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(Circulation. 1995;92:2627-2635.)
© 1995 American Heart Association, Inc.

Articles

Endothelial Modulation of ß-Adrenergic Dilation of Large Coronary Arteries in Conscious Dogs

Bijan Ghaleh, PhD; Marie-Luce Béa, PhD; Jean-Luc Dubois-Randé, MD; Jean-François Giudicelli, MD; Luc Hittinger, MD; Alain Berdeaux, MD

From the Département de Pharmacologie, Faculté de Médecine Paris-Sud (B.G., M-L.B., J.F.G., A.B.), the Service de Cardiologie (J.L.D.R.), and the Institut National de la Santé et de la Recherche Médicale (L.H.) U400, Hôpital Henri Mondor, Créteil, France.

Correspondence to Pr J.F. Giudicelli, Département de Pharmacologie, Faculté de Médecine Paris-Sud, 63, rue Gabriel Péri, 94276 Le Kremlin Bicêtre Cédex, France.

	Abstract

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Background Endothelium-derived relaxing factors have been described as important intermediates in ß-adrenergic vasodilation of resistance coronary vessels, but their involvement at the level of large epicardial coronary arteries remains controversial. Therefore, we examined the role of vascular endothelium in the ß-adrenergic–mediated vasodilation of large epicardial coronary arteries in conscious dogs.

Methods and Results Nine dogs were instrumented for measurement of left circumflex coronary artery diameter (CD) by sonomicrometry and coronary blood flow velocity (CBFv) with a Doppler technique in response to graded doses of isoproterenol (0.001 to 0.1 µg/kg IV bolus). Under control conditions, isoproterenol induced dose-dependent increases in CD and CBFv. When CBFv was kept constant at its baseline value by inflation of a cuff occluder, isoproterenol still induced dose-dependent increases in CD, but the latter were of lesser magnitude than those observed under normal CBFv conditions (110±20 versus 170±30 µm, respectively, ie, a reduction of 33% of the dilatory response at 0.1 µg/kg, P<.01). In the same dogs, the coronary endothelium was then mechanically removed at the site of CD measurement by a balloon angioplasty technique. After this procedure, the dose-dependent increases in CD induced by isoproterenol under either normal or controlled CBFv conditions were overimposable, and their magnitude was similar to that of the increases observed in the presence of an intact endothelium when CBFv was kept constant. After ß₁-adrenergic receptor blockade by atenolol (1 mg/kg), isoproterenol-induced increases in CD were abolished either when CBFv was kept constant or after endothelium removal.

Conclusions In conscious dogs, the direct stimulating effect of isoproterenol on ß₁-adrenergic receptors is endothelium-independent at the level of large coronary arteries. The endothelium reinforces the dilatory response to isoproterenol through an indirect, flow-dependent mechanism.

Key Words: endothelium-derived factors • receptors, adrenergic, beta • vasodilation

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The presence of ß-adrenergic receptors is well established in the coronary arteries of dogs,^{1 2 3} pigs,^{4 5} calves,⁶ monkeys,¹ and humans.⁷ Stimulation of these receptors at the level of large and small coronary arteries induces vasodilation, ie, increases in CD^{6 8} and CBF,^{9 10} respectively. These receptors play an important role in the regulation of coronary vascular tone, because ß-adrenergic receptor blockade induces vasoconstriction of the large coronary arteries,¹¹ especially during exercise in conscious dogs^{12 13} and in patients with vasospastic angina.¹⁴

The primary observation by Furchgott and Zawadzki¹⁵ for a role of the endothelium in acetylcholine-induced vascular relaxation has been extended in the past decade to demonstrate a critical role of the endothelium in mediating, either partially or entirely, the relaxations of isolated arteries to pharmacological agents such as ATP, ADP, serotonin, bradykinin, or thrombin.^{16 17} Similarly, physiological and mechanical stimuli leading to an increase in blood flow after dilation of resistance arterioles are able to induce dilation of large conductance arteries^{18 19} through an indirect endothelium-and flow-dependent mechanism.^{20 21} In this context and in view of the clinical prevalence of coronary vasospasms and atherosclerosis at the level of large coronary arteries, ie, pathophysiological situations in which endothelial dysfunctions have been reported,²² it is important to know whether the ability of large coronary arteries to dilate in response to ß-adrenergic stimulation depends on endothelium integrity.

