INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CORONARY ARTERIES located distal to chronic occlusion are dependent on the collateral circulation for their blood supply. Although collateral artery development following chronic occlusion may be sufficient to restore adequate blood flow to the collateral-dependent myocardium during rest, increased collateral vascular resistance and reduced maximal perfusion capacity ensue, causing impaired blood flow to the compromised myocardium under conditions of physiological stress such as exercise. Our laboratory (18) and others (16, 22, 24) have shown collateral-dependent arteries exhibit impaired vasomotor responsiveness that may have important implications in the regulation of blood flow to the collateral-dependent myocardium (23). Specifically, we previously reported that collateral-dependent canine coronary arteries exhibit impaired relaxation to the cAMP-dependent vasodilator adenosine, but not to the cGMP-dependent vasodilator sodium nitroprusside, compared with size-matched nonoccluded coronary arteries (18). On the other hand, several reports have documented that chronic exercise training produces specific alterations in coronary circulation that reduce vascular resistance and enhance myocardial blood flow capacity during maximal adenosine-induced vasodilatation (6, 11, 12). Additionally, Oltman et al. (15) found that exercise training enhances the sensitivity of porcine coronary arteries to adenosine. However, interactions and effects of chronic coronary occlusion and exercise training on adenosine-mediated relaxation have not been studied previously. Therefore, we hypothesized that the impaired relaxation in response to adenosine, but not sodium nitroprusside, reflects differential effects of chronic occlusion and exercise training on cAMP- versus cGMP-mediated relaxation. We specifically evaluated the effects of chronic coronary occlusion and exercise training on in vitro vasomotor responsiveness to the cAMP-dependent vasodilators adenosine, isoproterenol, and forskolin, and the cGMP-dependent vasodilator sodium nitroprusside in arterial segments of miniature swine. Furthermore, to investigate potential cellular mechanisms underlying alterations in adenosine responsiveness resulting from occlusion and exercise training, we simultaneously measured adenosine-induced changes in contractile tension and myoplasmic free Ca²⁺ concentration ([Ca²⁺]_m) in collateral-dependent and nonoccluded coronary arteries isolated from sedentary control and exercise-trained pigs.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental animals. Female Yucatan miniature swine, 7-8 mo of age, were obtained from the breeder (Charles River, Wilmington, MA) in six groups over a 4-yr period. Each group was maintained in an identical manner. Animals were ~14-15 mo of age and averaged 34.7 ± 6.9 kg (mean ± SD; range 25-52 kg) at the time of death. Animal protocols were approved by the University of Missouri Animal Care and Use Committee in accordance with the Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training set forth by the United States government.

Surgical procedures. Animals were preanesthetized with glycopyrrolate (0.004 mg/kg im) and midazolam (0.5 mg/kg im) after which surgical anesthesia was induced with ketamine (20 mg/kg im). Animals were intubated and anesthesia was maintained with 3% isoflurane and 97% O₂ during sterile surgery. A left lateral thoracotomy was performed in the fourth intercostal space and the underlying pericardium was opened. The proximal left circumflex coronary artery (LCx) was dissected free of surrounding tissue and an Ameroid constrictor (Research Instruments, Corvallis, OR), 2.5- to 3.5-mm inner diameter, was placed around the artery. The diameter of the occluder was selected upon visual inspection of the artery to provide a secure but nonconstrictive fit. The pericardium was then closed and the thoracotomy was repaired in tissue layers.

