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(Circulation. 1995;91:2655-2663.)
© 1995 American Heart Association, Inc.

Articles

Selective Impairment of Vagally Mediated, Nitric Oxide–Dependent Coronary Vasodilation in Conscious Dogs After Pacing-Induced Heart Failure

Gong Zhao, MD, PhD; Weiqun Shen, MD; Xiaobin Xu, MD; Manuel Ochoa; Robert Bernstein; Thomas H. Hintze, PhD

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

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

	Abstract

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Background Activation in conscious dogs of the carotid chemoreflex or cardiac receptors results in coronary vasodilation that is mediated by a vagal cholinergic mechanism. Our previous study showed that the coronary vasodilation following activation of carotid chemoreflex is also mediated by nitric oxide (NO). In addition, NO production is depressed after the development of heart failure. Therefore, we hypothesized that the coronary vasodilation after activation of reflexes that elicit efferent vagal coronary vasodilation would be blunted in conscious dogs after pacing-induced heart failure due to the disappearance of NO.

Methods and Results Mongrel dogs were chronically instrumented using sterile techniques for measurements of systemic hemodynamics and left circumflex coronary blood flow (CBF). Without the heart rate controlled, intra-atrial injection of veratrine (4 µg/kg) caused bradycardia (-36±3 beats per minute). With the heart rate controlled, veratrine increased CBF in a dose-dependent manner: for example, 4 µg/kg of veratrine increased CBF by 54±5% from 38±4.9 mL/min (P<.05). The increases in CBF induced by veratrine were markedly blunted by nitro-L-arginine (NLA). Activation of carotid chemoreflex by nicotine increased CBF by 121±9% from 32±4 mL/min (P<.05) with the heart rate controlled and caused bradycardia (-32±5 beats per minute) without the heart rate controlled. After the development of heart failure, in response to activation of carotid chemoreflex or cardiac receptors the coronary vasodilation was almost abolished (CBF increased by only 23±8% or 11±3%, P<.05 compared with control). There still was a marked bradycardia after injections of nicotine or veratrine (-50±11 or -48±7 beats per minute).

Conclusions Our results indicate that vagally mediated coronary vasodilation is selectively attenuated in conscious dogs after pacing-induced heart failure, whereas the vagally mediated bradycardia is preserved. Since muscarinic receptor–induced coronary vasodilation is mediated by NO, the disappearance of NO from blood vessels leads to a defect in the integrated neural regulation of coronary blood flow and myocardial function during heart failure.

Key Words: nicotine • carotid arteries • reflex • endothelium-derived factors • veratrine

	Introduction

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Since von-Bezold and Hirt¹ first reported that intravenous administration of veratrum alkaloids resulted in bradycardia and hypotension, a number of studies have been performed to investigate this Bezold-Jarisch reflex.^{2 3} It has been well documented that the sensory receptors of the Bezold-Jarisch reflex are located in the left ventricle of the heart and concentrated in the area supplied by the left circumflex coronary artery and that the afferent and efferent arms of the Bezold-Jarisch reflex are vagal.^{4 5} In addition to veratrum alkaloids, some endogenous substances, such as prostaglandin, bradykinin, and serotonin, also can elicit a Bezold-Jarisch–like reflex.^{2 3}

Activation of ventricular receptors not only results in bradycardia and hypotension but also causes coronary vasodilation that is mediated by a cholinergic mechanism in anesthetized⁶ and awake⁷ dogs. Similarly, activation of the carotid chemoreflex by nicotine causes parasympathetic cholinergic coronary vasodilation, which has recently been indicated to be mediated by NO.⁸

Several cardiovascular reflexes, including baroreflex and ventricular mechanoreflex, are impaired in patients with congestive heart failure and in experimental animals after the development of heart failure.^{9 10 11 12 13} Recently, Chen et al¹¹ and Brandle et al¹³ have demonstrated that the vagally mediated bradycardia and hypotension induced by activation of cardiac chemical receptors are not altered in the conscious dog after pacing-induced heart failure. However, their studies did not address changes in the coronary circulation induced by activation of cardiac receptors or the carotid chemoreflex after the development of heart failure. With this in mind, the goals of the present study were to determine (1) whether the coronary vasodilation after activation of chemically sensitive cardiac receptors was mediated by NO in conscious dogs and (2) whether reflex vagally mediated coronary vasodilation was altered in conscious dogs after the development of pacing-induced heart failure and whether this could be explained by a disappearance of nitric oxide (NO).

