The basis for EDTA-stimulation of methemoglobin reduction in hemolysates of human erythrocytes.

We have studied the stimulation by EDTA of methemoglobin reduction in hemolysates of human erythrocytes. The EDTA effect has been shown not to be the result of an allosteric interaction of EDTA with hemoglobin or the result of a photochemical reduction. The effect does not appear to be due to a direct interaction of free EDTA with either of the catalytic components of the erythrocyte methemoglobin reduction system. The EDTA stimulation seen in hemolysates is due to the formation of an iron-EDTA complex, which transfers electrons from the reductase to methemoglobin.     

Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide

Human neutrophils incubated with phorbol myristate acetate oxidized hemoglobin within the intact erythrocyte by a mechanism dependent on cell-cell contact but independent of phagocytosis. Spectrophotometric examination of the erythrocyte lysates revealed that the major component formed was methemoglobin along with small amounts of a species with spectral characteristics similar to choleglobin. Methemoglobin formation was directly related to the neutrophil concentration and the time of incubation. The addition of superoxide dismutase or catalase modestly inhibited the formation of methemoglobin, while a combination of the enzymes provided the most dramatic protection. Methemoglobin of hydroxyl radical or hypochlorous acid scavengers. Apparently, either O2.- or H2O2 alone was capable of mediating methemoglobin formation in the intact erythrocyte. Maintenance of the intraerythrocytic hemoglobin in its oxygenated state or its derivatization to carbon monoxyhemoglobin markedly inhibited methemoglobin formation. Blockade of the anion channels in the intact erythrocyte with sulfonated stilbenes inhibited O2.- but not H2O2 from oxidizing intracellular hemoglobin. It appears that neutrophil-derived O2.- and H2O2 can cross the erythrocyte membrane through the anion channel or diffuse directly into the intracellular space and react with oxyhemoglobin or deoxyhemoglobin to form a mixture of hemoglobin oxidation products within the intact cell.     

Curcumin inhibits nitrite-induced methemoglobin formation.

Curcumin protects hemoglobin from nitrite-induced oxidation to methemoglobin. The protection is not observed when curcumin is added after the autocatalytic stage of the oxidation of hemoglobin by nitrite. The ability of curcumin to scavenge superoxide may be responsible since superoxide is implicated in promoting the autocatalytic stage of oxidation of hemoglobin by nitrite.     

Hemoglobin vesicles containing methemoglobin and L-tyrosine to suppress methemoglobin formation in vitro and in vivo

Hemoglobin (Hb) vesicles have been developed as cellular-type Hb-based O(2) carriers in which a purified and concentrated Hb solution is encapsulated with a phospholipid bilayer membrane. Ferrous Hb molecules within an Hb vesicle were converted to ferric metHb by reacting with reactive oxygen species such as hydrogen peroxide (H(2)O(2)) generated in the living body or during the autoxidation of oxyHb in the Hb vesicle, and this leads to the loss of O(2) binding ability. The prevention of metHb formation by H(2)O(2) in the Hb vesicle is required to prolong the in vivo O(2) carrying ability. We found that a mixed solution of metHb and L-tyrosine (L-Tyr) showed an effective H(2)O(2) elimination ability by utilizing the reverse peroxidase activity of metHb with L-Tyr as an electron donor. The time taken for the conversion of half of oxyHb to metHb (T(50)) was 420 min for the Hb vesicles containing 4 g/dL (620 microM) metHb and 8.5 mM L-Tyr ((metHb/L-Tyr) Hb vesicles), whereas the time of conversion for the conventional Hb vesicles was 25 min by stepwise injection of H(2)O(2) (310 microM) in 10 min intervals. Furthermore, in the (metHb/L-Tyr) Hb vesicles, the metHb percentage did not reach 50% even after 48 h under a pO(2) of 40 Torr at 37 degrees C, whereas T(50) of the conventional Hb vesicles was 13 h under the same conditions. Moreover, the T(50) values of the conventional Hb vesicles and the (metHb/L-Tyr) Hb vesicles were 14 and 44 h, respectively, after injection into rats (20 mL/kg), confirming the remarkable inhibitory effect of metHb formation in vivo in the (metHb/L-Tyr) Hb vesicles.     

Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin.

The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte.     

Knockdown of tight junction protein claudin-2 prevents bile canalicular formation in WIF-B9 cells

The polarization of hepatocytes involves formation of functionally distinct sinusoidal (basolateral) and bile canalicular (apical) plasma membrane domains that are separated by tight junctions. Although various molecular mechanisms and signaling cascades including polarity complex proteins may contribute to bile canalicular formation in hepatocytes, the role of tight junction proteins in bile canalicular formation remains unclear. To investigate the role of the integral tight junction protein claudin-2 in bile canalicular formation, we depleted claudin-2 expression by siRNA in the polarized hepatic cell line WIF-B9 after treatment with or without phenobarbital. When WIF-B9 cells were treated with phenobarbital, claudin-2 expression and tight junction strands were markedly increased together with induction of canalicular formation with a biliary secretion function. Knockdown of claudin-2 prevented bile canalicular formation after treatment with or without phenobarbital. Furthermore, knockdown of claudin-2 caused a change from a hepatic polarized phenotype to a simple polarized phenotype, together with upregulation of pLKB1, pMAPK, pAkt and pp38 MAPK, but not pMLC, PTEN or cdc42, and an increase of intracellular vacuoles, which were present before bile canalicular formation. These results suggest that claudin-2 may affect not only the bile canalicular seal but also bile canalicular formation.     

NMR relaxation of protein and water protons in methemoglobin solutions.

