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Am J Physiol Heart Circ Physiol 284: H1693-H1701, 2003. First published January 9, 2003; doi:10.1152/ajpheart.00730.2002
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Vol. 284, Issue 5, H1693-H1701, May 2003

Cardiac enkephalins interrupt vagal bradycardia via delta 2-opioid receptors in sinoatrial node

Martin Farias III, Keith E. Jackson, Darice Yoshishige, and James L. Caffrey

Department of Integrative Physiology, University of North Texas Health Science Center, Fort Worth, Texas 76107


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Local cardiac opioids appear to be important in determining the quality of vagal control of heart rate. Introduction of the endogenous opioid methionine-enkephalin-arginine-phenylalanine (MEAP) into the interstitium of the canine sinoatrial node by microdialysis attenuates vagally mediated bradycardia through a delta -opioid receptor mechanism. The following studies were conducted to test the hypothesis that a delta 2-opiate receptor subtype mediates the interruption of vagal transmission. Twenty mongrel dogs were anesthetized and instrumented with microdialysis probes inserted into the sinoatrial node. Vagal frequency responses were performed at 1, 2, and 3 Hz during vehicle infusion and during treatment with the native agonist MEAP, the delta 1-opioids 2-methyl-4aa-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha -octahydroquinolino[2,3,3- g]isoquinoline (TAN-67) and [D-pen2,5]-enkephalin (DPDPE), and the delta 2 opioid deltorphin II. The vagolytic effects of intranodal MEAP and deltorphin were then challenged with the delta 1- and delta 2-opioid receptor antagonists 7-benzylidenenaltrexone (BNTX) and naltriben, respectively. Although the positive control deltorphin II was clearly vagolytic in each experimental group, TAN-67 and DPDPE were vagolytically ineffective in the same animals. In contrast, TAN-67 improved vagal bradycardia by 30-35%. Naltriben completely reversed the vagolytic effects of MEAP and deltorphin. BNTX was ineffective in this regard but did reverse the vagal improvement observed with TAN-67. These data support the hypothesis that the vagolytic effect of the endogenous opioid MEAP was mediated by delta 2-opioid receptors located in the sinoatrial node. These data also support the existence of vagotonic delta 1-opioid receptors also in the sinoatrial node.

TAN-67; heart rate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ROLE OF ENDOGENOUS OPIOID peptides in the local control of heart rate is not yet well understood. When administered exogenously, these peptides are effective modulators of cardiac vagal function. Weitzell et al. (31) first reported that enkephalin inhibited vagal transmission in isolated rabbit hearts. The inhibition was reversed by the nonselective opiate antagonist naloxone. Other investigators (3, 4, 10, 13, 22, 24) have observed that enkephalins suppressed vagal bradycardia in vivo, suggesting that enkephalins function as "governors" of vagal control.

Several enkephalin sequences are concentrated in the heart (32), including the heptapeptide methionine-enkephalin-arginine-phenylalanine (MEAP). MEAP attenuated vagally mediated bradycardia by >70% when infused intra-arterially in anesthetized dogs and did not appear to involve a direct interaction with the pacemaker cells (3, 4). The high affinity but nonselective opioid antagonist diprenorphine completely reversed the effect of MEAP, restored vagal control of heart rate, and indicated that opiate receptors were involved (3, 4).

Prejunctional vagal nerve terminals in the sinoatrial (SA) node and the nearby intracardiac parasympathetic ganglia were the most likely targets for MEAP. MEAP was delivered directly into the SA node by microdialysis to resolve these two potential targets. Intranodal MEAP attenuated vagally mediated bradycardia to the same extent as that observed during systemic infusion of the peptide and both nodal and systemic effects were reversed by the nodal delivery of diprenorphine (10). Collectively, these findings indicated that MEAP modulated vagal control of heart rate by acting on opioid receptors in the SA node, which were most likely located prejunctionally on vagal nerve terminals.