	Methods

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Surgical Preparation
Nineteen mongrel dogs (weighing 21 to 32 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 the descending 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 left ventricle for the measurement of left ventricular (LV) systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP) 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 coronary blood flow (CBF). A pair of pacing electrodes was sutured on the left ventricle for controlling heart rate and for long-term pacing. 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. 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.

On the day of the experiment, an intravenous catheter was inserted in a peripheral vein and attached to an infusion line for remote administration of drugs.

Recording Techniques
Arterial pressure was measured by connecting the previously implanted catheter to a strain-gauge transducer (P23 ID, Statham), and mean arterial pressure (MAP) was derived using a 2-Hz low-pass filter. LV pressure was measured from the solid-state pressure gauge, and LV dP/dt was calculated using a microprocessor set as a differentiator and having a frequency response flat to 700 Hz (LM 324, National Semiconductor). Left circumflex CBF 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. Late diastolic coronary resistance (LDCR) was chosen as the index of coronary vascular resistance since it is independent of the compressive effect of ventricular contraction on coronary microvessels and was calculated as the quotient of late diastolic arterial blood pressure and CBF. Heart rate was monitored from the pressure pulse interval using a cardiotachometer (Beckman Instruments).

Experimental Protocols
Effects of Activation of Cardiac Receptors by Veratrine
On the day of the experiment, with the dog (n=7) lying on the laboratory table quietly, when the hemodynamic parameters and CBF were stable, the experiments were begun. Veratrine (4 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 increased to 150 beats per minute. With the heart rate controlled, bolus intra-atrial injections of veratrine were given at doses of 1, 2, 4, and 8 µg/kg. To assess the role of NO in the response to veratrine, we infused nitro-L-arginine (NLA) (35 mg/kg); 30 minutes later veratrine at doses of 1, 2, 4, and 8 µg/kg was injected again with the heart rate controlled (150 beats per minute). Each measured variable was allowed to return to control for at least 10 minutes before the next injection was given.

Effects of Acetylcholine and Adenosine
Acetylcholine (5 µg/kg) and adenosine (0.5 µmol/kg) were administered intravenously with and without the heart rate controlled. After NLA, acetylcholine and adenosine were injected again with the heart rate controlled (150 beats per minute). Acetylcholine was used to test the blockade of NO synthesis by NLA, and acetylcholine and adenosine were used as coronary vasodilators, one endothelium-derived relaxant factor (EDRF) dependent and the other EDRF independent.

After the experiment, the heart rate was increased to 240 beats per minute for 4 to 5 weeks to allow heart failure to develop. After the development of heart failure, the above protocols were repeated 30 minutes after turning off the pacemaker, except that NLA was not given.

Effects of Activation of Carotid Chemoreflex Induced by Nicotine
Five dogs were reanesthetized with a short-acting barbiturate on the day before the experiment was performed. A midline cervical incision was performed, and a Tygon catheter was inserted into one of the common carotid arteries via the inferior thyroid artery so that the tip of the catheter was positioned just proximal to the bifurcation of the carotid arteries. On the subsequent day, with the dog in the conscious state, carotid chemoreflex activation was elicited by a bolus injection of nicotine in 1 mL saline into the carotid artery through the implanted catheter. Our previous results showed that 10 µg nicotine resulted in the maximal coronary vasodilation in the conscious dog⁸ ; therefore, 10 µg nicotine was chosen to activate the carotid chemoreflex during all experiments. The coronary vascular and hemodynamic responses to activation of carotid chemoreflex induced by nicotine were studied in spontaneous cardiac rhythm and with heart rate controlled (150 beats per minute) to avoid the influence of bradycardia on CBF induced by activation of carotid chemoreflex.