Hemoglobin (Hb) proton spins rapidly equilibrate among themselves after an initial excitation, and relax toward thermal equilibrium as a unit. In the diamagnetic form, spin diffusion to nearby methyl relaxation sinks can account for this. For metHb, four strong heme relaxation centers dominate, and spin diffusion must occur over long distances. A sizeable difference in protein T1 is found between H2O and D2O solutions, much more than for diamagnetic Hb, consistent with internal H2O acting as a spin carrier to the heme.     

Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes.

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65-70% of the rate with NADH. Added cytochrome b5 stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b5 and cytochrome b5 reductase determine the rate of methemoglobin reduction in hemolysates.     

Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improves myocardial function and coronary perfusion in senescent hearts.

Cardiovascular ageing is associated with an increase in cardiac susceptibility to ischaemia and reperfusion and production of reactive oxygen species has been suspected to be responsible for this age-associated particular vulnerability. To determine whether administration of antioxidant treatment could afford some protection against ischaemia and reperfusion during aging, isolated perfused hearts from adult and senescent rats were submitted to normoxia (180 min), prolonged low-flow ischaemia (15% of initial coronary flow;180 min) or low-flow ischaemia/reperfusion (45 min/30 min), without or with antioxidant enzymes (superoxide dismutase+catalase; 50IU/ml). Contractile function and coronary perfusion were measured and protein oxidation was quantitated in left ventricle after normoxia, ischaemia and ischaemia/reperfusion. Protein oxidation was higher in senescent than in adult hearts after ischaemia-reperfusion, in contrast to prolonged ischaemia. During prolonged ischaemia, antioxidant treatment prevented coronary vasoconstriction at both ages and delayed contractile dysfunction in senescent hearts but did not limit protein oxidation. During reperfusion, antioxidant treatment prevented coronary vasoconstriction and protein oxidation at both ages and considerably improved recovery of contractile function in senescent hearts. In conclusion, antioxidant treatment fully protects the senescent heart against ischaemia/reperfusion but not against prolonged ischaemia injury, indicating that oxidative stress plays a central role in the age-associated vulnerability to ischaemia-reperfusion.     

Oxidation of hemoglobin to methemoglobin in intact erythrocyte by a hydroperoxide induces formation of glutathionyl hemoglobin and binding of alpha-hemoglobin to membrane

Biochemical consequences of oxidation of hemoglobin (Hb) in intact human erythrocytes were studied. The incubation of washed erythrocyte with 1mM tert-butylhydroperoxide induced an increase in glutathionyl-Hb (G-Hb). The formation of G-Hb occurred linearly until 10 min in parallel with the formation of methemoglobin (metHb) after exhaustion of reduced glutathione. The results show that metHb, but not normal Hb, reacts with oxidized glutathione to form G-Hb. G-Hb was confirmed by immunoblotting with anti-glutathione antibody and the formation of G-Hb was accompanied by parallel decrease in beta-globin detected with a reversed phase HPLC. Electrophoretic studies showed time-dependent increase in membrane-associated alpha-Hb until 10 min, indicating that a part of unpaired alpha-Hb bound to the membrane. Pre-beta-globin increased despite the decrease in beta-globin and a part of the increase was independent of the decrease in beta-globin. Pre-beta-globin reacted with anti-glutathione antibody, but it differs from G-Hb in many features.     

Effect of glucocorticoids on the formation of methemoglobin in rats

Experiments on rats showed that treatment of rat with hydrocortisone, dexamethasone and diplacin for 14 days increases the content of methemoglobin in blood. In vitro experiments revealed that dexamethasone in a concentration of 10(-6) M increased the amount of methemoglobin in the whole blood and did not change its amount in hemolysate. The data obtained show the participation of erythrocyte membranes in methemoglobin-forming effect of the studied substances.     

MAP2 prevents protein aggregation and facilitates reactivation of unfolded enzymes.

It is well established that in addition to its functional role in cell motility, cell division and intracellular transport, cytoskeletal protein tubulin also possesses significant chaperone-like activity. In vitro studies from our laboratory showed that dimeric tubulin can prevent stress induced aggregation of substrate proteins, can resist thermal deactivation of enzymes and can also refold enzymes from their fully denatured state [Manna, T., Sarkar, T., Poddar, A., Roychowdhury, M., Das, K.P. & Bhattacharyya, B. (2001) J. Biol. Chem.276, 39742-39747]. Negative charges of the C-termini of both subunits of tubulin are essential for this chaperone-like property as the deletion of only beta-C-terminus or the binding of a 14-residue basic peptide P2 to the alpha-C-terminus completely abolishes this property [Sarkar, T., Manna, T., Bhattacharyya, S., Mahapatra, P., Poddar, A., Roy, S., Pena, J., Solana, R., Tarazona, R. & Bhattacharyya, B. (2001) Proteins Struct. Funct. Genet.44, 262-269]. Based on these results, one would expect that the microtubular proteins (MTP, tubulin with microtubular-associated proteins, i.e. MAPs bound to the C-terminus) should not possess any chaperone-like activity. To our surprise we noticed excellent chaperone-like activity of MTP. MTP prevents chemical and thermal aggregation of other proteins and can enhance the extent of refolding of fully unfolded substrate enzymes. Because MTP contains tubulin as well as several MAPs bound to the C-termini of tubulin, we fractionated and purified microtubular associated protein 2 (MAP2) and tau using phosphocellulose chromatography. Experiments with purified proteins demonstrated that it is the MAP2 of MTP that exhibits significant chaperone-like activity. This has been shown by the prevention of dithiothreitol-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase and regain of enzymatic activity during refolding of unfolded substrates. Tau, which shares a homologous C-terminal domain with MAP2, possesses no such activity.