tree of fully trained pigs are similar to that of humans. Hambrecht et al. (10) reported that the ACh-induced increase in coronary blood flow is enhanced in patients with coronary disease, following 4 wk of exercise training (suggesting that the coronary arterioles have enhanced endothelium-dependent dilation after training). In contrast, in these same trained humans, ACh induced constriction of the conduit coronary arteries in the patients both before and after exercise training.

It is not clear why the time course of endothelial adaptation in the coronary arteriolar circulation is not the same as that in the large coronary arteries. It is possible that the relative increases in shear stress within the coronary arterial tree change with time after the initiation of a training program so that, early in the adaptive process, shear stress is increased most in the conduit arteries. As these arteries remodel and become larger, the increases in shear stress may be more focused to the arterial microcirculation. In the absence of data concerning shear stress throughout the coronary arterial tree at rest and during exercise, it is difficult to evaluate these possibilities. It is important to emphasize that the magnitude and time course of endothelial adaptations in the coronary arterial tree may be different in the presence of coronary artery disease. For example, Griffin and colleagues (8) report that, in a porcine model of chronic coronary artery occlusion, exercise training for 16-20 wk produces enhanced endothelium-dependent dilation of conduit coronary arteries and improves endothelium-mediated dilation and eNOS expression in collateral-dependent arterioles (9).

In conclusion, the purpose of this study was to test the hypothesis that a short-term (7 days) exercise training program would induce increased endothelium-dependent relaxation in conduit coronary arteries but not coronary arterioles of pigs. These results, combined with current literature, suggest that endothelium-mediated control of conduit coronary artery diameter is enhanced early in the exercise-adaptive process (7-10 days) but returns to normal later in the training period, whereas endothelium-dependent control of arterioles is increased at a later time in the adaptive process. Coming full circle, the apparent discrepancy between the results among those who have examined the effects of exercise training on endothelium-dependent dilation in the coronary circulation of dogs and pigs are not due to a difference in the responses of these two species to exercise training. Rather, it appears that the differences were caused by the fact that coronary function was examined at different stages during a progressive adaptive process, stimulated by exercise training. Finally, it is important to emphasize that the present results, combined with those in the literature, indicate that the time course of progressive vascular adaptations induced by exercise training, in the absence of vascular disease, is not the same in all conduit arteries, even in those that are expected to have increased blood flow during exercise.


    ACKNOWLEDGEMENTS

The authors thank Pam Thorne, Tammy Strawn, and Denise Holiman for excellent technical contributions to this work and a host of student animal trainers for commitment and consistent hard work.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-52490 and a National Institutes of Health---National Research Service Award to C. R. Woodman (HL-09739).

Address for reprint requests and other correspondence: M. H. Laughlin, E102, Vet. Med. Bldg., Univ. of Missouri, Columbia, MO 65211 (E-mail: laughlinm{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.

September 13, 2002;10.1152/japplphysiol.00246.2002

Received 25 March 2002; accepted in final form 11 September 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 94(1):234-244
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