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(Circulation Research. 1997;80:868-876.)
© 1997 American Heart Association, Inc.

Articles

Short-term Exercise Training Enhances Reflex Cholinergic Nitric Oxide–Dependent Coronary Vasodilation in Conscious Dogs

Gong Zhao, Xiaoping Zhang, Xiaobin Xu, Manuel Ochoa, , Thomas H. Hintze

From the Department of Physiology, New York Medical College, Valhalla.

Correspondence to Thomas H. Hintze, PhD, Professor, Department of Physiology, New York Medical College, Valhalla, NY 10595.

	Abstract

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Abstract The effects of exercise training on the coronary vasodilation following activation of the Bezold-Jarisch reflex were examined in conscious dogs. Mongrel dogs were chronically instrumented using sterile techniques for measurements of systemic hemodynamics and left circumflex coronary blood flow (CBF). With the heart rate controlled (150 bpm), veratrine (0.5 to 20 µg/kg) caused dose-dependent increases in CBF; eg, 5 µg/kg of veratrine increased CBF by 61±6% from 31±1.3 mL/min (P<.05). After exercise training, the dose-response curve of CBF in response to veratrine was shifted to the left; eg, 5 µg/kg of veratrine increased CBF by 101±12% (P<.05 compared with control) from 34±2.3 mL/min. The enhanced coronary vasodilation was blunted by nitro-L-arginine (NLA, 35 mg/kg). In anesthetized dogs after exercise training, electrical stimulation of the left vagus nerve caused greater increases in CBF, and NLA inhibited increases in CBF. Acetylcholine, norepinephrine, angiotensin II, and bradykinin caused greater increases in NO₂^- production in coronary microvessels from exercise-trained dogs compared with those from normal dogs. Our results indicate that the coronary vasodilation following activation of the Bezold-Jarisch reflex is enhanced in conscious dogs after exercise training. Since electrical stimulation of the vagus nerve caused greater coronary vasodilation and since the agonists resulted in greater increases in NO production in coronary microvessels from exercise-trained dogs, the mechanism responsible for the enhanced coronary vasodilation following activation of the Bezold-Jarisch reflex is most likely due to the increased release of NO from the endothelial cells.

Key Words: Bezold-Jarisch reflex • adenosine • nitric oxide • nitro-L-arginine • exercise training

	Introduction

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Although the benefits of regular exercise on the cardiovascular system, including improved cardiac function, increased coronary reserve, lower incidence of coronary disorders, and more tolerance to cardiovascular diseases, have been recognized for a number of years, the mechanisms responsible for these benefits have not been elucidated.^{1 2 3 4 5} Since Furchgott and Zawadzki⁶ found that ACh-induced vasodilation in vitro was dependent on intact vascular endothelial cells, NO has been identified as one important EDRF. The synthesis of NO by vascular endothelium is intimately involved in the control of vascular tone and in the regulation of blood pressure.⁷

There is increasing evidence for enhanced NO-dependent vasodilation after long- and short-term exercise training.^{8 9 10 11 12} In vitro studies have indicated that exercise training enhances bradykinin-induced NO-dependent relaxation of porcine coronary arteries⁹ and increases endothelial NO synthesis in skeletal muscle arterioles of rats.^{11 12} Data from our laboratory have demonstrated that short-term exercise training enhances NO-dependent dilation of epicardial coronary arteries in conscious dogs,¹³ increases large coronary vascular NO production, and upregulates NO synthase gene expression in endothelial cells of the canine aorta.¹⁴ Our recent results have revealed that the coronary vasodilation induced by activation of carotid chemoreflex or the Bezold-Jarisch reflex (vagally mediated coronary vasodilation) is NO dependent, since NLA blocks such coronary vasodilation.^{15 16} Furthermore, vagally mediated coronary vasodilation is selectively impaired in conscious dogs after pacing-induced heart failure, whereas vagally mediated bradycardia is preserved.¹⁶ The mechanism responsible for the selective impairment of vagally mediated coronary vasodilation is related to the inability of the vascular endothelium to produce NO after heart failure.¹⁷

Our hypothesis, therefore, was that if NO is the mediator of vagally mediated vasodilation in the coronary circulation and if exercise training upregulates NO production in the coronary vascular endothelium, the coronary vasodilation following activation of the Bezold-Jarisch reflex would also be enhanced after exercise training. The goals of the present study were as follows: (1) to determine if exercise training enhances the coronary vasodilation following activation of the Bezold-Jarisch reflex in conscious dogs and (2) to determine the mechanisms responsible for the enhanced coronary vasodilation after exercise training.