To date, the involvement of endothelium in ß-adrenergic–mediated coronary vasodilation of large coronary arteries remains debated. Rubanyi and Vanhoutte²³ suggested that ß-adrenergic receptors may produce an endothelium-dependent vasorelaxation in large canine coronary arteries in vitro. In addition, Gray and Marshall²⁴ reported that ß-adrenergic–mediated relaxation was completely endothelium-dependent in rat thoracic aorta and suggested that the rise in cAMP could lead to the production of cGMP through stimulation of the endothelial constitutive NO synthase. In contrast, ß-adrenergic–induced relaxation has been described to be endothelium-independent in canine coronary arteries^{25 26 27} and not related to nitric oxide.²⁷ All these in vitro studies performed on large arteries are thus controversial. Moreover, the contribution of vascular endothelium to the dilation of large coronary arteries in response to stimulation of ß-adrenergic receptors has not yet been investigated in vivo.

The main goal of the present study was thus to clarify the relations between the coronary vascular endothelium and ß-adrenergic–induced dilation of large coronary arteries in conscious dogs. For this purpose, we used the combination of two complementary techniques. On the one hand, since arteriolar dilation and metabolic autoregulation could lead to an indirect endothelium- and flow-dependent dilation,^{17 28} responses to isoproterenol were investigated with and without controlling the CBF so as to evaluate the consequences of direct stimulation of ß-adrenergic receptors at the level of large coronary arteries. On the other hand, to examine the direct effect of isoproterenol on vascular endothelium, we compared, in the same dogs, the direct coronary dilating responses to isoproterenol in vivo, before and after endothelium removal, using a balloon angioplasty technique.^{17 29}

	Methods

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Animal Preparation
Nine male adult mongrel dogs weighing 20 to 31 kg were anesthetized with sodium pentobarbital (30 mg/kg IV), intubated, and ventilated with a respirator. Under sterile surgical conditions, a left thoracotomy through the fifth intercostal space was performed, and the heart was suspended in a pericardial cradle. Catheters were implanted in the descending thoracic aorta and in the pulmonary artery. A pair of ultrasonic dimension transducers, 5 MHz piezoelectric crystals (VD 5S, Triton Technology, Inc), was attached to a Dacron backing and sutured with Ethicon 5-0 suture (Ethicon, Inc) to opposing surfaces of the left circumflex coronary artery 2 to 4 cm from its origin. Care was taken during positioning of the transducers to limit dissection of and damage to any visible nerves, and proper alignment of the crystals was confirmed during surgery by monitoring of the ultrasonic signal with an oscilloscope. A 10-MHz Doppler flow probe (Crystal Biotech) and, downstream, a hydraulic occluder (Jones Instruments) were implanted distal to the dimension transducers. In seven of the nine dogs, a solid-state pressure transducer (model P7A, Konigsberg Instruments, Inc) was introduced into the left ventricle through the apical dimple and secured with purse-string sutures. The pericardium was left partially open, and all wires and catheters were passed subcutaneously to the back of the dog and brought through the skin between the scapulae. The pneumothorax was evacuated through a chest tube inserted in the sixth intercostal space. Cefazolin (1 g) and gentamicin (40 mg) were administered 30 minutes before incision and at the end of the surgery. The animal instrumentation and the ensuing experiments were performed in accordance with the official regulations of the French Ministry of Agriculture.

Measurement of Hemodynamic Parameters in Conscious Dogs
AP was measured with a Statham P23ID pressure transducer (Statham Instruments). LVP and its first derivative (LV dP/dt) were recorded from the Konigsberg gauge. Left circumflex CD was measured instantaneously with an ultrasonic transit-time dimension gauge with a resolution of ±40 µm (Triton Technology Inc, System 6 model 200), and mean CD was recorded as previously described.^{8 13 29} Left circumflex CBFv was measured with a Doppler flowmeter (Triton Technology Inc, System 6 model 200). The linearity of the relation between flow velocity and Doppler shift has been well demonstrated for this instrument. Data were recorded continuously on a multichannel electrostatic recorder (ES 2000, Gould Instruments Inc).

