calcimycin and Hemolysis

Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

Topics: Animals; Bacterial Proteins; Blotting, Western; Calcimycin; Cell Survival; Complement C9; Dose-Response Relationship, Drug; HCT116 Cells; Hemolysis; HSP70 Heat-Shock Proteins; Humans; Ionophores; K562 Cells; Leukemia, Erythroblastic, Acute; Pyridines; Rabbits; Recombinant Proteins; RNA Interference; Streptolysins; Thiazoles

High throughput methodologies that measure the distribution of osmotic fragilities in red blood cell populations have enabled the investigation of dynamic changes in red cell homeostasis and membrane permeability in health and disease. The common assumption in the interpretation of dynamic changes in osmotic fragility curves is that left or right shifts reflect a decreased or increased hydration state of the cells, respectively, allowing direct inferences on membrane transport from osmotic fragility measurements. However, the assumed correlation between shifts in osmotic fragility and hydration state has never been directly explored, and may prove invalid in certain conditions. We investigated here whether this correlation holds for red cells exposed to elevated intracellular calcium. The results showed that elevated cell calcium causes a progressive increase in osmotic fragility with minimal contribution from cell hydration (<8%). Loss of membrane area by the release of 160+/-40nm diameter (mean+/-SD) vesicles is shown to be a major contributor, but may not account for the full non-hydration component. The rest must reflect a specific calcium-induced lytic vulnerability of the membrane causing rupture before the cells attain their maximal spherical volumes. The implications of these findings are discussed.

Topics: Calcimycin; Calcium; Calcium Signaling; Cell Membrane Permeability; Cell Size; Cell-Derived Microparticles; Cells, Cultured; Clotrimazole; Erythrocytes; Hemolysis; High-Throughput Screening Assays; Humans; Microscopy, Electron, Transmission; Osmotic Fragility; Water

Sticholysin II (St II) is a toxin from the sea anemona Stichodactyla helianthus that produces erythrocytes lysis at low concentration and its activity depends on the presence of calcium. Calcium may act modifying toxin interaction with erythrocyte membranes or activating cellular processes which may result in a modified St II lytic action. In this study we are reporting that, in the presence of external K(+), extracellular calcium decreased St II activity on erythrocytes. On the other hand an increase of intracellular calcium promotes Sty II lytic activity. The effect of intracellular calcium was specifically studied in relation to membrane lipid translocation elicited by scramblases and how this action influence St II lytic activity on erythrocytes. We used 0.5 mmol/L calcium and 10 mmol/L A23187, as calcium ionophore, for scramblases activation and found increased St II activity associated to increase of intracellular calcium. N-ethyl maleimide (activator) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (inhibitor) were used as scramblases modulators in the assays which produced an increase and a decrease of the calcium effect, respectively. Results reported suggest an improved St II membrane pore-forming capacity promoted by intracellular calcium associated to membrane phospholipids translocation.

Topics: Calcimycin; Calcium; Circular Dichroism; Cnidarian Venoms; Erythrocyte Membrane; Erythrocytes; Ethylmaleimide; Hemolysis; Humans; Membrane Lipids; Osmosis; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

Cordyceps sinensis is a prized traditional Chinese medicine and its major component cordycepin is found to have anti-leukemia activities. However, its cytotoxicity in erythrocytes was unclear. To examine the effect of cordycepin on the induction of eryptosis (an apoptosis-like process in enucleated erythrocytes), flow cytometric assays based on membrane integrity and asymmetry were employed. For comparison, analyses were performed in parallel with two other anti-leukemia agents, indirubin 3'-monoxime (IDM) and As2O3. We found that at the IC50 against leukemia HL-60, cordycepin elicited eryptosis while IDM and As2O3 showed no erythrotoxicity in mouse erythrocytes. Mechanistically, cordycepin increased the [Ca2+]i and activated mu-calpain protease in a dose-dependent manner. Yet, no caspase-3 activation was observed in the cordycepin-treated erythrocytes. When extracellular Ca2+ was depleted, both the cordycepin-induced eryptosis and mu-calpain cleavage were suppressed. Our study therefore demonstrated for the first time that cordycepin induces eryptosis through a calcium-dependent pathway in the absence of mitochondria and caspase-3 activation.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Blotting, Western; Calcimycin; Calcium; Caspase 3; Cell Line, Tumor; Cell Survival; Deoxyadenosines; Dose-Response Relationship, Drug; Erythrocytes; Flow Cytometry; Hemolysis; HL-60 Cells; Humans; Indoles; Intracellular Fluid; Ionophores; Mice; Mice, Inbred BALB C; Oxides; Oximes

