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-opioid receptors regulate vagal bradycardia in canine sinoatrial node
Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 76107
Submitted 28 April 2003 ; accepted in final form 16 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-selective antagonist, naltrindole. We tested the hypothesis that vagal
responses to intranodal enkephalin are bimodal and that the polarity of the
response is both dose- and opioid receptor subtype dependent. Ultralow doses
of MEAP were introduced into the canine SA node by microdialysis. Heart rate
frequency responses were constructed by stimulating the right vagus nerve at
1, 2, and 3 Hz. Ultralow MEAP infusions produced a 50–100% increase in
bradycardia during vagal stimulation. Maximal improvement was observed at a
dose rate of 500 fmol/min with an ED50 near 50 fmol/min. Vagal
improvement was returned to control when MEAP was combined with the
-antagonist naltrindole. The dose of naltrindole (500 fmol/min) was
previously determined as ineffective vs. the vagolytic effect of higher dose
MEAP. When MEAP was later reintroduced in the same animals at nanomoles per
minute, a clear vagolytic response was observed. The
1-selective antagonist 7-benzylidenenaltrexone (BNTX)
reversed the vagal improvement with an ED50 near 1 x
10–21 mol/min, whereas the
2-antagonist naltriben had no effect through
10–9 mol/min. Finally, the improved vagal
bradycardia previously associated with nodal artery occlusion and endogenous
MEAP was blocked by the selective 1-antagonist BNTX. These
data support the hypothesis that opioid effects within the SA node are bimodal
in character, that low doses are vagotonic, acting on
1-receptors, and that higher doses are vagolytic, acting on
2-receptors.
heart rate; enkephalins; opiate receptors; microdialysis; parasympathetic nervous system
Low concentrations of enkephalins were identified in myocardial extracts soon after they were first isolated from brain homogenates (14). The mRNA for preproenkephalin was subsequently determined to be highly abundant in rat heart and comparable to that found in parts of the brain rich in opioids (13). The preproenkephalin mRNA was localized in isolated cardiomyocytes, which were also found to be able to translate the message into peptide and secrete the products into the surrounding culture medium (18, 30). The evaluation of cardiac opioid content suggested that there was substantially more opioid activity present in heart than accounted for by the primary proenkephalin product, methionine-enkephalin (2, 4, 7, 30). This discrepancy was explained in part when chromatographic analysis identified significantly higher concentrations of preproenkephalin and its partially processed intermediates in heart extracts (2, 19, 21, 34). In particular, unexpectedly high concentrations of the heptapeptide methionine-enkephalin-arginine-phenylalanine (MEAP) were identified.
The systemic infusion of MEAP interrupted the vagal control of heart rate, coronary blood flow, and contractile activity (6, 8). Opiate receptor participation was confirmed when each effect was prevented by pretreatment with the high-affinity nonselective opiate antagonist diprenorphine. The responsible opiate receptor appeared to be located prejunctionally because MEAP was ineffective when bradycardia was produced by the direct-acting muscarinic agonist methacholine. This observation was consistent with earlier reports that endogenous and exogenous opioids opposed the vagal control of heart rate, presumably through the inhibition of neurotransmitter release (23, 32). When MEAP was delivered directly into the interstitium of the SA node by microdialysis, the vagolytic effect was identical to that observed after systemic infusion (11), indicating that the opiate receptors were located within the SA node.
Careful analysis of the vagolytic effect indicated that the interruption of
vagal function within the SA node was mediated by -opiate receptors
(15). The response was
duplicated by the -agonist deltorphin and abrogated by the general
-antagonist naltrindole. The intranodal delivery of µ- and
-agonists and antagonists all failed to alter vagal function.
Surprisingly, when the endogenous nodal concentration of MEAP was elevated
after a "preconditioning-like" protocol and subsequent occlusion
of the SA node artery, vagal function was consistently improved and the
improvement was reversed by the same -antagonist, naltrindole. These
opposing observations suggested several potential explanations. First,
vagotonic responses at low doses may indicate that vagotonic responses are
more sensitive than companion vagolytic responses. Second, distinct
-receptor subtypes may mediate the two responses, with one of the
receptors (the vagotonic receptor) inaccessible by microdialysis. The third
explanation would include a combination of both, in which one of the two
responsible receptor subtypes was significantly more sensitive to the
peptide.