	Materials and Methods

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Surgical Preparation
Mongrel dogs (weighing 24 to 28 kg) were premedicated with acepromazine (0.3 mg/kg IM) and anesthetized with sodium pentobarbital (25 mg/kg IV) and then intubated and ventilated with room air. A thoracotomy was performed in the left fifth intercostal space using sterile surgical techniques. Tygon catheters (Cardiovascular Instruments) were placed in thedescending thoracic aorta and in the left atrial appendage for the measurement of pressures and injection of drugs. A solid-state pressure gauge (P 6.5, Konigsberg Instruments Inc) was placed in the apex of the LV for the measurement of LVSP and calculation of LV dP/dt. A Doppler flow transducer (Parks Medical Electronics Inc) was placed on the left circumflex coronary artery for measurement of CBF. A pair of pacing electrodes was sutured on the LV for controlling heart rate. The chest was closed in layers. The wires and the catheters were run subcutaneously and exited from the back of the dog's neck.

The dogs were allowed 10 to 14 days to recover fully and were trained to lie quietly on the laboratory table. On the day of the experiment, an intravenous catheter was inserted in a peripheral vein and attached to an infusion line to administer drugs without disturbing the dogs.

The protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the guiding principles for the use and care of laboratory animals of the National Institutes of Health and the American Physiological Society.

Recording Techniques
Arterial pressure was measured by connecting the previously implanted catheter to a strain-gauge transducer (Statham P23 ID), and MAP was derived using a 2-Hz low-pass filter. LV pressure was measured from the solid-state pressure gauge, and the first derivative of the LV pressure over time (LV dP/dt) was calculated using a microprocessor set as a differentiator and having a frequency response flat to 700 Hz (National Semiconductor LM 324). Left circumflex coronary blood flow was measured from the previously placed ultrasonic flow transducer using a pulsed Doppler flowmeter (System 6, Triton Technology), and mean CBF was derived using a 2-Hz low-pass filter. LDCR was chosen as the index of coronary vascular resistance, since it is least sensitive to the compressive effect of ventricular contraction on coronary microvessels and was calculated as the quotient of late diastolic arterial blood pressure and CBF.^{18 19} Heart rate was monitored from the pressure pulse interval using a cardiotachometer (Beckman Instruments).

Experimental Protocols
Effects of Activation of Bezold-Jarisch Reflex by Veratrine
On the day of the experiment, with the dog lying on the laboratory table quietly, when the hemodynamic parameters and CBF were stable, the experiments were begun. Veratrine (5 µg/kg) was administered as a bolus injection (1 mL) into the left atrium through the implanted catheter with the heart in spontaneous cardiac rhythm. Then the implanted pacing electrodes were attached to an external pacemaker (EV 3434, Pace Medical), and the heart rate was increased to 150 bpm. With the heart rate controlled, bolus intra-atrial injections of veratrine at doses of 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, and 20 µg/kg were given. Each measured variable was allowed to return to control for at least 10 minutes before the next injection was given.

Effects of ACh and Adenosine
ACh (5 µg/kg) and adenosine (0.5 µmol/kg) were administered intravenously, with the heart rate controlled. ACh and adenosine were used as coronary vasodilators, one EDRF dependent and the other EDRF independent.

Exercise Training Protocol
After the experiment, the dogs were trained to run on the treadmill at speeds of 9.4 to 10.4 km/h for 2 hours, twice a day (once in the morning and the other in the afternoon), for 9 to 10 days. After that, the above protocols were repeated. In some dogs (n=4), NLA (35 mg/kg) was infused intravenously, and 30 minutes later, veratrine (0.1 to 20 µg/kg), ACh (5 µg/kg), and adenosine (0.5 µmol/kg) were given again with the heart rate controlled.

