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1-opioid receptor stimulation reduces 2-opioid receptor responses in the SA node
University of North Texas Health Science Center, Fort Worth, Texas
Submitted 2 February 2006 ; accepted in final form 9 June 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-opioid receptors within the sinoatrial (SA) node. Higher doses activate 2-opioid receptors, interrupt vagal transmission (vagolytic), and reduce the bradycardia. Preconditioning-like occlusion of the nodal artery produced a vagotonic response that was reversed by the 1-antagonist 7-benzylidenaltrexone (BNTX). The following study tested the hypothesis that extended 1-opioid receptor stimulation reduces subsequent 2-receptor responses. The 2-agonist deltorphin II was introduced in the SA node by microdialysis to evaluate 2 responses before and after infusion of the 1-agonist TAN-67. TAN-67 reduced the vagolytic effect of deltorphin by two-thirds. When the 1-antagonist BNTX was combined with TAN-67, the deltorphin response was preserved, suggesting that attrition of the prior response was mediated by 1 activity. When TAN-67 was omitted in time control studies, some loss of 2 responses was apparent in the absence of the 1 treatment. This loss was also eliminated by BNTX, suggesting that the attenuation of the response after deltorphin alone was also the result of 1 activity. Additional studies tested TAN-67 alone in the absence of prior deltorphin. When time controls were conducted without the initial deltorphin treatment, a robust vagolytic response was observed. When TAN-67 preceded the delayed deltorphin, the vagolytic response was eroded, indicating an independent effect of TAN-67. BNTX infused afterward was unable to restore the 2 response. These data support the conclusion that the loss of the 2 response resulted from reduced 2 activity mediated by continued 1-receptor stimulation and not the arithmetic consequence of increased competition from that same 1 receptor.
sinoatrial node
Endogenous cardiac opioids are potent modulators of cardiovascular function with significant physiological and pathological influences. The mRNA for proenkephalin is concentrated in the heart (9, 14, 22, 23). The resulting precursor contains seven constituent opioid sequences, including four copies of methionine-enkephalin (ME) and one copy each of methionine-enkephalin-arginine-phenylalanine (MEAP), leucine-enkephalin (LE), and methionine-enkephalin-arginine-glycine-leucine. Despite its single copy representation, MEAP was consistently the most concentrated enkephalin in the heart (23). MEAP, ME, and LE all improved postischemic myocardial function, and all were vagolytic when introduced in the SA node by microdialysis (4, 11, 12). Opioids, however, can improve or reduce vagally mediated bradycardia. Nodal opioid receptors are probably located presynaptically on postganglionic vagal nerve terminals (3).
Opioid receptors are G protein-coupled receptors that commonly regulate neurotransmitter release from the neurons on which they are located (8, 15, 16). The native enkephalins found in heart are preferential agonists at the receptor. Pharmacological evidence suggests that there are two distinct -receptor subtypes despite biochemical analysis which has thus far only identified a single transcript (1, 12, 17, 21, 24). These two functional phenotypes of the receptor in the canine SA node modulate vagal transmission in opposite directions. Ultra-low-dose rates of MEAP facilitate vagal transmission (vagotonic), whereas higher concentrations reduce vagal transmission (vagolytic; see Ref. 4). The two opposing effects were blocked by subtype-specific antagonists, confirming their mediation by pharmacologically distinct phenotypes of the -opioid receptor.
The expression of similar excitatory and inhibitory pathways in cultured dorsal root ganglion cells was attributed to differences in receptor coupling. The polarity of this coupling could be shifted between inhibition and excitation by changing the local membrane environment (2). The expression of the proexcitatory environment was proposed to depend on positive feedback from activation of the excitatory pathway. The present study was based on related observations made in the SA node regarding the bimodal character of -receptor stimulation (4). In both model systems, the alternate coupling was concentration dependent with the excitation observed first at lower dose rates. Preliminary observations also suggested that sustained stimulation of the 1 receptor might reduce the efficacy of the opposing 2-receptor stimulation. The cardioprotection afforded by ischemic preconditioning is mediated in part by 1-receptor stimulation (18, 19). Intermittent perfusion of the SA node increases enkephalin locally and is accompanied by an 1-mediated increase in vagal transmission (4, 10). Preliminary studies also suggest that this intermittent coronary occlusion was also associated with a decrease in the opposing 2-mediated vagolytic activity. Thus cross talk between -receptor phenotypes may be common to both sensory and motor systems.