Training procedures. Eight weeks following surgery, pigs were familiarized with treadmill exercise over a 1- to 2-wk period. Animals were then randomly assigned to either a sedentary or exercise-trained group. Exercise-trained pigs underwent 16 wk of a progressive treadmill exercise training program adapted from Tipton et al. (29) and used extensively by Laughlin and colleagues (11, 12, 15). Sedentary pigs were restricted to their pens (6 × 12 ft) for an equivalent period of time. During the first week of the experimental protocol, a typical exercise training session included treadmill running at 3 miles/h for 20-25 min (endurance) and at 5 miles/h for 5-10 min (sprint). All exercise training sessions were preceded by a 5-min warm-up at 2.5 miles/h and followed by a 5-min cool-down at 2 miles/h. Speed and duration of the exercise training sessions were progressively increased so that during the last week of training, animals ran at 4-5.5 miles/h for 60 min and at 6 miles/h for 5-15 min. Grade of the treadmill was maintained at 0% throughout the experimental protocol. The progressive nature of the exercise training was dependent on the tolerance of each animal, and therefore ranges of running speed and duration presented represent differing abilities of the animals. Pigs were given positive reinforcement for exercise by being fed after each training bout.

Efficacy of training. Effectiveness of the exercise training program was determined by comparing skeletal muscle oxidative enzyme capacity and heart weight-to-body weight ratio of the exercise-trained versus sedentary animals. After the animals were euthanized, samples were taken from the deltoid and medial and lateral heads of the triceps brachii muscles, frozen in liquid N₂, and stored at 70°C until processed. Citrate synthase activity was measured from whole muscle homogenate using the spectrophotometric method of Srere (28).

Preparation of coronary arteries. After completion of the exercise training protocol or sedentary confinement, the animals were anesthetized with ketamine (30 mg/kg) and pentobarbital sodium (35 mg/kg). The hearts were removed, placed in ice-cold Krebs bicarbonate buffer (0-4°C), and weighed. Hearts were maintained in iced Krebs buffer during isolation of the occluded LCx and the nonoccluded left anterior descending (LAD) coronary arteries. Visual inspection at the Ameroid occluder during dissection of the LCx artery indicated 100% occlusion in all animals used in this study.

Concentration-response relationships in vitro. Coronary rings (3.5- to 4.0-mm axial length) were mounted on two stainless steel wires passed through the vessel lumen. One wire was fixed to a force transducer (Grass FT03, Grass Instruments, Quincy, MA) and the other to a micrometer microdrive (Stoelting/Prior Microdrive, Wood Dale, IL) to allow precise changes in circumferential length of the vessel. The mounted arterial ring was lowered into a 20-ml tissue bath containing Krebs bicarbonate buffer at 37 ± 0.5°C and aerated with 95% O₂-5% CO₂. Coronary rings were progressively stretched to the maximum of the length-developed tension relationship (L_max) in increments equal to 10% of the vessel's initial outer diameter. After each stretch, contraction was elicited with exposure to high K⁺ (80 mM), and L_max was defined as the circumferential length at which developed tension was <5% greater than the developed tension produced at the previous length. Arterial rings were allowed to equilibrate for 45-60 min at L_max before subsequent evaluation of pharmacological responsiveness. After equilibration, arterial rings were preconstricted with 30 µM PGF₂ and concentration-response relationships were determined by cumulative additions of concentrated stock solutions of vasodilatory agents directly to the tissue bath. Drug concentrations were increased when the response to the previous concentration had stabilized. Isometric contraction and relaxation responses were measured on a Grass polygraph recorder (Grass Instruments). Percent relaxation was defined as the percentage decline in developed tension of the agonist-mediated preconstriction. For this study, the phrase "impaired relaxation response" indicates a reduced sensitivity to the vasodilating agent not a reduction in the maximal relaxation response.