Recent studies identified the vagolytic response as 2 in
character. These studies unexpectedly provided support for distinct receptor
subtypes mediating the opposing vagolytic and vagotonic responses. The
vagolytic effect of MEAP was blocked by a 2-antagonist and
was unaltered by a 1-antagonist
(12). Although ineffective
vagolytically in that study, the 1-agonist
2-methyl-4aa-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a
-octahydroquinolino[2,3,3-g]isoquinoline
(TAN-67) produced a vagotonic response very similar to that observed
previously during nodal artery occlusion. The 1 character of
the vagotonic response to TAN-67 was further verified by its blockade with the
1-antagonist 7-benzylidenenaltrexone (BNTX). The assignment
of the 1-receptors as vagotonic might explain in part their
attributed role in preconditioning
(9,
22,
26–28).
Improved vagal function during ischemia would presumably be cardioprotective
by locally reducing myocardial work and oxygen demand in the area at risk
(1,
33). These observations led to
the proposal that ultralow doses of MEAP released during coronary arterial
occlusion improve vagal function by interacting with
1-opioid receptors.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The right and left cervical vagus nerves were isolated through a ventral midline incision. The nerves were double ligated with umbilical tape to abort afferent nerve traffic and were 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 muscle movements during the 10–15 min required for electrosurgical incision of the chest and removal of ribs 2–5. 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 and stabilize the heart.
A 25-gauge stainless steel spinal needle containing the microdialysis line was inserted into the long axis of the SA node at the junction of the superior vena cava and the right atrium. The needle was removed, and the probe was positioned with the dialysis window completely within the SA node (11). The microdialysis probes were fabricated from 1-cm lengths of dialysis fiber from a Clirans TAF 08 (Asahi Medical) artificial kidney. The dialysis tubing restricted the passage of solutes with molecular weights >36,000 and had a 200-µm inner diameter and a 220-µm outer diameter (OD). The inflow and outflow lines were constructed of hollow 170-µm-OD silica tubing (SGE, Austin, TX) sealed into the dialysis fiber with cyanoacrylate glue.
The dialysis line was perfused with saline (5 µl/min) for 1 h to permit the tissue to equilibrate before the experimental protocol began. Previous reports indicated that metabolites recovered from the interstitium had returned to a low basal steady state during the first hour after insertion of the probe (31). At the end of each experiment, norepinephrine (1 nmol/µl in saline and 0.1% sodium ascorbate) was introduced into the microdialysis line to confirm the position of the probe. When the probe was properly positioned in the SA node, the heart rate increased by 30–40 beats/min within 30 s. This brisk tachycardia was not observed when the probe was positioned in the atrial wall as little as 1–2 mm lateral to the node.
Protocol I: Is ultralow-dose MEAP vagotonic? After the 1-h
equilibration, the right vagus nerve was stimulated at a supramaximal voltage
(e.g., 15 V) at 1, 2, and 3 Hz for 15 s each. The new lower heart rate was
recorded when it reached a new steady state during the 15-s stimulus. Heart
rate/frequency-response curves were constructed. The heart rate was allowed
105 s for complete recovery after each stimulation frequency was completed.
Increasing doses of MEAP were then added to the perfusate in 10-fold
increments beginning with 5 fmol/min until a maximal vagotonic response was
observed. Each new dose was infused for 5 min at 5 µl/min, and the vagal
response was reevaluated. The dose was then washed out and perfused with
saline until the baseline vagal response was reconfirmed. Once the dose at
which a maximal vagal improvement was determined, that dose of MEAP was
combined with the nonselective -antagonist naltrindole (500 fmol/min)
for 5 min. The dose of naltrindole was previously determined as ineffective
versus the vagolytic effect of MEAP
(15). After 5-min exposure to
the combined agonist and antagonist, the vagus was again evaluated. The
combination was washed out, and the baseline vagal function was reestablished.
Next, a higher vagolytic dose of MEAP (5 nmol/min) was introduced into the
line to demonstrate a viable vagolytic response in the same animal. After
5-min exposure, the vagal function was reevaluated. In a second group of
animals the entire MEAP low-dose dose-response curve was constructed from
vagotonic to vagolytic (5 x 10–15 to 5
x 10–9 mol/min) in the same animals.
Protocol II: Are 1-receptors
responsible for the vagotonic effect of low-dose MEAP? In this protocol,
a low vagotonic dose of MEAP (500 fmol/min) was introduced into the nodal
perfusate. After 5 min, the vagal function was evaluated and the improvement
was recorded. After washout and reequilibration, increasing doses of the
1-specific antagonist BNTX were combined with MEAP in the
nodal perfusate for 5 min each. The initial dose of BNTX was 5 x
10–24 mol/min, and the subsequent doses were
increased in 1,000-fold increments. After blockade was obtained, the
combination was washed out and the effect of BNTX alone on vagal function was
evaluated.