Effects of Electrical Stimulation of Vagus Nerve
On the day of the experiment, some of the normal (n=6) and exercise-trained (n=4) dogs were anesthetized with sodium pentobarbital and ventilated (Harvard Apparatus). Through a midline cervical incision, the cervical vagal trunks were isolated bilaterally, doubly ligated, and sectioned to interrupt descending parasympathetic outflow to the heart. The cardiac end of the left vagus nerve was placed on bipolar electrodes and immersed in mineral oil for electrical stimulation. The vagus nerve was stimulated for 20 seconds using an electrical biphasic pulse of 0.4-ms duration in two ways, either with constant voltage (10 V) and frequencies from 2 to 20 Hz or with constant frequency (10 Hz) and voltage from 2 to 20 V. During the experiments, the heart rate was kept constant (150 bpm), and the dogs were given propranolol (1 mg/kg) to avoid the effects of changes in heart rate and contractility on CBF. In two exercise-trained dogs, the coronary vascular responses to electrical stimulation of the vagus nerve were evaluated before and after administration of NLA (35 mg/kg).

NO Production From Coronary Microvessels
Normal and exercise-trained dogs were killed with an overdose of pentobarbital sodium (50 mg/kg IV), and the hearts were obtained immediately. The myocardium was cut into small pieces, chopped using a McIlwain tissue chopper, and suspended in ice-cold phosphate-buffered saline. The resulting suspension was homogenized for 50 seconds at maximum speed. The homogenate was poured over a 100-µm nylon mesh sieve, and microvessels (including arterial, venous, capillary, and lymphatic vessels) were isolated using the method of Gerritsen and Printz,²⁰ Seyedi et al,²¹ and Sessa et al.¹⁴ NO production stimulated by ACh, NE, Ang II, and bradykinin was measured in coronary microvessels (wet weight, 20 mg) from normal and exercise-trained dogs. L-NAME was used to block NO production in vitro. The formation of NO was measured as nitrite using the Griess reaction and a spectrophotometer at 540-nm absorbance. Chronically instrumented dogs were used as the normal group. (Thus, throughout the text, control will refer to dogs before exercise training, exercise-trained will refer to the same control dogs, and normal dogs will refer to dogs instrumented but never exercised. This distinction is important, since the normal dogs were used before exercise training for stimulation of the vagus nerve or for the preparation of coronary microvessels for comparison with exercise-trained dogs.)

Chemicals
ACh, adenosine, NE, Ang II, and bradykinin were purchased from Sigma Chemical Co. NLA or L-NAME was obtained from Aldrich. Veratrine was purchased from K&K Laboratories.

Data Analysis
All data are presented as mean±SE. In in vivo studies, the responses are the peak after administration of each agent or electrical stimulation. The statistical significance of differences (the null hypothesis) was determined with a paired t test for each peak response, and differences between groups (ie, control and exercise-trained dogs, with n equal in both groups) were evaluated with repeated measures ANOVA. When the ratio of F values indicated a significant difference, significance was determined using Scheffé's test. The changes in NO production from canine coronary microvessels or for vagal stimulation (where the n was not equal) were determined with Student's t test. Significant changes were considered at a value of P<.05.

	Results

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Effects of Activation of Bezold-Jarisch Reflex by Veratrine
Effects of Veratrine on the Hemodynamics in Spontaneous Cardiac Rhythm
Bolus intra-atrial injection of veratrine (5 µg/kg) resulted in a typical cardiac inhibitory response, including hypotension, bradycardia, and decreases in LVSP and LV dP/dt. The actual changes in the hemodynamics and CBF are shown in Table 1. There were no significant increases in CBF because the extreme bradycardia and hypotension by veratrine elicited decreases in CBF in some of the dogs. These are consistent with our previous results.¹⁶

View this table: [in this window] [in a new window]   Table 1. Effects of Veratrine (5 µg/kg) on the Hemodynamics and CBF in Conscious Dogs Before and After Exercise Training

Effects of Veratrine on the Coronary Circulation and the Hemodynamics With the Heart Rate Controlled
Since veratrine resulted in a significant decrease in heart rate, which caused dramatic hypotension and affected the increases in CBF, the heart was paced at 150 bpm. With the heart rate controlled, veratrine caused dose-dependent increases in CBF and decreases in LDCR at doses of 0.5 to 20 µg/kg. Fig 1 shows a typical response to intra-atrial injection of veratrine at a dose of 2.5 µg/kg in the same conscious dog before and after exercise training. The actual changes in CBF and resistance are summarized in Fig 2. Veratrine also decreased MAP and LVSP. The changes in systemic hemodynamics induced by veratrine at a dose of 5 µg/kg are shown in Table 2 and were not significantly different before and after exercise training.