To explore the physiology of opioids in the SA node, an extended series of dose-response relationships with specific opioid agonists and antagonists were conducted to identify the responsible opioid receptor. Those studies have established a clear delta -receptor profile, indicating that the vagolytic effect of MEAP was mediated by delta -opioid receptors (13). The nodal delivery of MEAP and the delta 2-agonist deltorphin II produced equipotent vagolytic responses and both effects were reversed by the delta -antagonist naltrindole. The µ- and kappa -agonists had no effect on vagally mediated bradycardia, and µ- and kappa -antagonists were ineffective versus MEAP (13). These data strongly indicated that delta -opioid receptors within the SA node were responsible for the vagolytic effect of MEAP.

Although the distinct transcripts corresponding to delta -receptor subtypes have not been isolated (1, 9, 17), there is considerable functional and pharmacological evidence for the existence of distinct delta 1- and delta 2-receptor-mediated responses (1, 15, 25, 28-30, 33). The nature of subtype-specific actions on cardiac function is not well defined but Schultz et al. (27) demonstrated that pretreatment with the selective delta 1 agonist 2-methyl-4aa-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha -octahydroquinolino[2,3,3-g]isoquinoline (TAN-67) significantly reduced infarct size in the ischemic rat heart. The cardioprotection conferred by TAN-67 was subsequently reversed with the use of the selective delta 1 antagonist 7-benzylidenenaltrexone (BNTX). Chien et al. (5) also reported that delta 1-agonists helped to preserve the viability of multiorgan preparations. Because the activation of cholinergic receptors has also been implicated in cardioprotection (34), a potential link between opioids and vagal function might be physiologically important. However, the vagolytic action of added MEAP cited above would be difficult to reconcile with reported cardioprotective effects of cholinergic stimulation.

The application of a preconditioning-like protocol to the SA node artery stimulated a reproducible increase in the endogenous MEAP recovered by dialysis from the nodal interstitium (14). In contrast to the vagolytic effect of exogenously administered MEAP, the rise in endogenous MEAP was accompanied by a consistent enhancement of vagally mediated bradycardia. The delta -antagonist naltrindole reversed the vagotonic effect and suggested participation by delta -opiate receptors (14). An opioid-mediated increase in vagal function during arterial occlusion makes a role in cardioprotection mechanistically easier to explain. An increase in cholinergic stimulation during oxidative stress could reduce tissue loss by lowering metabolic demand locally.

These collected observations suggest the hypothesis that different subtypes of the delta -receptor (delta 1 and delta 2) may mediate respectively the opposing vagotonic and vagolytic effects of opioids. Consistent with the suggestion that the vagotonic effect is mediated by delta 1-receptors, Shultz et al. (27) reported that TAN-67 reduced resting heart rate in the rat. In contrast, delta -activation with the use of administered enkephalin in the dog produced a clear attenuation of vagal bradycardia. These opposing observations would be compatible if the vagolytic activity in the dog is mediated by delta 2-receptors. The two subtypes of delta -receptors may serve distinctly different roles in the regulation of heart rate.

The purpose of these studies was to test the hypothesis that delta 2-opioid receptors in the SA node were responsible for the vagolytic effect of the cardiac opioid MEAP and to rule out the participation of delta 1-opioid receptors. This was accomplished with two strategies. In one strategy, the vagolytic effects of MEAP and the delta 2-agonist deltorphin II were first demonstrated, and the endogenous opioid MEAP was then challenged with delta 1- and delta 2-selective antagonists. In the second, the vagolytic effects of MEAP and deltorphin II were compared with those of the selective delta 1-agonists [D-pen2,5]- enkephalin (DPDPE) and TAN-67. This endeavor arose as a result of previous studies, which established a role for delta -receptors in the vagolytic actions of MEAP. The efficacy of deltorphin II in those studies suggested the vagolytic effect might involve a delta 2 response, but the definitive comparisons were not available.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments conformed to the Guide for the Care and the Use of Laboratory Animals published by the National Institutes of Health.

Surgical preparation. Twenty Mongrel dogs were anesthetized with pentobarbital sodium, intubated, and mechanically ventilated with room air. Fluid-filled catheters were inserted into the femoral artery and vein and then advanced into the descending aorta and inferior vena cava, respectively. The arterial line was attached to a pressure transducer (model PD23XL; Statham) to monitor heart rate and blood pressure continuously online (PowerLab). The venous line was used to administer additional anesthetic as needed. Arterial blood gases were monitored with a blood gas analyzer (Instrumentation Laboratories) and the PO2 (90-120 mmHg), pH (7.34-7.45), and PCO2 (35-45 mmHg) were adjusted to normal with supplemental oxygen, bicarbonate, or by altering the minute volume.