Simultaneous measures of [Ca²⁺]_m and tension. In separate studies, [Ca²⁺]_m and developed tension were measured simultaneously in arterial rings utilizing a specially designed microfluorometry apparatus (18). Denuded arterial rings were mounted on two stainless steel wires, one fixed to a force transducer (Kulite Semiconductor Products, Leonia, NJ) and the other attached to a lever driven by a digital micrometer to permit precise changes in circumferential length of the vessel. The mounted arterial ring was lowered into a heated superfusion chamber of a microfluorometry system (Nikon Diaphot, Nikon, Garden City, NY) and stretched to L_max as determined by repeated exposure to high K⁺ (60 mM) at increasing vessel diameter. Excitation light from a 150-W xenon arc lamp was passed through a rotating filter wheel (50-ms intervals) containing alternating 340- and 380-nm interference filters. Fluorescence emission at 510 nm was synchronized with the appropriate excitation wavelength by a photodetector mounted on the filter wheel and was reflected to a photomultiplier tube with a dichroic mirror. Fluorescence was analyzed with an analog fluorescence signal processor and an analog-to-digital converter. The change in fluorescence ratio (F₃₄₀/F₃₈₀) was representative of relative [Ca²⁺]_m following off-line subtraction of vessel autofluorescence. Autofluorescence was determined at the end of each experiment by superfusion in physiological saline solution (PSS) containing 2 mM Mn²⁺, followed by bathing the vessel in PSS containing 2 mM Mn²⁺ plus 10 µM ionomycin to quench intracellular fura 2-AM. Fluorescence and contractile data were sampled every 5 s. Data acquisition and transformations were performed using AxoBASIC 1.0 software (Axon Instruments, Foster City, CA) customized for multichannel data acquisition.

Solutions. The Krebs buffer contained (in mM) 131.5 NaCl, 5 KCl, 1.2 NaH₂PO₄, 1.2 MgCl₂, 2.5 CaCl₂, 11.2 glucose, 13.5 NaHCO₃, and 0.025 EDTA. High K⁺ solutions were produced by equimolar substitution of KCl for NaCl. Vessels used for fura 2-AM experiments were stored and rinsed in sterile modified Eagle's minimal essential storage media that contained (in mM) 135 NaCl, 5 KCl, 0.34 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, 10 glucose, 2.6 NaHCO₃, 0.44 KH₂PO₄, 20 HEPES, and (volume for volume) 0.02 amino acids, 0.01 vitamins, 0.002 phenol red, and 0.01 penicillin/streptomycin, including 2% horse serum. The solution pH was adjusted to 7.2 at 23°C using NaOH and then passed through a sterile filter producing a final pH of 7.4. The fura 2-AM loading solution contained 10 µM fura 2-AM, 0.5% cremophor, and 5% BSA. The loading solution was vortexed for 1 min and then sonicated for an additional 1 min to increase solubilization of fura 2-AM. Drugs were obtained from Sigma Chemical (St. Louis, MO) unless otherwise noted. Endothelin-1 was purchased from Peninsula Laboratories (Belmont, CA), ionomycin from Calbiochem (La Jolla, CA), and PGF₂ from Upjohn (Kalamazoo, MI).

Statistical analysis. Heart weight-to-body weight ratio and citrate synthase activity were evaluated using the unpaired Student's t-test. Dimensional characteristics of coronary arterial rings were compared using ANOVA and the Student-Newman-Keuls correction for multiple comparisons. Concentration-response relationships in the presence of vasodilating agents are presented as percentages of maximal relaxation from preconstricted values and were analyzed using ANOVA for repeated measures and the Student Newman-Keuls correction for multiple comparisons. The concentration of agonist causing 50% of the maximal relaxation response was designated EC₅₀ and was calculated for each artery. The individual EC₅₀ values were averaged for LAD and LCx and compared using unpaired Student's t-tests; sedentary versus exercise-trained LCx EC₅₀ values were also compared using unpaired t-tests. The combined tension and [Ca²⁺]_m experiments are presented as the reduction in grams of developed tension and fluorescence ratio, respectively. Reductions in developed tension and [Ca²⁺]_m were analyzed using the unpaired Student's t-test. For all analyses, P  0.05 was considered significant. Data are presented as means ± SE, and n represents the number of animals. When more than one vascular ring from a single coronary artery was used in identical protocols, the responses from these rings were averaged before data analyses were conducted.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Training status. Effectiveness of the 16-wk exercise training program was demonstrated by significant (P < 0.05) increases in skeletal muscle oxidative enzyme capacity and an increased heart weight-to-body weight ratio in exercise-trained animals. Citrate synthase activity increased significantly in the deltoid muscle (20.6 ± 1.4 vs. 16.5 ± 0.7 µmol · min¹ · g¹) as well as the medial head (17.9 ± 0.9 vs. 15.7 ± 0.6 µmol · min¹ · g¹) and lateral head (18.6 ± 1.2 vs. 15.2 ± 0.7 µmol · min¹ · g¹) of the triceps brachii muscle in exercise-trained (n = 31) versus sedentary animals (n = 31), respectively. Heart weight-to-body weight ratio increased in exercise-trained versus sedentary pigs (6.7 ± 0.2 vs. 5.7 ± 0.2 g/kg, respectively; P < 0.05).