Experimental Protocols
All experiments were conducted at least 2 to 3 weeks after the initial surgery, when the dogs were healthy and trained to lie quietly on their right side on the experimental table. Vasodilation of large and small coronary arteries of the circumflex vascular bed was assessed before inclusion of each dog in the protocol to ensure that vasomotion of the epicardial artery and coronary flow reserve were not impeded by the surgical procedure. Release of a 20-second coronary occlusion was associated with a 321±27% increase in CBFv followed by a delayed increase in large coronary artery diameter that reached 5.9±1.1%. The increase in large coronary artery diameter in response to nitroglycerin (10 µg/kg IV bolus) reached 6.3±1.1% in these dogs. Each drug was administered only when the effects of the previous one had completely disappeared, ie, when all parameters had returned to their corresponding control values.

After basal hemodynamic parameters had been obtained, six dogs received increasing doses of isoproterenol (0.001 to 0.1 µg/kg IV bolus). To prevent flow-dependent mechanisms in the observed responses to isoproterenol in the same dogs, the coronary effects of isoproterenol (0.01 to 0.1 µg/kg) were studied again while CBF was controlled by inflation of a cuff occluder.^{6 19}

In eight dogs, ß₁-adrenergic receptor blockade was achieved by administration of atenolol (1 mg/kg IV).³⁰ Isoproterenol was administered when CBF was either normal (0.001 to 0.3 µg/kg) or maintained at its baseline value by an inflated cuff occluder (0.03 to 0.3 µg/kg).

Three days after the last drug administration, the endothelium of epicardial coronary artery was removed at the level of crystal attachment according to the previously described technique.^{17 29 31} For this purpose, dogs were lightly anesthetized with propofol (200 mg IV) and 0.5% halothane. Under aseptic conditions, an incision was made to expose the right carotid artery. An 8F left coronary guiding catheter (Schneider Climo) was inserted through the right carotid artery and positioned in the left coronary ostium under fluoroscopic guidance. A balloon angioplasty catheter (Thruflex, Medtronic) was inserted through the guiding catheter into the left circumflex coronary artery into the area of the piezoelectric crystals. To avoid distension of the coronary artery, care was taken to calibrate the balloon catheter both according to the external coronary diameter measured by ultrasonic transit-time gauge and by estimation of the internal diameter after serial injections of contrast medium (Iopamidol, Schering Laboratories) into the left coronary ostium. Consequently, the size of the balloon was 2.5 or 3 mm. The balloon was inflated with 1 mL air, and the catheter was gently moved back and forth three times over the entire segment from the proximal circumflex coronary artery to the crystal area. The balloon was then deflated, the catheter was withdrawn, and the dog was allowed to fully recover. This procedure causes deendothelialization on each side of the crystals, leaving the distal circumflex, the left anterior descending, and the septal arteries intact, as previously demonstrated by histological and pharmacological studies.²⁹ Two to 4 days after endothelium removal, ie, before any significant endothelial regeneration had occurred in this preparation,^{17 29} the protocols described previously were repeated in the same dogs. The adequacy of endothelium removal was verified with reactive hyperemia after the release of a 20-second coronary occlusion (flow-mediated, endothelium-dependent dilation) and administration of acetylcholine (0.3 µg/kg) and nitroglycerin (1 µg/kg). Before endothelium removal, acetylcholine, reactive hyperemia, and nitroglycerin significantly increased the CD by 120±10, 180±30, and 200±30 µm, respectively, from 2950±140 µm. After endothelium removal, acetylcholine and reactive hyperemia induced increases in CD that were strongly reduced (-75.9±3.4% and -91.7±2.4%, respectively, P<.001), whereas responses to nitroglycerin in the deendothelialized segment were only slightly affected (-8.5±2.4%, P<.05).

Drugs
Drugs used were acetylcholine (Sigma Chimie), atenolol (ICI Pharma), isoproterenol (Winthrop), and nitroglycerin (Laboratoires Besins-Iscovesco).