The right and left vagus nerves were isolated in the cervical region through a midline surgical incision and tied off tightly with umbilical tape and were returned to their position in the neck for later retrieval. A single dose of succinylcholine (1 mg/kg) was administered intravenously to temporarily reduce involuntary muscle movements during the 10-15 min required for the electrosurgical incision of the right thorax and removal of right ribs 2-5. The pericardium was opened and the upper margins were sutured to the body wall to provide a pericardial cradle.

A 27-gauge stainless steel cannula was used to introduce the microdialysis probe into the SA node. To confirm the probe placement in the SA node, norepinephrine (1 x 10-9 mol/µl) was introduced into the microdialysis probe. The observation of a brisk 30- to 40-beat increase in heart rate provided a functional confirmation of the probe location within the SA node. Prior studies (14) have determined that deliberate repositioning of the probe as little as 2 mm lateral to the node eliminates the norepinephrine-mediated tachycardia. The microdialysis probe was constructed from a single 1-cm length of dialysis fiber (220 µm OD, 200 µm ID) and hollow silica inflow and outflow tubes (120 µm ID, 170 µm OD). The dialysis tubing permits molecules with a molecular weight of 36,000 or less to freely cross from the lumen into the nodal interstitium. This technique allows one to both alter and sample the local nodal interstitial environment while minimizing alterations in systemic hemodynamics and reflex compensations.

Protocols. These experiments were conducted to demonstrate that the delta 2-opioid receptor subtype was responsible for the vagolytic effect of nodal enkephalins. Two strategies were employed. In the first strategy, the influence of the delta -subtype-specific agonists DPDPE, TAN-67, and deltorphin II was compared for their vagolytic action. In the second strategy, a vagolytic effect of the endogenous agonist MEAP was established and then the ability of subtype-selective antagonists (BNTX and naltriben) to reverse this effect were evaluated. All treatments were introduced locally into the interstitium of the SA node by microdialysis at a flow rate of 5 µl/min.

Previous studies (13) revealed that deltorphin II (1.5 × 10-9 mol/min) blocked vagally mediated bradycardia. The vagolytic effect of deltorphin II was successfully reversed by the delta -selective antagonist naltrindole. These findings suggested participation of a delta 2-opioid receptor in this effect. This study will determine the subtype of delta -opioid receptor responsible for the inhibition of vagally mediated bradycardia by MEAP.

Protocol 1. This protocol tested whether the intranodal administration of delta 1-selective agonists was capable of interrupting vagal bradycardia. After microdialysis probe insertion, the SA node was perfused (5 µl/min) with saline for 60 min. After this period of equilibration, control vagal responses were obtained by stimulating the right vagus nerve at 1, 2, and 3 Hz. The nerve was stimulated at a supramaximal voltage for 15 s, followed by 1 min 45 s for recovery. Deltorphin II was then infused (5 µl/min) into the SA node for 5 min to establish a functional vagolytic effect. The effective dose used for deltorphin II (1.5 × 10-9 mol/min) was determined previously (13). Once established, the effect of deltorphin II served as a positive control in cases where the subsequent agonists under evaluation were without effect. After this procedure, dose responses were constructed for the selective delta 1-agonist DPDPE or TAN-67. Doses were selected to provide molar equivalent ranges (0.05-5 × 10-9 mol/min) to those previously determined to be vagolytic for MEAP and deltorphin II (13). Each dose of each agent was infused for 5 min before evaluating the vagus nerve. After each dose evaluation, the agent was washed out for 15 min and vagal function was retested to ensure that it had returned to normal. The length of washout was based on previous experiments (13). At the end of the TAN-67 protocol, this agent was combined with the delta 1-antagonist BNTX to determine whether the unexpected improvement in vagal function was mediated by a delta 1-opioid receptor.

