cytochalasin-b and Hemolysis

Abstract  Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.

Topics: Aldehydes; Anemia, Sickle Cell; Cell Membrane; Cytochalasin B; Erythrocytes, Abnormal; Hematologic Tests; Hemoglobins; Hemolysis; Humans; Hydrogen-Ion Concentration; Polymerization; Prognosis; Sucrose; Temperature; Urea

Some researches have shown that melatonin (MLT) has effects on the erythrocyte deformability and on the osmotic fragility which, currently, seem to be heterogeneous and fragmentary.. This work wished to evaluate in vitro the possible modifying action of MLT at pharmaceutical doses on the osmotic fragility of rat blood samples treated with cytochalasin B (CB). The variation of the lactate dehydrogenase (LDH) levels have been measured because LDH is an enzyme which is considered an important marker of hemolysis. Working in a strongly reducing environment has been necessary in order to avoid possible antioxidant actions of MLT, measuring the plasma levels of malondialdehyde and total phospholipids in order to highlight possible MLT actions which are not related to its recognized antioxidant properties.. The data show a possible MLT action which strengthens the CB action on the osmotic fragility and on the membrane deformability of the erythrocytes.. Under the adopted working conditions, a direct relation of MLT with the biochemical dynamics of the cytoskeleton-mediated processes can be suggested. Further studies will be needed to clarify the mechanisms and the extent of the observed phenomena.

Topics: Actin Cytoskeleton; Actins; Animals; Antioxidants; Biomarkers; Cytochalasin B; Erythrocyte Deformability; Erythrocyte Membrane; Erythrocytes; Hemolysis; L-Lactate Dehydrogenase; Malondialdehyde; Melatonin; Osmotic Fragility; Phospholipids; Protein Structure, Secondary; Rats; Rats, Wistar

Standard models for carrier-mediated nonelectrolyte transport across cell membranes do not explain sugar uptake by human red blood cells. This means that either (1) the models for sugar transport are incorrect or (2) measurements of sugar transport are flawed. Most measurements of red cell sugar transport have been made over intervals of 10 s or greater, a range which may be too long to measure transport accurately. In the present study, we examine the time course of sugar uptake over intervals as short as 5 ms to periods as long as 8 h. Using conditions where transport by a uniform population of cells is expected to be monophasic (use of subsaturating concentrations of a nonmetabolizable but transported sugar, 3-O-methylglucose), our studies demonstrate that red cell sugar uptake is comprised of three sequential, protein-mediated events (rapid, fast, and slow). The rapid phase is more strongly temperature-dependent than the fast and slow phases. All three phases are inhibited by extracellular (maltose or phloretin) or intracellular (cytochalasin B) sugar-transport inhibitors. The rate constant for the rapid phase of uptake is independent of the 3-O-methylglucose concentration. The magnitude (moles of sugar associated with cells) of the rapid phase increases in a saturable manner with [3-O-methylglucose] and is similar to (1) the amount of sugar that is retained by red cell membrane proteins upon addition of cytochalasin B and phloretin and (2) the d-glucose inhibitable cytochalasin B binding capacity of red cell membranes. These results are consistent with the hypothesis that previous studies have both under- and overestimated the rate of erythrocyte sugar transport. These data support a transport mechanism in which newly bound sugars are transiently sequestered within the translocation pathway where they become inaccessible to extra- and intracellular water.

Topics: 3-O-Methylglucose; Binding Sites; Biological Transport, Active; Cytochalasin B; Erythrocyte Membrane; Extracellular Fluid; Glucose Transporter Type 1; Hemolysis; Humans; Hypotonic Solutions; Intracellular Fluid; Maltose; Models, Biological; Models, Chemical; Monosaccharide Transport Proteins; Phloretin; Temperature; Time Factors; Tritium

Red blood cell (RBC) swelling and membrane hole formation in hypotonic external media were studied by measuring the time-dependent capacitance, C, and the conductance, G, in the beginning of the beta-dispersion range. At high and moderate osmolarities of the external solution the capacitance reaches a steady-state whereas at low osmolarities it reveals a biphasic kinetics. Examination of RBC suspensions exposed to different concentrations of HgCl(2) demonstrates that water transport through mercury-sensitive water channel controls RBC swelling. Unlike the capacitance, an increase in the conductance to a stationary level is observed after a certain delay. A comparison of G(t) curves recorded for the suspensions of the intact cells and those treated with cytochalasin B or glutaraldehyde demonstrates the significant effect of the membrane viscoelasticity on the pore formation. It is shown that the stretched membrane of completely swollen RBC retains its integrity for a certain time, termed as the membrane lifetime, t(memb). Therefore, the resistivity of RBCs to a certain osmotic shock may be quantified by the distribution function of RBC(t(memb)).

