Ischemic preconditioning depends on interaction between mitochondrial KATP channels and actin cytoskeleton. (1/305)

Both mitochondrial ATP-sensitive K+ (KATP) channels and the actin cytoskeleton have been proposed to be end-effectors in ischemic preconditioning (PC). For evaluation of the participation of these proposed end effectors, rabbits underwent 30 min of regional ischemia and 3 h of reperfusion. PC by 5-min ischemia + 10-min reperfusion reduced infarct size by 60%. Diazoxide, a mitochondrial KATP-channel opener, administered before ischemia was protective. Protection was lost when diazoxide was given after onset of ischemia. Anisomycin, a p38/JNK activator, reduced infarct size, but protection from both diazoxide and anisomycin was abolished by 5-hydroxydecanoate (5-HD), an inhibitor of mitochondrial KATP channels. Isolated adult rabbit cardiomyocytes were subjected to simulated ischemia by centrifuging the cells into an oxygen-free pellet for 3 h. PC was induced by prior pelleting for 10 min followed by resuspension for 15 min. Osmotic fragility was assessed by adding cells to hypotonic (85 mosmol) Trypan blue. PC delayed the progressive increase in fragility seen in non-PC cells. Incubation with diazoxide or pinacidil was as protective as PC. Anisomycin reduced osmotic fragility, and this was reversed by 5-HD. Interestingly, protection by PC, diazoxide, and pinacidil could be abolished by disruption of the cytoskeleton by cytochalasin D. These data support a role for both mitochondrial KATP channels and cytoskeletal actin in protection by PC.  (+info)

Decreased plasma membrane thiol concentration is associated with increased osmotic fragility of erythrocytes in zinc-deficient rats. (2/305)

Zinc deficiency leads to pathological signs that are related to impaired function of plasma membrane proteins. The purpose of this study was to assess the effect of dietary zinc status on the sulfhydryl (SH) content of erythrocyte plasma membranes and erythrocyte function. Three experiments were performed. In the first, immature male rats were fed for 21 d either a low-zinc (<1.0 mg/kg) diet free choice (-ZnAL), an adequate-zinc (100 mg/kg) diet free choice (+ZnAL), or the adequate-zinc diet limited to the intake of -ZnAL pair-mates (+ZnPF). Tail blood was sampled to measure osmotic fragility and SH concentration of erythrocyte membrane proteins. The zinc-deficient rats were then repleted for 2 d and erythrocytes assayed for fragility and SH content. In the second experiment blood was sampled at 3-d intervals to determine the time course of change in fragility and SH concentration. In the third experiment the SH concentration of erythrocyte band 3 protein and the binding of zinc to isolated plasma membranes were measured. SH concentration decreased from approximately 75 nmol/mg protein to 68 nmol/mg protein during 21 d of depletion and returned to control level within 2 d of repletion. There was an inverse relationship between osmotic fragility and SH concentration of erythrocyte membrane proteins. Maximal decrease in SH occurred within 6 d of consuming the low-zinc diet. The SH content of band 3 protein isolated from deficient rats was also significantly lower than that of pair-fed controls (45 vs. 51 nmol/mg protein). The zinc-binding affinity of plasma membrane proteins tended to be decreased by zinc deficiency. In summary, low-zinc status lowers the plasma membrane SH concentration, and the decreased reducing potential is inversely related to osmotic fragility, and presumably, with impaired volume recovery of erythrocytes.  (+info)

Extracellular ATP stimulates volume decrease in Necturus red blood cells. (3/305)

This study examined whether extracellular ATP stimulates regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The hemolytic index (a measure of osmotic fragility) decreased with extracellular ATP (50 microM). In contrast, the ATP scavenger hexokinase (2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, the ATP-dependent K+ channel antagonist glibenclamide (100 microM) increased the hemolytic index, and this inhibition was reversed with ATP (50 microM). We also measured cell volume recovery in response to hypotonic shock electronically with a Coulter counter. Extracellular ATP (50 microM) enhanced cell volume decrease in a hypotonic (0.5x) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml) inhibited cell volume recovery. The inhibitory effect of hexokinase was reversed with the Ca2+ ionophore A-23187 (1 microM); it also was reversed with the cationophore gramicidin (5 microM in a choline-Ringer solution), indicating that ATP was linked to K+ efflux. In addition, glibenclamide (100 microM) and gadolinium (10 microM) inhibited cell volume decrease, and the effect of these agents was reversed with ATP (50 microM) and A-23187 (1 microM). Using the whole cell patch-clamp technique, we found that ATP (50 microM) stimulated a whole cell current under isosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide (100 microM), and gadolinium (10 microM) inhibited whole cell currents that were activated during hypotonic swelling. The inhibitory effect of apyrase was reversed with the nonhydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (50 microM), and the effect of glibenclamide or gadolinium was reversed with ATP (50 microM). Finally, anionic whole cell currents were activated with hypotonic swelling when ATP was the only significant charge carrier, suggesting that increases in cell volume led to ATP efflux through a conductive pathway. Taken together, these results indicate that extracellular ATP stimulated cell volume decrease via a Ca2+-dependent step that led to K+ efflux.  (+info)

