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Ultra-low-dose methionine-enkephalin-arginine-phenylalanine improves vagal transmission (vagotonic) and decreases heart rate via [[delta].sub.1]-opioid receptors within the sinoatrial (SA) node. Higher doses activate [[delta].sub.2]-opioid receptors, interrapt vagal transmission (vagolytic), and reduce the bradycardia. Preconditioning-like occinsion of the nodal artery produced a vagotonic response that was reversed by the [[delta].sub.1]-antagonist 7-benzylidenaltrexone (BNTX). The following study tested the hypothesis that extended [[delta].sub.1]-opioid receptor stimulation reduces subsequent [[delta].sub.2]-receptor responses. The [[delta].sub.2]-agonist deltorphin II was introduced in the SA node by microdialysis to evaluate [[delta].sub.2] responses before and after infusion of the [[delta].sub.1]-agonist TAN-67. TAN-67 reduced the vagolytic effect of deltorphin by two-thirds. When the [[delta].sub.1]-antagonist BNTX was combined with TAN-67, the deltorphin response was preserved, suggesting that attrition of the prior response was mediated by [[delta].sub.1] activity. When TAN-67 was omitted in time control studies, some loss of [[delta].sub.2] responses was apparent in the absence of the 81 treatment. This loss was also eliminated by BNTX, suggesting that the attenuation of the response after deltorphin alone was also the result of [[delta].sub.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 [[delta].sub.2] response. These data support the conclusion that the loss of the [[delta].sub.2] response resulted from reduced [[delta].sub.2] activity mediated by continued [[delta].sub.1]-receptor stimulation and not the arithmetic consequence of increased competition from that same [[delta].sub.1] receptor. sinoatrial node

Methionine-enkephalin-arginine-phenylalanine (MEAP) introduced into the interstitium of the canine sinoatrial (SA) node by microdialysis interrupts vagal bradycardia. In contrast, raising endogenous MEAP by occluding the SA node artery improves vagal bradycardia. Both are blocked by the same [delta]-selective antagonist, naltrindole. We tested the hypothesis that vagal responses to intranodal enkephalin are bimodal and that the polarity of the response is both doseand 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 E[D.sub.50] near 50 fmol/min. Vagal improvement was returned to control when MEAP was combined with the [delta]-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 [[delta].sub.1]-selective antagonist 7-benzylidenenaltrexone (BNTX) reversed the vagal improvement with an E[D.sub.50] near 1 x [10.sup.-21] mol/min, whereas the [[delta].sub.2]-antagonist naltriben had no effect through [10.sup.-9] mol/min. Finally, the improved vagal bradycardia previously associated with nodal artery occlusion and endogenous MEAP was blocked by the selective [[delta].sub.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 [[delta].sub.1]-receptors, and that higher doses are vagolytic, acting on [[delta].sub.2]-receptors. heart rate; enkephalins; opiate receptors; microdialysis; parasympathetic nervous system

Ultra-low-dose methionine-enkephalin-arginine-phenylalanine improves vagal transmission (vagotonic) and decreases heart rate via δ1-opioid receptors within the sinoatrial (SA) node. Higher doses activate δ2-opioid receptors, interrupt vagal transmission (vagolytic), and reduce the bradycardia. Pre-conditioning-like occlusion of the nodal artery produced a vagotonic response that was reversed by the δ1-antagonist 7-benzylidenaltrexone (BNTX). The following study tested the hypothesis that extended δ1-opioid receptor stimulation reduces subsequent δ2-receptor responses. The δ2-agonist deltorphin II was introduced in the SA node by microdialysis to evaluate δ2 responses before and after infusion of the δ1-agonist TAN-67. TAN-67 reduced the vagolytic effect of deltorphin by two-thirds. When the δ1-antagonist BNTX was combined with TAN-67, the deltorphin response was preserved, suggesting that attrition of the prior response was mediated by δ1 activity. When TAN-67 was omitted in time control studies, some loss of δ2 responses was apparent in the absence of the δ1 treatment. This loss was also eliminated by BNTX, suggesting that the attenuation of the response after deltorphin alone was also the result of δ1 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 E2 response. These data support the conclusion that the loss of the δ2 response resulted from reduced δ2 activity mediated by continued δ1-receptor stimulation and not the arithmetic consequence of increased competition from that same receptor. [ABSTRACT FROM AUTHOR]