View larger version (73K): [in this window] [in a new window]   Figure 1. An actual recording showing the changes in CBF and arterial pressure (AP) induced by veratrine (VER) at a dose of 2.5 µg/kg in the same conscious dog before (left) and after (right) exercise training. Before exercise training, VER caused increases in CBF and decreases in AP. After exercise training, the same dose of VER caused greater increases in CBF, whereas the hypotensive responses to VER were unchanged. HR indicates heart rate.

View larger version (26K): [in this window] [in a new window]   Figure 2. Changes in CBF and LDCR following activation of the Bezold-Jarisch reflex with veratrine in conscious dogs before and after exercise training. Values are mean±SE. Groups are as follows: control group, n=5 at doses of 0.01 and 0.05 µg/kg and n=10 at doses of 0.1 to 20 µg/kg; exercise-trained group, n=5 at doses of 0.005 to 0.05 µg/kg and n=8 at doses of 0.1 to 20 µg/kg; and exercise trained plus NLA group (Ex+NLA), n=4. *P<.05 compared with baseline; +P<.05 compared with before exercise training; and #P<.05 compared with before NLA.

View this table: [in this window] [in a new window]   Table 2. Effects of Veratrine, ACh, and Adenosine on the Hemodynamics and Coronary Circulation in Conscious Dogs With Heart Rate Controlled Before and After Exercise Training

Effects of Veratrine in Conscious Dogs After Exercise Training
After the exercise training, the dogs' cardiovascular and coronary circulatory functions were not altered. Hemodynamics, CBF, and resistance before and after exercise training are shown in Table 1. There were slight alterations in baseline CBF and in baseline LDCR (3.18±0.37 versus 2.62±0.17 mm Hg/mL per minute); however, these changes did not reach statistical significance.

After exercise training with spontaneous cardiac rhythm, the hemodynamic responses to intra-atrial injection of veratrine at a dose of 5 µg/kg were not altered. The actual changes in the hemodynamics are summarized in Table 1. There were no significant increases in CBF (Table 1). There was still a marked bradycardia following veratrine injection in conscious dogs after exercise training (Table 1).

Fig 1 shows greater increases in CBF in response to intra-atrial injection of veratrine at a dose of 2.5 µg/kg in the same dog after exercise training, whereas the hypotensive responses to veratrine were not significantly different. After exercise training, with the heart rate controlled, the coronary vasodilation induced by veratrine at doses of 0.01 to 5 µg/kg was significantly enhanced (Fig 2). The hemodynamic changes in response to injection of veratrine at a dose of 5 µg/kg were not altered after exercise training (Table 2).

In four chronically instrumented conscious dogs, we observed the changes in CBF in response to veratrine at a dose of 5 µg/kg on days 1 and 10 after the dogs were fully recovered from the surgery. There were no significant differences in the baseline hemodynamics and CBF on days 1 and 10. With the heart rate controlled, 5 µg/kg of veratrine increased CBF by 18±5 mL/min from 31±2 mL/min on day 1 and by 19±4 mL/min from 31±3 mL/min on day 10.

Effects of NLA on the Responses to Veratrine With the Heart Rate Controlled in Conscious Dogs After Exercise Training
To determine if the enhanced coronary vasodilation following activation of cardiac receptors is NO dependent, in four exercise-trained dogs, 35 mg/kg of NLA was given to block NO synthase. After intravenous infusion of NLA, with the heart rate controlled, there were significant elevations of MAP and LVSP but no significant changes in LV dP/dt (Table 2). NLA had no significant effects on baseline CBF or baseline LDCR. NLA significantly attenuated the coronary vascular responses to intra-atrial injections of veratrine. After NLA, with the heart rate controlled, the elevations in CBF and the reductions in LDCR induced by veratrine were essentially abolished. The changes in CBF and LDCR induced by veratrine before and after NLA are summarized in Fig 2. The hemodynamic responses to veratrine at a dose of 5 µg/kg before and after NLA are shown in Table 2.

