INTRODUCTION

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DURING EXERCISE, coronary blood flow increases to provide the myocardium with an adequate supply of oxygen and nutrients as well as to remove metabolic by-products. This increase in coronary blood flow is mediated primarily by a reduction in coronary vascular resistance. It has previously been shown that the vasodilator capacity of coronary resistance arteries (7) and coronary blood flow capacity are increased by a program of endurance exercise training (4, 5). The mechanism accounting for the improved vasodilation is not well understood; however, recently published data indicate that the training effect is at least in part endothelium dependent, perhaps involving an increased production and release of endothelium-derived nitric oxide (7, 11, 14).

A potential mechanism accounting for the training-induced increase in endothelium-mediated vasodilation in vessels isolated from exercise-trained animals is an upregulation of the expression of the endothelial cell nitric oxide synthase (ecNOS) gene. Although exercise training has been shown to elicit an increase in the expression of ecNOS mRNA in canine aorta (11), the effect of exercise training on the expression of this gene in coronary resistance arteries is not known. Given that the primary site of resistance in the coronary circulation is resistance arteries <150 µm in diameter (2), it is important that the effect of exercise training on ecNOS gene expression be determined in these vessels.

The purpose of this study was to develop a method by which ecNOS mRNA expression could be detected and quantified in single coronary resistance arteries and to determine whether the gene coding for ecNOS is upregulated by exercise training. It was hypothesized that ecNOS mRNA expression would be greater in single coronary resistance arteries isolated from exercise-trained pigs compared with sedentary controls.

	METHODS

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Experimental animals. Before initiation of this study, approval was received from the Animal Care and Use Committee at the University of Missouri. The experimental animals were adult female Yucatan miniature swine (n = 9) purchased from a commercial breeder (Charles River). The pigs were 8-12 mo of age and weighed 25-40 kg. All of the pigs were housed in the animal care facility in the Department of Veterinary Biomedical Sciences in a room maintained at 20-23°C with a 12:12 h light-dark cycle.

Training program. All of the pigs were familiarized with running on a motorized treadmill and randomly assigned to an exercise-trained (ET; n = 5) or sedentary control (Sed; n = 4) group for 16 wk. Pigs assigned to the ET group ran 5 days/wk for 16 wk using a previously published protocol (7). The intensity and duration of the exercise bouts were increased weekly to maximize the training stimulus (7). Pigs assigned to the Sed group were restricted to their enclosures (2 × 4 m pens) and did not exercise. At the conclusion of the 16-wk training program, the ET and Sed pigs performed a graded intensity treadmill exercise test to exhaustion to assess exercise capacity. The efficacy of the training protocol was assessed from measurements of run time to exhaustion, heart weight-to-body weight ratio, and citrate synthase activity in the deltoid muscle (12).

Coronary vessel preparation. At the end of the 16-wk training period, ET and Sed pigs were sedated with ketamine (30 mg/kg im) and anesthetized with pentobarbital sodium (35 mg/kg iv). After an intravenous infusion of heparin, hearts were removed and placed in iced (4°C) Krebs bicarbonate buffer solution. Single coronary resistance arteries (50-100 µm ID, unpressurized) in the region of the left anterior descending coronary artery (LAD) were dissected, quick frozen in liquid nitrogen, and stored separately in ribonuclease (RNase)-free microcentrifuge tubes at 80°C. Four to six coronary resistance arteries were collected from each pig.

Preparation of vessel lysate. A crude lysate was prepared from each resistance artery by homogenizing the vessel in a LiCl buffer solution. Specifically, 50 µl of a lysis buffer solution [100 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 8.0), 500 mM LiCl, 10 mM EDTA (pH 8.0), 1% LiDS, and 5 mM dithiothreitol (DTT)] were added to each frozen microvessel sample and vortexed vigorously until the vessel was completely dissociated. The lysate was then spun for 60 s in a microcentrifuge (14,000 g) to remove any insoluble material.

mRNA isolation from crude lysate. Poly(A)⁺ RNA was isolated from the crude lysate with paramagnetic oligo(dT) polystyrene beads [Dynabeads oligo(dT)₂₅, Dynal]. Specifically, the lysate prepared in the previous step was transferred to a siliconized RNase-free microcentrifuge tube containing 20 µl of prewashed Dynabeads. The lysate and Dynabeads were gently mixed and incubated for 15 min at 20°C. The beads were washed twice with 100 µl of 10 mM Tris · HCl (pH 8.0), 0.15 mM LiCl, 1 mM EDTA, and 0.1% LiDS and three times with 100 µl of 10 mM Tris · HCl (pH 8.0), 0.15 M LiCl, and 1 mM EDTA. Poly(A)⁺ RNA was eluted from the Dynabeads by adding 10 µl RNase-free H₂O and heating the samples for 2 min at 65°C.