The existence of a single -receptor transcript and two functional opposing responses suggested that interconversion between the two phenotypes might be physiologically important. An interconversion during preconditioning might shift the balance of responses in favor of a cardioprotective 1-subtype. Preliminary observations indicated a reduction in the intensity of 2-mediated vagolytic responses after the exposure of the SA node to extended 1-receptor stimulation. The following studies were designed to test whether extended exposure to the cardioprotective 1-opioid TAN-67 would reduce the intensity of subsequent 2-mediated vagolytic responses. The protocol also tested whether the declining 2 response resulted from disappearance of the response or from masking of the response by an increasing 1 response. In summary, experimental protocols were conducted to test the hypothesis that extended 1-opioid receptor stimulation downregulates 2-receptor responses.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Surgical preparation. Thirty mongrel dogs of either gender weighing 15–25 kg were assigned at random to various experimental protocols. The animals were anesthetized with pentobarbital sodium (32.5 mg/kg), intubated, and mechanically ventilated initially at 225 ml·min–1·kg–1 with room air. Fluid-filled catheters were inserted in the right femoral artery and vein and advanced into the descending aorta and inferior vena cava, respectively. The arterial line was attached to a Statham PD23 XL pressure transducer to monitor heart rate and arterial pressure during the remainder of the surgical preparation. The venous line was used to administer supplemental anesthetic, as required. The acid-base balance and the blood gases was determined regularly with an Instrumental Laboratories Blood Gas Analyzer. The PO2 (90–120 mmHg), pH (7.35–7.45), and PCO2 (30–40 mmHg) were adjusted to normal by administering supplemental oxygen or bicarbonate or by modifying the minute volume.
The right and left cervical vagus nerves were isolated through a ventral midline surgical incision. The nerves were double ligated with umbilical tape to prevent afferent nerve traffic during electrical stimulation. The isolated nerves were then returned to the prevertebral compartment for later retrieval. Surgical anesthesia was carefully monitored, and a single dose of succinylcholine (1 mg/kg) was administered intravenously to temporarily reduce involuntary movements during the 10–15 min required for electrosurgical incision of the chest. The costosternal cartilage for ribs two to five was severed to permit access to the thoracic cavity, and the heart was exposed from the right aspect. The pericardium was opened, and the dorsal pericardial margins were sutured to the body wall to support the heart. The left femoral artery was isolated, and a high-fidelity-catheter pressure transducer (Millar) was inserted and advanced in the abdominal aorta to measure heart rate and blood pressure continuously on-line thereafter (PowerLab).
Nodal microdialysis.
The SA node is visualized at the junction of superior vena cava and the right atrium. A 25-gauge stainless steel needle containing a microdialysis line was inserted in the center of the SA node along its long axis (5, 11). The needle was removed, and the probe was then positioned so that the dialysis window was completely within the substance of the SA node. The microdialysis probe was constructed of a single 1-cm length of dialysis fiber from a Clirans TAF08 (Asahi Medical) artificial kidney (200 µm ID, 220 µm OD) and a hollow silica glass fiber inflow and outflow. The dialysis tubing permits molecules with a molecular mass of 
35,000 kDa to cross from the lumen in the nodal interstitium. This technique allows the precise introduction of agents directly in the nodal interstitium for extended periods without provoking complicating systemic reflexes. After placement of the probe in the SA node, the preparation was allowed to equilibrate for 1 h while perfused with saline at 5 µl/min.
Materials.
Deltorphin II was obtained from American Peptide (Sunnyvale, CA). 7-Benzylidenaltrexone (BNTX) and 2-methyl-4a
-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a-octahydro-quinolino[2,3,3-
]isoquinoline (TAN-67) were obtained from Tocris Cookson (Ellisville, MO). The doses of TAN-67 and deltorphin administered in the subsequent protocols were applied at dose rates approximating the ED100 based on prior dose responses (6, 12).
Statistical methods. All data were expressed as means ± SEs. Differences were evaluated by ANOVA with repeated measures where appropriate, and the post hoc analysis was performed with Tukey's test for multiple comparisons and Dunnett's test for comparison between control and treatments. Differences determined to occur by chance with P < 0.05 were deemed statistically significant.
Protocol 1: The 1-agonist TAN-67 reduces subsequent deltorphin II-mediated 2 vagolytic response.