Effects of ACh
Our previous results have shown that controlling the heart rate (150 bpm) does not affect the coronary vasodilation response to ACh¹⁶ ; therefore, we studied only the coronary vasodilation in response to ACh with the heart rate controlled. Bolus intravenous injection of ACh (5 µg/kg) resulted in significant increases in CBF (160±19%) and decreases in LDCR (-70±2%). A typical response to ACh is shown in Fig 3. Group data are summarized in Table 2. ACh also caused decreases in LVSP and MAP and reflex increases in LV dP/dt. After exercise training, ACh at a dose of 5 µg/kg still caused increases in CBF (164±13%) and decreases in LDCR (-69±2%), which were not significantly different from those of the controls (Table 2), consistent with our previous results.¹³ After NLA, the coronary vasodilation and the hypotension in response to ACh were significantly attenuated, whereas the decreases in LVSP were unchanged, as shown in Table 2. The changes in LV dP/dt in response to ACh were not significantly different from baseline. CBF increased by only 69±11%, and LDCR decreased by -39±9% (both P<.05 compared with before NLA).

View larger version (53K): [in this window] [in a new window]   Figure 3. An actual recording of CBF response to ACh at a dose of 5 µg/kg in the same dog before and after exercise training. There is no significant difference between before and after exercise training.

Effects of Adenosine
With the heart rate controlled, adenosine (0.5 µmol/kg) increased CBF by 172±9% and decreased LDCR by 69±1%. Adenosine also reduced MAP and LVSP and increased heart rate and LV dP/dt. The changes in hemodynamic and coronary circulation in response to adenosine with the heart rate controlled are shown in Table 2. After exercise training, the coronary vasodilation produced by adenosine was not altered. CBF was increased by 182±22%, and LDCR was decreased by 68±3% (both P>.05 compared with before exercise training). The changes in hemodynamics and the coronary vasodilation in response to adenosine are shown in Table 2. NLA did not affect the coronary vasodilation in response to adenosine (Table 2). In addition to the decreased elevation in LV dP/dt, the other hemodynamic parameters were not altered after NLA, as shown in Table 2.

CBF Response to Electrical Stimulation of the Left Cervical Vagus Nerve in Anesthetized Dogs
In normal anesthetized dogs, electrical stimulation of the left vagus nerve resulted in frequency- and intensity-dependent increases in CBF, as shown in Fig 4. For instance, 10-V 10-Hz electrical stimulation increased CBF by 54±8% from 22±3 mL/min (P<.05). In exercise-trained dogs, the same electrical stimuli caused greater increases in CBF, as shown in Fig 4. Ten-volt 10-Hz stimulation increased CBF by 93±11% (P<.05 compared with normal dogs) from 24±3 mL/min. To determine whether the enhanced coronary vasodilation during vagal stimulation is NO dependent, NLA (35 mg/kg) was used to block NO synthase in two exercise-trained dogs. After NLA, the coronary vasodilation induced by vagal stimulation was significantly attenuated. A 10-V 10-Hz stimulus increased CBF by only 38% from 28±1 mL/min.

View larger version (21K): [in this window] [in a new window]   Figure 4. Changes in CBF in response to electrical stimulation of the left cervical vagal trunk in anesthetized dogs. The dogs were given propranolol, and the heart rate was kept at constant 150 bpm. Values are mean±SE. Ex indicates exercise-trained dogs. +P<.05 compared with normal dogs.

NO Production From Coronary Microvessels
There were no statistically significant differences in the basal nitrite production in coronary microvessels from normal (n=13) and exercise-trained (n=6) dogs used in the present study (70±5 versus 96±19 pmol/mg tissue). In coronary microvessels from normal dogs, ACh, NE, Ang II, and bradykinin caused concentration-dependent increases in nitrite production. The actual increases in nitrite production in coronary microvessels from both normal and exercise-trained dogs are shown in Figs 5 and 6. Nitrite production by coronary microvessels from exercise-trained dogs in response to ACh, NE, Ang II, and bradykinin was significantly greater (P<.05) compared with that from normal dogs. For instance, 10^-6 mol/L of ACh, NE, and bradykinin caused 60%, 146%, and 168% greater increases, respectively, in nitrite production in coronary microvessels from exercise-trained dogs compared with normal dogs. Ang II (10^-7 mol/L) resulted in an 88% greater increase in nitrite production by coronary microvessels from exercise-trained dogs (P<.05 compared with normal dogs). The increased nitrite production in response to the highest (10^-5 mol/L) dose of ACh, NE, Ang II, and bradykinin was eliminated by L-NAME, as shown in Table 3. This indicates that the increased release of nitrite in response to the agonists reflects upregulation of NO production by the vascular endothelium. The enhanced nitrite production in response to bradykinin, NE, and Ang II was also blocked by HOE 140 (bradykinin 2 receptor antagonist), suggesting that bradykinin 2 receptors mediate NO production by bradykinin, NE, and Ang II.