In seven dogs after pacing-induced heart failure, a catheter was inserted into one of the common carotid arteries after anesthesia with thiamylal sodium (15 mg/kg) on the day before the experiment. On the next day, with the dog lying quietly on the experimental table and after we turned off the pacemaker for at least 30 minutes, 10 µg nicotine was given to activate carotid chemoreflex with the heart in spontaneous cardiac rhythm and with the heart rate controlled (150 beats per minute).

In four of the dogs, after chronic rapid pacing for 3 weeks, a catheter was placed into the common carotid artery on the day before the experiment. On the day of the experiment, with the chronic pacing stopped for at least 30 minutes, the dog was given 10 µg nicotine with the heart in spontaneous cardiac rhythm or with the heart rate controlled (150 beats per minute).

Chemicals
Acetylcholine and adenosine were purchased from Sigma Chemical Co. NLA was obtained from Aldrich Chemical Co. Veratrine was obtained from K&K Lab.

Statistical Analysis
All data are presented as mean±SEM. The responses are the peak responses after administration of each agent. The statistical significance of differences was determined with paired t test for each peak response, and multiple comparisons were evaluated with repeated-measures ANOVA. When the ratio of F values indicated a significant difference, this ratio was converted to a t-distribution using a Scheffé's test. Significant changes were considered at the P<.05 level.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of Activation of Cardiac Receptors by Veratrine
Effects of Veratrine on Hemodynamics in Spontaneous Cardiac Rhythm
Bolus intra-atrial injection of veratrine (4 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 caused by veratrine elicited decreases in CBF in some of the dogs.

View this table: [in this window] [in a new window]   Table 1. Effects of Veratrine (4 µg/kg) on Hemodynamics in Conscious Dogs Before and After Pacing-Induced Heart Failure

Effects of Veratrine on Coronary Circulation and Hemodynamics With Heart Rate Controlled
Because veratrine resulted in a significant decrease in heart rate that affected the increases in CBF, the heart was paced at 150 beats per minute so that the effects of the bradycardia induced by veratrine on CBF were avoided. With the heart rate controlled, veratrine caused dose-dependent increases in CBF and decreases in LDCR. The actual changes in the blood flow and resistance are shown in Fig 1. After intra-atrial injections of veratrine at doses of 1, 2, 4, and 8 µg/kg, CBF increased by 17±3%, 35±7%, 54±5%, and 71±7% (all P<.05) from 38±4.9 mL/min, respectively, and LDCR decreased by 16±2.9%, 31±2.9%, 41±2.1%, and 48±1.7% (all P<.05) from 2.21±0.18 mm Hg · mL^-1 · min^-1 (Fig 1). Veratrine also decreased MAP and LVSP. The changes in hemodynamics induced by veratrine at doses of 4 µg/kg are summarized in Table 2.

View larger version (18K): [in this window] [in a new window]   Figure 1. Plots of effects of activation of cardiac receptors by veratrine on coronary blood flow (CBF) and late diastolic coronary resistance (LDCR) in conscious dogs. Values are given as mean±SEM for control (n=7), nitro-L-arginine (NLA) (n=7), and heart failure (HF) (n=4 to 6). *P<.05, compared with control.

View this table: [in this window] [in a new window]   Table 2. Effects of Veratrine, Acetylcholine, and Adenosine on Hemodynamics in Conscious Dogs With Heart Rate Controlled Before and After Pacing-Induced Heart Failure

Effects of NLA on Responses to Veratrine With Heart Rate Controlled
The results from our laboratory showed that 30 mg/kg of NLA blocked approximately 60% of increases in CBF induced by acetylcholine (5 µg/kg).^{8 14} In the present study, 35 mg/kg of NLA was used to block NO synthase. After intravenous infusion of NLA (35 mg/kg), there were significant elevations in MAP and LVSP and a reduction in heart rate 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 significantly different. The changes in CBF and LDCR induced by veratrine before and after NLA administration are summarized in Fig 1. The hypotensive responses to the smaller doses of veratrine were not altered by NLA, whereas the hypotensive responses to the larger doses of veratrine were attenuated by NLA. The changes in LVSP and LV dP/dt induced by veratrine were not altered by NLA.