Data Analysis
Data shown are mean values±SEM. All hemodynamic parameters were measured at baseline and at the time of their respective maximal effect. Results are expressed as either absolute or percent changes from baseline values. Sequential changes of mean values were evaluated by a two-way ANOVA for repeated measures. When overall differences were detected, individual comparisons were made by a paired t test with Bonferroni's correction.^{32 33} Comparisons of the effects of isoproterenol in the absence and in the presence of atenolol, while the CBF was controlled or not controlled, and before versus after deendothelialization were performed on the absolute variations of the investigated parameter by a two-way ANOVA for repeated measures followed, if necessary, by a paired t test with Bonferroni's correction. Comparisons between experimental protocols in which the number of animals varied were performed by use of an unbalanced repeated-measures model designed for missing observations.

Because of a slight but significant reduction of the response to nitroglycerin after endothelium removal, an index of large coronary artery dilation (R_{CD iso/nitro}) was calculated as the ratio of absolute change in mean CD induced by isoproterenol to the corresponding absolute change induced by 1 µg/kg of nitroglycerin.³⁴ The consequences of endothelium removal for the responses of the large coronary artery to isoproterenol were then assessed by comparing these ratios calculated before and after endothelium removal by a two-way ANOVA for repeated measures followed by a paired t test with Bonferroni's correction.

All these statistical analyses were performed on an IBM-compatible personal computer using BMDP statistical software (BMDP). A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Fig 1 illustrates the coronary responses to bolus injections of isoproterenol (0.1 µg/kg) and nitroglycerin (1 µg/kg) before and 3 days after endothelium removal in one typical dog. Table 1 summarizes the cardiac, systemic, and coronary hemodynamic responses to isoproterenol, and Fig 2 illustrates the changes in mean CD induced by isoproterenol under normal and controlled CBF conditions before and after endothelium removal in six dogs.

View larger version (54K): [in this window] [in a new window]   Figure 1. Recording of phasic and mean CD (CD and CD, respectively), phasic and mean CBFv (CBFv and CBFv, respectively), and phasic AP with intravenous bolus administration of isoproterenol (0.1 µg/kg) (left) and nitroglycerin (1 µg/kg) (right), before and 3 days after (A and C, respectively) endothelium removal (B and D, respectively). In this example, the increase in CD with isoproterenol was attenuated by 50% after endothelium removal, whereas the dilatory response to nitroglycerin remained almost unchanged.

View this table: [in this window] [in a new window]   Table 1. Baseline Hemodynamic Values and Peak Changes Induced by Isoproterenol Before and After Endothelium Removal With and Without Coronary Blood Flow Control in Conscious Dogs

View larger version (12K): [in this window] [in a new window]   Figure 2. Graph showing absolute changes in mean CD in response to administration of increasing doses of isoproterenol measured before (open symbols) and after (closed symbols) endothelium removal while CBF was normal (left) or controlled (right). Under normal conditions, responses of large coronary arteries were significantly reduced after endothelium removal. Under controlled flow, the dilatory responses of these arteries were not affected by endothelium removal. *P<.01 vs corresponding value before endothelium removal.

Effects of Isoproterenol Under Normal Conditions and Controlled CBF
Before Endothelium Removal
Under normal CBF conditions, administration of increasing doses of isoproterenol induced significant and dose-dependent increases in heart rate, LV dP/dt, CBFv, CD, and decreases in mean arterial pressure and LVP.

When CBF was maintained constant at its control level with a cuff occluder, the effects of isoproterenol on coronary and systemic hemodynamic parameters were not significantly affected compared with control conditions except for the increases in CD, which were significantly attenuated (-61.5±14.8%, -41.5±10.2%, and -33.3±8.9% from corresponding values under control conditions after administration of isoproterenol at 0.01, 0.03, and 0.1 µg/kg, respectively).

After Endothelium Removal
These experiments were performed in the same animals. As shown in Table 1, endothelium removal induced no significant changes in basal hemodynamic and coronary parameters except for mean arterial pressure and mean CD, which both were increased by 8%. Furthermore, endothelium removal did not significantly modify the effects of isoproterenol on CBFv and systemic hemodynamics under either normal or controlled CBF conditions.