View larger version (20K): [in this window] [in a new window]   Figure 5. Changes in nitrite production in response to acetylcholine and bradykinin in coronary microvessels from normal and exercise-trained dogs. The changes in nitrite production were calculated as the value stimulated by the drugs minus the control value. Values are mean±SE. *P<.05 compared with the control value; +P<.05 compared with normal.

View larger version (21K): [in this window] [in a new window]   Figure 6. Changes in nitrite production in response to norepinephrine and angiotensin II in coronary microvessels from normal and exercise-trained dogs. The changes in nitrite production were calculated as the value stimulated by drugs minus the control value. *P<.05 compared with the control value; +P<.05 compared with normal.

View this table: [in this window] [in a new window]   Table 3. Changes in Nitrite Production by Coronary Microvessels From Exercise-Trained Dogs in Response to Different Agonists

	Discussion

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The most important finding of the present study is that reflex NO-dependent coronary vasodilation is enhanced in conscious dogs after exercise training. This conclusion is supported by the observation that the coronary vasodilation following activation of the Bezold-Jarisch reflex by veratrine is enhanced in conscious dogs after exercise training. The mechanism responsible for the enhanced coronary vasodilation after exercise training is related to upregulation of NO production from the coronary vascular endothelium, as evidenced by increased coronary vasodilation in response to electrical stimulation of the vagus nerve and by enhanced NO production in response to agonists in coronary microvessels from exercise-trained dogs.

There is increasing evidence for the enhancement of endothelium-dependent vasodilation after long-term (10- to 16-week) exercise training.^{8 9 10 11 12} For instance, Delp and colleagues^{8 10} reported that the maximal vasodilator responses to ACh were greater in rings of abdominal aorta from exercise-trained normal⁸ and hypothyroid¹⁰ rats compared with sedentary rats. Muller et al⁹ found that the endothelium-dependent relaxation in response to bradykinin was enhanced in coronary resistance arteries from exercise-trained pigs and that N^G-monomethyl-L-arginine caused greater inhibition of bradykinin-induced relaxation of coronary resistance arteries from exercise-trained pigs relative to arteries from sedentary pigs. In addition, the data obtained by Sun et al¹¹ and Koller et al¹² showed that short-term (3- to 4-week) exercise training also enhanced endothelium- and flow-dependent dilation in rat skeletal muscle arterioles and that this resulted from increased release of both NO and prostaglandins.¹² Our previous results from conscious dogs indicated that short-term (7-day) exercise training enhanced ACh-induced dilation or reactive dilation of epicardial coronary arteries, which is mediated by NO.¹³ In contrast, in the present study, we did not find that short-term exercise training increased the ACh-induced increase in CBF or fall in late diastolic vascular resistance. These observations are consistent with our previous study by Wang et al.¹³

It is well established that in addition to hypotension and bradycardia, activation of the Bezold-Jarisch reflex results in reflex cholinergic coronary vasodilation,^{22 23} which we have shown is mediated by NO in conscious dogs.¹⁶ We have further found that the coronary vasodilation following activation of the Bezold-Jarisch reflex is selectively impaired in conscious dogs after pacing-induced heart failure because of the disappearance of NO from vascular endothelial cells.^{16 17} The present results indicate that short-term exercise training enhances the coronary vasodilation following activation of the Bezold-Jarisch reflex in conscious dogs. This enhanced coronary vasodilation is NO dependent, as evidenced by abolition of coronary vasodilation in response to veratrine after administration of NLA. These data already indicate, quite opposite the effects of heart failure, that exercise potentiates reflex cholinergic NO-dependent coronary vasodilation in conscious dogs.

It is generally accepted that at least 4 to 12 weeks of training is necessary to result in significant adaptive structural changes in the heart and the coronary vascular beds of dogs.^{3 18 24} In addition, resting bradycardia is used as an important index of training in conscious animals.⁹ Some in vitro studies showed that in addition to NO-dependent mechanisms, other factor(s) might be involved in the enhancement of vasodilation of rat aorta after long-term (at least 10-week) exercise training.^{8 10} To avoid the involvement of other factor(s) and long-term training, we designed a short-term training protocol to determine the effects of short-term exercise training on endothelium-dependent dilation of epicardial coronary arteries in the conscious dog; our results revealed that 7 days of exercise training did enhance the endothelium-dependent dilation of epicardial coronary arteries in conscious dogs.¹³ The present results have shown that the hemodynamics and coronary circulation were not altered and, most important, that there was no resting bradycardia in our conscious dogs after 9 to 10 days of training. Therefore, the enhanced coronary vasodilation following activation of the Bezold-Jarisch reflex is not due to exercise-induced adaptive structural changes in coronary vessels or conditioning.