After equilibration for 1 h, the right cervical vagus nerve was stimulated at a supramaximal voltage (15 volts) for 15 s at low (1–2 Hz) and high (3–4 Hz) frequencies selected to produce, respectively, 10–20 and 30–40 beats/min decreases in heart rate. Two minutes were allowed for recovery between the two sequential stimulation frequencies. After recovery from the control stimulations, deltorphin II (1.67 x 10–9 mol/min), a selective 2-receptor agonist, was added to the dialysis inflow. The vagal stimulations (low and high) were repeated after 5 min of deltorphin II to quantify the initial 2 vagolytic response before exposure to TAN-67. This test of efficacy was designated 2-5 to indicate the time in the protocol. After determining the 2 response, deltorphin was discontinued, and the system was washed out with saline (45–60 min) until the control vagal responses were restored. TAN-67 (1.67 x 10–9 mol/min), the selective 1-receptor agonist, was then introduced in the dialysis inflow. Vagal stimulations were repeated at 15-min intervals for 1 h. Deltorphin II (1.67 x 10–9 mol/min) was then reintroduced in the microdialysis infusion; 2 responses were retested and designated 2-155. The deltorphin was discontinued and washed out (45–60 min).
Protocol 2: 1 blockade with BNTX prevents the loss of 2 response.
The purpose of this study was to test whether the loss of 2 response was mediated by 1-receptor stimulation. After the initial 1 h of equilibration, vagal stimulations were performed at low and high frequencies to obtain control values as described above. Deltorphin II (1.67 x 10–9 mol/min) was added to the dialysis inflow. The two right vagal stimulations were repeated after 5 min of deltorphin II treatment. This protocol was similar to protocol 1 except that the BNTX was combined with TAN-67 in the dialysis inflow at an equimolar dose rate. Vagal stimulations were repeated at 15-min intervals for 1 h. Deltorphin II (1.67 x 10–9 mol/min) was then reintroduced in the perfusate, and the 2-receptor response was assessed 5 min later.
Protocol 3: Vehicle, duration, and repeated deltorphin II (controls).
The purpose of this study is to test whether the duration of the protocol, the repeated vagal stimulation, and/or the repeated exposure to deltorphin influenced the subsequent deltorphin-mediated 2 vagolytic responses. This protocol was identical to the first 150 min in protocol 1 except vehicle (saline) was substituted for TAN-67 during the treatment period. After 2-155, deltorphin was discontinued but was introduced again 25 min later for another vagal test designated 2-180 to evaluate the progression of changes in the 2 response.
Protocol 4: Influence of 1 blockade on 2 response.
The purpose of this study was to test whether apparent contributions of duration of protocol and/or deltorphin to the erosion of the 2 response observed in protocol 3 depended on 1-receptor activity. This protocol was identical to the control in protocol 3 except that the 1-antagonist BNTX was added to the dialysis inflow for 60 min instead of saline. BNTX was then continued throughout the following 2 h, and 2 challenges were conducted at 2-155 and 2-180.
Protocol 5: Vehicle, duration, and naive deltorphin II (controls).
The purpose of this study was to remove the influence of prior deltorphin exposure on the 2 response and to evaluate the influence of the protocol alone. This protocol is similar to protocol 3 except initial exposure to deltorphin (2-5) was omitted. Vehicle was perfused for 2.5 h with vagal stimulations every 15 min during the 2nd h as in protocol 3. Deltorphin was evaluated at 2-155 and 2-180. After the deltorphin wash out after 2-180, BNTX was introduced (1.67 nmol/min) for 5 min, and the right vagus nerve was stimulated to evaluate the effects of 1-receptor blockade with BNTX alone. BNTX and deltorphin were then introduced together (1:1) for 5 min, and the two-step vagal stimulation designated 2-250 was conducted to determine (by subtraction) the contribution of any opposing 1-mediated (vagotonic) response to any decline in the 2 response. The treatments were then discontinued, the area was washed and the vagal responses were tested periodically during the washout.
Protocol 6: TAN-67 and naive deltorphin.