Protocol 2. This protocol was designed to test whether vagolytic effects of MEAP and deltorphin II were blocked by a selective delta 2-opioid receptor antagonist and not by a selective delta 1-opioid receptor antagonist. MEAP and deltorphin II (1.5 × 10-9 mol/min) were introduced into the interstitium of the SA node, and vagal stimulations were performed as previously described to establish the vagolytic effect of each. After washout of these initial tests, MEAP was combined with increasing doses of the selective delta 1-antagonist BNTX or the selective delta 2-antagonist naltriben. At the end of the protocol, the specific subtype was further confirmed by combining deltorphin II with the maximum effective dose of one or the other antagonist. As predicted by the hypothesis, the delta 2-antagonist naltriben should overcome the vagolytic effect of MEAP and deltorphin and verify participation of the delta 2-opioid receptor. BNTX should not reverse the vagolytic effect of MEAP or deltorphin indicating the absence of participation by delta 1-opioid receptors.

Materials. MEAP and deltorphin II were synthesized by American Peptide (Sunnyvale, CA). TAN-67, DPDPE, and BNTX were obtained from Tocris Cookson (Ellisville, MO). Naltriben was obtained from Sigma (St. Louis, MO).

Statistical methods. All data were expressed as means ± SE. Differences were evaluated with ANOVA for repeated measures. Individual treatment differences were determined by post hoc analysis with Tukey's test for multiple comparisons. Differences determined to occur by chance with a probability of P < 0.05 were accepted as statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Twenty dogs were randomly assigned to various protocols employing delta 1- and delta 2-agonists and antagonists. Table 1 represents the resting cardiovascular parameters for all animals across all treatments. There were no significant differences in heart rate or blood pressure among groups before treatment. Resting heart rate and blood pressure were also unaltered by any of the opioid agonists and antagonists, regardless of the dose.

                              
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  		Table 1.   Cardiovascular indexes

Deltorphin vagolysis. Deltorphin II was used as a positive control to demonstrate the functional integrity of the system in each animal before other agents were tested. This pretest also served to verify the appropriate placement of the dialysis probe in the proximity of the nodal opiate receptors responsible for the interruption of vagal bradycardia. The nodal administration of deltorphin II (1.5 × 10-9 mol/min) reduced vagally mediated bradycardia by 75-85% at all vagal frequencies employed and was significantly different from control.

DPDPE dose responses. In this protocol, DPDPE was introduced directly into the SA node to rule out the participation of delta 1-opioid receptors in the opioid-mediated interruption of vagal bradycardia. Control vagal stimulations during vehicle infusion produced a normal graded decline in heart rate at all vagal frequencies used (Fig. 1). The nodal delivery of DPDPE had no effect on heart rate during the vagal frequency response as indicated by the superimposition of the DPDPE and vehicle responses (Fig. 1, bottom two traces). The vagolytic effect of deltorphin II is illustrated in the top trace. The complete dose responses for all three frequencies are illustrated in Fig. 2.


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  		Fig. 1.   The heart rate/frequency response [in beats/min (bpm)] is mediated by right vagal nerve stimulation during the nodal delivery of deltorphin II (1.5 × 10-9 mol/min) and [D-pen2,5]enkephalin (DPDPE) (5 × 10-9 mol/min) by microdialysis. The data illustrated are for the maximal dose of DPDPE employed in its dose-response curve. * P < 0.05, significantly different from control.



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  		Fig. 2.   Change in heart rate produced during right vagal stimulation during exposure (5 min) to increasing doses of the delta 1-selective opioid agonist DPDPE. A: 1 Hz; B: 2 Hz; C: 3 Hz. The units for the doses listed within the bars are 0.05, 0.15, 0.5 1.5, and 5.0 × 10-9 mol/min. Deltorphin (1.5 × 10-9 mol/min) was included as a positive control. All treatments were infused into the sinoatrial node of the dog via microdialysis. * P < 0.05, significantly different from control.