To take into account the reduction to nitroglycerin responsiveness after endothelium removal that is the consequence of a partial decrease in the reactivity of coronary smooth muscle cells and of the increase in basal CD,²⁹ we calculated the ratios of the absolute changes in CD induced by isoproterenol to the corresponding absolute change induced by nitroglycerin (R_{CD iso/nitro}).³⁴ The increases in mean CD induced by isoproterenol were attenuated after endothelium removal (-51.1±9.3%, -42.0±6.7%, and -35.4±3.8% at 0.01, 0.03, and 0.1 µg/kg, respectively, all P<.01) (Figs 1 and 2). In addition, the increases in CD induced by isoproterenol were overimposable either when the endothelium was intact but CBF controlled or when the CBF was normal but the endothelium removed (eg, R_{CD iso/nitro}, 0.57±0.08 at 0.1 µg/kg of isoproterenol when the endothelium was intact but CBF controlled versus 0.55±0.05 when the endothelium was removed but CBF normal). Finally, the increases in CD induced by isoproterenol after endothelium removal were also overimposable when CBF was either normal or controlled (eg, R_{CD iso/nitro}, 0.55±0.05 versus 0.60±0.05 at 0.1 µg/kg of isoproterenol under normal and controlled flow, respectively, after endothelium removal).

Effects of Isoproterenol Under ß₁-Adrenergic Receptor Blockade
Eight dogs were investigated before endothelium removal. Because of an unfunctional cuff occluder, experiments performed while CBF was controlled are reported in seven animals only. Finally, after endothelium removal, experiments were performed in six dogs.

After atenolol administration, there were decreases in basal CD (-2.7±0.6% from 2980±140 µm, P<.01), LV dP/dt (-14.8±2.4% from 3147±268 mm Hg/s, P<.001), and heart rate (-8.5±2.1% from 88±5 beats per minute, P<.01). Basal mean arterial pressure, LVP, and CBFv did not change significantly.

Table 2 summarizes the cardiac, systemic, and coronary hemodynamic responses to isoproterenol, and Fig 3 illustrates the changes in mean CD induced by isoproterenol after ß₁-adrenergic receptor blockade. As expected, changes in heart rate, mean arterial pressure, and CBFv in response to isoproterenol were significantly reduced after atenolol compared with normal conditions. In addition, LV dP/dt did not change after isoproterenol administration up to 0.3 µg/kg, confirming the adequacy of ß₁-adrenergic receptor blockade.

View this table: [in this window] [in a new window]   Table 2. Baseline Hemodynamic Values and Peak Changes Induced by Isoproterenol in Dogs Pretreated With Atenolol (1 mg/kg) Before (Normal and Controlled Flow) and After (Normal Flow) Endothelium Removal

View larger version (13K): [in this window] [in a new window]   Figure 3. Graph illustrating the changes in mean CD in response to administration of increasing doses of isoproterenol after ß1-adrenergic receptor blockade with atenolol (1 mg/kg). The isoproterenol-induced coronary vasodilation in the presence of endothelium and normal flow () was abolished by controlling the CBF () and after endothelium removal ().

Before endothelium removal, administration of increasing doses of isoproterenol induced dose-dependent increases in CD. When CBF was kept constant, isoproterenol-induced dilation of large coronary arteries was completely abolished.

After endothelium removal, isoproterenol-induced dilation of large coronary arteries was also completely abolished.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Despite numerous reports demonstrating that ß- adrenergic stimulation induces vasorelaxation in vitro^{23 24 25 26 27} and vasodilation of large coronary arteries in dogs⁸ and calves,⁶ the involvement of vascular endothelium in such dilation has not been fully assessed in vivo. Since CBF increases after ß-adrenergic stimulation and since the endothelium may also induce dilation of the large coronary vessels through a flow-dependent mechanism, we investigated this issue through two different approaches: (1) by maintaining CBF at its baseline value to suppress the flow-dependent component of the coronary vasodilator effects of isoproterenol and (2) by performing a coronary deendothelialization that abolishes a potential direct endothelium-dependent component of the drug's effects.