Targeted disruption of the beta adducin gene (Add2) causes red blood cell spherocytosis in mice. (4/305)

Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). In a comprehensive assay of gene expression, we show the ubiquitous expression of alpha- and gamma-adducins in contrast to the restricted expression of beta-adducin. beta-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created beta-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of beta-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. beta-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of beta-adducin in RBCs leads to decreased membrane incorporation of alpha-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in gamma-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.  (+info)

Elimination of anemia-inducing substance by cyclic plasma perfusion of tumor-bearing rabbits. (5/305)

We carried out a fundamental study to search for a therapeutic modality that would remove the anemia-inducing substance (AIS) from the plasma of cancer patients because it is thought to be one of the substances responsible for anemia and immunodeficiency in advanced cancer patients. Using AIS isolated from the plasma of patients with advanced ovarian carcinoma, we confirmed that adsorption of AIS to noncoated charcoal was nonspecific and high. Moreover, it was verified that VX2 carcinoma-bearing rabbits are an optimal experimental model for plasma perfusion. The data obtained on day 40 after transplantation (hemoglobin, 9.1+/-2.1 g/dl; osmotic pressure inducing RBC lysis, 137+/-11 mosmol/kg; lymphocyte stimulation index, 8.8+/-8.6; and RBC fragility-inducing activity, 40+/-9 mosmol/kg) proved similar to the hematological findings in patients with cancer cachexia. A 1-h plasma perfusion (3 ml/min) through noncoated charcoal was performed in tumor-bearing rabbits, and it resulted in the restoration of RBC fragility-inducing activity and suppression of lymphocyte blast formation to pretransplantation values. When plasma perfusion was performed every 3 days, RBC fragility-inducing activity, which increased again 3 days after perfusion, was diminished, and RBC osmotic resistance was within the normal range from the fourth perfusion onward. These results showed that cyclic plasma perfusion is effective in sustained removal of RBC fragility-inducing factor from plasma, suggesting that it might have the potential for clinical application.  (+info)

Relation between erythrocyte reduced glutathione and glutamate concentrations in Korean Jindo dogs with erythrocytes possessing hereditary high activity of Na-K-ATPase and a high concentration of potassium. (6/305)

The concentrations of sodium, potassium, reduced glutathione (GSH) and free amino acids and Na-K-ATPase activity in erythrocytes were examined in 35 purebred Jindo dogs in Korea. The incidence of Jindo dogs with a high potassium concentration and high activity of Na-K-ATPase in erythrocytes (HK phenotype) was 25.7%. The erythrocyte GSH concentration in HK Jindo dogs varied widely, from 2.45 to 12.38 mmol/l of RBCs, and was positively correlated with the erythrocyte glutamate concentration. These results indicate that HK Jindo dogs have normal to very high levels of erythrocyte GSH, which might result from the varying quantity of Na-dependent glutamate influx in the erythrocytes.  (+info)

Bis-(N-maleimidomethyl) ether: an antisickling reagent. (7/305)

The interaction of bis-(N-maleimidomethyl) ether with oxyhemoglobin results in covalent linkages of both maleimide groups, converting them to succinyl derivatives of beta93 Cys and beta97 His at their sulfhydryl and imidazolyl side chains, respectively. The resultant hemoglobin is stable, and reveals a left-shifted oxyhemoglobin equilibrium curve in which cooperativity is abolished. This reagent readily traverses the red cell membrane and prevents the sickling reaction upon deoxygenation. It appears to affect none of the activities of the red cell enzymes adversely, nor does it appear to affect the red cell membrane. Since there are several defined effects on the stereochemical status of the molecule conferred by interaction with bis-(N-maleimidomethyl) ether, the precise mechanism of the anitsickling effect remains to be elucidated. A more subtle perturberant will be required to specify a precise antisickling effect. By use of bis-(N-maleimidomethyl) ether a precise locus on the beta chain of human hemoglobin S can be perturbed to produce the desired effect.  (+info)

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure. (8/305)

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.  (+info)