Red blood cells (RBCs) are reservoirs of vasodilatory, antiaggregatory, and antiinflammatory lipid mediators-epoxyeicosatrienoic acids (EETs). This study addresses the formation and release of erythrocyte-derived EETs in response to ATP receptor stimulation that may represent an important mechanism regarding circulatory regulation.. Erythrocyte EET formation and release were investigated by incubating rat RBCs in physiological salt solution with agents that effected ATP release via P2 receptor stimulation of phospholipase A2 and epoxygenase-like activities with activation of the ATP secretory mechanism. EETs were analyzed by gas and liquid chromatography-mass spectrometry.. EETs were released from rat RBCs: 14,15-, 11,12-, 8,9- and 5,6-EETs in a ratio of 1.2:1.0:0.9:0.8. EETs were produced by epoxidation of arachidonic acid catalyzed by hemoglobin. Spontaneous release of EETs, 0.66+/-0.14 ng per 10(9) RBCs, was dose-dependently increased by an ATP analog, BzATP, and inhibited by P2X(7) receptor antagonists. 5 microM ATP increased release of EETs over 20% to 0.83+/-0.15 ng per 10(9) RBCs; 10 microM BzATP tripled the amount of EET release to 1.87+/-0.20 ng per 10(9) RBCs. EET release by ATP or BzATP was not associated with hemolysis. Carbenoxolone, a gap junction inhibitor that inhibits ATP release, and glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), which is required for ATP release, inhibited the spontaneous and stimulated EET release from RBCs.. EETs are produced and released from RBCs via a mechanism that is mediated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, pannexin-1 and CFTR.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenosine Triphosphate; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Carbenoxolone; Chromatography, High Pressure Liquid; Erythrocytes; Gas Chromatography-Mass Spectrometry; Glyburide; Group IV Phospholipases A2; Hemolysis; Ionophores; Male; Niflumic Acid; Phospholipases A; Phospholipases A2; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Spectrometry, Mass, Electrospray Ionization

Previous studies have shown that ferriprotoporphyrin IX (FP) and non-heme iron have a marked inhibitory effect on the Ca(2+)-Mg(2+)-ATPase activity of isolated red cell membranes, the biochemical counterpart of the plasma membrane Ca(2+) pump (PMCA). High levels of membrane-bound FP and non-heme iron have been found in abnormal red cells such as sickle cells and malaria-infected red cells, associated with a reduced life span. It was important to establish whether sublytic concentrations of FP and non-heme iron would also inhibit the PMCA in normal red cells, to assess the possible role of these agents in the altered Ca(2+) homeostasis of abnormal cells. Active Ca(2+) extrusion by the plasma membrane Ca(2+) pump was measured in intact red cells that had been briefly preloaded with Ca(2+) by means of the ionophore A23187. The FP and nonheme iron concentrations used in this study were within the range of those applied to the isolated red cell membrane preparations. The results showed that FP caused a marginal inhibition ( approximately 20%) of pump-mediated Ca(2+) extrusion and that non-heme iron induced a slight stimulation of the Ca(2+) efflux (11-20%), in contrast to the marked inhibitory effects on the Ca(2+)-Mg(2+)-ATPase of isolated membranes. Thus, FP and non-heme iron are unlikely to play a significant role in the altered Ca(2+) homeostasis of abnormal red cells.