TAN-67 dose responses. In the absence of an effect as observed with DPDPE, it is difficult to say with confidence that the agent successfully crossed the dialysis membrane into the interstitium. In this regard, a second selective delta 1-opioid receptor agonist, TAN-67, was used in a second group of animals to provide further evidence that delta 1-opioid receptors were not vagolytic. During vehicle infusions, control vagal stimulations produced a normal graded decline in heart rate as the frequency of stimulation was increased (Fig. 3, middle trace). Deltorphin II produced a vagolytic response similar to that observed (80% inhibition) in the prior group (Fig. 3, top trace). The administration of TAN-67 into the SA node had no vagolytic effect during the vagal frequency response at any dose employed. Rather, TAN-67 produced a greater vagal bradycardia as the dose was increased (Fig. 3, bottom trace). The maximum effect was observed at the 1.5 × 10-9 mol/min (Fig. 4) with an apparent ED50 of 1.0 × 10-10 mol/min. The maximal improvement at 1.5 × 10-9 mol/min was 28-37% and was significantly different from control at all vagal frequencies.


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  		Fig. 3.   The heart rate/frequency response is mediated by right vagal nerve stimulation during the nodal delivery of vehicle, deltorphin II (1.5 × 10-9 mol/min), 2-methyl-4aa-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha -octahydroquinolino[2,3,3-g]isoquinoline (TAN-67) (5 × 10-9 mol/min) or 7-benzylidenenaltrexone (BNTX) alone (5 × 10-9 mol/min), and TAN-67 (1.5 × 10-9 mol/min) combined with an equimolar dose of BNTX. * P < 0.05, significantly different from control.



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  		Fig. 4.   A-C: change in heart rate produced during right vagal stimulation during exposure (5 min) to increasing doses of the delta 1-selective opioid agonist TAN-67. The units for the doses listed within the bars are 0.05, 0.15, 0.5, 1.5, and 5.0 × 10-9 mol/min. Deltorphin (1.5 × 10-9 mol/min) was included as a positive control. All treatments were infused into the sinoatrial node of the dog via microdialysis. * P < 0.05, significantly different from control.

Acting on the presumption that the vagotonic effect of TAN-67 was perhaps mediated by a delta 1-receptor, TAN-67 (1.5 × 10-9 mol/min) was then combined with the delta 1-antagonist BNTX (1.5 × 10-9 mol/min) and infused directly into the SA node via microdialysis. BNTX effectively prevented the vagotonic effect of TAN-67 because the vagally mediated bradycardia during the combined infusion was similar to control values (Fig. 3, middle trace). The administration of BNTX alone had no effect on vagal bradycardia and once again produced values that were similar to control. Vagal stimulations were performed after washout of each treatment and were again similar to control values.

MEAP versus naltriben dose responses. In the second strategy, deltorphin II and the endogenous cardiac opioid MEAP were introduced into the SA node at vagolytically effective doses. Then each agonist was subsequently combined with selective delta 1- and delta 2-antagonists to verify which delta -receptor subtype was responsible for the interruption of vagal bradycardia. The control frequency response is illustrated in Fig. 5, bottom traces. The vagolytic effects of deltorphin II and MEAP are illustrated in the two top traces. Increasing doses of the selective delta 2-opioid receptor antagonist naltriben were combined with MEAP in the dialysis perfusate. Naltriben progressively reversed the effect of MEAP and restored vagal regulation of heart rate to control (Fig. 6). The reversal was obtained with an ID50 of ~1.5 × 10-10 mol/min and a maximal effect near molar parity with the agonist (1.5 × 10-9 mol/min). The similar blockade of the deltorphin and MEAP effects is illustrated among the bottom traces in Fig. 5 for the last dose in the naltriben dose-response curve. Perfusion with the highest dose of naltriben alone was similar to control indicating that naltriben had no effect on vagal function independent of it ability to obstruct the access of MEAP and deltorphin II to nodal delta 2-receptors.


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  		Fig. 5.   The heart rate/frequency response is mediated by right vagal nerve stimulation during the nodal delivery of vehicle, deltorphin II (1.5 × 10-9 mol/min), methionine-enkephalin-arginine-phenylalanine (MEAP) (1.5 × 10-9 mol/min), and MEAP or deltorphin II (1.5 × 10-9 mol/min) combined with an equimolar dose of naltriben (NTB), and naltriben (5 × 10-9 mol/min) alone. * P < 0.05, significantly different from control.