Topics: Aquaporins; Cell Size; Culture Media; Cytochalasin B; Elasticity; Electric Capacitance; Electric Conductivity; Erythrocytes; Glutaral; Hemolysis; Humans; Hypotonic Solutions; Kinetics; Osmolar Concentration; Time Factors; Water

A multitarget functional bioassay was optimized as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes, mainly to release elastase detected by p-nitroanilide (pNA) formation. Using this bioassay, 100 fractionated extracts of 96 plants were screened, with results presented in a manner that links recorded biological activity to phylogenetic information. The plants were selected to represent a major part of the angiosperms, with emphasis on medicinal plants, Swedish anti-inflammatory plants, and plants known to contain peptides. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. The extract of Digitalis purpurea enhanced pNA formation, and digitoxin, the active compound, was isolated and identified. Plant extracts that exhibited potent nonselective inhibition (>80% inhibition) were evaluated further for direct inhibition of isolated elastase and trypsin enzyme. The inhibitory effect of most tested extracts on the isolated enzyme elastase was similar to that of PAF- and fMLP-induced pNA formation. Compared to trypsin, inhibition of elastase by extracts of Rubus idaeus and Tabernaemontana dichotoma was significantly higher (80% and 99%, respectively). Inhibition of trypsin by the extract of Reseda luteola was high (97%). Orders such as Lamiales and Brassicales were shown to include a comparably high proportion of plants with inhibitory extracts.

Topics: alpha-Amylases; Anti-Inflammatory Agents; Benzoylarginine Nitroanilide; Biological Assay; Biological Products; Cytochalasin B; Digitalis; Digitonin; Digitoxin; Dose-Response Relationship, Immunologic; Hemolysis; Humans; Lamiaceae; Leukocyte Elastase; Magnoliopsida; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Peptides; Plant Proteins; Plants, Medicinal; Platelet Activating Factor; Superoxides; Sweden; Tabernaemontana; Trypsin Inhibitors; Violaceae

Topics: Animals; Antibodies; Cell Adhesion; Complement System Proteins; Cytochalasin B; Erythrocyte Aging; Erythrocytes; Fluorescent Antibody Technique; Hemolysis; Humans; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin G; Phagocytosis; Rabbits; Rh-Hr Blood-Group System

The mechanism by which human monocytes increase the osmotic fragility of red cells sensitized with Rhesus alloantibodies anti-D was studied in vitro. Both the increase in osmotic fragility and the lysis of red cells by monocytes were enhanced by cytochalasin B and were inhibited by hydrocortisone. These effects were similar to the effects of these agents on lysosomal enzyme release by monocytes. However, hydrocortisone was completely ineffective when added 1 h after mixing monocytes and sensitized red cells. This indicates that the damage responsible for the fragility increase and lysis is completed within 1 h and suggests that it is due to lysosomal enzymes released by the monocytes. Since for the full expression of the osmotic fragility increase and lysis an incubation time much longer than 1 h is required, it appears that the latter phenomena are the non-specific sequelae of damage inflicted upon the red cell by released lysosomal enzymes.

Topics: Cytochalasin B; Erythrocytes; Hemolysis; Humans; Hydrocortisone; Isoantibodies; Monocytes; Osmotic Fragility; Rh-Hr Blood-Group System; Time Factors

Topics: Adult; Animals; Chromatography, Gel; Complement System Proteins; Cytochalasin B; Dithiothreitol; Erythrocytes; Glucuronidase; Hemolysis; Humans; Immune Sera; Immunoelectrophoresis; L-Lactate Dehydrogenase; Leukocytes; Lysosomes; Polyethylene Glycols; Rabbits

Topics: Animals; Complement System Proteins; Cytochalasin B; Cytoplasm; Edetic Acid; Glucuronidase; Hemolysis; Humans; L-Lactate Dehydrogenase; Leukocytes; Lysosomes; Male; Phagocytosis; Snakes; Venoms; Zymosan