Methionine-enkephalin-arginine-phenylalanine (MEAP) introduced into the interstitium of the canine sinoatrial (SA) node by microdialysis interrupts vagal bradycardia. In contrast, raising endogenous MEAP by occluding the SA node artery improves vagal bradycardia. Both are blocked by the same δ-selective antagonist, naltrindole. We tested the hypothesis that vagal responses to intranodal enkephalin are bimodal and that the polarity of the response is both doseand 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 EDso near 50 fmol/min. Vagal improvement was returned to control when MEAP was combined with the δ-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 δ[sub 1]-selective antagonist 7-benzylidenenaltrexone (BNTX) reversed the vagal improvement with an EDso near 1 × 10[sup -21] mol/min, whereas the δ[sub 2]-antagonist naltriben had no effect through 10[sup -9] mol/min. Finally, the improved vagal bradycardia previously associated with nodal artery occlusion and endogenous MEAP was blocked by the selective δ[sub 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 δ[sub 1]-receptors, and that higher doses are vagolytic, acting on δ°2-receptors. [ABSTRACT FROM AUTHOR]

Background: Intermittent hypoxia training (IHT) produces robust myocardial protection against ischemia-reperfusion induced infarction and arrhythmias. Blockade of this cardioprotection by antagonism of either β1-adrenergic or δ-opioid receptors (δ-OR) suggests autonomic and/or opioidergic adaptations., Purpose: To test the hypothesis that IHT shifts cardiac autonomic balance toward greater cholinergic and opioidergic influence., Methods: Mongrel dogs completed 20d IHT, non-hypoxic sham training, or IHT with the δ-OR antagonist naltrindole (200μg/kgsc). The vagolytic effect of the δ-OR agonist met-enkephalin-arg-phe delivered by sinoatrial microdialysis was evaluated following IHT. Sinoatrial, atrial and left ventricular biopsies were analyzed for changes in δ-OR, the neurotrophic monosialoganglioside, GM-1, and cholinergic and adrenergic markers., Results: IHT enhanced vagal bradycardia vs. sham dogs (P<0.05), and blunted the δ2-OR mediated vagolytic effect of met-enkephalin-arg-phe. The GM-1 labeled fibers overlapped strongly with cholinergic markers, and IHT increased the intensity of both signals (P<0.05). IHT increased low and high intensity vesicular acetylcholine transporter labeling of sinoatrial nodal fibers (P<0.05) suggesting an increase in parasympathetic arborization. IHT reduced select δ-OR labeled fibers in both the atria and sinoatrial node (P<0.05) consistent with moderation of the vagolytic δ2-OR signaling described above. Furthermore, blockade of δ-OR signaling with naltrindole during IHT increased the protein content of δ-OR (atria and ventricle) and vesicular acetylcholine transporter (atria) vs. sham and untreated IHT groups. IHT also reduced the sympathetic marker, tyrosine hydroxylase in ventricle (P<0.05)., Summary: IHT shifts cardiac autonomic balance in favor of parasympathetic control via adaptations in opioidergic, ganglioside, and adrenergic systems., Competing Interests: 5.0 Conflicts of Interest and Ethical Standards No conflicts of interest, financial or otherwise, are declared by the authors. Experimentation on animals was conducted in accordance with the Guide to the Care and Use of Laboratory Animals (U.S. National Research Council Publication 85–23, revised 2011). Approval for testing on animals was obtained through the Institutional Animal Care and Use Committee of the University of North Texas Health Science Center at Fort Worth. This work was supported by research grants AT-003598 from the U.S. National Institutes of Health, and 03-04-49065 from the University of North Texas Health Science Center (UNTHSC). JAE was supported by a predoctoral fellowship from UNTHSC Graduate School of Biomedical Sciences’ Minority Opportunities in Research and Education program., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Brief interruptions in coronary blood flow precondition the heart, engage delta-opioid receptor (DOR) mechanisms and reduce the damage that typically accompanies subsequent longer coronary occlusions. Repeated short occlusions of the sinoatrial (SA) node artery progressively raised nodal methionine-enkephalin-arginine-phenylalanine (MEAP) and improved vagal transmission during subsequent long occlusions in anesthetized dogs. The DOR type-1 (DOR-1) antagonist, BNTX reversed the vagotonic effect. Higher doses of enkephalin interrupted vagal transmission through a DOR-2 mechanism. The current study tested whether the preconditioning (PC) protocol, the later occlusion or a combination of both was required for the vagotonic effect. The study also tested whether evolving vagotonic effects included withdrawal of competing DOR-2 vagolytic influences. Vagal transmission progressively improved during successive SA nodal artery occlusions. The vagotonic effect was absent in sham animals and after DOR-1 blockade. After completing the PC protocol, exogenously applied vagolytic doses of MEAP reduced vagal transmission under both normal and occluded conditions. The magnitude of these DOR-2 vagolytic effects was small compared to controls and repeated MEAP challenges rapidly eroded vagolytic responses further. Prior DOR-1 blockade did not alter the PC mediated, progressive loss of DOR-2 vagolytic responses. In conclusion, DOR-1 vagotonic responses evolved from signals earlier in the PC protocol and erosion of competing DOR-2 vagolytic responses may have contributed to an unmasking of vagotonic responses. The data support the hypothesis that PC and DOR-2 stimulation promote DOR trafficking, and down regulation of the vagolytic DOR-2 phenotype in favor of the vagotonic DOR-1 phenotype. DOR-1 blockade may accelerate the process by sequestering newly emerging receptors.

