Influence of Nitric Oxide on Vascular, Metabolic, and Contractile Responses to Dobutamine in In Situ Canine Hearts

Abstract

The left anterior descending coronary arteries of 30 anesthetized, open-chest dogs were perfused via an extracorporeal circuit. Coronary blood flow (CBF), myocardial oxygen consumption (MVO₂), and segmental shortening (SS) were measured. Studies were performed with coronary perfusion pressure (CPP) or CBF constant. With CPP constant, effects of intracoronary (IC) infusions of dobutamine (2.5, 5.0, or 10.0 [micro sign]g/min) were evaluated alone (control) and after inhibition of nitric oxide (NO) synthase with N^{G-nitro-L-arginine} methyl ester (L-NAME). With CBF constant, a NO donor (sodium nitroprusside [SNP] 80 [micro sign]g/min IC) or nitroglycerin [NTG] 40 [micro sign]g/min IC) or a releaser of endogenous NO (acetylcholine [ACh]; 20 [micro sign]g/min IC) was infused along with dobutamine. Increases in CBF during dobutamine and isoproterenol were compared before and after blockade of beta_1-adrenergic receptors with atenolol. Dobutamine caused proportional, dose-dependent increases in CBF, MVO₂, and SS, which were not altered by L-NAME. Administration of the NO donors or ACh during dobutamine markedly decreased CPP, but only ACh also reduced SS and MVO₂. These latter effects persisted after L-NAME. Atenolol blunted increases in CBF by dobutamine more than those by isoproterenol. We conclude that endogenous NO did not modulate the coronary vasodilation or the increases in myocardial contractility and MVO₂ during dobutamine. In addition, neither SNP nor NTG altered myocardial contractility or MVO₂ in dobutamine-stimulated myocardium, whereas ACh had a negative inotropic effect in dobutamine-stimulated myocardium that was independent of NO. Implications: Endogenous nitric oxide (NO) did not modulate increases in coronary blood flow, myocardial contractility, or myocardial oxygen consumption during intracoronary infusions of dobutamine. The NO donors sodium nitroprusside and nitroglycerin had no effect on contractility or oxygen consumption in dobutamine-stimulated myocardium. Acetylcholine had negative inotropic effect in dobutamine-stimulated myocardium that was independent of NO.

(Anesth Analg 1998;87:994-1001)

Endothelium-derived nitric oxide (NO) has been shown to play an important role in the coronary vasodilation elicited by a variety of physiological stimuli ^[1]. Treatment with a NO synthase (NOS) inhibitor blunts the coronary vasodilation during inotropic stimulation with isoproterenol, which implies that NO is involved in the response ^[2,3]. Similar findings were obtained when isoproterenol was preceded by the selective beta_1-adrenergic blocker atenolol ^[3]. This suggests that isoproterenol causes release of NO via stimulation of the coronary beta_2-adrenergic receptors, rather than secondary to the increases in contractility and O₂ demand accompanying stimulation of the myocardial beta_1-adrenergic receptors. If so, inotropic drugs that do not have significant beta_2-adrenergic stimulatory properties should cause increases in coronary blood flow (CBF) that are independent of NO. This hypothesis was evaluated by comparing the changes in CBF during dobutamine before and after treatment with the NOS inhibitor N^{G-nitro-L-arginine} methyl ester (L-NAME). Although dobutamine, like isoproterenol, is a beta-receptor agonist, its ability to cause proportional increases in CBF and myocardial oxygen consumption (MVO₂) suggests that its effects are essentially confined to the myocardial beta_1-receptors^[4].

Intracoronary (IC) infusions of the NO donors sodium nitroprusside (SNP) and nitroglycerin (NTG) and of the stimulator of endogenous NO release acetylcholine (ACh) cause marked coronary vasodilation but does not alter segmental shortening (SS) (or MVO₂) in nonstimulated canine myocardium ^[5]. Observations obtained in vitro suggest that beta-adrenergic stimulation may sensitize the myocardium to the negative inotropic effects of NO ^[6]. Thus, an additional objective was to evaluate the effects of SNP, NTG, and ACh on SS and MVO₂ in dobutamine-stimulated myocardium. Finally, a series of studies was performed using the selective beta_1-receptor antagonist atenolol to verify that dobutamine had minimal beta_2-receptor activity in the dose range used in the present study.

An extracorporeal system was used to selectively perfuse the left anterior descending coronary artery (LAD) in in situ canine hearts. This approach permitted both constant-pressure and constant-flow studies and facilitated the use of selective IC infusions of drugs, which minimized their systemic effects.