Effects of Veratrine in Dogs After Pacing-Induced Heart Failure
After 4 to 5 weeks of chronic rapid pacing, the dogs developed severe congestive heart failure, accompanied by edema, dyspnea, and ascites. After the development of heart failure, LVSP, LV dP/dt, and MAP significantly decreased, whereas LVEDP and heart rate were significantly increased. The changes in the hemodynamics and the coronary circulation after pacing-induced heart failure are shown in Table 3. There were slight increases in baseline CBF and decreases in LDCR; however, those changes were not significantly different from that of healthy dogs.

View this table: [in this window] [in a new window]   Table 3. Changes in Hemodynamics and Coronary Circulation in Conscious Dogs After Pacing-Induced Heart Failure

After the development of heart failure, in spontaneous cardiac rhythm, the hemodynamic responses to intra-atrial injection of veratrine at a dose of 4 µg/kg were not altered. The actual changes in the hemodynamics are shown in Table 1. There were no significant increases in CBF (Table 1). There was still a marked bradycardia after veratrine injection in dogs after pacing-induced heart failure (Table 1 and Fig 2).

View larger version (28K): [in this window] [in a new window]   Figure 2. Bar graphs of changes in heart rate (HR) induced by activation of the carotid chemoreflex by nicotine and cardiac receptors by veratrine in conscious dogs before and after pacing-induced heart failure. Values are given as mean±SEM. *P<.05, compared with baseline; #P<.05, compared with healthy dogs (normal).

After the development of heart failure, with the heart rate controlled, the coronary vasodilation induced by veratrine was significantly attenuated. Veratrine at doses of 1, 2, 4, and 8 µg/kg increased CBF by only 3.8±1.2%, 5.0±0.5%, 11±3%, and 25±5% (all P<.05, compared with before heart failure) from 43±4 mL/min, and LDCR was reduced by 2.6±1.6%, 8.9±2.4%, 23±3%, and 34±4% (all P<.05, compared with before heart failure) from 1.86±0.17 mm Hg · mL^-1 · min^-1 (Fig 1). The hemodynamic changes in response to injections of veratrine were not altered after the development of heart failure (Table 2).

Effects of Acetylcholine
Bolus intravenous injection of acetylcholine (5 µg/kg) resulted in significant increases in CBF and decreases in LDCR. The actual changes in the blood flow and resistance are shown in Fig 3. In spontaneous cardiac rhythm, after injection of acetylcholine, CBF increased and LDCR was reduced (Fig 3; both P<.05). Acetylcholine also caused decreases in MAP and LVSP and increases in heart rate and LV dP/dt. Controlling the heart rate did not affect the coronary vascular and hemodynamic responses to intravenous injection of acetylcholine (both P>.05, compared with spontaneous cardiac rhythm). The changes in hemodynamics are shown in Table 2.

View larger version (34K): [in this window] [in a new window]   Figure 3. Bar graphs of changes in coronary blood flow (CBF) and late diastolic coronary resistance (LDCR) induced by intravenous injection of acetylcholine (5 µg/kg) in conscious dogs. Values are given as mean±SEM (n=7). C indicates control; HRC, heart rate controlled (150 beats per minute); NHRC, nitro-L-arginine with heart rate controlled; HF, pacing-induced heart failure; and HFHRC, heart failure with heart rate controlled. *P<.05, compared with control; #P<.05, compared with HRC.

NLA significantly attenuated the coronary vasodilation induced by acetylcholine. After NLA, the elevation in CBF and the reduction in LDCR in response to acetylcholine were significantly smaller following intravenous injection of acetylcholine, as shown in Figure 3. NLA did not affect the reduction in LVSP produced by acetylcholine, whereas the decreases in MAP response to acetylcholine were smaller. After NLA, acetylcholine did not result in significant changes in LV dP/dt (Table 2).

After the development of heart failure, the coronary vasodilation induced by acetylcholine, with or without the heart rate controlled, also was significantly attenuated. The actual changes in CBF and LDCR are shown in Fig 3. The hemodynamic changes in response to acetylcholine with the heart rate controlled are summarized in Table 2. In spontaneous cardiac rhythm, the hypotensive effect in response to acetylcholine was smaller (-26±4 versus -39±4 mm Hg, P<.05), and the reflex tachycardia and increases in LV dP/dt were abolished (3±12 beats per minute and -84±44, both P>.05, compared with baseline).