The purpose of this study was to evaluate the efficacy of TAN-67 alone. The protocol was designed to test, by omission of deltorphin at 2-5, the influence of TAN-67 alone and whether the decline in the 2 vagolytic response depended on an interaction between the initial exposure to deltorphin (2-5) and the subsequent addition of TAN-67. The initial two-step vagal stimulation was conducted followed by 2 h of TAN-67 perfusion at the rate of 1.67 x 10–9 mol/min. The 1st h was conducted to simulate the 2-5 exposure to deltorphin and its washout. In the 2nd h, the right vagus nerve was tested at 15-min intervals as described in the other protocols to evaluate progressive effects of TAN-67. TAN-67 was discontinued and followed by deltorphin challenges applied in a sequence equivalent to 2-155, 2-180, and at 2-250 with and without BNTX as described in protocol 3.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-agonist pretreatment (TAN-67) reduces subsequent 2 vagolytic responses (deltorphin II).
As shown in Fig. 1, deltorphin produced a significant vagolytic response when first introduced in the nodal interstitium (2-5). The degree of inhibition was similar at both low (76%) and high (80%) frequencies. Vagal effects similar to control were reestablished after 45–60 min of washout. The 60 min, 1-treatment with TAN-67 gradually produced significant though modest increases in the negative chronotropic effect of vagal stimulation during the 60-min treatment period. The control responses were reestablished after 20 min of washout with vehicle. The subsequent 2 vagolytic evaluation (2-155) with deltorphin was dramatically attenuated in magnitude compared with the earlier 2-evaluation (2-5) conducted before the TAN-67 pretreatment. Although a clear vagolytic response remains apparent, the vagolytic response was no longer statistically different from control. The degree of inhibition was reduced from >75% before TAN-67 to <25% after TAN-67 regardless of the stimulation frequency tested. A matched washout of the deltorphin was again followed by restoration of the vagal effects to values not different from control. These results led to the suggestion that 1-stimulation reduced the subsequent 2-mediated vagolytic response.
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1 blockade prevents the TAN-67-mediated erosion of subsequent 2 vagolytic responses.
Figure 2 shows that deltorphin produced a significant vagolytic response when first introduced in the nodal interstitium (2-5). The degree of inhibition, although less robust than in protocol 1, was similar at both low (62%) and high (57%) frequencies. Vagal effects similar to control were reestablished after 45–60 min of washout. The vagotonic effect of TAN-67 observed in protocol 1 was eliminated when TAN-67 was combined with the 1-antagonist BNTX. After 20 min of washout with vehicle, the response was likewise not different from control. Unlike the previous treatment without BNTX (Fig. 1), the second 2 evaluation at 2-155 remained significantly different from control and was not different from that observed at the beginning (2-5) of the protocol. Washout of the deltorphin was again followed by restoration of vagal transmission to that observed at the beginning of the experiment. These results further supported the thesis that the erosion of the 2-mediated vagolytic effect observed after TAN-67 in protocol 1 was mediated by activation of 1 receptors.
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2 response observed in protocol 1 occurs in the absence of added TAN-67. Thus vehicle was substituted for TAN-67 during the treatment period. The initial vagolytic effect of deltorphin was similar to the initial response in the first protocol, with percent inhibitions of 83 and 55%, respectively, at the lower and higher frequency of stimulation. Surprisingly, after a vehicle-only infusion for a time interval matching the TAN-67 treatment period in protocol 1, there was similar though less complete loss in the 2-mediated vagolytic effect of deltorphin. The resulting inhibition was still different from control, but the degree of inhibition was now reduced to 64 and 36%. The intermediate response prompted us to conduct an additional 2 evaluation. Deltorphin was reintroduced 25 min later, and the vagus was retested. In this case, the vagolytic responses had eroded further to 42 and 22% inhibition and were no longer different from control. Thus the protocol itself and/or the prior exposure to deltorphin appeared to have contributed to the loss of the 2 vagolytic response.
Protocol 4: Influence of 1 antagonist on the untreated 2 response.
The purpose of this study was to test whether 1 blockade prevents loss of the 2 response observed in the time control protocol. In this study, BNTX was introduced in the SA node by microdialysis. After 5 min of exposure to BNTX, the vagal stimulations were repeated, and there was no significant difference between this response and the control response. BNTX was then combined with deltorphin for 5 min, and the vagus was retested. A typical deltorphin-mediated vagolytic response was observed (Fig. 3). Deltorphin was discontinued, and protocol 3 was then repeated with BNTX added throughout. In this case, the two later 2 evaluations (2-155 and 2-180) were convincingly very similar to the initial evaluation (2-5). There was no loss apparent in the two subsequent vagolytic responses, and all three were significantly different from control. These data led to the suggestion that the loss of the 2 response in the time control was also mediated by activation of 1 receptors. Thus either deltorphin has intrinsic 1 activity or it provoked the release of or facilitated the activity of an endogenous 1 agonist.