Methods

This study was approved by our institutional animal investigation committee. Experiments were performed on 30 conditioned mongrel dogs of either sex (weight 22.7-30.0 kg). Anesthesia was induced with an IV bolus injection of thiopental (15 mg/kg) and was maintained by a continuous infusion of fentanyl and midazolam (12 [micro sign]g [center dot] kg^-1 [center dot] h^-1 and 0.6 mg [center dot] kg^-1 [center dot] h^-1 IV, respectively). After tracheal intubation, the lungs were mechanically ventilated with fractional inspired O₂ concentration (FIO₂) equal to 1.0. The volume and rate of the ventilator were established to maintain PaCO₂ and pH at physiological levels. Muscle paralysis was obtained with an IV injection of vecuronium bromide 0.1 mg/kg with supplements at 0.05 mg [center dot] kg^-1 [center dot] h^-1 to facilitate mechanical ventilation. Body temperature was maintained at 38[degree sign]C with a heating pad. Heparin (400 U/kg with supplementation) was used for anticoagulation.

After a left thoracotomy in the fourth intercostal space, the LAD was perfused via an extracorporeal system ^[5]. This system contained a pressurized reservoir that was supplied with arterial blood. CBF was measured by using an in-line Doppler flow transducer (Transonics Systems, Inc., Ithaca, NY). The flow values were used to calculate the coronary blood concentration for dobutamine (drug concentration per milliliter = drug infusion rate divided by CBF) and to calculate MVO₂. Coronary perfusion pressure (CPP) was measured via a small-diameter tube positioned at the orifice of the perfusion cannula.

Measurements of aortic, left atrial, and left ventricular pressures; left ventricular dP/dt_max; and heart rate were obtained using standard methods ^[5]. A continuous record of hemodynamic variables was obtained on an eight-channel physiological recorder (Model 2800S; Gould, Cleveland, OH).

The anterior interventricular vein was cannulated for samples of effluent from the LAD-perfused myocardium ^[5]. MVO₂ was calculated using the Fick equation. Oxygen extraction was calculated by dividing the arteriovenous oxygen difference by arterial oxygen content and used as an index of the effectiveness of metabolic vasodilation. An oxygen cost ratio was calculated by dividing the inotropically induced percent increase in MVO₂ by the inotropically induced percent increase in SS.

Measurements of SS, an index of local myocardial contractility, were obtained with a pair of ultrasonic crystals implanted into the LAD-perfused myocardium to a subendocardial depth ^[4,5]. SS was calculated from the formula: (Equation 1) where EDL and ESL are the end-diastolic and end-systolic lengths, respectively.

In Series 1 (n = 7), the effects of dobutamine were evaluated before and after L-NAME. After >or=to45 min recovery from surgical preparation, control measurements were obtained with CPP set to 80 mm Hg and maintained at this level throughout the study. Dobutamine was infused into the LAD stepwise at three infusion rates: 2.5, 5.0, and 10.0 mg/min. These rates for dobutamine were chosen because they produced arterial blood concentrations (Table 1) that span the clinical range ^[4]. When stable values for monitored local hemodynamic variables, e.g., SS and CBF, indicated that steady-state conditions had been attained at each dose of dobutamine (2-3 min), measurements were obtained. Isotonic saline was used to dilute dobutamine to 5.0 mg/min, which resulted in infusion rates of 0.5-2.0 mL/min. Preliminary studies demonstrated that infusion of the saline vehicle alone at these low flow rates had no effect on CBF or SS. After the final dose of dobutamine, the infusion pump was stopped, and >or=to20 min was allowed for recovery. L-NAME was then infused at the rate of 300 mg/min IC for 15 min to inhibit the production of NO from the vascular endothelium. The efficacy of L-NAME was verified by its ability to attenuate the increases in CBF by ACh (20 [micro sign]g/min IC), an endothelium-dependent vasodilator, while having no effect on those by SNP (80 mg/min IC), an endothelium-independent vasodilator. The doses for SNP and ACh were the largest that could be used without causing aortic hypotension. Vasodilator reserve was assessed using a maximally dilating infusion of adenosine (8 mg/min IC). The post-L-NAME infusions of dobutamine were always administered after the pre-L-NAME infusions because L-NAME has been demonstrated to inhibit NOS activity in the coronary circulation for as long as 24 h.¹

Table 1: Effect of Graded Intracoronary Infusions of Dobutamine on Systemic Hemodynamic Variables Before and After L-NAME

(¹) Smith TP Jr, Canty JM Jr. N^{G-nitro-L-arginine} methyl ester (LNAME) is a potent and noncompetitive inhibitor of acetylcholine induced vasodilation in conscious dogs [abstract]. FASEB J 1992;6:A1257.