Effects of Adenosine
In spontaneous cardiac rhythm, adenosine (0.5 µmol/kg) increased CBF by 71±12 mL/min (P<.05) from 30±2 mL/min and decreased LDCR by 70±3% from 2.86 mm Hg · mL^-1 · min^-1 (both P<.05). Adenosine also reduced MAP (22±4 mm Hg, P<.05) and LVSP (21±5 mm Hg, P<.05) and increased heart rate (77±5 beats per minute, P<.05) and LV dP/dt (926±86 mm Hg/s, P<.05). Controlling heart rate did not affect the coronary vascular and the hemodynamic responses to intravenous injection of adenosine. With the heart rate controlled, adenosine caused increases in CBF and decreases in LDCR by 71±16 mL/min and 66±3% (both P>.05, compared with in spontaneous cardiac rhythm). The hemodynamic changes in response to adenosine with the heart rate controlled are shown in Table 2.

After NLA, the coronary vasodilation produced by adenosine was not altered. CBF increased by 82±19 mL/min and LDCR decreased by 67±2% (both P>.05, compared with before NLA). The hemodynamic changes in response to adenosine are shown in Table 2. Other than the decreased elevation in LV dP/dt, the other hemodynamic parameters were not affected by NLA.

After the development of heart failure, no changes occurred in the coronary vascular responses to adenosine. With or without the heart rate controlled, adenosine increased CBF by 51±3 and 69±7 mL/min and decreased LDCR by 59±2% and 68±3% (all P>.05, compared with before heart failure). In spontaneous cardiac rhythm, the reduction in MAP and LVSP induced by adenosine was not altered (-14±3 and -11±3 mm Hg, P>.05, compared with before heart failure), whereas, the reflex elevations in heart rate and LV dP/dt produced by adenosine were abolished after heart failure (-4±12 beats per minute and 94±108 mm Hg/s, P>.05, compared with baseline). The hemodynamic changes in response to adenosine with heart rate controlled are shown in Table 2.

Effects of Activation of Carotid Chemoreflex Induced by Nicotine in Healthy Conscious Dogs
Activation of carotid chemoreflex induced by the intracarotid injection of nicotine elicited significant bradycardia and hypertension in healthy conscious dogs. A bolus intracarotid artery injection of nicotine at a dose of 10 µg significantly increased CBF by 131±9% from 28±2.52 mL/min and decreased LDCR by 41±6% from 3.11±0.24 mm Hg · mL^-1 · min^-1 (both P<.05). Controlling the heart rate did not influence the coronary vascular response to nicotine. For example, 10 µg nicotine caused a significant elevation in CBF by 121±17% from 32±4 mL/min and a reduction in LDCR by 36±5% from 3.05±0.35 mm Hg · mL^-1 · min^-1 (both P<.05, compared with in spontaneous cardiac rhythm). The actual changes in CBF and LDCR are shown in Fig 4. The hemodynamic changes in response to nicotine with or without heart rate controlled are summarized in Table 4.

View larger version (30K): [in this window] [in a new window]   Figure 4. Bar graphs of changes in coronary blood flow (CBF) and late diastolic coronary resistance (LDCR) by activation of carotid chemoreflex induced by nicotine (10 µg) in conscious dogs with heart rate controlled (150 beats per minute). Values are given as mean±SEM. Healthy dogs (normal), n=5; pacing for 3 weeks, n=4; HF, pacing-induced heart failure, n=7. *P<.05, compared with normal.

View this table: [in this window] [in a new window]   Table 4. Effects of Activation of Carotid Chemoreflex Induced by Nicotine (10 µg) on Hemodynamics in Conscious Dogs Before and After Pacing-Induced Heart Failure

Effects of Activation of Carotid Chemoreflex Induced by Nicotine in Dogs After Chronic Rapid Pacing for 3 Weeks
In four dogs whose heart was paced at 240 beats per minute for 3 weeks, in spontaneous cardiac rhythm, 10 µg of nicotine still caused significant increases in CBF by 27±5 mL/min from 38±5 mL/min and decreases in LDCR by 35±4% from 2.11±0.23 mm Hg · mL^-1 · min^-1 (both P>.05, compared with before chronic rapid pacing). Controlling the heart rate did not influence the coronary vascular response to nicotine. The increases in CBF and decreases in LDCR induced by nicotine (10 µg) shown in Fig 4 are with the heart rate controlled (both P>.05, compared with in spontaneous cardiac rhythm). The hemodynamic changes in response to nicotine are summarized in Table 4.

