Zhenhai Yao

Morphine Mimics Preconditioning via Free Radical Signals and Mitochondrial KATP Channels in Myocytes

BackgroundWe tried to determine whether morphine mimics preconditioning (PC) to reduce cell death in cultured cardiomyocytes and whether opioid &dgr;1 receptors, free radicals, and KATP channels mediate this effect. Methods and ResultsChick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, and O2 and CO2 tension were controlled. Cardiomyocyte viability was quantified with propidium iodide (5 &mgr;mol/L), and production of free radicals was measured with 2′,7′-dichlorofluorescin diacetate. PC with 10 minutes of simulated ischemia before 10 minutes of reoxygenation or morphine (1 &mgr;mol/L) or BW373U86 (10 pmol/L) infusion for 10 minutes followed by a 10-minute drug-free period before 1 hour of ischemia and 3 hours of reoxygenation reduced cell death to the same extent (*P <0.05) (PC, 20±1%, n=7*; morphine, 32±4%, n=8*; BW373U86, 21±6%; controls, 52±5%, n=8). Like PC, morphine and BW373U86 increased free radical production 2-fold before ischemia (0.35±0.10, n=6*; 0.41±0.08, n=4* versus controls, 0.15±0.05, n=8, arbitrary units). Protection and increased free radical signals during morphine infusion were abolished with either the thiol reductant 2-mercaptopropionyl glycine (400 &mgr;mol/L), an antioxidant; naloxone (10 &mgr;mol/L), a nonselective morphine receptor antagonist; BNTX (0.1 &mgr;mol/L), a selective opioid &dgr;1 receptor antagonist; or 5-hydroxydecanoate (100 &mgr;mol/L), a selective mitochondrial KATP channel antagonist. ConclusionsThese results suggest that direct stimulation of cardiocyte opioid &dgr;1 receptors leads to activation of mitochondrial KATP channels. The resultant increase of intracellular free radical signals may be an important component of the signaling pathways by which morphine mimics preconditioning in cardiomyocytes.

Signal Transduction of Opioid-induced Cardioprotection in Ischemia-Reperfusion

Background Morphine reduces myocardial ischemia–reperfusion injury in vivo and in vitro. The authors tried to determine the role of opioid &dgr;1 receptors, oxygen radicals, and adenosine triphosphate–sensitive potassium (KATP) channels in mediating this effect. Methods Chick cardiomyocytes were studied in a flow-through chamber while pH, flow rate, oxygen, and carbon dioxide tension were controlled. Cell viability was quantified by nuclear stain propidium iodide, and oxygen radicals were quantified using molecular probe 2′,7′-dichlorofluorescin diacetate. Results Morphine (1 &mgr;m) or the selective &dgr;-opioid receptor agonist BW373U86 (10 pm) given for 10 min before 1 h of ischemia and 3 h of reoxygenation reduced cell death (31 ± 5%, n = 6, and 28 ± 5%, n = 6 [P < 0.05], respectively, 53 ± 6%, n = 6, in controls) and generated oxygen radicals before ischemia (724 ± 53, n = 8, and 742 ± 75, n = 8 [P < 0.05], respectively, vs. 384 ± 42, n = 6, in controls, arbitrary units). The protection of morphine was abolished by naloxone, or the selective &dgr;1-opioid receptor antagonist 7-benzylidenenaltrexone. Reduction in cell death and increase in oxygen radicals with BW373U86 were blocked by the selective mitochondrial KATP channel antagonist 5-hydroxydecanoate or diethyldithiocarbamic acid (1000 &mgr;m), which inhibited conversion of O2− to H2O2. The increase in oxygen radicals was abolished by the mitochondrial electron transport inhibitor myxothiazol. Reduction in cell death was associated with attenuated oxidant stress at reperfusion. Conclusion Stimulation of &dgr;1-opioid receptors generates oxygen radicals via mitochondrial KATP channels. This signaling pathway attenuates oxidant stress and cell death in cardiomyocytes.

Ischemia reperfusion injury, preconditioning and critical illness.

The purpose of this review is to describe in more detail ischemia reperfusion injury and preconditioning, and to speculate on the potential role of preconditioning in the care of critically ill patients. Current hemodynamic treatment of hypotension and hypoperfusion in critically ill patients is directed at ensuring essential organ perfusion by maintaining intravascular volume and cardiac output, and ensuring adequate oxygen delivery by maintaining arterial oxygen partial pressure and hemoglobin levels. However, morbidity and mortality remain high and new approaches to critically ill patients are required. Treatments are needed that can protect against organ ischemia during periods of low blood flow. In recent years, there has been a growing appreciation of the importance of ischemia reperfusion injury. Ischemia associated with reperfusion may result in greater injury than ischemia alone. Ischemic preconditioning is used to describe the protective effect of short periods of ischemia to an organ or tissue against longer periods of ischemia. Although first described in the myocardium, there is now evidence that this phenomenon occurs in a wide variety of organs and tissues, including the brain and other nervous tissue such as the retina and spinal cord, liver, stomach, intestines, kidney, and the lungs. Preconditioning therapy may offer a new avenue of treatment in critically ill patients. Both traditional preconditioning methods and pharmacologic agents that mimic or induce such preconditioning may be used in the future. Clinical trials of pharmacologic agents are underway in patients with coronary artery disease. Further trials of such methods and agents are needed in critically ill patients suffering from sepsis or multiorgan system failure.