In Series 2 (n = 7), the effects of SNP were evaluated during dobutamine administration with CBF constant. Baseline measurements were initially obtained with CPP set at 80 mm Hg. With CPP maintained at this level, dobutamine was infused at 10.0 [micro sign]g/min IC, and the responses evaluated. During the infusion of dobutamine, SNP was infused at 80 [micro sign]g/min IC, and its effects were assessed while CBF was maintained at the dobutamine-induced increased level. A constant level of CBF was accomplished by reducing CPP manually as necessary to offset SNP-induced coronary vasodilation. The use of constant-flow conditions during SNP avoided the possibility of increases in myocardial contractility secondary to increases in CBF per se, i.e., Gregg's phenomenon ^[7], and it also ensured that the arterial concentration for dobutamine remained constant. A similar protocol was used in Series 3 (n = 4) and Series 4 (n = 8) to assess the effects of an infusion of NTG (40 [micro sign]g/min IC) and ACh (20 [micro sign]g/min IC), respectively, in the presence of dobutamine. In the initial four studies of Series 4, ACh reduced SS in dobutamine-stimulated myocardium (Table 2). To evaluate the role of endogenous NO in this response, in the remaining studies, a second infusion of ACh during dobutamine was performed after treatment with L-NAME, administered as described for Series 1.

Table 2: Effects of Sodium Nitroprusside (SNP), Nitroglycerin (NTG), and Acetylcholine (ACh) on Segmental Shortening and Oxygen Consumption in Myocardium Stimulated with Dobutamine (DOB)

Series 5 (n = 4) was performed to assess the relative beta_2-adrenergic vasodilator potencies of dobutamine and isoproterenol in the coronary circulation. With CPP maintained at 80 mm Hg, the dose-dependent changes in CBF during IC infusions of dobutamine (2.5, 5.0, 10, and 25 [micro sign]g/min) and isoproterenol (2.5, 5.0, 10, and 25 ng/min) were assessed before and after treatment with atenolol at a dose of 100 [micro sign]g/min for 10 min. The infusions of dobutamine and isoproterenol were performed randomly. In Series 5, no measurements of SS or MVO₂ were obtained.

After each experiment, Evans blue dye was injected into the LAD to identify its perfusion territory, and the heart was stopped with KCl and removed. The dyed tissue was excised and weighed. The average weight of the LAD perfusion territory was 34 +/- 2 g.

Statistical analysis was performed using two-way analysis of variance for repeated measures in combination with the Student-Newman-Keuls test and the Student's t-test for paired samples ^[8]. P < 0.05 was considered significant.

Results

(Table 1) shows that the graded intracoronary infusions of dobutamine before and after L-NAME had no effect on systemic hemodynamic variables (mean aortic pressure, mean left atrial pressure, and heart rate) or on arterial blood gases, but that they increased dP/dt_max. The stability of the systemic hemodynamic variables during the selective intracoronary infusions of drugs was a consistent finding throughout the study.

(Figure 1) presents the dobutamine-induced changes in local cardiac variables before and after L-NAME. Dobutamine caused dose-related increases in SS, accompanied by proportional increases in MVO₂, i.e., the O₂ cost ratio remained equal to unity. CBF increased in proportion to MVO₂, resulting in no change in oxygen extraction across the entire dose range. These increases in CBF occurred at constant CPP; thus, they reflected the induced decreases in vascular resistance. L-NAME had no independent effect on the cardiac variables. There was no interaction between the effects of dobutamine and L-NAME.

Figure 1: Dose-related effects of intracoronary dobutamine on coronary blood flow (CBF), myocardial oxygen consumption (MVO₂), oxygen extraction (EO₂), and segmental shortening (SS) before and after N^{G-nitro-L-arginine} methyl ester (L-NAME). Findings were similar before and after L-NAME. Values are mean +/- SE. P < 0.05 versus *control, [dagger]2.5, [double dagger]5.0.

(Figure 2) summarizes the changes in CBF by ACh and SNP before and after L-NAME. L-NAME blunted the increases in CBF by ACh (252% +/- 39% vs 60% +/- 12%), whereas it had no effect on the increases in CBF by SNP (132% +/- 10% vs 145% +/- 18%). Adenosine caused nearly fivefold increases in CBF (from 103 +/- 11 to 494 +/- 54 mL [center dot] min^-1 [center dot] 100 g^-1).