Effects of Activation of Carotid Chemoreflex Induced by Nicotine in Dogs After Pacing-Induced Heart Failure
After the development of heart failure, in spontaneous cardiac rhythm, activation of carotid chemoreflex induced by nicotine (10 µg) still caused a significant bradycardia that was even larger than that induced by nicotine in healthy dogs (Fig 2). This was accompanied by decreases in MAP, LVSP, and LV dP/dt (Table 4). The bradycardia was so great that the heart was arrested for as long as 10 seconds in some of the dogs; these even resulted in decreases in CBF. To avoid the effect of the bradycardia on CBF induced by nicotine, the heart was paced at 150 beats per minute. With the heart rate controlled, intracarotid injection of nicotine at a dose of 10 µg increased CBF by only 23±8% from 48±5 mL/min and reduced LDCR by 15±5% from 1.75±0.23 mm Hg · mL^-1 · min^-1 (both P<.05, compared with before heart failure). The increases in CBF and decreases in LDCR were significantly different from those induced by nicotine before heart failure (Fig 4). The hemodynamic changes in response to activation of carotid chemoreflex induced by nicotine are shown in Table 4.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The most important finding of the present study is that vagally mediated coronary vasodilation is selectively depressed in conscious dogs after pacing-induced heart failure. This conclusion is supported by the observation that the coronary vasodilation induced by activation of carotid chemoreflex or cardiac receptors is blunted in conscious dogs after pacing-induced heart failure, whereas the bradycardia caused by stimulation of carotid chemoreflex or cardiac receptors remains preserved.

The coronary vasodilation produced by activation of cardiac receptors was first observed in anesthetized dogs by Feigl in 1975⁶ ; then, Zucker et al⁷ confirmed it in conscious dogs. The coronary vasodilation induced by veratridine was abolished by both atropine and vagotomy, indicating that the coronary vasodilation elicited by activation of cardiac receptors is mediated by a vagal reflex. The present results have demonstrated that the coronary vasodilation induced by activation of cardiac receptors after intra-atrial injections of veratrine is mediated by an NO-dependent mechanism, as evidenced by the attenuated coronary vasodilation in response to veratrine after administration of NLA (an inhibitor of NO synthase). It was not surprising to us that NLA markedly blunted the coronary vasodilation caused by veratrine since Broten et al¹⁵ reported that the coronary vasodilation caused by vagal stimulation could be inhibited by nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthase). In addition to inhibiting NO synthase, however, L-NAME as used by Broten et al¹⁵ has been found to be a muscarinic antagonist.¹⁶ Recently, we have indicated that NLA, which has no interaction with muscarinic receptors, significantly inhibits the coronary vasodilation induced by activation of carotid chemoreflex or vagal stimulation. The decreased coronary vascular response to vagal stimulation by NLA was reversed by L-arginine.⁸ Moreover, very recently, we have found that NLA partly attenuates the coronary vasodilation induced by intravenous injections of prostacyclin (PGI₂).¹⁷ It is well known that PGI₂ is one of the endogenous stimulators of cardiac receptors.³ Therefore, our results further support the conclusion that NO plays an important role in parasympathetic control of CBF.

The method for inducing heart failure used by us in the present study—rapid chronic ventricular pacing for 4 to 5 weeks—was originally developed by Whipple et al¹⁸ and expanded by Coleman et al.¹⁹ This method has been used in a number of laboratories^{20 21 22 23} to create a model of dilated cardiomyopathy. This model of heart failure is characterized by reduction in inotropic state, resting hypotension and tachycardia, LV dilation, ascites, and edema.^{20 21 22 23} The changes in hemodynamics after pacing-induced heart failure in the present study are consistent with these previous studies.