Coronary vessel dimensions and characteristics. Dimensions of coronary arterial rings used in this study are presented in Tables 1 and 2. Arterial rings are divided into those used for concentration-response relationships and those used for simultaneous [Ca²⁺]_m and tension experiments. No significant differences were observed between LAD and LCx arteries within exercise-trained or sedentary groups for arterial rings used in the concentration-response relationships (Table 1) or in rings used for fura 2-AM experiments (Table 2). The luminal diameter of rings used for concentration-response relationships (Table 1) and the outer diameter of rings used in fura 2-AM experiments (Table 2) were slightly larger in the exercise-trained LAD artery relative to the sedentary LCx artery. However, no comparisons of vasomotor responsiveness between these two groups of vessels were performed in this study.

Relaxation responses to adenosine and isoproterenol. We evaluated concentration-dependent relaxation to the cAMP-dependent vasodilators adenosine (10⁷ to 10³ M) and isoproterenol (3 × 10⁸ to 3 × 10⁵ M) in arterial rings preconstricted with 30 µM PGF₂. Relaxation responses to both adenosine (Fig. 1A) and isoproterenol (Fig. 2A) were significantly impaired (P < 0.05) in the collateral-dependent LCx compared with the nonoccluded LAD artery in sedentary animals. The EC₅₀ for LCx arterial rings was significantly greater than that for LAD rings in response to adenosine (30.1 ± 11.1 vs. 6.7 ± 1.3 µM) and isoproterenol (165 ± 35 vs. 80 ± 11 nM) in LCx versus LAD arteries, respectively. Importantly, exercise training reversed the impaired relaxation response to adenosine (Fig. 1B) and isoproterenol (Fig. 2B) in LCx arteries. Further evaluation of EC₅₀ values revealed no statistical differences between LCx arteries isolated from sedentary versus exercise-trained animals in response to adenosine (30.1 ± 11.1 vs. 17.5 ± 8.4 µM) or isoproterenol (165 ± 35 vs. 103 ± 19 nM). Subsequent control experiments conducted in our laboratory (data not shown) indicate that normal nonoccluded animals do not demonstrate regional differences (LCx vs. LAD) in relaxation responses to adenosine. Thus these data provide evidence that the nonoccluded artery from the same heart may prove useful for comparison of the effects of chronic occlusion and exercise training on collateral-dependent vasculature.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent relaxation responses to cAMP-dependent vasodilator adenosine in collateral-dependent left circumflex (LCx) and nonoccluded left anterior descending (LAD) arteries isolated from sedentary (A) and exercise-trained (B) animals. Arteries were preconstricted with 30 µM PGF2. Adenosine-induced relaxation was significantly impaired in LCx vs. LAD arteries in sedentary animals. Concentration yielding 50% relaxation response (EC50) was significantly greater in LCx vs. LAD arterial rings in response to adenosine in sedentary animals (inset). Exercise training reversed this impaired adenosine-mediated relaxation. Values are means ± SE. NS, not significant. *P < 0.05 vs. LAD.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent relaxation responses to cAMP-dependent vasodilator isoproterenol in collateral-dependent LCx and nonoccluded LAD arteries isolated from sedentary (A) and exercise-trained (B) animals. Arteries were preconstricted with 30 µM PGF2. Isoproterenol-induced relaxation was significantly impaired in LCx vs. LAD arteries in sedentary animals. EC50 was significantly greater in LCx vs. LAD arterial rings in response to isoproterenol in sedentary animals (inset). Exercise training reversed this impaired isoproterenol-mediated relaxation. Values are means ± SE. *P < 0.05 vs. LAD.