Figure 2: Effects of acetylcholine (ACh) and sodium nitroprusside (SNP) on coronary blood flow before and after N^{G-nitro-L-arginine} methyl ester (L-NAME). L-NAME blunted the increases in coronary blood flow by ACh but not by SNP. Values are mean +/- SE. *P < 0.05 versus before L-NAME.

The cardiac effects of SNP, NTG, and ACh in dobutamine-stimulated myocardium are presented in Table 2. Under constant-flow conditions, both SNP and NTG caused marked reductions in CPP (reflecting proportional decreases in vascular resistance), but neither drug changed SS or MVO₂ from their dobutamine-induced increased levels. However, although ACh also reduced CPP in dobutamine-stimulated myocardium, these responses were accompanied by proportional reductions in SS and MVO₂. L-NAME blunted the ACh-induced decreases in CPP in the dobutamine-stimulated myocardium, but it did not affect the decreases in SS and MVO₂.

(Figure 3) presents the effects of atenolol on the increases in CBF by dobutamine and isoproterenol. Atenolol attenuated the dose-dependent increases in CBF caused by both drugs. However, at all but the smallest dose, this effect was less during the isoproterenol than the dobutamine infusion. This was reflected in the greater values for the ratio of the post- to preatenolol increases in CBF during isoproterenol. Atenolol caused reductions in the baseline values for CBF and dP/dt (_max), but it had no effect on those for heart rate or mean aortic pressure (Table 3). Atenolol abolished the dobutamine- and isoproterenol-induced increases in dP/dt (_max).

Figure 3: Dose-dependent increases in coronary blood flow by dobutamine and isoproterenol before and after atenolol. The value in parentheses is the ratio of the drug-induced increases in coronary blood flow after atenolol to those before atenolol. Atenolol blunted the dobutamine and isoproterenol-induced increases in coronary blood flow. At all but the smallest dose, the ratio of the post- to predrug increases in flow was greater for isoproterenol than for dobutamine. Atenolol reduced the baseline value for coronary blood flow by 26%. Values are mean +/- SE. P < 0.05 versus *preatenolol and [dagger]dobutamine.

Table 3: Effects of Dobutamine and Isoproterenol on Systemic Hemodynamic Variables Before and After Atenolol

Discussion

Before L-NAME, dobutamine caused proportional increases in SS and MVO (₂). The accompanying increases in CBF were, in turn, proportional to those in MVO₂, which maintained oxygen extraction constant. This implies that metabolic vasodilating mechanisms were intact and that dobutamine had only a minor, if any, direct influence on the coronary resistance vessels, i.e., its effects in the heart were mediated primarily via the beta_1-(myocardial) adrenergic receptors. A predominant beta_1-receptor action for dobutamine is also suggested from its minimal coronary vasodilating effect after atenolol (Figure 3).

L-NAME did not blunt the increases in CBF or alter their relationship to MVO₂ during the dobutamine infusion, which implies that endogenous NO was not necessary for the functional hyperemic responses. These findings are in accord with those of Altman et al. ^[9] and Matsunaga et al. ^[10], who evaluated the role of endogenous NO during the coronary functional hyperemias accompanying treadmill exercise and atrial pacing, respectively. Matsunaga et al. ^[10] presented evidence that suggests that augmented adenosine release preserves the pacing-induced increase in CBF after treatment with a NOS inhibitor. Whether a similar mechanism operated during the infusions of dobutamine after L-NAME in the present study remains to be determined.

Our findings contrast with those from previous studies indicating that isoproterenol-induced coronary vasodilation is attenuated after NOS inhibition ^[2,3]. This may be because isoproterenol has a greater ability than dobutamine to activate the beta_2-(vascular) adrenergic receptors. A link between beta_2-adrenergic vasodilation and NO formation and has been demonstrated in a variety of vascular preparations ^[3,11].

Changes in CBF reflect adjustments in tone at the level of the small arteries and arterioles, where most of the vascular resistance (>or=to95%) resides ^[12]. Thus, our findings provide no insight into the role of the vascular endothelium in beta-receptor-mediated dilation of the large epicardial coronary arteries. Ghaleh et al. ^[13] showed that isoproterenol caused dilation of the left circumflex coronary artery (assessed with ultrasonic crystals) in conscious dogs via a mechanism involving the vascular beta_1-adrenergic receptors, but not the vascular endothelium; however, the endothelium reinforced this mechanism through an indirect, flow-dependent effect. The dominance of the beta_1-receptors in the large epicardial coronary arteries contrasts with that of the beta_2-receptors in the coronary resistance vessels ^[7].