Oltman et al²⁵ reported that long-term (16- to 22-week) exercise training enhanced relaxing responses to adenosine in rings of large porcine coronary arteries, whereas the results obtained by Muller et al⁹ showed that the same exercise training did not affect the vasodilator responses to adenosine in porcine coronary resistance arteries. The present results indicate that the coronary vasodilation in response to adenosine is not altered in conscious dogs after short-term (9- to 10-day) exercise training, consistent with the finding of Muller et al. Furthermore, the coronary vasodilation in response to adenosine is unchanged after administration of NLA in conscious dogs after exercise training, consistent with our previous results from normal dogs.¹⁶ Thus, the enhanced coronary vasodilation following activation of the Bezold-Jarisch reflex in the present study is not likely due to the changed sensitivity of the coronary vascular smooth muscle.

An early study²⁶ showed in conscious dogs that 4 weeks of exercise training caused nonspecific enhanced coronary vascular sensitivity to vasoconstrictors or vasodilators, which was dependent on an intact nervous system, since left stellate ganglionectomy abolished the enhanced reactivity. If the enhanced coronary vasodilation after exercise training in the present study is due to the changes in receptors, in the central nervous system, or in the efferent activity of vagal nerves, the outflow of vagus nerves would be increased. To eliminate this possibility, we used electrical stimulation to control the efferent activity of the vagus nerve. Vagal stimulation caused cholinergic coronary vasodilation,²⁷ which has been indicated by others²⁸ and us¹⁵ to be mediated by NO. The present results confirmed that vagal stimulation results in frequency- and intensity-dependent increases in CBF in normal anesthetized dogs, consistent with our previous results.¹⁵ We have further found that similar frequency and intensity of electrical stimulation of the vagus nerve causes greater increases in CBF in anesthetized dogs after exercise training. These were significantly attenuated by NLA, suggesting that the enhanced coronary vasodilation in response to vagal stimulation after exercise training is NO dependent. In addition to activation of the vagus nerve, electrical stimulation of the cervical vagus also activates sympathetic efferents to the heart, thereby resulting in ß-adrenergic coronary vasodilation.²⁷ To avoid the effects of ß-adrenergic coronary vasodilation, we used propranolol to block ß₁ and ß₂ receptors. Therefore, ß-adrenergic mechanisms were not responsible for the enhanced coronary vasodilation in response to vagal stimulation in our studies. It should be pointed out that the enhanced coronary vasodilation in response to vagal stimulation could not rule out the possibility of involvement of the afferent activity of the vagi. However, the present results have demonstrated that the reflex control of heart rate by the Bezold-Jarisch reflex is unchanged in conscious dogs after exercise training (Table 1). These results strongly suggest that the enhanced coronary vasodilation after exercise training is not simply due to altered vagal efferent activity.

Broten et al²⁸ and our previous study¹⁵ demonstrated in normal dogs that the coronary vasodilation in response to electrical stimulation of the vagus was mediated by NO, since L-NAME and NLA blocked the coronary vasodilation induced by vagal stimulation. We further found that the coronary vasodilation following activation of carotid chemoreflex¹⁵ or the Bezold-Jarisch reflex¹⁶ was also mediated by NO, as indicated by the abolition of the vagally mediated coronary vasodilation after administration of NLA.

