We examined the ability of ACh to mimic ischemic preconditioning in cardiomyocytes and the role of ATP-sensitive potassium (KATP) channels and mitochondrial reactive oxygen species (ROS) in mediating this effect. Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, PO2, and PCO2 were controlled. Cell viability was quantified with propidium iodide (5 microM), and production of ROS was measured using 2', 7'-dichlorofluorescin diacetate. Data were expressed as means +/- SE. Preconditioning with 10 min of ischemia followed by 10 min of reoxygenation or 10 min of ACh (1 mM) followed by a drug-free period before 1 h of ischemia and 3 h of reoxygenation reduced cell death to the same extent [preconditioning 19 +/- 2% (n = 6, P < 0.05) ACh 21 +/- 5% (n = 6, P < 0.05) vs controls 42 +/- 5% (n = 9)]. Like preconditioning, ACh increased ROS production threefold before ischemia [0.60 +/- 0.16 (n = 7, P < 0.05) vs. controls, 0.16 +/- 0. 03 (n = 6); arbitrary units]. Protection and increased ROS production during ACh preconditioning were abolished with 5-hydroxydecanoate (5-HD, 100 microM), a selective mitochondrial K(ATP) channel antagonist, and the thiol reductant 2-mercaptopropionyl glycine (2-MPG, 1 mM), an antioxidant [cell death: 5-HD+ACh 37 +/- 7% (n = 5), 2-MPG+ACh 47 +/- 6% (n = 6); ROS signals: 5-HD+ACh 0.09 +/- 0.03 (n = 5), 2-MPG+ACh 0.01 +/- 0.04 (n = 4)]. In addition, ACh-induced ROS signaling was blocked by the mitochondrial site III electron transport inhibitor myxothiazol (0.02 +/- 0.07, n = 5). These results demonstrate that activation of mitochondrial K(ATP) channels and increased ROS production from mitochondria are important intracellular signals that participate in ACh-induced preconditioning in cardiomyocytes.
Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti-P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti-P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti-P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti-P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti-Pselectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti-P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti-P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti-Pselectin aptamer may be useful as a novel therapeutic agent for SCD. (Blood. 2011; 117(2):727-735) IntroductionSickle cell disease (SCD) is caused by a point mutation of the -globin chain, but its pathophysiology is extremely complex and heterogeneous. A salient clinical feature of this disorder is vasoocclusive crisis, which is a major cause of morbidity and mortality in SCD patients; repetitive crises could eventually lead to multiorgan damage in the long term. 1 Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells have been implicated as critical pathologic events for the development of vaso-occlusion. Much attention has been directed to identifying adhesion molecules involved in cell-cell interactions.Endothelial cell P-selectin, a member of the selectin family of cell adhesion molecules, 2 plays a key role in leukocyte recruitment as well as the adhesion of sickle RBCs to the endothelium. 3,4 Presynthesized P-selectin is stored in the Weibel-Palade bodies in endothelial cells and rapidly translocated to the cell surface in response to extracellular stimuli such as hypoxia. 5 Expression levels of P-selectin are elevated in patients with SCD. 6,7 The interactions between P-selectin and its ligands are likely to contribute to cell adhesion between multiple types of cells, which results in the impairment of microvascular circulation presumably involved in the development of painful vaso-occlusive episodes. 4,8 Several antiadhesion compounds have been tested for their ability to inhibit sickle RBC adhesion to endothelial cells, however, no agents are currently used...
The hallmark of sickle cell disease (SCD) is the polymerization of deoxygenated sickle hemoglobin (HbS). In SCD patients, one strategy to reduce red blood cell (RBC) sickling is to increase HbS oxygen affinity. Our objective was to determine if low concentrations of nitric oxide (NO) gas would augment the oxygen affinity of RBCs containing homozygous HbS (SS). Blood containing normal adult hemoglobin (AA) or SS RBCs was incubated in vitro in the presence of varying concentrations of NO up to 80 ppm, and oxygen dissociation curves (ODCs) were measured. In addition, blood was obtained from three AA and nine SS volunteers, before and after breathing 80 ppm NO in air for 45 min, and the ODCs were measured. Exposure of SS RBCs to 80 ppm NO in vitro for 5 min or longer decreased the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen (P 50 ), an average of 15% (4.8 Ϯ 1.7 mmHg mean Ϯ SE; P Ͻ 0.001). The increase in SS RBC oxygen affinity correlated with the NO concentration. The P 50 of AA RBCs was unchanged ( P Ͼ 0.1) by 80 ppm NO. In SS volunteers breathing 80 ppm NO for 45 min, the P 50 decreased ( P Ͻ 0.001) by 4.6 Ϯ 2.0 mmHg. 60 min after NO breathing was discontinued, the RBC P 50 remained decreased in five of seven volunteers in whom the ODC was measured. There was no RBC P 50 change ( P Ͼ 0.1) in AA volunteers breathing NO. Methemoglobin (Mhb) remained low in all subjects breathing NO (SS Mhb 1.4 Ϯ 0.5%), and there was no correlation ( r ϭ 0.02) between the reduction in P 50 and the change in Mhb. Thus, low concentrations of NO augment the oxygen affinity of sickle erythrocytes in vitro and in vivo without significant Mhb production. These results suggest that low concentrations of NO gas may offer an attractive new therapeutic model for the treatment of SCD. ( J. Clin. Invest. 1997. 100: 1193-1198.)
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