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  		Fig. 6.   A-C: change in heart rate produced during right vagal stimulation during exposure (5 min) to increasing doses of the delta 2-antagonist, naltriben combined with MEAP (1.5 × 10-9 mol/min). The units for the doses listed in the bars are 0.05, 0.15, 0.5, 1.5, and 5.0 × 10-9 mol/min. Deltorphin (1.5 × 10-9 mol/min) was included as confirmation of the delta 2-character of the naltriben blockade. All treatments were infused into the sinoatrial node of the dog via microdialysis. * P < 0.05, significantly different from control.

MEAP versus BNTX dose responses. The selective delta 1-opioid receptor antagonist BNTX was used to confirm that the vagolytic effect of MEAP was mediated by delta 2- and not by delta 1-opioid receptors. This was achieved by combining increasing doses of BNTX with an effective vagolytic dose of MEAP (1.5 × 10-9 mol/min). The rationale presumed that if naltriben identified a functional delta 2 response, then combining MEAP with increasing doses of BNTX would find BNTX ineffective or much less effective than naltriben. The bottom two traces in Fig. 7 illustrate the control bradycardia response in this group and the absence of an effect of BNTX alone. The 50-70% inhibition by both MEAP and deltorphin II is indicated among the top traces in Fig. 7. When BNTX was combined with MEAP or deltorphin II, the resulting curves were very similar to those for MEAP and deltorphin alone (Fig. 7, top traces). BNTX had no effect on the vagolytic properties of either MEAP or deltorphin. The complete dose-response curves for BNTX versus MEAP are described in Fig. 8. Although a subtle reversal of the effect of MEAP might be suggested from these data, the observed bradycardia was never different from MEAP alone. The absence of an effect of BNTX versus both MEAP and the delta 2 agonist, deltorphin II further supports the exclusive delta 2 character of the vagolytic effect.


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  		Fig. 7.   Heart rate/frequency response is mediated by right vagal nerve stimulation during the nodal delivery of vehicle, deltorphin II (1.5 × 10-9 mol/min), MEAP (1.5 × 10-9 mol/min), and MEAP or deltorphin II (1.5 × 10-9 mol/min) combined with an equimolar dose of BNTX, and BNTX (5 × 10-9 mol/min) alone. * P < 0.05, significantly different from control.



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  		Fig. 8.   A-C: change in heart rate produced during right vagal stimulation during exposure (5 min) to increasing doses of the delta 1-antagonist BNTX combined with a fixed dose of MEAP (1.5 × 10-9 mol/min). The units for the doses listed in the bars are 0.05, 0.15, 0.5, 1.5, 5.0 × 10-9 mol/min. Deltorphin (1.5 × 10-9 mol/min) was included as added confirmation of the absent delta 1-receptor participation in the response. All treatments were infused into the sinoatrial node of the dog via microdialysis. * P < 0.05, significantly different from control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data reported above support the primary hypothesis that the vagolytic effect of the endogenous opioid MEAP on heart rate is mediated by delta 2-opioid receptors in the SA node. This conclusion is based on the observation that vagolytic response to MEAP was duplicated by the delta 2-agonist deltorphin II when the delta 1- agonists DPDPE and TAN-67 were both vagolytically ineffective in the same animals. Participation by delta 2-receptors was verified further by demonstrating the vagolytic effect of MEAP was reversed by the delta 2-antagonist naltriben and unaltered by equimolar doses of the delta 1-antagonist BNTX. The delta 1-character of the vagolytic effect of MEAP was rigorously determined earlier (13), and the current findings suggest that the vagolytic effect was mediated by delta 2-receptors without a measurable delta 1-receptor contribution.

Deltorphin II served as positive control in these experiments to confirm the location of the dialysis probe within functional reach of the nodal opioid receptors responsible for the vagolytic response. The absence of a response when introducing agents by microdialysis can be ambiguous because it is often difficult to verify that every agent has successfully crossed the dialysis membrane into the interstitium in biologically effective concentrations. In this instance, functionally similar but molecularly distinct delta 1-agonists were used to reduce the probability of interference with diffusion due to molecular charge, adsorption, or solubility. In this case, both DPDPE and TAN-67 are delta 1-agonists but DPDPE is a modified peptide and TAN-67 is a heterocyclic isoquinoline. This dramatically reduces the probability that the absence of a delta 1-effect resulted from a failure to reach the target due to adsorption or failure to diffuse freely.