Protocol III: Do 2-receptors
participate in the vagotonic effect of low-dose MEAP? In this protocol, a
low vagotonic dose of MEAP (500 fmol/min) was introduced into the nodal
perfusate. After 5 min, the vagal function was evaluated and the improvement
was recorded. After washout and reequilibration, increasing doses of the
2-specific antagonist naltriben were combined with MEAP in
the nodal perfusate for 5 min each. The initial dose of naltriben was 5
x 10–24 mol/min, and the subsequent doses
were increased in 1,000-fold increments through 5 x
10–9 mol/min. After the dose response was
completed, the combination was washed out and the effect of naltriben alone on
vagal function was evaluated.
Protocol IV: Do 1-receptors mediate
the vagotonic effects of nodal artery occlusion? A preconditioning-like
protocol similar to that used in earlier studies
(16) was used to raise
endogenous MEAP and to produce a vagotonic effect. Because this effect was
previously blocked with the nonselective -antagonist naltrindole, this
protocol was conducted to determine whether this vagotonic effect was more
specifically dependent on the 1-receptor subtype. A suture
was placed around the SA node artery to allow for a reversible arterial
occlusion. A 10-min initial dialysate collection was made. Leucine-arginine
(30 nmol/µl) was then added to the microdialysis perfusate and continued
thereafter as a competitive inhibitor to reduce the enzymatic breakdown of
MEAP (24) and to conform to
the prior experimental design
(16). After 5-min preinfusion,
a 10-min control collection was made and then the SA node artery was
temporarily occluded to produce four preconditioning-like 10-min periods of
reduced blood flow. Each occlusion was followed by a 10-min reperfusion. Two
30-min occlusions separated by one 30-min reperfusion period followed the
preconditioning-like sequences. After the preconditioning-like protocol was
completed, the right vagus nerve was stimulated for 15 s at 3 Hz. The vagus
nerve was then reevaluated at 15 and 20 min of each of the three subsequent
30-min periods. During the third period (second 30-min occlusion) BNTX was
added to the perfusate between the 15 min and 20 min stimulations.
Materials. MEAP was synthesized by American Peptide (Sunnyvale, CA). Naltrindole and naltriben were obtained from Sigma RBI (St. Louis, MO). BNTX was obtained from Tocris (Ellisville, MO).
Data Analyses. The data are expressed as means ± SE. Differences were evaluated by analysis of variance, and multiple post hoc comparisons were made with Tukey's test (GB-STAT; Dynamic Microsystems, Silver Spring, MD). Repeated-measures designs were used where appropriate, and P < 0.05 was accepted as a statistically significant difference.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Protocol I: Is ultralow-dose MEAP vagotonic? The results of these
experiments are illustrated in Figs.
1 and
2. In the first group of
experiments, the dose of MEAP was gradually increased until a consistent
improvement was observed. An improvement was observed at 50 fmol/min, and the
effect was always distinguishable at 500 fmol/min
(Fig. 1, bottom
curve). At that juncture, an equimolar dose of the nonselective
-antagonist naltrindole was combined with the MEAP in the nodal
perfusate. The antagonist reversed the vagotonic effect and restored the vagal
bradycardia to control. The agonist and antagonist were washed out, and a dose
of MEAP previously determined to be vagolytic (5 nmol/min) was introduced into
the node. As observed in the top curve of
Fig. 1, this higher dose of
MEAP nearly eliminated vagal control of heart rate. Of note, the dose of
naltindole used was previously demonstrated to be completely ineffective
versus the vagolytic effect of MEAP
(15). A full low-dose dose
response was conducted in the second group of animals and is illustrated in
Fig. 2. In this group of
animals, the effect of 5 fmol/min was inconsistent and not significantly
different from control. However, a statistically significant improvement in
vagal function was observed at 50 fmol/min, which reached an apparent maximum
at 500 fmol/min. Higher doses of MEAP gradually reduced vagal function back
toward the control response, and a vagolytic response was again clearly
evident when the dose was raised to 5 nmol/min. The higher doses of MEAP were
washed out, and the maximal vagotonic dose (500 fmol/min) was reintroduced to
demonstrate that the vagotonic response was reproducible and could be
restored. When combined with an equimolar dose of naltrindole, the vagotonic
effect at each frequency was returned to the original control response.
