Darice Yoshishige

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.

Autonomic control of heart rate in dogs treated chronically with morphine

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.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.

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.

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 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.

Intrinsic cardiac enkephalins & vagal control

Abstract Met-enkephalin-arg-phe (MEAP) has been identified in acid extracts of canine heart. The effects of synthetic MEAP on the vagal control of heart rate and atrial contractility were investigated in anesthetized dogs. Arterial MEAP (3 nmol/min/kg) inhibited right vagal bradycardia by two thirds. Postinfusion responsiveness to vagal stimulation returned to normal with an estimated half-time of 2–3 min. Inhibition by MEAP was reversed by the high affinity opiate antagonist, diprenorphine at pmolar concentrations. Since MEAP did not alter the negative chronotropic effect of methacholine, the effective site must reside in the efferent vagal tract proximal to nodal muscarinic receptors. Left vagal stimulation dramatically suppresses left atrial contractile activity. Infused MEAP also interrupts this negative inotropic response through a diprenorphine sensitive mechanism. Increasing MEAP infusions (0.09 – 3.00 nmol/min/kg) produced a graded suppression of vagal bradycardia with an ED50 near 0.3 nmol/min/kg. Heart rate dose responses for methionine-enkephalin were shifted to the right of MEAP and required approximately 3 X the dose to produce the same effect. The data suggest that the intrinsic cardiac enkephalin, MEAP can regulate vagal control of heart rate at physiologically achievable concentrations and may serve as a local regulator of the parasympathetic/myocardial interface.