Relaxation response to forskolin. We also evaluated cAMP-dependent vasodilatation using the receptor-independent adenylyl cyclase activator forskolin (10⁹ to 10⁵ M). In contrast to our findings using adenosine and isoproterenol, concentration-dependent relaxation responses to forskolin were not different between the collateral-dependent LCx artery and the nonoccluded LAD artery in sedentary animals (Fig. 3A). The EC₅₀ values for LCx and LAD arteries were not significantly different in response to forskolin. Furthermore, these relaxation responses were not altered with exercise training (Fig. 3B).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent relaxation responses to membrane-permeant, cAMP-dependent vasodilator forskolin in collateral-dependent LCx and nonoccluded LAD arteries isolated from sedentary (A) and exercise-trained (B) animals. Arteries were preconstricted with 30 µM PGF2. Forskolin-induced relaxation was not significantly different between LCx and LAD arteries in sedentary or exercise-trained animals. EC50 values were not significantly different in LCx vs. LAD arterial rings in response to forskolin in sedentary or exercise-trained animals (inset). Values are means ± SE.

Relaxation response to sodium nitroprusside. In addition to our evaluation of cAMP-mediated relaxation, we also assessed concentration-dependent relaxation in response to the cGMP-dependent vasodilator sodium nitroprusside (10⁹ to 10⁴ M). Our findings demonstrate that relaxation responses were not significantly different in the LCx and LAD arteries in sedentary animals (Fig. 4A). The EC₅₀ values for LCx and LAD arterial rings in response to sodium nitroprusside were not significantly different. Furthermore, these relaxation responses were not altered with exercise training (Fig. 4B).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent relaxation responses to cGMP-dependent vasodilator sodium nitroprusside in collateral-dependent LCx and nonoccluded LAD arteries isolated from sedentary (A) and exercise-trained (B) animals. Arteries were preconstricted with 30 µM PGF2. Sodium nitroprusside-induced relaxation was not significantly different between LCx and LAD arteries in sedentary or exercise-trained animals. EC50 values were not significantly different in LCx vs. LAD arterial rings in response to sodium nitroprusside in sedentary or exercise-trained animals (inset). Values are means ± SE.