It has been demonstrated that several cardiovascular reflexes are impaired after development of heart failure, including baroreflex^{11 24} and ventricular mechanoreflexes.^{12 13} Although the mechanism(s) of baroreflex dysfunction after heart failure are still unclear, there is evidence to suggest that this abnormality is multifactorial. Studies have indicated that the depression of the baroreflex after heart failure is due to both afferent and efferent abnormalities.²⁴ In addition to the afferent and efferent abnormalities, the downregulation of cardiac ß-receptors may also be involved in the abnormal sinoatrial nodal response to baroreceptor unloading.²⁵ There is also evidence that vagally mediated ventricular mechanoreflex is blunted after heart failure, which appears to be due to abnormalities in cardiopulmonary baroreceptors or in the central nervous system.^{12 13} On the other hand, the results from one laboratory have indicated that the control of heart rate by stimulation of cardiac receptors is not altered in conscious dogs after pacing-induced heart failure.^{11 13} However, these authors did not determine the changes in the control of coronary circulation after activation of cardiac receptors. Our results have demonstrated that the coronary vasodilation induced by activation of cardiac receptors after intra-atrial injections of veratrine is significantly attenuated in conscious dogs after pacing-induced heart failure. In marked contrast, no changes in the bradycardia in response to veratrine were observed, consistent with the results of other studies.^{11 13} In addition, our results also have indicated that the coronary vasodilation induced by activation of carotid chemoreflex is markedly blunted after pacing-induced heart failure. Although the afferents of carotid chemoreflex and cardiac receptors are different, the efferents to the heart for both reflexes are vagal, and stimulation of both reflexes causes coronary vasodilation. Thus, our results clearly indicate that vagally mediated coronary vasodilation is depressed in conscious dogs after pacing-induced heart failure.

There are several mechanisms for abnormal vagally mediated control of the coronary circulation after pacing-induced heart failure. One possibility is that the reactivity of the coronary vessels to all the vasodilators is depressed. However, the coronary vasodilation induced by adenosine in the present study was not affected by administration of NLA or after pacing-induced heart failure.

Second, the abnormalities in vagally mediated coronary vasodilation after heart failure could be present at the receptor level, in the central nervous system, or in the efferent nerve. There is evidence for excessive Na⁺,K⁺-ATPase activity as a mechanism for decreased arterial baroreceptor activity.²⁴ The decreased coronary vasodilation in response to activation of the carotid chemoreflex or cardiac receptors in the present study probably is not due to the abnormalities in the receptor level, central nervous system, or efferent nerve. Our results and those of others^{11 13} show that the bradycardia in response to veratridine or veratrine remains maintained after pacing-induced heart failure. Our results indicated that the bradycardia in response to activation of the carotid chemoreflex by nicotine not only was preserved after heart failure but also was even greater than before heart failure. These data strongly suggest that the abnormality in coronary vasodilation in response to activation of the carotid chemoreflex and cardiac receptors after heart failure do not occur at the receptor level, in the central nervous system, or in the efferent nerve.