Topics: Biological Transport; Ca(2+) Mg(2+)-ATPase; Calcimycin; Calcium; Cell Size; Enzyme Inhibitors; Erythrocyte Membrane; Hemin; Hemolysis; Humans; Iron; Kinetics; Osmotic Pressure

Amphiphiles which induce either spiculated (echinocytic) or invaginated (stomatocytic) shapes in human erythrocytes, and ionophore A23187 plus Ca(2+), were studied for their capacity to induce shape alterations, vesiculation and hemolysis in the morphologically and structurally different lamprey and trout erythrocytes. Both qualitative and quantitative differences were found. Amphiphiles induced no gross morphological changes in the non-axisymmetric stomatocyte-like lamprey erythrocyte or in the flat ellipsoidal trout erythrocyte, besides a rounding up at higher amphiphile concentrations. No shapes with large broad spicula were seen. Nevertheless, some of the 'echinocytogenic' amphiphiles induced plasma membrane protrusions in lamprey and trout erythrocytes, from where exovesicles were shed. In trout erythrocytes, occurrence of corrugations at the cell rim preceded protrusion formation. Other 'echinocytogenic' amphiphiles induced invaginations in lamprey erythrocytes. The 'stomatocytogenic' amphiphiles induced invaginations in both lamprey and trout erythrocytes. Surprisingly, in trout erythrocytes, some protrusions also occurred. Some of the amphiphiles hemolyzed lamprey, trout and human erythrocytes at a significantly different concentration/membrane area. Ionophore A23187 plus Ca(2+) induced membrane protrusions and sphering in human and trout erythrocytes; however, the lamprey erythrocyte remained unperturbed. The shape alterations in lamprey erythrocytes, we suggest, are characterized by weak membrane skeleton-lipid bilayer interactions, due to band 3 protein and ankyrin deficiency. In trout erythrocyte, the marginal band of microtubules appears to strongly influence cell shape. Furthermore, the presence of intermediate filaments and nuclei, additionally affecting the cell membrane shear elasticity, apparently influences cell shape changes in lamprey and trout erythrocytes. The different types of shape alterations induced by certain amphiphiles in the cell types indicates that their plasma membrane phospholipid composition differs.

Topics: Abietanes; Animals; Anion Exchange Protein 1, Erythrocyte; Calcimycin; Calcium; Carboxylic Acids; Cell Size; Chlorpromazine; Cytoskeleton; Detergents; Diterpenes; Erythrocytes; Glucosides; Hemolysis; Humans; Ionophores; Lampreys; Phenanthrenes; Polyethylene Glycols; Quaternary Ammonium Compounds; Tissue Fixation; Trout

Fluorinated anesthetics can profoundly alter plasma membrane structure and function, potentially impacting cell injury responses. Because major surgery often precipitates acute renal failure, this study assessed whether the most commonly used fluorinated anesthetic, isoflurane, alters tubular cell responses to toxic and hypoxic attack.. Mouse proximal tubule segments were incubated under control conditions or with a clinically relevant isoflurane dose. Cell viability (lactate dehydrogenase release), deacylation (fatty acid, such as C20:4 levels), and adenosine triphosphate (ATP) concentrations were assessed under one or more of the following conditions: (a) exogenous phospholipase A2 (PLA2) or C20:4 addition, (b) Ca2+ overload (A23187 ionophore), (c) increased metabolic work (Na ionophore), and (d) hypoxia- or antimycin A-induced attack. Isoflurane's effect on NBD phosphatidylserine uptake (an index of plasma membrane aminophospholipid translocase activity) was also assessed.. Isoflurane alone caused trivial deacylation and no lactate dehydrogenase release. However, it strikingly sensitized to both PLA2- and A23187-induced deacylation and cell death. Isoflurane also exacerbated C20:4's direct membrane lytic effect. Under conditions of mild ATP depletion (Na ionophore-induced increased ATP consumption; PLA2-induced mitochondrial suppression), isoflurane provoked moderate/severe ATP reductions and cell death. Conversely, under conditions of maximal ATP depletion (hypoxia, antimycin), isoflurane conferred a modest cytoprotective effect. Isoflurane blocked aminophospholipid translocase activity, which normally maintains plasma membrane lipid asymmetry (that is, preventing its "flip flop").. Isoflurane profoundly and differentially affects tubular cell responses to toxic and hypoxic attack. Direct drug-induced alterations in lipid trafficking/plasma membrane orientation and in cell energy production are likely involved. Although the in vivo relevance of these findings remains unknown, they have potential implications for intraoperative renal tubular cell structure/function and how cells may respond to superimposed attack.