Simultaneous tension and [Ca²⁺]_m. In additional experiments designed to elucidate potential cellular mechanisms underlying alterations in adenosine responsiveness resulting from chronic coronary occlusion and exercise training, we simultaneously measured adenosine-mediated changes in developed tension and [Ca²⁺]_m in collateral-dependent LCx and nonoccluded LAD arteries isolated from sedentary and exercise-trained pigs. Figure 5 represents our experimental protocol in which vessels were preconstricted with endothelin-1 (10 and 100 nM) and then treated with both submaximal (10 µM) and maximal (100 µM) concentrations of adenosine.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Representative experiment showing simultaneous measures of contractile tension and change in myoplasmic free Ca2+ concentration ([Ca2+]m) using F340/F380 fluorescence ratio during exposure to endothelin-1 and adenosine. Experiments were performed on both collateral-dependent LCx and nonoccluded LAD arteries.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Absolute decline in grams of contractile tension (A) and change in [Ca2+]m (B; F340/F380 fluorescence ratio) during exposure to 10 and 100 µM adenosine in collateral-dependent LCx and nonoccluded LAD arteries isolated from sedentary animals. Arteries were preconstricted with 100 nM endothelin-1. Adenosine-induced reductions in contractile tension and [Ca2+]m were significantly impaired in LCx vs. LAD arteries in sedentary animals. Values are means ± SE. *P < 0.05 vs. LCx.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Absolute decline in grams of contractile tension (A) and [Ca2+]m (B; F340/F380 fluorescence ratio) during exposure to 10 and 100 µM adenosine in collateral-dependent LCx and nonoccluded LAD arteries isolated from exercise-trained animals. Arteries were preconstricted with 100 nM endothelin-1. Adenosine-induced reductions in contractile tension and [Ca2+]m were not significantly different between LCx and LAD arteries in exercise-trained animals. Values are means ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, we demonstrate that chronic coronary artery occlusion impairs relaxation responses to the cAMP-dependent vasodilators adenosine and isoproterenol but not to forskolin or the cGMP-dependent vasodilator sodium nitroprusside. These findings support the previous observation in our laboratory using a canine model of chronic coronary occlusion that adenosine-induced but not sodium nitroprusside-induced relaxation was significantly impaired in the collateral-dependent LCx artery (18). Although the vasodilatory response to isoproterenol in previous work by our laboratory was not diminished in the canine collateral-dependent LCx artery (18), Sellke et al. (23, 25) documented impaired isoproterenol-mediated relaxation of microvessels isolated from porcine collateral-dependent myocardium. Therefore, impaired vasodilatory responses to isoproterenol in our present study but not in previous work from our laboratory may reflect species differences in adaptations to coronary occlusion (porcine vs. canine model). Our finding, that relaxation in response to the cGMP-dependent vasodilator sodium nitroprusside was not impaired in the collateral-dependent LCx artery of sedentary animals, is supported by previous research in our laboratory (18) as well as another (24). Sellke and colleagues (24) documented that relaxation in response to the cGMP-dependent vasodilator nitroglycerin was not different between collateral-dependent and nonoccluded canine coronary arteries. Both of these nitrovasodilators (sodium nitroprusside and nitroglycerin) activate soluble guanylyl cyclase and increase intracellular cGMP, in turn activating downstream cGMP-dependent protein kinase. Thus our work, in concert with previous findings, supports the assertion that the cGMP signal-transduction pathway is not impaired in vasculature subjected to chronic coronary occlusion. Furthermore, this pathway appears unaffected by exercise training in our model.

Adenosine-induced vasodilatation in vascular smooth muscle is proposed to occur through the activation of adenylyl cyclase via coupling of A₂ receptors to the guanine nucleotide-binding stimulatory (G_s) protein (5, 9).¹ Similarly, relaxation in response to isoproterenol involves coupling of -adrenoceptors and the G_s protein for activation of adenylyl cyclase (9). In contrast, forskolin, a membrane-permeant, receptor-independent vasodilator, stimulates adenylyl cyclase directly (21). The activation of adenylyl cyclase by any of these agonists increases intracellular cAMP levels, which in turn stimulates cAMP-dependent protein kinase (10). Our results of impaired coronary artery relaxation responses to adenosine and isoproterenol but not to forskolin suggest potential downregulation of adenosine (A₂) and -adrenergic receptors, impaired receptor-G_s protein coupling, and/or subsequent G_s activation of adenylyl cyclase after chronic coronary occlusion. Previously, Sellke et al. (25) documented impaired relaxation responses to both isoproterenol and forskolin in microvessels isolated from porcine collateral-dependent myocardium. The discrepancy between this report (25) and our present findings may result from differences in vessel size (microvessels vs. conduit vessels) or duration of occlusion before isolation of vessels (5-7 wk vs. 24 wk). Interestingly, pulmonary arteries isolated from chronically hypoxic rats exhibited impaired vasorelaxation responses to isoproterenol, but not to forskolin (17), and furthermore demonstrated -adrenoceptor downregulation in the presence of unaltered adenylyl cyclase or G_s protein activity (26). Similar cellular mechanisms may account for impaired relaxation to isoproterenol but not to forskolin in our study. On the other hand, additional mechanisms independent of the adenylyl cyclase signaling cascade may mediate differential relaxation responses of receptor-dependent (adenosine and isoproterenol) versus receptor-independent (forskolin) agonists (27). Alternatively, unaltered relaxation responses to forskolin may also result from differential access of receptor agonists and/or associated G proteins versus forskolin to intracellular pools of adenylyl cyclase in coronary smooth muscle distal to occlusion.

