No abstract

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.

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.

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.

T. Mallet. Acetoacetate augments ␤-adrenergic inotropism of stunned myocardium by an antioxidant mechanism. Am J Physiol Heart Circ Physiol 284: H1340-H1347, 2003. First published December 19, 2002 10.1152/ajpheart.00473.2002.-Blunted ␤-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 ␤-adrenergic signaling has never been reported. To test the hypothesis that acetoacetate restores contractile performance and ␤-adrenergic inotropism of stunned myocardium, postischemic Krebs-Henseleitperfused 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 ϩ 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 acetoacetatevs. isoproterenol-treated hearts (P Ͻ 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 ␤-adrenergic mechanisms. Thus acetoacetate increased contractile performance and potentiated ␤-adrenergic inotropism in stunned myocardium without increasing energy reserves, suggesting its antioxidant character is central to its beneficial actions.isoproterenol; glutathione; adenosine 3Ј,5Ј-cyclic monophosphate; nicotinamide adenine dinucleotide phosphate; citrate POSTISCHEMIC MYOCARDIAL STUNNING is produced in large part by cytotoxic oxygen and nitrogen metabolites generated upon coronary reperfusion (6). The resulting superoxide, H 2 O 2 , hydroxyl radical, and peroxynitrite (2, 12, 39) modify a variety of cellular components (5) to produce stunning. Inotropic responses to ␤-adrenergic stimulation are dampened in stunned myocardium (38, 44), perhaps resulting from oxidative damage to protein components of the ␤-adrenergic signaling cascade.Pyruvate, a natural metabolic fuel in myocardium, markedly increased ␤-adrenergic responsiveness of stunned myocardium and preserved energy stores (44). Pyruvate also restored GSH/GSSG redox potential (43), the princ...