Vascular shear stress increases when blood flow or blood viscosity increases or when vessel diameter decreases ^[1]. An increase in shear stress is a potent stimulus for endothelial NO synthesis and release. The lack of effect of L-NAME implies that there was no overall increase in shear stress across the coronary circulation during the dobutamine infusion. This can be explained by increases in vessel diameter that were sufficient to offset the effect of an increased flow rate.

The failure of NOS inhibition to reduce baseline CBF has been demonstrated previously ^[5,14], and it has been explained by the emergence of an alternate metabolic mechanism, e.g., endogenous adenosine, which preserves the myocardial oxygen supply-demand balance when the tonic influence of NO is blocked.

The NO donors used in this study, SNP and NTG, have limitations that warrant consideration. SNP contains five cyanide groups, which are also released in the blood ^[15]. Normally, these cyanide groups are converted into the relatively nontoxic compounds cyanmethemoglobin and thiocyanate. However, high blood concentrations of SNP can overload this conversion mechanism, resulting in accumulation of cyanide ions, which can combine with cytochrome oxidase, thus impairing tissue respiration. This was improbable in the present study because brief IC administrations of SNP were used. Unlike SNP, NTG requires specific enzymes for the release of NO. A deficiency in these enzymes in the coronary resistance vessels limits this release, which explains the less pronounced coronary vasodilating effect for NTG compared with SNP shown herein (Table 2) and in previous studies ^[5].

Our findings during SNP and NTG administration provides evidence that significant, although physiologically relevant, increases in tissue concentration for NO (as implied by the appreciable coronary vasodilator responses) have no effect on contractility in beta-receptor-stimulated myocardium. These results extend our previous observations obtained in nonstimulated myocardium using the same basic canine preparation and experimental approach ^[5].

The ability of ACh and vagal stimulation to depress ventricular function during beta-receptor activation has been shown previously in dogs ^[16,17] and humans ^[18]. This phenomenon was originally attributed to the ability of ACh to bind to a muscarinic receptor and to inhibit adenylate cyclase via a G protein, leading to a reduction in cAMP concentration and, ultimately, to a decrease in Ca²⁺ influx ^[16]. More recently, a role for a NO/cGMP pathway has been postulated ^[17,18]. However, the failure of L-NAME to inhibit the ACh-induced negative inotropic response in dobutamine-stimulated myocardium in the present study argues against an important involvement for this pathway.

Our findings with ACh seem to conflict with those of Hare et al ^[17], which showed that the vagally mediated reduction in contractility in dobutamine-stimulated myocardium was diminished with a NOS inhibitor. One possible explanation for this apparent discrepancy is that the increases in cardiac NO concentration during vagal stimulation were greater than those produced by ACh administration. Another possibility is that vagal stimulation caused the release of additional factor(s), which potentiated the negative inotropic effects of NO.

The present findings pertain to NO synthesized in the coronary endothelium via the constitutive NOS pathway. Another form of NOS (so-called inducible NOS) has been identified in a variety of cell types, e.g., neutrophils and myocytes, after induction by immunological stimuli, such as cytokines and endotoxin ^[19]. NO production is more extended and at much higher concentrations via the inducible NOS pathway ^[19]. Evidence for inducible NOS has been obtained in myocardial samples from patients with dilated cardiopathy, ischemic heart disease, valvular heart disease, and cardiac allograft rejection ^[20-22]. A recent study showed that the administration of a NOS inhibitor potentiated the positive inotropic effect of beta-adrenergic receptor stimulation in patients with heart failure due to idiopathic dilated cardiomyopathy ^[23]. This suggests that excessive cardiac NO levels may contribute to the reduced beta-adrenergic responsiveness frequently observed in patients with heart failure.

Studies conducted in vitro have demonstrated that NO can have a direct depressive effect on tissue oxygen use, which is attributable to its binding to the hememoiety of cytochrome enzymes in the mitochondrial electron-transport chain ^[24]. Previous investigations have shown that this effect may not apply to unstimulated myocardium exposed in vivo to physiologically relevant concentrations of NO ^[5,14]. The present findings extend these observations to myocardium under beta-receptor stimulation with dobutamine.

In conclusion, the present study demonstrates that endogenous NO does not modulate the coronary vasodilation or the increases in myocardial contractility and MVO₂ during inotropic stimulation with dobutamine. Doses of NO donors sufficient to cause marked reductions in coronary vasomotor tone have no apparent effect on contractility and MVO₂ in dobutamine-stimulated myocardium. ACh has a negative inotropic effect in dobutamine-stimulated myocardium that is independent of NO.

We appreciate the expert technical assistance of Derrick L. Harris, BS.