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Protocol II: Are 1-receptors
responsible for the vagotonic effect of low-dose MEAP? The results of
this experiment are presented in Fig.
3. The control vagal responses are illustrated in the top group of
curves. As in protocol I, the nodal introduction of MEAP at 500
fmol/min produced a significant increase in vagal bradycardia
(Fig. 3, bottom
curve). Once the response was established, the peptide was washed out and
the return to the control response was confirmed. Increasing doses of the
selective 1-antagonist BNTX were then combined with MEAP in
the nodal perfusate. As indicated, 5 x 10–24
mol/min (5 ymol/min) did not alter the vagotonic effect. However, 5 x
10–21 mol/min (5 zmol/min) completely restored
vagal function to control (Fig.
3, top curves). Raising the concentration of BNTX another
1,000-fold had no additional effect on the vagal response. In addition, the
later introduction of BNTX alone at 500 nmol/min had no effect on vagal
function independent of the ability of BNTX to oppose MEAP, indicating that
BNTX is not itself vagolytic.
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Protocol III: Do 2-receptors
participate in the vagotonic effect of low-dose MEAP? The summary of this
experiment is presented in Fig.
4. As in protocol II, the introduction of MEAP at 500
fmol/min improved vagal function by >50%
(Fig. 4, bottom
curve). Subsequent combinations with increasing doses of the
2-receptor antagonist naltriben (5 x
10–24 through 5 x
10–9 mol/min) had no effect on the existing
vagotonic response. After the agonist-antagonist combinations were washed out,
the addition of naltriben alone (5 x 10–9
mol/min) also had no effect on the control vagal function.
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Protocol IV: Do 1-receptors mediate
the vagotonic effects of nodal artery occlusion? In these experiments, a
series of nodal artery occlusions and reperfusions were conducted
(Fig. 5). This protocol was
previously determined to increase nodal MEAP and improve vagal function in a
-opioid receptor-dependent manner. When vagal function was evaluated
during the first 30-min occlusion, the vagal bradycardia at 3 Hz was improved
by about one-third. This vagal evaluation was repeated 5 min later to
demonstrate that repeated stimulation does not alter the response. During the
second stimulation, a very similar improvement was evident. The two
stimulations applied during the subsequent 30-min reperfusion were both
reduced and very similar to the postconditioning, preocclusion control and to
one another. When the artery was reoccluded, the vagal function once again
improved to a value comparable to those recorded during the first 30-min
occlusion. BNTX (5 x 10–18 mol/min) was then
added to the dialysis inflow after the vagal response was recorded at 15 min.
BNTX did not alter the resting heart rate. However, when the vagus was
retested 5 min later at 20 min of occlusion, the observed bradycardia was
reduced to a value below the original control, indicating that the improved
function was mediated by a 1-receptor.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-receptor antagonist,
naltindole (15,
16). This led to the
hypothesis that the two opposing responses were mediated by different
-receptor subtypes and that the subtype responsible for the vagotonic
response was far more sensitive to ultralow doses of the native peptide. The vagal response to MEAP was clearly bimodal in character. MEAP improved vagal function at doses well below those needed to interrupt vagal function. The dose responses may overlap one another, providing an explanation for a central dose range in which the opposing influences balance one another with no net effect. This would explain the failure to observe an enhanced vagal response at the beginning of earlier dose-response studies focused on vagolytic actions (15). In those prior studies vagolytic effects were largely absent between 5 and 50 pmol/min, which coincides well with the waning of the vagotonic effect observed here. The difference in the sensitivity of these two responses to reversal by naltrindole further confirmed their fundamental differences in sensitivity to enkephalin. Naltindole produced a complete reversal of the vagotonic effect at 500 fmol/min, whereas the threshold for reversal of the vagolytic effect was 100 pmol/min (15).
In addition to the difference in sensitivity, the opposing responses also
appear to be mediated by different receptor subtypes. The blockade of both
responses by naltrindole strongly suggests that both were mediated by
-receptors. A recent report presented evidence that the vagolytic
response was mediated by 2-receptors
(12). Those authors found that
the vagolytic response was blocked by the 2-antagonist
naltriben and unaltered by the 1-antagonist BNTX. The data
reported here complement those findings and illustrate that the vagotonic
response is mediated by 1-receptors. In this case, the
vagotonic response was blocked by BNTX and unaltered by naltriben. The
sensitivity to the blockade by BNTX was remarkable. The 1
character was suggested earlier when the 1-agonist TAN-67
produced an improved vagal response that was subsequently reversed by BNTX
(12). Therefore, these data do
collectively support the hypothesis that the vagotonic and vagolytic effects
of MEAP are mediated by 1- and 2-receptor
subtypes, respectively, and that the 1 response is far more
sensitive.