Opioids introduced at reperfusion (R) following ischemia (I) reduce infarct size much like postconditioning, suggesting the hypothesis that postconditioning increases cardiac opioids and activates local opioid receptors. Anesthetized male rats subjected to 30 min regional I and 3 h R were postconditioned with three cycles of 10 s R and 10 s reocclusion at onset of R. Naloxone (NL), its peripherally restricted analog naloxone methiodide, delta-opioid receptor (DOR) antagonist naltrindole (NTI), kappa-opioid receptor antagonist norbinaltorphimine (NorBNI), and mu-opioid receptor (MOR) antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were administered intravenously 5 min before R. The area at risk (AAR) was comparable among groups, and postconditioning reduced infarct size from 57 +/- 2 to 42 +/- 2% (P < 0.05). None of the antagonists alone altered infarct size. All antagonists abrogated postconditioning protection at higher doses. However, blockade of infarct sparing by postconditioning was lost, since tested doses of NL, NTI, NorBNI, and CTAP were lowered. The efficacy of NorBNI declined first at 3.4 micromol/kg, followed sequentially by NTI (1.1), NL (0.37), and CTAP (0.09), suggesting likely MOR and perhaps DOR participation. Representative small, intermediate, and large enkephalins in the AAR were quantified (fmol/mg protein; mean +/- SE). I/R reduced proenkephalin (58 +/- 9 vs. 33 +/- 4; P < 0.05) and sum total of measured enkephalins, including proenkephalin, peptide B, methionine-enkephalin, and methionine-enkephalin-arginine-phenylalanine (139 +/- 17 vs. 104 +/- 7; P < 0.05) compared with shams. Postconditioning increased total enkephalins (89 +/- 8 vs. 135 +/- 5; P < 0.05) largely by increasing proenkephalin (33 +/- 4 vs. 96 +/- 7; P < 0.05). Thus the infarct-sparing effect of postconditioning appeared to involve endogenously activated MORs and possibly DORs, and preservation of enkephalin precursor synthesis in the AAR.

Delta-opioid receptors (DORs) are associated with ischemic preconditioning and vagal transmission in the sinoatrial (SA) node and atria. Although functional studies suggested that DORs are prejunctional on parasympathetic nerve terminals, their precise location remains unconfirmed. DORs were colocalized in tissue slices and synaptosomes from the canine right atrium and SA node along with cholinergic and adrenergic markers, vesicular acetylcholine transporter (VAChT), and tyrosine hydroxylase (TH). Synapsin I immunofluorescence verified the neural character of tissue structures and isolated synaptosomes. Acetylcholine and norepinephrine measurements suggested the presence of both cholinergic and adrenergic synaptosomes. Fluorescent analysis of VAChT and TH signals indicated that >80% of the synapsin-positive synaptosomes were of cholinergic origin and <8% were adrenergic. DORs colocalized 75-85% with synapsin in tissue slices from both atria and SA node. The colocalization was equally strong (85%) for nodal synaptosomes but less so for atrial synaptosomes (57%). Colocalization between DOR and VAChT was 75-85% regardless of the source. Overlap between DOR and TH was uniformly low, ranging from 8% to 17%. Western blots with synaptosomal extracts confirmed two DOR-positive bands at molecular masses corresponding to those reported for DOR monomers and dimers. The abundance of DOR was greater in nodal synaptosomes than in atrial synaptosomes, largely attributable to a greater abundance of monomers in the SA node. The abundant nodal and atrial DORs predominantly associated with cholinergic nerve terminals support the hypothesis that prejunctional DORs regulate vagal transmission locally within the heart.