Third, the abnormalities in vagally mediated coronary vasodilation after pacing-induced heart failure could be due to abnormal efferent transmission. It is well known that coronary vascular response during activation of the carotid chemoreflex^{26 27} or stimulation of ventricular receptors^{6 7} is mediated by vagal cholinergic fibers, since the coronary vasodilation following activation of carotid chemoreflex or cardiac receptors was abolished by atropine or vagotomy. Our previous results⁸ and the present results indicate that activation of carotid chemoreflex or cardiac receptors resulted in NO-dependent coronary vasodilation, as evidenced by attenuation of the coronary vasodilation in response to activation of carotid chemoreflex or cardiac receptors after administration of NLA. There is increasing evidence that indicates that NO-mediated vasodilation is depressed in patients with congestive heart failure^{28 29} and in experimental animals after heart failure.^{30 31} The data from our laboratory showed that endothelium-mediated control of the coronary circulation was attenuated in conscious dogs after pacing-induced heart failure.³¹ Reactive dilation of the large coronary artery is a typical flow-dependent, endothelium-mediated response because it was abolished by holding the flow constant^{32 33} or after removal of endothelium in the dog.³⁴ NO is the mediator responsible for the reactive dilation, since it is completely abolished after administration of NLA³⁵ or N-monomethyl-L-arginine.³⁶ Our previous results³¹ and the present results show that coronary vasodilation induced by acetylcholine was also markedly blunted after pacing-induced heart failure. Moreover, there is direct evidence for depression of NO after heart failure in that the production of nitrite from both large coronary arteries and microvessels from failing hearts is significantly less than that from healthy hearts.³¹ From these data, we propose that depression of NO is responsible for decreased coronary vasodilation in response to activation of the carotid chemoreflex or cardiac receptors in the conscious dog after pacing-induced heart failure. In four conscious dogs, we observed a normal coronary vascular response to activation of carotid chemoreflex after rapid pacing for 3 weeks. These results indicated that the increases in CBF and decreases in LDCR in response to activation of carotid chemoreflex were still maintained (Fig 4). The dogs subjected to rapid pacing for 3 weeks did not develop overt heart failure (Table 4) and had no clinical signs of heart failure. This suggests that NO-mediated vasodilation is not depressed until after the development of overt heart failure. Murray and Vatner²⁶ observed the coronary vascular response to activation of carotid chemoreflex in conscious dogs after development of pressure-overload right ventricular hypertrophy induced by chronic pulmonic stenosis. Their results showed that the early coronary vasodilation in response to activation of carotid chemoreflex was not altered after right ventricular hypertrophy, whereas the late coronary vasoconstriction in response to activation of carotid chemoreflex was attenuated. However, our results showed that the coronary vasodilation during activation of carotid chemoreflex by nicotine was not altered after chronic pacing for 3 weeks (mild cardiac dysfunction), whereas the coronary vasodilation in response to activation of carotid chemoreflex was significantly blunted after overt heart failure (chronic pacing for 4 to 5 weeks).

In addition to the changes in coronary vascular response to activation of carotid chemoreflex, the results obtained by those authors indicated that the bradycardia in response to activation of carotid chemoreflex by nicotine was enhanced in conscious dogs after right ventricular hypertrophy. Chen et al¹¹ and Brandle et al¹³ observed that the bradycardia in response to activation of cardiac chemical receptors by veratridine or prostacyclin was potentiated in conscious dogs after pacing-induced heart failure. Our results also demonstrated that the bradycardia induced by activation of carotid chemoreflex was enhanced in conscious dogs after pacing-induced heart failure (Fig 2). In some dogs in our study, the heart was arrested for as long as 10 seconds after intracarotid injection of nicotine after heart failure. The mechanism(s) of enhanced bradycardia in response to activation of carotid chemoreflex after heart failure was not determined in the present study. We have found in a previous study an increased bradycardia in response to intravenous injection of prostacyclin in conscious dogs after pacing-induced heart failure. This increased bradycardia is mediated by a cholinergic mechanism, as evidenced by abolition of the bradycardia after administration of atropine.³⁷

Both carotid chemoreflex and Bezold-Jarisch reflexes may have a compensatory role to increase oxygen delivery (via coronary vasodilation) and to decrease oxygen demand (via bradycardia) of the heart. It has been reported that Bezold-Jarisch reflex is activated under some pathophysiological conditions such as coronary ischemia, myocardial infarction, and aortic stenosis² and may have a protective role for the heart. The impaired vagally mediated coronary vasodilation after heart failure suggests that one of these protective roles is abolished because of the disappearance of NO-mediated vasodilation.

In summary, our results demonstrate that (1) the coronary vasodilation in response to activation of cardiac receptors is mediated by an NO-dependent mechanism in the conscious dog and (2) the coronary vasodilation in response to activation of carotid chemoreflex or cardiac receptors is selectively impaired in conscious dogs after pacing-induced heart failure, whereas the bradycardia induced by both reflexes is preserved. These results suggest that there is a selective impairment of vagal control of CBF after the development of heart failure due to the inability of the endothelium to produce NO.

	Acknowledgments

 
This study was supported by grants PO-1-HL-43023, RO-1-50142, and 53053 from the National Heart, Lung, and Blood Institute. Dr Zhao was supported by a Fellowship from the American Heart Association, New York State Affiliate.

Received October 31, 1994; accepted December 13, 1994.

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