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2 response observed in protocol 3. In this protocol, the initial deltorphin exposure was omitted, but the remainder of the 3-h protocol through 2-180 was replicated. The two 2-receptor evaluations at 2-155 and 2-180 were both significantly different from control. In fact, the initial 2 challenge at 155 min was equal or better than that observed in any of the prior experiments at 5 or at 155 min, suggesting the vagolytic response may have even gained strength during the protocol. When deltorphin was retested 25 min later, a degree of attrition was clearly evident, but the percent inhibition was still significantly different from control, suggesting that the exposure to deltorphin 30 min earlier had already initiated the erosion of the response. At this point, deltorphin was washed, out and the vagal responses were tested before and after BNTX to evaluate the effect of 1 blockade alone. In each case, the resultant decline in heart rate was not different from that observed at the start of the experiment. When BNTX was combined with deltorphin for a third 2 evaluation, the response was eroded slightly further but was still significantly different from control. These data supported the suggestion that, when untreated, the 2 response remains strong during the protocol and that prior deltorphin must have contributed to the loss of the 2 response observed in protocols 1 and 3.
Protocol 6: TAN-67 and naive deltorphin.
The purpose of this protocol was to evaluate the contribution of TAN-67 to the loss of the 2 response in the absence of a prior exposure to deltorphin. Perfusion (60 min) with TAN-67 was substituted for the initial exposure to deltorphin and subsequent wash. TAN-67 was then continued for a 2nd h, and vagal function was tested at 15-min intervals for 1 h. The vagal responses during TAN-67 treatment were significantly elevated when compared with control. Once again, the increase was modest. TAN-67 was discontinued and washed out. When deltorphin was introduced for the first time, intermediate vagolytic responses were recorded. This response was less than those observed at 2-5 in protocol 1 and clearly weaker that those parallel responses observed at 2-155 in protocol 5 without TAN-67. The subsequent 2-challenge conducted 25 min later resulted in a further erosion of the response, which was now closer yet to the control. As in protocol 5, BNTX added afterward had no apparent effect on the control or vagolytic responses. The failure of BNTX to restore the vagolytic response indicates the erosion of the 2 response is not the result of a reversible arithmetic increase in the 1 vagotonic response. Thus TAN-67 was able to reduce the 2-mediated vagolytic effect in the absence of prior exposure to deltorphin.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-opioid receptor stimulation reduces 2-opioid receptor responses in the SA node. This hypothesis was formulated from preliminary observations in the SA node that dose responses to 1 agonists were followed by a dramatic reduction in the subsequent 2-mediated vagolytic responses. In the current study, prior exposure to the 1-agonist TAN-67 eroded the vagolytic effect of the 2-agonist deltorphin II. The heterologous participation by 1 receptors was verified by demonstrating that pretreatment with the selective 1-antagonist BNTX effectively prevented the loss of the 2 response. Unexpectedly, a qualitatively similar erosion in the vagolytic effect of deltorphin was observed in time controls in which TAN-67 was omitted, suggesting a complex interaction between the 2-agonist deltorphin, the evaluation protocol, and the 2 receptor.
These initial results posed a potential conflict in interpreting the results. The attrition in the response may have been the result of the duration of the protocol itself or the prior exposure to the 2-agonist deltorphin. The smaller, though qualitatively similar, progressive reduction in the 2 response observed when TAN-67 was omitted suggested that 2 stimulation was capable of eroding its own response in the absence of 1-receptor stimulation. Downregulation of opiate receptors mediated by opioid peptides is a well-recognized phenomenon. However, the prior studies in the SA node with MEAP had provided little evidence for tachyphylaxis of the vagolytic responses (7). The earlier observation might be attributed in part to reported differences in the selectivity of deltorphin and MEAP (4, 12). MEAP is a mixed 1/2 agonist, and deltorphin is considered a more selective 2 agonist. However, the homologous quality of the desensitization was placed in doubt when the apparent effect of deltorphin alone was also blocked by the 1-antagonist BNTX, indicating that the loss of the vagolytic response in the absence of TAN-67 was also mediated by 1-receptor stimulation. At that point, the collected observations suggested that deltorphin was functionally active at the 1 receptor or that the protocol itself facilitated endogenous 1 activity.