First-strand cDNA synthesis. First-strand cDNA synthesis was performed in a 20-µl volume using reverse transcriptase (RT) and oligo(dT)_12-18 to prime the reaction (Superscript Preamplification System, GIBCO-BRL Life Technologies). Specifically, the 10 µl of mRNA sample eluted from the beads were transferred to a fresh RNase-free microcentrifuge tube. One microliter of oligo(dT)_12-18 (0.5 µg/µl) and 40 U of RNase inhibitor (40 U/µl, Boehringer Mannheim) were added to the samples, mixed gently, incubated for 10 min at 70°C, and placed on ice. Two microliters of polymerase chain reaction (PCR) buffer [200 mM Tris · HCl (pH 8.4), 500 mM KCl], 2 µl of 25 mM MgCl₂, 1 µl of 10 mM deoxynucleotide triphosphate (dNTP), and 2 µl of 0.1 M DTT were then added to each sample. The samples were mixed, briefly spun in a microcentrifuge (14,000 g), and incubated in a 42°C H₂O bath for 5 min. At the conclusion of the 5-min incubation, 200 U of RT (200 U/µl, Superscript II, GIBCO-BRL Life Technologies) were added to each sample, and the reaction was allowed to proceed for 50 min at 42°C. At the end of the 50-min incubation, the reaction was terminated by incubating each sample in a 70°C H₂O bath for 15 min. The RNA template was digested by adding 2 U of RNase H (GIBCO-BRL Life Technologies) and incubating the mixture in a 37°C H₂O bath for 20 min.

PCR. Five microliters of the RT cDNA sample were used to perform a PCR reaction in a 50-µl volume containing buffer [50 mM KCl, 20 mM Tris · HCl (pH 8.4), and 4 mM MgCl₂], 0.2 µM of each primer, 0.2 mM dNTP, and 2.5 U of Taq DNA polymerase (Promega). The sense and antisense ecNOS primers were a modification of the primers used previously by Tracie et al. (13). Each primer was 21 nucleotides in length (sense, 5'-GTG TTT GGC CGA GTC CTC ACC-3'; antisense 5'-CTC CTG CAA GGA AAA GCT CTG-3'). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers (Stratagene) were 24 nucleotides in length (sense, 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3'; antisense, 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3'). The PCR reaction was initiated with a denaturation step at 94°C (5 min) and an annealing step at 60°C (5 min). This was followed by 35 cycles at 72 (2 min), 94 (1 min), and 60°C (1 min). The PCR reaction was terminated with a final step at 72°C (10 min). The PCR amplified products were electrophoresed on a 1.5% agarose gel and visualized with ethidium bromide staining.

Positive controls. Cultured human aortic endothelial cells (HAEC) and RNA isolated from porcine aorta were used as positive controls to test the specificity of the ecNOS and GAPDH primers. Total cellular RNA was isolated from HAEC and porcine aorta by the method of Chomczynski and Sacchi (3). The RNA preparations were quantitated by absorbance at 260 nm, and integrity was assessed via 1.5% agarose gel electrophoresis and ethidium bromide staining. A first-strand cDNA synthesis reaction was performed as described above for the coronary resistance arteries using 1 µg total RNA. ecNOS or GAPDH was amplified via PCR using the cycling conditions described above.

Identification of PCR products. PCR-amplified products from isolated coronary resistance arteries, HAEC, and porcine aorta were electrophoresed on 1.5% agarose gel and visualized with ethidium bromide staining. The ecNOS and GAPDH PCR fragments were identified by size, and their identity was confirmed by direct nucleotide sequencing of gel purified (Qiagen) material (1).

Negative controls. To confirm that the PCR reaction did not amplify genomic DNA, the following control experiment was performed. Poly(A)⁺ RNA was isolated from single coronary resistance arteries (n = 2) as described previously. Poly(A)⁺ RNA was eluted from the Dynabeads by adding 20 µl of RNase-free H₂O, and first-strand cDNA synthesis was performed using 10 µl of the mRNA sample. The remainder of the mRNA sample (10 µl) was used in a first-strand cDNA synthesis reaction in the absence of RT. Five microliters of the RT cDNA samples were used to perform a PCR in a 50-µl volume as described previously. The PCR reactions were evaluated with 1.5% agarose gel electrophoresis and ethidium bromide staining.