Topics: Adenosine Triphosphate; Amphotericin B; Anesthetics, Inhalation; Animals; Arachidonic Acid; Calcimycin; Cell Death; Energy Metabolism; Fatty Acids, Nonesterified; Hemolysis; In Vitro Techniques; Ionophores; Isoflurane; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Male; Mice; Phospholipases A; Phospholipases A2

Complement-induced procoagulant alteration of red blood cell (RBC) membranes in paroxysmal nocturnal haemoglobinuria (PNH) was examined. Microvesicles, deficient in acetylcholinesterase, were generated and released from PNH RBC upon complement activation. The microvesicles generated from complement-activated PNH RBC accelerated factor Xa-dependent plasma coagulation more than those generated from RBC by the treatment with ionophore A23187. When assessed by factor Xa-catalysed prothrombin activation, complement activation enhanced procoagulant properties of both normal and PNH RBC similarly, although PNH RBC were lysed but normal RBC were not. This enhancement of factor Xa-dependent prothrombinase activity of complement-activated RBC was inhibited by the treatment of the RBC with annexin V, a protein with binding affinity for anionic phospholipids especially for phosphatidylserine (PS). Neither the enhanced procoagulant properties of RBC nor apparent RBC population with annexin V-binding affinity were demonstrated before complement activation in any of the four PNH patients studied. PS-externalized PNH RBC and microvesicles may contribute to the removal of PNH RBC from the circulation. We conclude that although PNH RBC do not constantly exhibit enhanced procoagulant properties in vivo, complement activation induces a procoagulant alteration of RBC membranes with microvesicle formation, potentially contributing to the thrombogenesis in PNH.

Topics: Annexin A5; Blood Coagulation; Calcimycin; CD59 Antigens; Complement System Proteins; Erythrocyte Membrane; Factor Xa; Hemoglobinuria, Paroxysmal; Hemolysis; Humans

Maitotoxin (MTX), a putative Ca(2+) channel activator produced by the dinoflagellate Gambierdiscus toxicus showed extremely potent hemolytic and ichthyotoxic activities. Hemolysis of 1% mouse blood cell suspension in saline occurred at 15 nM of MTX. The activity was enhanced six-fold in the presence of 10 microM of Ca(2+) and completely blocked by EDTA2Na, indicating its dependency on external Ca(2+). The MTX-induced hemolysis was little affected by L-type Ca(2+) channel blockers (diltiazem, nifedipine, verapamil) but was strongly inhibited by calmodulin blockers (prenylamine and chlorpromazine) or a phospholipase A2 inhibitor (quinacrine). MTX was mimicked by a calcium ionophore, calcimycin. Based on these results, a series of cellular events triggered by MTX were presumed to occur in the following sequence: increased Ca(2+) entry in cells, activation of calmodulin, promotion of phospholipase A2 activity, and finally destruction of cell membrane resulting from hydrolysis of membrane lipids. The sensitivity of blood cells to MTX varied significantly, dependent on the animal sources. Nucleated blood cells of carps and chickens were 100 times more resistant than those of mammals. LC(50) of MTX to freshwater fish Tanichthys albonubes in Ca(2+) free media (pH 8) was 5 nM but was markedly lowered to 3 pM by raising pH to 8 and increasing Ca(2+) concentration to 2 mM. In a marine environment MTX was 2000 times more toxic to fish than 42-di-hydrobrevetoxin-B (PbTx-3), one of the best known ichthyotoxins of red-tide origins.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcimycin; Calcium; Calcium Channel Blockers; Fish Diseases; Fishes; Hemolysis; Hydrogen-Ion Concentration; Lethal Dose 50; Magnesium; Marine Toxins; Mice; Oxocins; Quinacrine