The present study demonstrates that (1) ß-adrenergic–mediated dilation of large coronary arteries is mainly due to a direct stimulating effect of isoproterenol on ß₁-adrenergic receptors located on coronary vascular smooth muscle and (2) the endothelium reinforces the dilatory response of isoproterenol through an indirect flow-dependent mechanism. In agreement with an earlier study,⁸ these data underline the role of the vascular endothelium in the ß-adrenergic–mediated vasodilation in physiological conditions and suggest that an alteration of the coronary endothelium may limit the dilatory response of large coronary arteries after ß-adrenergic stimulation.

In our experiments, local deendothelialization of the circumflex coronary artery at the site of crystal attachment was achieved by a balloon angioplasty procedure, as shown by the almost complete abolition of coronary dilation induced by reactive hyperemia and acetylcholine, thus confirming previous data.^{17 21 29 35} In contrast, and as previously described,^{17 29 36} responses to nitroglycerin were only slightly reduced, which probably reflects a small residual alteration of the coronary vascular medial layer. It is important to note, however, that the cardiac and systemic hemodynamic effects induced by isoproterenol were not affected by the balloon angioplasty procedure, whether or not the CBF was controlled.

As expected, administration of graded doses of isoproterenol induced dose-dependent dilations of large coronary arteries and concomitant increases in CBF when the endothelium was intact, in agreement with previous studies.^{6 8 10} These dilatory responses of large coronary arteries to isoproterenol are the consequence of at least two phenomena,¹⁶ ie, a direct dilatory response due to stimulation of ß-adrenergic receptors located at the level of coronary smooth muscle cells and/or endothelium, and an indirect flow-dependent dilation due to the simultaneous increase in CBF. To discriminate between these two mechanisms, we repeated the same experiments while controlling CBF by using a cuff occluder.^{18 19} This maneuver attenuated the ß-adrenergic dilatory responses to isoproterenol on large coronary arteries, demonstrating that at least part of the observed increase in CD is the consequence of a flow-dependent mechanism, whereas the observed remaining dilation is due to a direct stimulating effect of the drug on coronary ß-adrenergic receptors. Finally, the observed changes in CD were not related to modifications of the intraluminal pressure, since mean arterial pressure values measured at the time of CD assessment did not differ among the various protocols, ie, before and after endothelium removal (Fig 1), with and without constant flow.

To assess the involvement of vascular endothelium in the latter direct effect of isoproterenol, we compared the responses obtained before and after endothelium removal. Endothelium removal reduced the dilatory effect of isoproterenol on large coronary arteries, and the responses obtained in such conditions were similar whether or not the CBF was controlled. This indicates that the flow-controlling procedure has not modified the mechanical properties of the vessel. Moreover, the remaining dilatory responses, which are overimposable on those obtained before endothelium removal when CBF was controlled, are obviously the result of a direct relaxant effect of isoproterenol at the level of epicardial coronary smooth muscle cells, thus demonstrating that the direct isoproterenol-induced dilation of large coronary arteries is endothelium-independent in the conscious dog. Thus, it appears that vascular endothelium reinforces the direct dilating responses of isoproterenol only through an indirect flow-dependent mechanism. This finding does not support the previous report by Vatner et al⁶ concluding that there was a flow-independent dilation of large coronary arteries after administration of isoproterenol, prenalterol, and pirbuterol in conscious calves. These authors suggested that the magnitude of the coronary dilation they observed might not have reached the threshold necessary to produce an endothelium-dependent dilation.¹⁸ In our study, the approximately twofold increase in CBF may have been enough to elicit an endothelium-dependent dilation in the present study, and this, in addition to differences in the experimental design and in the species used,³⁷ could explain the discrepancy between our study and that by Vatner et al.⁶ In the conscious dog, Parent et al¹⁰ postulated that endothelium was of major importance through the release of nitric oxide in ß-adrenergic–mediated dilation of small coronary arteries and that a receptor-operated mechanism rather than a flow-dependent phenomenon was involved. However, this study investigated the contribution of nitric oxide to the ß-adrenergic–mediated dilation of resistance coronary arteries, whereas the present study considered the role of endothelium at the level of large coronary arteries. Previous studies^{12 13} have demonstrated opposite responses of large and small coronary arteries to ß-adrenergic receptor blockade. Indeed, the present conclusions are supported by previous in vitro studies conducted in large canine coronary arteries.^{25 26 27}