Brief hypoxia conditions monocytes to protect reperfused cardiocytes against cell death via the CD11b receptor

BACKGROUND Traditionally leukocytes have been viewed as the mediators and effectors of cell injury after tissue ischemia and reperfusion through the indiscriminate release of toxic cytokines and oxygen free radicals. This can be detrimental to functioning of the transplanted heart when reperfused after implantation. Paradoxically, evidence now suggests that certain cytotoxic cytokines and even oxygen free radicals can be cytoprotective in smaller concentrations. This study sought to determine whether cultured human monocytes can be pre-conditioned by brief hypoxia to protect cardiomyocytes from cell death after hypoxia and re-oxygenation. METHODS Cultured human monocytes were exposed to transient hypoxia (10 minutes), after which we determined CD11b expression using flow cytometry. The 3 control groups comprised immunoglobulin G-negative controls, fMLP-positive controls, and virgin monocyte (VM) controls. We studied chick embryonic ventricular myocytes in a flow-through chamber while controlling flow rate, pH, O(2), and CO2 tension. We quantified cardiomyocyte viability using propidium iodide (5 micromol/liter). Cell systems of cardiomyocytes alone, cardiomyocytes and VM, human monocytes exposed to transient hypoxia before coculture with cardiomyocytes (PCHM-cardiomyocyte), and PCHM cocultured with anti-CD11b antibodies for 30 minutes before coculture with cardiomyocytes (CDHM-cardiomyocyte) were subjected to 1 hour of hypoxia and 3 hours of re-oxygenation, and cell death was determined. The experiment was repeated and the cell systems fixed, stained, and examined for monocytes adhering to cardiomyocytes. RESULTS CD11b expression increased significantly with both transient hypoxia and fMLP (18.39% +/- 4.116%, n = 5, and 37.04% +/- 7.783%, n = 5, respectively, p < 0.05 vs VM 100%, n = 5). Coculture of cardiomyocytes with VM had no effects on cardiocyte death (40.6% +/- 6.1%, n = 6) compared with controls (46.5% +/- 4.0%, n = 10). The PCHM cocultured with cardiomyocytes significantly decreased cardiomyocyte death (25.2% +/- 4.7%, n = 6, p < 0.05). This protection was abrogated by the addition of CD11b-blocking antibodies to PCHM before coculture with cardiomyocytes (51.0% +/- 6.1%, n = 6, p < 0.05). The PCHM showed increased adhesion to cardiomyocytes (5.4 +/- 0.38/high-power [HP] field vs 0.67 +/- 0.24/HP field in VM, p < 0.05). The increased adhesion was abolished by CD11b-blocking antibody (0.78 +/- 0.28 vs 0.67 +/- 0.24 cells/HP field, p < 0.05). CONCLUSIONS These results suggest that monocytes activated by transient hypoxia protect cardiomyocytes during hypoxia and re-oxygenation through expression of CD11b receptors. These cells seem to adhere to myocytes through this receptor to achieve this effect. The exact mechanism is unclear and requires further study. Autologous recipient monocytes may be pre-conditioned to protect the donor heart during reperfusion.

Acetylcholine attenuates cardiomyocyte oxidant stress during simulated ischemia and reoxygenation

We wanted to determine whether oxygen radicals open the mitochondrial ATP-dependent potassium channels (KATP) during an ischemic period to reduce cell death and oxidant stress. Chick embryonic cardiomyocytes were used. Cell viability was quantified with propidium iodide (5 µM), and free radicals was measured using 2′,7′-dichlorofluorescin diacetate. Preconditioning was produced by 10 min of simulated ischemia followed by 10 min of reoxygenation. Acetylcholine (1 mM), infused for 10 min instead of preconditioning, reduced cell death similarly (24 ± 5%, n = 7 and 18 ± 2%, n = 7, respectively, vs. controls, 49 ± 6%, n = 8). In control series, 60 min of simulated ischemia and 3 h of reoxygenation generated free radicals more than 300% above the baseline (ischemia: 3.63 ± 0.58, reoxygenation: 3.66 ± 0.47, n = 8). Preconditioning and acetylcholine markedly attenuated the oxidant stress during simulated ischemia (1.18 ± 0.41, n = 6 and 1.34 ± 0.60, n = 7 vs. controls 3.63 ± 0.58, n = 8) and re-oxygenation (1.23 ± 0.36, n = 6 and 1.50 ± 0.59, n = 7 vs. controls 3.66 ± 0.47, n = 8). The protection of acetylcholine was abolished with pretreatment with the antioxidant thiol reductant 2-mercaptopropionyl glycine and posttreatment with 5-hydroxydecanoate, a selective mitochondrial KATP channel antagonist (37 ± 7%, n = 7). These results demonstrate that oxygen radicals open mitochondrial KATP channels, which mediates the acetylcholine-induced preconditioning effect, and that stimulation of this signaling pathway attenuates oxidant stress.