The subsequent protocols that omitted the initial exposure to deltorphin provided clear evidence of deltorphin's involvement in the erosion of the vagolytic response. In the absence of the initial evaluation with deltorphin at 5 min, the subsequent vagolytic response at 155 min was as strong or stronger than those observed at 5 min or at 155 min in any of the previous four protocols. This finding revealed that, in the absence of TAN-67 and/or prior deltorphin, the intensity of the 2-mediated vagolytic response was maintained throughout the extended perfusion of the SA node with vehicle. The strong vagolytic response in the absence of prior deltorphin suggested that prior exposure to deltorphin had contributed to the subsequent erosion of the vagolytic response. Thus deltorphin was a contributory factor and most likely exerted its influence through interaction with 1 receptors. The vagotonic 1-receptor-mediated responses in this system are significantly more sensitive (fM vs. pM) than their 2-mediated vagolytic counterparts (4, 5). Thus, despite the preference of deltorphin for 2 receptors, a small degree of cross-reactivity with the 1 receptor might have been sufficient to initiate the heterologous response.
The combination of deltorphin and TAN-67 produced the greatest decline in the subsequent vagolytic responses. Thus the final protocol was conducted without the initial exposure to deltorphin at 5 min to determine whether TAN-67 was effective without prior deltorphin. After 2 h of exposure to the 1 agonist, the degree of inhibition during the initial 2 evaluation at 155 min was significantly different from control but was also significantly reduced (ANOVA: P < 0.02) compared with the vagolytic response observed after 2.5 h of vehicle (protocol 5). This result indicated that, in the absence of treatment, the potency of the 2 response remains strong as the protocol proceeds. Thus TAN-67 alone produced a qualitatively similar although intermediate response compared with the result observed at 2-155 after the single exposure to deltorphin at 5 min. In both protocols 5 and 6, a subsequent 2 evaluation 30 min later at 2-180 resulted in vagolytic responses slightly weaker than the preceding response at 2-155. Thus, at the dose rates employed, the combined effects of deltorphin and TAN-67 were greater than with either agent applied alone, suggesting that the integrated time/dose effect of each was submaximal. Because both the combination and deltorphin alone were each blocked by the 1 antagonist, it seems likely that both deltorphin and TAN-67 activated the same 1 pathway. Whether deltorphin has intrinsic 1 activity or provokes the release of an endogenous 1 agonist remains to be determined.
The paired 2 evaluations at 155 and 180 min provide additional support for the 1 character of the heterologous response. In the three protocols (Figs. 4
–6) without prior 1 blockade, the latter two evaluations consistently demonstrate a reduced intensity in the second of the two vagolytic responses. In protocol 4 (Fig. 3), when the same evaluations were preceded by BNTX throughout, the second vagal response at 180 min was nearly identical to its predecessor. Thus the ostensibly homologous 2 effect appears instead to be heterologously mediated by the opposing receptor subtype. However, because there is no molecular evidence for -receptor subtypes, receptor phenotypes might be more appropriate terminology.
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1 blockade with BNTX prevented the erosion of the vagolytic response, the same agent was unable to acutely restore the lost vagolytic response when added afterward. Explanations for the diminished vagolytic response had included the potential emergence or strengthening of a competing vagotonic response mediated by 1 receptors. The net effect of increased opposition would have then masked the vagolytic response, which would have appeared as a net loss in the 2 response. In that case, the addition of BNTX should have immediately restored the original vagolytic response by withdrawing the competing vagotonic contribution to expose the underlying 2 response. TAN-67 reduced the 2-mediated vagolytic effect, and the lost vagolytic response was not restored by the subsequent blockade of the nodal 1 receptors. This result indicated that the reduced vagolytic response was not the result of an increased vagotonic response. Thus, by inference, the results favor some variant of heterologous uncoupling, desensitization, or downregulation.