Semiquantitative PCR. Relative differences in ecNOS expression in vessels isolated from ET and Sed pigs were assessed using a modification of the semiquantitative PCR assay reported previously by Martin et al. (6). Specifically, 5 µl of the RT cDNA sample were used to perform a PCR reaction in a 50-µl volume as described above with two modifications. First, the reaction was spiked with 10 µCi of [³²P]CTP. Second, the PCR assay was terminated after 25 rounds. This time point was selected based on preliminary data indicating that the 25th round fell within the linear range of the PCR assay for both the ecNOS and GAPDH primer sets (data not shown). The PCR-amplified products were electrophoresed on a 1.5% agarose gel and visualized with ethidium bromide staining. The bands corresponding to ecNOS and GAPDH were excised from the gel and transferred to separate scintillation vials. The agarose samples were dissolved in 1 ml QX1 solution (Qiagen) and counted for 1 min in 10 ml of scintillation fluid using a Packard 1600CA liquid scintillation counter. The ecNOS-to-GAPDH ratio was determined in four to six coronary resistance arteries per pig, and a mean ecNOS-to-GAPDH ratio was calculated.

Data analysis. All values are means ± SE. Between-group differences in run time, citrate synthase activity, heart weight-to-body weight ratio, and ecNOS-to-GAPDH ratio were assessed using Student's t-tests for unpaired observations. Statistical significance was set at the P  0.05 probability level.

	RESULTS

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Indexes of training. The efficacy of the exercise-training protocol was assessed from measurements of deltoid muscle citrate synthase activity, heart weight-to-body weight ratio, and run time to exhaustion during the maximal exercise test. After 16 wk of training ET pigs ran significantly longer than the Sed controls (Table 1). In addition, deltoid muscle citrate synthase activity and heart weight-to-body weight ratio were significantly higher in the ET group.

HAEC and porcine aorta. The results of the positive control experiment are shown in Fig. 1. Agarose gel electrophoresis of the PCR products revealed single bands corresponding to ecNOS (341 bp) and GAPDH (600 bp) for both HAEC and porcine aorta. Gel purification and nucleotide sequencing of the PCR products confirmed that the 341-bp fragment amplified from the cultured cells and the porcine aorta sample was ecNOS (100% sequence homology with Sus scrofa NOS, gene accession no. U33832). Nucleotide sequencing revealed that the 600-bp fragment was GAPDH (98% homologous with porcine GAPDH, gene accession no. U48832). These findings confirmed the specificity of the primers for use in the microvessel experiments.

View larger version (129K): [in this window] [in a new window]   Fig. 1.   Polymerase chain reaction (PCR) detection of endothelial cell nitric oxide synthase (ecNOS, 341 bp, lanes 1 and 3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 600 bp, lanes 2 and 4) mRNA in cultured human aortic endothelial cells (HAEC) and porcine aorta. PCR products were electrophoresed on a 1.5% agarose gel and visualized with ethidium bromide staining. Lanes 1 and 2, PCR products from HAEC; lanes 3 and 4, PCR products from porcine aorta. MW, 123-bp molecular-weight marker.

Coronary resistance arteries. PCR fragments obtained using cDNA made from single coronary resistance arteries (n = 7) and the primers specific for ecNOS and GAPDH revealed single bands corresponding to ecNOS and GAPDH (Fig. 2). Nucleotide sequencing of the PCR products confirmed that the 341-bp fragment was ecNOS as indicated by sequence homology with S. scrofa NOS (gene accession no. U33832). Nucleotide sequencing revealed that the 600-bp fragment was porcine GAPDH (gene accession no. U48832). These data indicate that both sets of primers can be used simultaneously to coamplify their respective products in the same reaction from an individual coronary resistance artery.

View larger version (95K): [in this window] [in a new window]   Fig. 2.   PCR detection of ecNOS (341 bp) and GAPDH (600 bp) mRNA in single coronary resistance arteries (50-100 µm, unpressurized). PCR products were electrophoresed on a 1.5% agarose gel and visualized with ethidium bromide staining. Lanes 1-7, 7 different coronary resistance arteries.

View larger version (101K): [in this window] [in a new window]   Fig. 3.   PCR detection of ecNOS (341 bp) and GAPDH (600 bp) mRNA in single coronary resistance arteries (50-100 µm, unpressurized). First-strand cDNA synthesis reaction was performed in presence or absence of reverse transcriptase. RT(+), reverse transcriptase present; RT(), reverse transcriptase absent. PCR products were electrophoresed on a 1.5% agarose gel and visualized with ethidium bromide staining.

Effect of training. The effect of training on the expression of ecNOS and GAPDH in single coronary resistance arteries is shown in Fig. 4. GAPDH levels were unchanged by training. In contrast, ecNOS mRNA levels were greater in vessels isolated from the ET group than in vessels isolated from the Sed controls. Figure 5 shows the corresponding mean data obtained after 25 rounds of PCR. Compared with the Sed group, the ecNOS-to-GAPDH ratio was significantly greater in the ET group.

View larger version (138K): [in this window] [in a new window]   Fig. 4.   PCR detection of ecNOS (341 bp) and GAPDH (600 bp) mRNA in single coronary resistance arteries (50-100 µm, unpressurized). Lanes 1-3, coronary resistance arteries isolated from a sedentary (Sed) pig; lanes 4-6, coronary resistance arteries isolated from an exercise-trained (ET) pig. PCR products were electrophoresed on a 1.5% agarose gel and visualized with ethidium bromide staining.