The cardiac enkephalin, methionine-enkephalin-arginine-phenylalanine (MEAP), alters vagally induced bradycardia when introduced by microdialysis into the sinoatrial (SA) node. The responses to MEAP are bimodal; lower doses enhance bradycardia and higher doses suppress bradycardia. The opposing vagotonic and vagolytic effects are mediated, respectively, by delta(1) and delta(2) phenotypes of the same receptor. Stimulation of the delta(1) receptor reduced the subsequent delta(2) responses. Experiments were conducted to test the hypothesis that the delta-receptor interactions were mediated by the monosialosyl ganglioside GM-1. When the mixed agonist MEAP was evaluated after nodal GM-1 treatment, delta(1)-mediated vagotonic responses were enhanced, and delta(2)-mediated vagolytic responses were reduced. Prior treatment with the delta(1)-selective antagonist 7-benzylidenaltrexone (BNTX) failed to prevent attrition of the delta(2)-vagolytic response or restore it when added afterward. Thus the GM-1-mediated attrition was not mediated by delta(1) receptors or increased competition from delta(1)-mediated vagotonic responses. When GM-1 was omitted, deltorphin produced a similar but less robust loss in the vagolytic response. In contrast, however, to GM-1, the deltorphin-mediated attrition was prevented by pretreatment with BNTX, indicating that the decline in response after deltorphin alone was mediated by delta(1) receptors and that GM-1 effectively bypassed the receptor. Whether deltorphin has intrinsic delta(1) activity or causes the release of an endogenous delta(1)-agonist is unclear. When both GM-1 and deltorphin were omitted, the subsequent vagolytic response was more intense. Thus GM-1, deltorphin, and time all interact to modify subsequent delta(2)-mediated vagolytic responses. The data support the hypothesis that delta(1)-receptor stimulation may reduce delta(2)-vagolytic responses by stimulating the GM-1 synthesis.

Growing evidence suggests that cardiac enkephalins and their receptors are involved in ischemic preconditioning (IPC). Because there is no evidence for vesicular storage of small bioactive enkephalins in the heart, studies were designed to test the hypothesis that ischemia depletes cardiac enkephalins and that IPC preserves the same enkephalins by accelerating their processing from the larger proenkephalin precursor (PEP) pool. The precursors and two bioactive representatives, Met-enkephalin (ME) and Met-enkephalin-Arg-Phe (MEAP), were separated by size-exclusion chromatography and quantified by radioimmunoassay. Isolated perfused rat hearts were prepared and exposed to global ischemia. After 30 min of global ischemia and 40 min of reflow, the PEP pool was reduced (from 17.99 +/- 1.52 to 14.20 +/- 2.38 pmol/g wet wt), MEAP increased by 53%, and ME declined by 68%. The sum of the two smaller peptides was unchanged (9.78 +/- 0.83 vs. 9.33 +/- 2.81). Thus the total enkephalin peptide content was not altered (27.77 +/- 1.69 vs. 24.10 +/- 4.75). Peptide distribution after ischemia and reflow was also unaltered by pretreatment with peptidase inhibitors. However, when the hearts were preconditioned, the PEP pool remained significantly lower and both of the bioactive peptides, MEAP and ME, were elevated (+49% and +86%, respectively). The decline in the PEP pool was prevented by peptidase inhibition and the rise in MEAP was exaggerated. In separate protocols, synthetic enkephalins (ME, MEAP, and Leu-enkephalin) were added to the coronary inflow before 30 min of global ischemia and throughout the subsequent reflow. The added enkephalins (10(-8) M) had no inotropic effect on baseline function but completely prevented the mechanical dysfunction observed in untreated controls during reflow. Thus IPC appears to increase available bioactive enkephalins (MEAP + ME) within the heart by enhancing synthesis of precursors and their subsequent processing from the PEP pool.

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-pen(2,5)]-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.