Agents that increase cellular levels of cAMP or cGMP are proposed to induce vascular smooth muscle relaxation by reducing intracellular Ca²⁺ concentrations and/or Ca²⁺ sensitivity of the contractile elements (10, 19). Therefore, to gain further insight into possible underlying mechanisms responsible for our findings of impaired cAMP-mediated vasodilatation and its reversal with exercise training, we measured changes in developed tension and [Ca²⁺]_m simultaneously. Our findings from these experiments (Fig. 6A) confirmed our earlier findings (Fig. 1A) of impaired relaxation in the collateral-dependent LCx in sedentary animals and was reversed with exercise training (Fig. 7A). Importantly, [Ca²⁺]_m closely paralleled the reduction in developed tension in both sedentary (Fig. 6B) and exercise-trained animals (Fig. 7B). Thus the impaired relaxation observed in the collateral-dependent LCx artery to both 10 and 100 µM adenosine treatments appears to have been mediated at least partially by a diminished reduction in [Ca²⁺]_m level.

In addition to reducing intracellular Ca²⁺ concentrations, cyclic nucleotides are also postulated to mediate a Ca²⁺ desensitization of myosin light chain phosphorylation of the contractile apparatus (3, 13, 31). Chen and Rembold (3) reported that cAMP- or cGMP-mediated relaxation of arterial strips depolarized with high KCl was associated with a reduction in tension without a significant change in Ca²⁺ influx or intracellular Ca²⁺ levels. On the other hand, cyclic nucleotide-mediated relaxation of agonist-stimulated contraction produced a proportional reduction in intracellular Ca²⁺ and developed tension. These investigators (3) suggest that in situations such as agonist-stimulated contraction, cyclic nucleotide-induced activation of K⁺ channels and subsequent hyperpolarization decreases Ca²⁺ influx at L-type channels and therefore reduces intracellular Ca²⁺. In contrast, with KCl depolarization, where K⁺ channel activation would have less effect on membrane potential, cyclic nucleotides appear to induce relaxation through the dissociation of tension and intracellular Ca²⁺ levels (3). These conclusions (3) support our data, which show that arterial rings preconstricted with the agonists PGF₂ and endothelin-1 demonstrate corresponding reductions in developed tension and [Ca²⁺]_m in the presence of the cAMP-dependent vasodilator adenosine, with no significant change in Ca²⁺ sensitivity (data not shown).

In the present study, we also report that exercise training reverses the impaired adenosine- and isoproterenol-mediated vasodilatation so that relaxation responses of the collateral-dependent and nonoccluded vasculature to these agonists are not different. These findings suggest chronic exercise training enhances the sensitivity of vasculature distal to chronic occlusion to the cAMP-dependent vasodilators adenosine and isoproterenol. Although exercise training reversed the impaired adenosine- and isoproterenol-mediated vasodilatation, unpaired Student's t-test comparisons of EC₅₀ values revealed no statistical differences between LCx arteries isolated from sedentary versus exercise-trained animals. Thus our data demonstrate that exercise training reversed the impaired relaxation responses to adenosine and isoproterenol in the collateral-dependent LCx artery when compared with the nonoccluded LAD isolated from the same heart, although comparisons of the LCx artery isolated from sedentary and exercise-trained animals were not different. The inability of sedentary versus exercise-trained LCx artery vasodilatory responses to reach statistical significance may be attributed to inherent differences in the coronary and collateral circulations of these animals at the start of the experimental protocol, as documented previously (4). Furthermore, control experiments conducted in our laboratory (data not shown) indicate that normal, nonoccluded animals do not demonstrate regional differences (LCx vs. LAD) in relaxation responses to adenosine. Therefore, the comparison of relaxation responses of collateral-dependent and nonoccluded arteries of the same heart is a unique characteristic of our model of chronic occlusion, providing an "internal" control vessel to compare the effects of occlusion and exercise training on vessel reactivity without the confounding variability observed in between-animal comparisons.