Human erythrocytes either untreated or Mg2+(-)loaded by means of A23187 in the presence of 3 or 12 mM MgCl2 were haemolysed by freezing and thawing. In the haemolysates the concentration of total Mg2+ (by atomic absorption spectrophotometry), free Mg2+ (by a Mg2+(-)sensitive electrode) and ATP were determined. The increase in the free Mg2+ concentration was also measured when titrating the haemolysates with MgCl2. The experiments yielded a total Mg2+ buffer capacity of 4.85mM with a Ka of 0.9 mM-1, indicating that 2,3 bisphosphoglycerate was the main Mg2+ buffer in haemolysates of erythrocytes which had been ATP-depleted during preparation. The activity coefficient of free Mg2+ in erythrocytes was the same as in the chloride-based calibration solutions.

Topics: Buffers; Calcimycin; Erythrocytes; Hemolysis; Humans; Linear Models; Magnesium; Magnetic Resonance Spectroscopy; Phosphorus

A new water-soluble, orally absorbable de-N-acetyl-lysoganglioside (WILD20), breakdown product of the monosialoganglioside GM1, was found to influence some parameters of neutrophil response to inflammation stimuli. Superoxide anion production appears inhibited, along with neutrophil killing properties. A block of both pathways of arachidonic acid cascade and PAF was also found, as well as neutrophil ICAM-1-mediated adhesion to endothelial cells. Of particular interest was the significant reduction of neutrophils observed at the site of inflammation, whichever agonist was used. The effects on neutrophil physiology found in normal or in pathological conditions, are in favour of a WILD20-related inhibitory effect on neutrophil contribution to inflammation.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Calcimycin; Cell Adhesion; Chemotaxis, Leukocyte; Eicosanoids; Erythrocytes; Gangliosides; Hemolysis; Humans; In Vitro Techniques; Inflammation; Leukocyte Count; Mice; Neutrophils; Phagocytosis; Platelet Activating Factor; Platelet Aggregation Inhibitors; Superoxides

The effect of intracellular Ca2+ on high pressure-induced hemolysis of human erythrocytes was examined. Red cells were incubated with Ca2+ (0.01-1 mM) in the presence of ionophore A23187. The Ca(2+)-loaded cells were subjected to a pressure of 200 mPa. Treatment with 0.1 mM Ca2+ had the greatest suppressive effect on the hemolysis. On removal of intracellular Ca2+, red cells showed a morphological change from echinocytes to normal discocytes but the hemolysis remained unaltered. Measurement of intracellular K+ and viscosity demonstrated that the suppressive effect of Ca2+ on the hemolysis is irreversible and is largely associated with the increase of intracellular viscosity induced by K+ efflux.

Topics: Blood Viscosity; Calcimycin; Calcium; Cells, Cultured; Erythrocytes; Hemolysis; Humans; Hydrostatic Pressure; Intracellular Fluid; Kinetics; Osmolar Concentration; Potassium; Sodium Chloride; Temperature; Time Factors

In vitro effects of thyroxine on erythrocyte deformability and mechanical fragility were observed. Deformability of erythrocytes was improved in a dose dependent manner by thyroxine. Mechanical hemolysis was found to be lower if thyroxine was included in erythrocyte suspensions at concentrations close to the physiological levels (10(-9)M). These changes might be related to the alterations of intracellular calcium concentration, as in the erythrocyte suspensions containing 10(-9)M thyroxine, intracellular calcium concentration was found to be 30 times lower than the control suspensions which did not contain thyroxine. Thyroxine also reduced the mechanical hemolysis ratio in calcium loaded cells. These observations suggest that thyroxine might play some role in the regulation of the mechanical properties of erythrocytes which might be mediated via the effects on calcium metabolism.