Although TAN-67 had no vagolytic effect, it produced a consistent improvement in vagal bradycardia and thus provided additional direct evidence that TAN-67 had reached the nodal interstitium. The delta 1-opioid receptor antagonist BNTX subsequently reversed the TAN-67-mediated vagal improvement. Thus delta 1-receptors were present in the SA node and were vagotonic rather than vagolytic. These observations suggested that the opioid modulation of vagal function is bimodal with opposite poles of the response mediated by different subtypes of the delta -receptor.

Selectivity issues: TAN-67 and DPDPE. The existence of delta -receptor subtypes has been based entirely on biological responses that can be distinguished by agonists and antagonists reported as selective for the respective subtypes (1, 13, 25, 28-30, 33). Each receptor subtype stimulated responses that were reversed by agonists preferential to that subtype. Mixed results were obtained when cross-tolerance or cross-desensitization experiments were conducted (1, 21, 29). A single receptor transcript has been isolated, and attempts to identify distinct receptor proteins associated with delta 1- and delta 2-mediated responses have been as yet unsuccessful (1, 9, 17). Contradictory findings in some isolated systems in vitro support the suggestion that differences in coupling, agonist concentration or local membrane conditions may determine whether delta 1-, delta 2-, or mixed responses are evident (7).

Subtype-specific responses have been used to quantify the relative delta -selectivity of various agents. DPDPE and deltorphin II have been widely employed respectively as preferential delta 1- and delta 2-agonists. Each has ~80- to 100-fold selectivity for its respective receptor subtype in antinociceptive and binding studies (6, 8, 30). Antagonists for each receptor subtype have been characterized as well. BNTX and naltriben currently serve respectively as prototypical delta 1- and delta 2-antagonists (15, 25).

DPDPE reportedly has some mixed delta 2-agonist activity in some biological systems (33). This aspect might complicate the interpretation of the absent response with DPDPE during vagal stimulations and may help to explain the difference observed between DPDPE and TAN-67. Because delta 2-opioid receptors were clearly vagolytic, the absence of a response to DPDPE would suggest either the absence of delta 1-receptors or the absence of a delta 1-effect on vagal function. If DPDPE has measurable delta 2-activity, one might expect to see a vagolytic response at the high end of the dose-response curve. TAN-67, which is significantly more selective for delta 1-opioid systems (6, 16), improved vagal bradycardia by 35% and was reversed by BNTX. This suggests that delta 1-receptors were present and they did alter vagal function through an apparent delta 1-mechanism. If DPDPE acted on both delta 1- and delta 2-receptors simultaneously, the opposing vagotonic and vagolytic actions may have cancelled out one another. In summary, selective activation of delta 1-receptors had no demonstrable vagolytic effect. In contrast, delta 1-receptors appeared to facilitate vagal function.

The normal role of cardiac opioids in the autonomic control of the heart remains unclear, but some of the details have begun to resolve. The presence of significant mRNA for proenkephalin in heart and the prodigious capability of the heart to degrade enkephalin suggest the cardiac enkephalins function primarily as a local paracrine hormones. The current studies reported here have concentrated on interactions with vagal control of heart rate. Earlier studies (3, 4, 10, 13, 22, 24, 31) both in vivo and in isolated heart models demonstrated that opioids attenuated a variety of cardiac parasympathetic responses during vagal nerve stimulation. The delta 2-mediated interruption of vagal bradycardia is consistent with the traditional view of opioids as inhibitory neuromodulators. The apparent bimodal character of delta -receptor activation though not often acknowledged is also not that unusual (7, 26). Because distinct delta 1- and delta 2-receptor proteins have not been isolated, opposing responses in the same tissue presents some interesting mechanistic questions. One proposal suggested that the local membrane environment determined the functional expression of opposing opioid receptor responses by regulating how the receptors were coupled to their respective second messenger systems (7). How this local environment and the balance of these responses participate in normal heart rate control remains to be determined.