Blunted beta-adrenergic inotropism in stunned myocardium is restored by pharmacological (N-acetylcysteine) and metabolic (pyruvate) antioxidants. The ketone body acetoacetate is a natural myocardial fuel and antioxidant that improves contractile function of prooxidant-injured myocardium. The impact of acetoacetate on postischemic cardiac function and beta-adrenergic signaling has never been reported. To test the hypothesis that acetoacetate restores contractile performance and beta-adrenergic inotropism of stunned myocardium, postischemic Krebs-Henseleit-perfused guinea pig hearts were treated with 5 mM acetoacetate and/or 2 nM isoproterenol at 15-45 and 30-45 min of reperfusion, respectively, while cardiac power was monitored. The myocardium was snap frozen, and its energy state was assessed from phosphocreatine phosphorylation potential. Antioxidant defenses were assessed from GSH/GSSG and NADPH/NADP(+) redox potentials. Stunning lowered cardiac power and GSH redox potential by 90 and 70%, respectively. Given separately, acetoacetate and isoproterenol each increased power and GSH redox potential three- to fivefold. Phosphocreatine potential was 70% higher in acetoacetate- vs. isoproterenol-treated hearts (P < 0.01). In combination, acetoacetate and isoproterenol synergistically increased power and GSH redox potential 16- and 7-fold, respectively, doubled NADPH redox potential, and increased cAMP content 30%. The combination increased cardiac power four- to sixfold vs. the individual treatments without a coincident increase in phosphorylation potential. Potentiation of isoproterenol's inotropic actions endured even after acetoacetate was discontinued and GSH potential waned, indicating that temporary enhancement of redox potential persistently restored beta-adrenergic mechanisms. Thus acetoacetate increased contractile performance and potentiated beta-adrenergic inotropism in stunned myocardium without increasing energy reserves, suggesting its antioxidant character is central to its beneficial actions.

Short-term morphine stimulates vagal bradycardia. This led us to propose the hypothesis that chronically administered morphine would down-regulate myocardial muscarinic receptor systems. Dogs received morphine continuously for 2 weeks through an s.c. catheter, and cellular aspects of parasympathetic control of the heart were examined. Contrary to expectations, morphine increased muscarinic receptor density in the right atrium and left ventricle by 17 and 34%, respectively, with no change in the apparent affinity of the receptor (K(D)). Morphine also increased the expression of the G protein G(ialpha) by 115 and 233%, respectively, in right atrial and left ventricular sarcolemmal membranes. Morphine increased ventricular and atrial G(salpha) to a much lesser degree (49 and 25%). Morphine failed to alter basal or maximally stimulated (forskolin plus MnCl(2)) adenylate cyclase activity. The maximum cyclase activation by isoproterenol and the maximum inhibition by carbachol were similarly unaltered by morphine. Morphine reduced the ventricular but not atrial norepinephrine. Both long- and short-term morphine lowered tissue epinephrine content, suggesting that short-term morphine reduces extraneuronal uptake. Potential systemic and cellular models for myocardial adaptation to morphine are proposed, including sequential sympathetic and parasympathetic compensations.

The vagotonic effect of chronic morphine on the parasympathetic control of the heart was examined in dogs treated with morphine for 2 wk. Because normal vagal function is critical to myocardial stability, the study was conducted to evaluate for potential impairments following chronic vagal stimulation. The hypothesis that persistent vagal outflow would result in a loss of vagal reserve and reduced vagal control of heart rate was tested. Heart rate and the high-frequency variation in heart rate (power spectral analysis) declined shortly after initiation of subcutaneous morphine infusion. A progressive bradycardia correlated well with the rising plasma morphine. The resting bradycardia (57 beats/min) was maintained through day 2 and was accompanied by a significant parallel increase in vagal effect and a decline in the intrinsic heart rate (160 vs. 182 beats/min). A compensatory increase in the ambient sympathetic control of heart rate was evident on day 2 and was supported by an increase in circulating catecholamines. The lowered intrinsic heart rate and elevated sympathetic activity were maintained through day 10 despite a return of the resting heart rate and plasma catecholamines to pretreatment values. These observations suggested that chronic morphine alters either the intrinsic function of the sinoatrial node or reduces the postvagal tachycardia normally attributed to nonadrenergic, noncholinergic agents. Both acute and chronic morphine depressed the rate of development of bradycardia during direct vagal nerve stimulation without altering the rate of recovery afterward. This last observation suggests that acute morphine reduces the rate of acetylcholine release. Results provide insight into the mechanisms that maintain vagal responsiveness. The results are also relevant clinically because opiates are increasingly prescribed for chronic pain and opiate abuse is currently in resurgence.