Topics: Adult; Calcimycin; Calcium; Erythrocyte Deformability; Hemolysis; Humans; In Vitro Techniques; Male; Osmotic Fragility; Thyroxine

The lateral mobilities of erythrocyte membrane proteins and terminal complement complexes (TCC) were measured on C-treated erythrocyte ghosts by the technique of fluorescence redistribution after photobleaching. Results showed that the lateral diffusion coefficient of the bulk membrane proteins decreased with the assembly of TCC on the membrane at low C dose and was significantly reduced with assembly of the full membrane attack complex (C5b-9), even in the absence of cell lysis. At high serum doses, the mobility of the membrane proteins increased slightly above that of the control cells. The diffusion coefficients of the TCC on the erythrocyte membrane range from 1.18 to 4.37 x 10(-11) cm2/s, values characteristic of anchored membrane proteins. Spectrin-depletion of the C-lysed erythrocytes results in 25- and 45-fold increases in the diffusion coefficients of the membrane proteins and the C5b-9 complex, respectively. Conversely, oxidative cross-linking of spectrin by diamide reduced the diffusion coefficients of both membrane and C proteins. These studies indicate that the deposition of TCC on an erythrocyte can result in a substantial change in the physical and structural properties of the target membrane, aside from the creation of functional lesions. The low mobilities of the terminal complexes on the target membrane suggest possible interactions with cytoskeletal elements or with anchored membrane proteins.

Topics: Animals; Calcimycin; Complement Membrane Attack Complex; Complement System Proteins; Cross-Linking Reagents; Cytoskeletal Proteins; Diffusion; Diphenylhexatriene; Erythrocyte Membrane; Fluorescence Polarization; Hemolysis; Humans; Membrane Lipids; Membrane Proteins; Rabbits; Sheep; Spectrin

During an examination of the effects of shear and of the Ca2+ ionophore A23187 on Ca2+ entry into erythrocytes of rats and humans, we noted that rat erythrocytes were much more sensitive to Ca2+-induced hemolysis than the human cells. An examination of the effect of Ca2+ on transglutaminase, a cytosolic enzyme in the erythrocyte which cross-links membrane proteins and renders cells less deformable, demonstrated a correlation between enzyme activity and Ca2+-induced hemolysis. Both rat and human cells subjected to shear-induced Ca2+ entry exhibited increased enzyme activity and altered membrane protein SDS-PAGE patterns. Twenty micromolar A23187 with Ca2+ at concentrations above 80 microM caused hemolysis of rat erythrocytes. In contrast to human erythrocytes, under these conditions no membranes were recoverable from rat erythrocytes. At lower concentrations of Ca2+ (25 and 50 microM), however, rat erythrocytes maintained integrity, and exhibited enhanced transglutaminase activity and cross-linking of membrane proteins. The rat enzyme can be activated 30% by 10 microM Ca2+, while 50 microM Ca2+ was necessary to achieve a similar activation of the enzyme from human red blood cells. In studies of shear-stimulated Ca2+ uptake by erythrocytes the rat red cell enzyme was more readily activated. The SDS-PAGE pattern of rat red cell membranes after a 30 sec shear showed specific changes in protein banding, including the appearance of bands greater than 330 kDa. Changes in protein banding were also apparent in cytosolic proteins. This work supports the view that shear-induced Ca2+ entry activates transglutaminase that leads to cross-linking of membrane components, a loss of cell integrity, and eventual cell death.

Topics: Animals; Blood Proteins; Calcimycin; Calcium; Drug Resistance; Erythrocytes; Hemolysis; Humans; Male; Rats; Stress, Mechanical; Transglutaminases

Ca2+ does not promote crosslinking of proteins nor stimulate proteolysis in goat and sheep erythrocyte membranes. Neither crosslinking nor proteolysis was observed even when the goat erythrocytes were loaded with calcium with the help of calcium ionophore A23187. Membrane-free human erythrocyte hemolysate, however, induced Ca2+-dependent crosslinking in goat erythrocyte membranes.

