δ-Opioid receptors: Pivotal role in intermittent hypoxia-augmentation of cardiac parasympathetic control and plasticity.

BACKGROUND: Intermittent hypoxia training (IHT) produces robust myocardial protection against ischemia-reperfusion induced infarction and arrhythmias. Blockade of this cardioprotection by antagonism of either ß1-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.

Repeated arterial occlusion, delta-opioid receptor (DOR) plasticity and vagal transmission within the sinoatrial node of the anesthetized dog.

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.

Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation.

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.

Cholinergic location of delta-opioid receptors in canine atria and SA node.

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 monosialosyl ganglioside GM-1 reduces the vagolytic efficacy of delta2-opioid receptor stimulation.

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.

Ischemic preconditioning increases the bioavailability of cardiac enkephalins.

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.

Acetoacetate augments beta-adrenergic inotropism of stunned myocardium by an antioxidant mechanism.

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.

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

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.