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Comparison of ecNOS-to-GAPDH ratio in Sed and ET pigs. Values are means ± SE for 4 Sed and 5 ET pigs. * ecNOS-to-GAPDH ratio was significantly greater in ET pigs (P = 0.008).

	DISCUSSION

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The purpose of this study was to test the hypothesis that ecNOS mRNA expression is upregulated in single coronary resistance arteries isolated from ET pigs. The key finding of the study was that the ecNOS-to-GAPDH ratio was significantly greater in coronary resistance arteries isolated from ET pigs compared with Sed controls.

Coronary resistance arteries (50-100 µm, unpressurized) isolated from the region of the LAD were selected for use in this study because previously published data indicated that exercise training enhances endothelium-dependent vasodilation in vessels of this size (7). This training adaptation was attributed to greater production of nitric oxide because it was abolished by treatment with arginine analogs (7). In addition, this size of coronary artery was selected because it represents a primary site of resistance in the coronary circulation (2).

Although these vessels have been documented to contribute greatly to coronary vascular resistance, their small size and limited mRNA content make the detection of cell-specific gene expression a technically difficult endeavor. Therefore, to perform the training study, we first had to develop a method based on the PCR for assessing ecNOS mRNA expression in single coronary resistance arteries. The specificity of the primers and the thermal cycling conditions used were initially determined with RNA isolated from cultured HAEC and porcine aorta. The HAEC were chosen as a positive control, since a large amount of RNA could be isolated and purified. The porcine aorta preparation was used as a positive control that also provided a large quantity of RNA in a tissue harvested from the animal model to be studied.

Initial efforts using previously published primer sequences for ecNOS (13) failed to amplify a single specific band corresponding to the predicted size of ecNOS. Because we could directly sequence the PCR fragments obtained in this study, subcloning of the PCR products was not necessary. Therefore the nucleotide sequences coding for restriction sites built into the original primers (13) were deleted. When the modified primers were used in the PCR reaction, agarose gel electrophoresis revealed a single band corresponding to the predicted size for ecNOS (341 bp) in both HAEC and porcine aorta (Fig. 1). In addition, a single band corresponding to the predicted size of GAPDH (600 bp) was amplified using the GAPDH primers (Fig. 1).

The identity of the PCR fragments was confirmed by gel purification and DNA sequencing. The 341-bp fragment obtained in these experiments shared a high degree of sequence homology with porcine (100%), bovine (96%), and human (94%) ecNOS. The 600-bp PCR fragment from Yucatan pigs was 98% homologous with porcine GAPDH.

Because several isoforms of NOS have been cloned and sequenced (10), we compared the sequence of the PCR product generated in this study to all known sequences of NOS. The closest match obtained with a nonendothelial isoform was with human retinal NOS (64%). The low level of sequence homology with nonendothelial isoforms further confirmed that the primer was specifically amplifying ecNOS in these preparations. Consequently, the primers were accepted for use in the training study.

The gels shown in Figs. 2 and 3 indicate that ecNOS can be successfully detected in single isolated coronary resistance arteries alone or coamplified with GAPDH. GAPDH is a constitutively expressed gene that is not affected by training (11). Consequently, expressing ecNOS levels relative to GAPDH allowed for relative comparisons between vessels isolated from ET and Sed pigs (Figs. 4 and 5).

The ecNOS-to-GAPDH ratio in coronary resistance arteries isolated from ET pigs was significantly greater than the ecNOS-to-GAPDH ratio obtained for the Sed controls (Fig. 5). The increased expression of ecNOS mRNA may provide the mechanism for the enhanced endothelium-dependent vasodilation in coronary resistance arteries isolated from ET pigs previously reported by Muller et al. (7).

The signal initiating the observed increase in ecNOS gene expression in ET animals is not known; however, Sessa et al. (11) have previously proposed that the signal may involve increased blood flow velocity and shear stress. This possibility is supported by recently published data indicating that chronic exposure to conditions of high blood flow via arteriovenous fistula increased ecNOS gene expression in the aorta of rats (8).

In conclusion, the results of this study indicate that RT-PCR can be used on single coronary resistance arteries to detect cell-specific gene expression. The method can be applied to mRNA of any protein that has been cloned and sequenced. We have shown in the present study that endurance exercise training induces an upregulation of ecNOS gene expression in porcine coronary resistance arteries. The increased mRNA content is likely to contribute to the training-induced increase in nitric oxide production and the enhanced endothelium-dependent vasodilation observed in porcine coronary resistance arteries. Further studies are required to determine the molecular mechanisms that mediate the increase in ecNOS expression.