What purpose do these delta -subtypes serve in modulating heart rate during normal homeostasis? When endogenous nodal MEAP was elevated during occlusion of the nodal artery, vagal bradycardia was improved (14). The vagotonic effect was blocked by the general delta -antagonist naltrindole, and the vagal improvement was quantitatively very similar to that observed during administration of TAN-67 in this current report. Because the latter was blocked by BNTX, both responses may have been mediated by delta 1-receptors. The coupling hypothesis cited above (7) also suggested that one side of the bimodal response was far more sensitive to agonist. The hypothesis argued that the positive coupling to adenylate cyclase through the G protein Gsalpha predominated at physiologically very low opioid concentrations. Thus the vagotonic effect associated with nodal artery occlusion would be consistent with the bimodal hypothesis if the modest increases in nodal MEAP also observed during occlusion (14) improved the efficiency of vagal transmission through delta 1-receptors much like TAN-67. The activation of delta 1-receptors during arterial insufficiency might serve to stabilize the heart by improving local vagal function and thereby reducing local oxygen demand and consequent irritability.

At the other end of the spectrum, vasovagal syncope poses a different threat to the organism during stressful circumstances. In this regard, higher rates of opioid release, combined with the activation of delta 2-opioid receptors may suppress vagal function when that activity is inappropriately intense. Thus at higher concentrations the more widely recognized neuroinhibitory coupling to adenylate cyclase through the inhibitory G protein GIalpha might predominate with the opioids now serving as inhibitory governors of vagal activity. In accord with this proposed hypothesis, one might argue that the delta 1 activity provides a background environment of neurofacilitatory activity, whereas the delta 2-receptors provide a more episodic governor-like function.

The opioid receptor systems may also be of significance during cardiovascular pathologies such as myocardial infarction and congestive heart failure. Evidence that delta 1-receptors mediate preconditioning suggested that these receptors might be therapeutically valuable during myocardial infarction (27). Nodal MEAP recovered in the dialysate was elevated during a series of brief nodal artery occlusions. As indicated above, this increase in nodal MEAP was accompanied by an improved vagal function (14) that in retrospect may have been mediated by delta 1-receptors. Healthy vagal influences have been associated with better survival statistics after myocardial infarction (2, 18). The activation of delta 1-receptors could enhance vagal function during myocardial infarction, and by slowing the heart, decrease work output and energy demand (23, 34). This would then reduce the damage caused by free radicals and help to maintain cellular integrity (23).

The observation that delta 2-opioid receptors are vagolytic suggests that their actions may be pathological for instance during sustained excess. Circulating endogenous opioids rise significantly during congestive heart failure (11). The vagolytic action of these peptides may contribute to cardiac dysfunction and the rise in sympathetic activity. In support of this hypothesis, delta -opioid antagonists restored vagal function in atrial preparations from failing human hearts (19). However, the characterization of delta 1- and delta 2-receptor effects on heart rate during cardiovascular disease remains to be elucidated and may hold significant clinical potential.

In conclusion, the current results suggested that the endogenous cardiac enkephalin MEAP attenuated vagal bradycardia via delta 2-opioid receptors concentrated within the canine SA node. The data above also support the presence of delta 1-opioid receptors in the SA node that appear to facilitate vagal transmission. Whether delta 1- and delta 2-opioid receptors in the SA node are located prejunctionally on vagal nerve terminals and whether these receptors modify the release of acetylcholine both remain to be verified directly and as such constitute important future directions.


    ACKNOWLEDGEMENTS

This research was supported by the Texas Higher Education Coordinating Board Advanced Research Program Grant 130-0039-2001 and by National Research Service Award 1-F31-HL-7133401 (to M. Farias).


    FOOTNOTES

Address for reprint requests and other correspondence: J. L. Caffrey, Univ. of North Texas Health Science Center, Dept. of Integrative Physiology, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (E-mail: jcaffrey{at}hsc.unt.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 9, 2003;10.1152/ajpheart.00730.2002

Received 27 August 2002; accepted in final form 20 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 284(5):H1693-H1701
0363-6135/03 $5.00 Copyright © 2003 the American Physiological Society


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