Topics: Animals; Blood Proteins; Calcimycin; Calcium; Erythrocyte Membrane; Erythrocytes; Goats; Hemolysis; Humans; Membrane Proteins; Species Specificity

The protective effects on mast cell activation were compared between a new antiallergic agent, KP-136 and disodium cromoglycate (DSCG), both of which inhibited the immunological degranulation of rat peritoneal mast cells. The IC50 was 0.03 micrograms/ml for KP-136 and 4.7 micrograms/ml for DSCG. KP-136 predominantly acted on the early stage of mast cell activation processes and inhibited the immunological increase in 45Ca uptake. KP-136 also inhibited A23187- and heat-induced degranulation and heat-induced hemolysis. In addition, KP-136 was effective on phospholipase A2-induced degranulation, although the compound did not directly affect the enzyme activity. In all tests for comparison, KP-136 and DSCG had similar profiles of action and the parallel experiments indicated that KP-136 was a more potent inhibitor of mast cell activation than DSCG, having a DSCG-like membrane stabilizing activity.

Topics: Animals; Calcimycin; Calcium; Chromones; Cromolyn Sodium; Female; Hemolysis; Hot Temperature; Immunosuppressive Agents; Mast Cells; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; Tetrazoles

In hemolysates of red cells from hypertensive patients the proteolytic activity of calpain is expressed at a rate approximately three fold higher than in red cells of normotensive subjects. Susceptibility to lysis upon exposure to ionophore A23187 and calcium, conditions that increase intracellular calpain activity, is also significantly enhanced in erythrocytes of hypertensive patients. In inside-out vesicles prepared from erythrocytes of these patients band 3 region undergoes a high extent of phosphorylation which is 1.5 fold higher than that occurring in control red cells from normotensive subjects. This increased phosphorylation can be reproduced in inside-out vesicles from erythrocytes of normal subjects following pretreatment with calpain. Taken together, these results suggest that the presence in erythrocytes of hypertensive subjects of an unregulated calpain dependent proteolytic activity may affect the structure of plasma membranes and determine an increased phosphorylation of intrinsic membrane proteins.

Topics: Anion Exchange Protein 1, Erythrocyte; Calcimycin; Calcium; Calpain; Erythrocyte Membrane; Hemolysis; Humans; Hypertension; Kinetics; Phosphorylation; Reference Values

The effects of intracellular calcium, or Cac, on the Na permeability of human red blood cells were examined during 3-h incubations with the Ca ionophore A23187 and varied external Ca, Cao. Above 3 microM Cao, Nac increased significantly as ATP decreased. Maintenance of normal ATP with vanadate did not prevent the gain of Nac. Similar amounts of Nac were gained in 3 h by ouabain-treated cells exposed to the K ionophore valinomycin or by cells osmotically shrunken. Cells shrunken with sucrose also exhibited partial loss of Kc. When the cells with increased Nac were subsequently transferred to Na-free, high-K medium, the Nac and Kc that had changed slowly over 3 h returned toward normal within 10 min. The development of irreversible high cation permeability in shrunken cells was not prevented by a variety of transport inhibitors. These observations and cell volume distributions suggest that prolonged shrinkage induces a subpopulation of cells to become highly cation permeable, or "prolytic". The major effect of Cac on Na permeability appears to be an indirect consequence of cell shrinkage due to KCl loss.