Previous observations reported by Oltman et al. (15) demonstrated enhanced sensitivity of large epicardial arteries to adenosine, but decreased sensitivity to sodium nitroprusside, in normal exercise-trained versus sedentary miniature swine. We did not observe statistically significant training-induced reductions in sodium nitroprusside sensitivity in the current study, potentially due to differences in models (normal vs. occluded) or levels of exercise training between studies. Training-enhanced sensitivity to adenosine supports earlier work by Laughlin and colleagues (12) demonstrating that constant-pressure myocardial blood flow during maximal adenosine vasodilatation was significantly enhanced in exercise-trained swine. DiCarlo et al. (6) also documented an enhanced maximum coronary blood flow response to intracoronary infusion of adenosine in conscious, exercise-trained dogs. Although the etiology of this enhanced sensitivity is unknown, recent findings (30) demonstrate that chronic exercise training promotes capillary angiogenesis and arteriolar growth, and in turn increases coronary blood flow and capillary transport reserve in the porcine myocardium. Sellke and colleagues (25) suggest enhanced angiogenesis may contribute to the preservation of vascular responses and improved myocardial perfusion and viability. Furthermore, the potential increase in shear stress in response to increased coronary blood flow following exercise training may cause alterations in receptor expression, receptor-G_s protein coupling, and/or subsequent G_s activation of adenylyl cyclase to enhance adenosine- and isoproterenol-mediated sensitivity of the collateral-dependent LCx artery. Thus whereas chronic coronary occlusion appears to produce impaired cAMP-mediated vasodilatation, exercise training reverses this impaired relaxation and therefore may optimize blood flow to the myocardium and vasculature distal to chronic occlusion under conditions of increased adenosine release.

Conclusions and implications. Our findings demonstrate that chronic coronary artery occlusion impairs receptor-dependent but not receptor-independent cAMP-mediated relaxation. Furthermore, cGMP-dependent relaxation responses are not altered by chronic coronary artery occlusion. Evidence is presented indicating that impaired cAMP-dependent relaxation is paralleled by diminished reduction of [Ca²⁺]_m levels in collateral-dependent arteries. Importantly, impaired reductions in developed tension and [Ca²⁺]_m are reversed with exercise training so that relaxation responses of the collateral-dependent and nonoccluded arteries are not different. The impact of alterations in the vasomotor reactivity of the large collateral-dependent LCx artery has both potential physiological and clinical significance. Blood flow to the chronically occluded main artery is maintained via collateral circulation (7, 20) and has been reported to quantitatively approach the magnitude of typical nonoccluded flow in this vessel (7, 20). Furthermore, the presence of disease has been reported to increase the contribution of epicardial arteries to total coronary vascular resistance and therefore blood flow regulation (8). Previous studies have also demonstrated that both ischemia (1) and exercise (14) increase epicardial concentrations of adenosine, implicating a role for adenosine in the control of coronary blood flow by epicardial arteries of chronically occluded hearts. Taken together, these findings suggest that the observed increased sensitivity to adenosine in the occluded LCx artery following exercise training may give rise to enhanced blood flow to the collateral-dependent myocardium.