The present study also demonstrates that although neurohumoral and metabolic factors may also contribute, dilation of large coronary arteries to isoproterenol after ß₁-adrenergic receptor blockade is mediated mainly by a flow- and endothelium-dependent mechanism and that ß₂-adrenergic receptors do not play a significant role in these conductance vessels. Since we did not observe any residual dilation of the epicardial coronary arteries after atenolol whether or not CBF was kept constant or after endothelium removal, we considered that further blockade of ß₂-adrenergic receptors with a selective blocker was not useful in vivo. Although the presence of ß₂-adrenergic receptors has previously been documented in canine,³⁷ bovine,⁶ and human⁷ coronary arteries, in which their stimulation leads to a weak but significant vasorelaxation,^{38 39 40 41} Krauss et al²⁵ clearly showed that the distribution of these receptors is heterogeneous in the coronary vessels, being negatively correlated to the diameter of the vessel. Hence, ß₂-adrenergic receptors play an important role in the control of coronary resistance vessels, in which they predominate,^{9 42} whereas ß₁-adrenergic receptors for the most part predominate on large epicardial coronary arteries,^{2 5 7 40 43} and the present in vivo study confirms these observations.

Clinical Implications
The present study follows a recent one from our group demonstrating that the endothelium plays a key role in the mediation of epicardial coronary artery dilation during exercise and in protection of these vessels against the constrictor effect of endogenous catecholamines.²⁹ Indeed, mechanical removal of the endothelium unmasks the constrictor effect of exercise-induced release of catecholamines at the level of ₁-adrenergic receptors located on large epicardial coronary arteries.¹³ In the present study, we addressed the role of vascular endothelium in ß-adrenergic–mediated dilation of these vessels, since we previously reported that blockade of these receptors with either propranolol or atenolol induced a significant constriction of coronary arteries in dogs at exercise.^{12 30} This experimental observation agreed with the description of paradoxical exacerbation in coronary vasospasms during ß-adrenergic receptor blockade in patients with vasotonic angina⁴⁴ and suggested that the well-known impairment of endothelial functions in atherosclerotic patients^{45 46} might play a pivotal role in such a pathogenesis.

In this study, we demonstrate that the endothelium reinforces the direct coronary dilating responses of epicardial coronary arteries to isoproterenol through an indirect flow-dependent mechanism. Such a mechanism, however, is impaired during atherosclerosis,^{22 47} and it is likely that during ß-adrenergic receptor blockade in atherosclerotic patients, the loss of endothelial flow-mediated dilation is associated with a deleterious reinforcement of -adrenergic tone. Consequently, the prevention of exercise-induced coronary stenosis reported by Bortone et al¹⁴ after propranolol administration might be explained by a relative preservation of the flow-mediated dilation mechanism in their patients, which might have overridden the vasoconstrictive action of epicardial coronary arteries due to ß-adrenergic receptor blockade, as observed at rest in their study. Since there are different stages of endothelial dysfunction at the level of coronary arteries,²² impairment of the flow-mediated dilation could be a critical event in atherosclerotic patients under ß-adrenergic receptor blockade. This also suggests that future development of anti-ischemic agents that could reduce the imbalance between myocardial oxygen supply and demand without detrimental action on vasomotion of epicardial coronary arteries might be important for the treatment of patients with coronary artery disease.

Conclusions
The present investigation demonstrates that direct stimulation of ß-adrenergic receptors is endothelium-independent at the level of large coronary arteries and is primary due to the stimulation of ß₁-adrenergic receptors in conscious dogs. The coronary endothelium reinforces the dilatory response to isoproterenol through an indirect flow-dependent mechanism resulting from the simultaneous increase in CBF.

	Selected Abbreviations and Acronyms

 

AP = arterial blood pressure CBF = coronary blood flow CBFv = coronary blood flow velocity CD = coronary artery external diameter LVP = left ventricular pressure

	Acknowledgments

 
This work was supported by grant 930403 from the Institut National de la Santé et de la Recherche Médicale, France. The authors are greatly indebted to Alain Bizé for his excellent technical assistance.

Received November 16, 1994; revision received June 7, 1995; accepted June 12, 1995.

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