Most important, our recent studies have indicated that vagally mediated NO-dependent coronary vasodilation is selectively impaired in conscious dogs after pacing-induced heart failure, whereas vagally mediated bradycardia is preserved.¹⁶ The impairment of vagally mediated NO-dependent coronary vasodilation after heart failure is due to the inability of the vascular endothelium to produce NO.¹⁷ The present results demonstrate that exercise training enhances the coronary vasodilation following activation of the Bezold-Jarisch reflex in conscious dogs or during vagal stimulation in anesthetized dogs. The enhanced coronary vasodilation in response to activation of the Bezold-Jarisch reflex or vagal stimulation is mediated by NO, as indicated by significant attenuation of the enhanced coronary vasodilation after administration of NLA. The direct evidence for the involvement of NO in the enhanced coronary vasodilation is from our observations of NO production in coronary microvessels. ACh activates muscarinic receptors to cause release of NO from the endothelial cells. To rule out the effects of altered muscarinic receptor function, we also used a number of other agonists whose effects are not mediated by muscarinic receptors (bradykinin, NE, and Ang II) to stimulate the release of NO from endothelial cells. The present results clearly demonstrate that bradykinin, NE, and Ang II result in greater increases in NO production in coronary microvessels from exercise-trained dogs. The increased NO production in response to the agonists was significantly blocked by L-NAME, indicating that the increased NO production in coronary microvessels from exercise-trained dogs most likely reflects upregulation of NOS in the vascular endothelium. In addition, our previous study showed that short-term exercise training increased NO synthase gene expression in the endothelial cells of the aorta from exercise-trained dogs.¹⁴ Therefore, the enhanced coronary vasodilation in response to activation of the Bezold-Jarisch reflex or vagal stimulation is most likely due to the upregulation of NO production in coronary vascular endothelial cells.

It was surprising to us and somewhat inconsistent that there were no significant changes in CBF response to intravenous injection of ACh in conscious dogs after exercise training. This is, however, similar to our previous results.¹³ We observed only the enhanced dilation of epicardial coronary arteries in our previous study. We proposed that this was due to the greater sensitivity to NO in large coronary arteries than in small coronary arteries. In the present study, we prolonged the exercise training and increased the intensity in an attempt to observe changes in CBF response to ACh in small coronary arteries (CBF reflects the function of small coronary arteries). However, we still could not observe changes in the CBF response to ACh. Although ACh has been widely used to activate muscarinic receptors and to release NO, the effects of exogenous ACh may differ substantially from the effects of ACh released from cholinergic nerve endings. Exogenous ACh could enter the systemic circulation to cause systemic hemodynamic effects, whereas ACh released from nerve endings only exerts its effects locally. Our recent results showed that NLA could inhibit by >80% the coronary vasodilation following activation of the carotid chemoreflex¹⁵ or the Bezold-Jarisch reflex,¹⁶ whereas the coronary vasodilation following intravenous injection of ACh (5 µg/kg) was inhibited by only 40% to 60%.^{15 16}

The Bezold-Jarisch reflex may have a protective role in the heart. It has been reported that the Bezold-Jarisch reflex is activated under some pathophysiological conditions, such as coronary ischemia, myocardial infarction, and aortic stenosis.²⁹ In a recent study, Hornig et al³⁰ have found that 4 weeks of daily handgrip training significantly enhances endothelium-mediated flow-dependent dilation in the forearm of patients with heart failure, most likely by increased endothelial release of NO. The beneficial effect of physical training on endothelial function is restricted to the trained extremity and is lost 6 weeks after cessation of training. Our results suggest that exercise training, by increasing vagally mediated NO-dependent coronary vasodilation, contributes to a protective mechanism in the heart. This could be one of the benefits of exercise training on the cardiovascular system.

In summary, the present results demonstrate that (1) the coronary vasodilation following activation of the Bezold-Jarisch reflex is enhanced in conscious dogs after exercise training, whereas the bradycardia is unchanged, and (2) the mechanism responsible for the enhanced coronary vasodilation after exercise training is most likely due to enhanced NO production in coronary vascular endothelial cells.

	Selected Abbreviations and Acronyms

 

ACh = acetylcholine Ang II = angiotensin II CBF = coronary blood flow EDRF = endothelium-derived relaxing factor L-NAME = NG-nitro-L-arginine methyl ester LDCR = late diastolic coronary resistance LV = left ventricle (ventricular) LVSP = left ventricular systolic pressure MAP = mean arterial pressure NE = norepinephrine NLA = nitro-L-arginine

	Acknowledgments

 
This study was supported by grants PO-1-HL-43023, RO-1-50142, and RO-1-53053 from the National Heart, Lung, and Blood Institute (Dr Hintze) and a Grant-in-Aid (96-134, Dr Zhao) and a Fellowship (96-106, Dr Zhang) from the American Heart Association, New York State Affiliate, Inc. HOE-140 was generously supplied by Hoechst Marion Roussel.

	Footnotes

 
This manuscript was sent to Peter Libby, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received April 24, 1996; accepted March 19, 1997.

	References

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