Topics: Adult; Biological Transport; Calcimycin; Calcium; Cell Membrane Permeability; Chlorides; Egtazic Acid; Electric Conductivity; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Membrane Fluidity; Membrane Lipids; Membrane Potentials; Potassium; Sodium

Ca2+ was introduced into fresh and ATP-depleted chicken erythrocytes through the aid of the inophore A23187. Intracellular Ca2+ (10-40 mM) induced fusion in ATP-depleted cells after 30-60 min incubation at 37 degrees C, but not in fresh cells. Fresh cells underwent a higher degree of haemolysis than ATP-depleted cells after accumulation of Ca2+. Uptake of Ca2+ was the same in these two systems. Intracellular Ca2+ induced rearrangement of intramembranous particles, as revealed by freeze-etching studies. The intramembranous particles in the protoplasmic face of fractured membranes obtained from fresh cells incubated with 1 mM of Ca2+ were more scattered and their density was lower than in control cells. Incubation with higher concentrations of Ca2+ (10-40 mM) induced transient changes in the intramembranous particles' density with the appearance of protrusions and depressions on the protoplasmic and exoplasmic faces of the fractured membranes, respectively. These effects were reversible upon removal of Ca2+ by washing the cells with ethyleneglycol bis(alpha-aminoethylether)-N,N'-tetraacetic acid; rearrangement of intramembranous particles was less evident after accumulation of Ca2+ in ATP-depleted cells, whose fractured membranes did not contain any protrusions or depressions. Transferring Ca2+-loaded cells to the cold caused the formation of large smooth areas devoid of intramembranous particles in the protoplasmic face of the fractured membranes. Cells containing Ca2+ appeared spherical, and removal of Ca2+ restored the normal oval shape of chicken erythrocytes.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Calcimycin; Calcium; Chickens; Erythrocytes; Freeze Etching; Hemolysis; Kinetics; Microscopy, Electron

Topics: Anti-Bacterial Agents; Calcimycin; Calcium; Child; Child, Preschool; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Muscular Dystrophies; Osmolar Concentration; Potassium; Sodium

1. Obelin, the Ca(2+)-activated luminescent protein from the hydroid Obelia geniculata, was sealed inside pigeon erythrocyte ;ghosts' in order to investigate effects on their permeability of different methods of preparation and of the bivalent cation ionophore A23187. 2. Changes in free Ca(2+) within the ;ghosts' were studied by following the rate of luminescence of obelin. The possibility that the obelin might have been released from the ;ghosts' during an experiment was investigated by studying the release of inulin and pyruvate kinase from the ;ghosts'. Less than 10% of the inulin or pyruvate kinase sealed within the ;ghosts' was released under any of the experimental conditions. 3. Triton X-100 (0.1-10%, v/v) made the ;ghosts' highly permeable to Ca(2+). In the presence of 1mm-Ca(2+) and Triton, 95-100% of the obelin was utilized within 10-20s. 4. A time-course of resealing ;ghosts' at 37 degrees C showed that over a period of 90min, the ;ghosts' became gradually less permeable to Ca(2+). ;Ghosts' which remained at 0 degrees C retained only a small concentration of obelin and ATP, and were highly permeable to Ca(2+). 5. Erythrocyte ;ghosts' resealed for 30min at 20 degrees C rather than 37 degrees C were more permeable to Ca(2+), as shown by the fact that 92% of the obelin in the ;ghosts' was utilized during the first 60s after the addition of 1mm-Ca(2+), as opposed to 44% for ;ghosts' resealed at 37 degrees C. 6. Haemolysis at pH6.0 rather than 7.0 resulted in ;ghosts' which were highly permeable to Ca(2+) after resealing for 60min at 37 degrees C. Of the obelin in the ;ghosts', produced by haemolysis at pH6.0, 90% was utilized in the first 60s after the addition of 1mm-Ca(2+) compared with 23% for ;ghosts' produced at pH7.0. 7. The bivalent cation ionophore A23187 increased the permeability of the ;ghosts' to Ca(2+). Maximum effects of the ionophore (16mug/ml) were obtained by preincubating the ;ghosts' with the ionophore A23187 (16mug/ml) in the presence of a low concentration of Mg(2+) and in the absence of Ca(2+).

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Cell Membrane Permeability; Columbidae; Erythrocytes; Hemoglobins; Hemolysis; Hydrogen-Ion Concentration; In Vitro Techniques; Inulin; Ionophores; Luminescent Measurements; Luminescent Proteins; Polyethylene Glycols; Proteins; Pyruvate Kinase; Temperature; Time Factors