The last question addressed was whether the changing vagotonic responses
associated with the occlusion and reperfusion of the SA node artery were
likewise mediated by 1-receptors. Prior studies indicated
that the improvement associated with occlusion was accompanied by an increased
recovery of MEAP and that the improved response was reversed by naltrindole
(16). In this case, the
extraordinary selectivity of BNTX was used to probe this response. This too
appears to be mediated by 1-receptors, because the response
was completely reversed by a very low concentration of intranodal BNTX.
How are these opposing responses mediated? Bimodal actions of opioids,
although often unacknowledged, are not uncommon
(25). The receptors in
question may be located on different cellular targets. The vagolytic response
was presumed to be mediated through the prejunctional inhibition of
acetylcholine release. This assumption was based on the failure of MEAP to
alter the bradycardia following the addition of the direct-acting cholinergic
methacholine (8). Although the
2-receptor is probably located on the vagal nerve terminals,
MEAP may act indirectly on an intermediate cell or interneuron. The case for
the 1-receptor is less clear because the pre- or
postjunctional character of the response has not been addressed. However, no
improved vagal function was noted when MEAP and methacholine were combined
(8). The data thus far are,
however, consistent with the hypothesis suggested by Crain and Shen
(10) to explain the bimodal
effects of opioids on the action potential duration in cultured dorsal root
ganglion cells. They argued convincingly that subtypes of the opioid receptor
in the same cell were coupled to adenylate cyclase through opposing
stimulatory and inhibitory G proteins. They also reported that the excitatory
receptors, like the 1-receptors here, were far more
sensitive to agonist. Because alterations in the cAMP within vagal nerve
terminals can modify acetylcholine release, the bimodal effects of MEAP on
vagal function may represent a functional expression of Crain and Shen's
observations in cultured neurons.
The normal function of these opposing receptors remains to be determined.
The ubiquitous presence of enkephalin-degrading aminopeptidases in tissue and
blood suggests that the cardiac enkephalins function primarily as paracrine
hormones (5). The higher
sensitivity associated with the vagotonic effect might suggest that the
primary function of the cardiac enkephalins is one of facilitation. The
release of enkephalin during hypoxia may serve to stabilize the myocardium
during metabolic stress by increasing vagal function, reducing work, and
decreasing local oxygen demand
(1,
33). Consistent with this
suggestion, opioid receptors have been implicated in the cardioprotection
afforded by ischemic preconditioning
(20,
22,
29) and preconditioning
improves the efferent vagal component of the baroreflex
(1). Several investigators
demonstrated that opioids reduce the area of injury after coronary occlusion
in a manner similar to ischemic preconditioning
(9,
20,
22,
26–29).
In addition, opioid antagonists will prevent this effect and, furthermore,
opiate antagonists abort the beneficial effect of ischemic preconditioning
(9,
22,
26,
27). Particularly consonant
with the current findings, 1-receptors were specifically
implicated in opioid-mediated preconditioning based on positive
preconditioning-like results with TAN-67 and their reversal with BNTX
(26,
27).
On the other end of the spectrum, the vagolytic effect of MEAP is less easily rationalized. Impaired vagal function is often viewed as a liability (17), and strategies that improve vagal function have demonstrated therapeutic efficacy (3). However, too much of a good thing may also have undesirable consequences. The electrical stability of the intracardiac nervous system is the result of a delicate balance of multiple inputs, and an inappropriately intense vagal response under the wrong circumstances could be proarrhythmic. Similarly threatening to survival, intense unopposed vagal stimulation can trigger vasovagal syncope. Therefore, the vagolytic effect of MEAP may serve the organism as a governor of vagal activity at the other extreme.
In summary, the data presented here support the hypothesis that
ultralow-dose enkephalin improves vagal function by activation of
1-receptors within the interstitium of the SA node.
Furthermore, these same 1-receptors appear to be involved in
the vagotonic effect associated with nodal artery occlusion and
reperfusion.
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DISCLOSURES |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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H909–H914, 1998.1- but not µ- or -opioid receptors.
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2187–2195, 1997.[ISI][Medline]
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of vagal transmission in rabbit isolated heart. Naunyn
Schmiedebergs Arch Pharmacol 328:
186–190, 1984.[ISI][Medline]
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