In conclusion, the relatively selective 2-agonist deltorphin was used to evaluate the erosion of vagolytic responses after 1-receptor treatment with TAN-67. The erosion of the vagolytic response was greatest in protocol 1 in which the SA node was exposed to both TAN-67 and deltorphin early in the experiment. Both of these opioids contributed to the desensitization; both were less effective when added alone. Together they were blocked by the 1-antagonist BNTX. The common role of the 1 receptor and the apparent additive character of the responses suggested that TAN-67 and deltorphin mediated the desensitization through a common mechanism. Whether deltorphin has intrinsic 1 activity or provoked the release of an endogenous 1 agonist remains to be determined. The failure of BNTX to restore the vagolytic response afterward suggested that the lost vagolytic response was not the result of emerging competition from an opposing 1-mediated vagotonic response. Finally, in the absence of any prior intervention, the 2-mediated vagolytic response was very strong through the duration of the protocol. This suggested a dynamic shift of the -receptor coupling in favor of 2 respones during the course of the experiment. In this model system, repeated occlusion of the SA node artery in a preconditioning-like protocol raised enkephalins locally and improved vagal transmission (1). The improvement in vagal function in the ischemic region might thus preserve the ischemic myocardium by reducing work and oxygen demand locally. Because 1 receptors and improved vagal transmission have been associated with the cardioprotective mechanisms (18–20), a shift toward 2 mechanisms may represent an undesirable outcome. In contrast, the 1-mediated reduction in 2 responses may reinforce further the cardioprotective efficacy of 1 agonists like TAN-67. Finally, the recovery of the 2 response after downregulation may reflect a mechanism by which these opioid receptors contribute to the gradual closure of the initial window of cardioprotection that follows ischemic preconditioning. Thus time, TAN-67, and deltorphin II all interact to modify the subsequent 2-mediated vagolytic responses. The specific mechanism by which the 1- and 2-coupled opioid receptors interact with each other still remains to be determined.
The capability of endogenous opioids to modify cardiac responses to vagal stimulation is clear; however, the role of receptors in the normal physiology of the heart is less clear. There is a significant degree of plasticity in vagal function that may be attributed in some part to the observed plasticity inherent in the expression of opposing -receptor phenotypes. For instance, some of the increase in vagal control associated with exercise training may result from a shift in -receptor balance in favor of vagotonic 1 receptors. Likewise, an intense 2-mediated vagolytic response might contribute to the arrhythmic susceptibility of the heart after myocardial infarction. In this regard, it is interesting that morphine is intensely vagotonic and has long been employed therapeutically in acute coronary care. Thus the physiological consequences of shifts in the -receptor balance are very much in need of careful investigation.
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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.
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1-and 2-opioid receptor subtypes in the human neuroblastoma cell line SK-N-BE: no evidence for distinct molecular entities. Biochem Pharmacol 59: 915–925, 2000.[CrossRef][ISI][Medline]-opioid receptors regulate vagal bradycardia in canine sinoatrial node. Am J Physiol Heart Circ Physiol 285: H1332–H1339, 2003.2-opioid receptors in sinoatrial node. Am J Physiol Heart Circ Physiol 284: H1693–H1701, 2003.1-receptor antagonist: 7-benzylidenaltrexone. Eur J Pharmacol 218: 195–196, 1992.[CrossRef][ISI][Medline]1- but not µ- or 
-opioid receptors. Circulation 97: 1282–1289, 1998.1-opioid receptor agonist, reduces infarct size via activation of Gi/o proteins and KATP channels. Am J Physiol Heart Circ Physiol 274: H909–H914, 1998.-opioid agonists by naltrindole, and its benzofuran analog (N.TB) in mice: evidence for -opioid receptor subtypes. J Pharmacol Exp Ther 257: 676–680, 1991.-receptor as a model. Ann Rev Pharmacol Toxicol 36: 379–401, 1996.[CrossRef][ISI][Medline]This article has been cited by other articles:
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  S. H. Deo, M. A. Barlow, L. Gonzalez, D. Yoshishige, and J. L. Caffrey Repeated Arterial Occlusion, Delta-Opioid Receptor (DOR) Plasticity and Vagal Transmission Within the Sinoatrial Node of the Anesthetized Dog Experimental Biology and Medicine, January 1, 2009; 234(1): 84 - 94. [Abstract] [Full Text] [PDF]
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 S. Davis, S. H. Deo, M. Barlow, D. Yoshishige, M. Farias, and J. L. Caffrey The monosialosyl ganglioside GM-1 reduces the vagolytic efficacy of {delta}2-opioid receptor stimulation Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2318 - H2326. [Abstract] [Full Text] [PDF]
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