phosphorus-radioisotopes and Anemia--Sickle-Cell

Deoxygenation (DO) of sickle cell anemia red blood cells (SS cells) induces membrane permeabilization to Ca2+, Na+, and K+ and cell dehydration mostly through the activation of the Ca(2+)-dependent K+ channels. We show that DO of both SS cells and normal red blood cells was accompanied by a nonspecific dephosphorylation of membrane proteins. After treatment with a protein kinase C activator (phorbol myristate acetate) or a phosphoprotein phosphatase inhibitor (okadaic acid), the level of membrane protein phosphorylation in deoxygenated cells was maintained higher or equal, respectively, to that of the oxygenated controls. We found that these drugs in SS cells (1) inhibited by 40% the DO-stimulated net Ca2+ uptake, without affecting the DO-stimulated Ca2+ influx, suggesting that they activated the Ca2+ efflux; (2) slightly increased the DO-induced Na+ uptake and decreased the DO-induced K+ loss; and (3) prevented the DO-induced cell dehydration. Both drugs are known to stimulate both phosphorylation and activity of the Ca pump and of the Na/H antiport. Inhibition of SS cell dehydration might be due to an activation of the Ca pump preventing [Ca2+]i elevation responsible for the stimulation of the K+ channels and/or to an activation of the Na/H exchange resulting in cell water gain.

Topics: Adenosine Triphosphate; Anemia, Sickle Cell; Biological Transport; Calcium; Calcium-Transporting ATPases; Desiccation; Erythrocyte Membrane; Erythrocytes; Ethers, Cyclic; Humans; In Vitro Techniques; Kinetics; Membrane Proteins; Okadaic Acid; Oxygen; Phosphates; Phosphorus Radioisotopes; Potassium; Protein Kinase C; Protein Tyrosine Phosphatases; Reference Values; Sodium; Tetradecanoylphorbol Acetate

Topics: Anemia, Sickle Cell; Animals; DNA; Genes; Genetic Engineering; Humans; Nucleic Acid Hybridization; Phosphorus Radioisotopes; RNA

Sickle-cell-anaemia erythrocytes (SS cells) are known to have a high Ca2+ content (particularly the dense cell fraction) and to take up Ca2+ on deoxygenation. It has been reported that this high Ca2+ was responsible for the activation of the Ca2+-dependent K+ loss, and of the Ca2+-sensitive polyphosphoinositide phospholipase C (PIC) in dense SS cells. We found that, either in the total population of SS cells or in the light or dense fractions, the content of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was not changed, whereas that of phosphatidylinositol 4-phosphate was increased and that of phosphatidic acid (PtdOH) was decreased compared with normal (AA) erythrocytes. Deoxygenation-induced Ca2+ entry into SS cells did not change the concentration or, in 32P-prelabelled cells, the radioactivity of polyphosphoinositides and PtdOH. It also failed to induce the formation of inositol 1,4,5-trisphosphate, the product of PtdIns(4,5)P2 hydrolysis by PIC, which was measured by an original method using ion-pair reverse-phase h.p.l.c. Thus there was no evidence of an endogenous Ca2+ effect on the PIC activity in SS cells, in agreement with the demonstration that the excess Ca2+ in SS cells is compartmentalized into internal vesicles and unavailable as free Ca2+. The 32P incorporation in polyphosphoinositides and PtdOH was markedly higher in SS than in AA cells, but this increase was the same in both dense and light SS cells. The increase in the turnover of these phospholipids in SS cells is consistent either with an activation of the lipid kinases and phosphatases or with perturbation in the metabolic compartmentation of these lipids.

Topics: Adenosine Triphosphate; Anemia, Sickle Cell; Calcium; Chromatography, High Pressure Liquid; Erythrocytes; Erythrocytes, Abnormal; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Lipids; Oxidation-Reduction; Phosphatidic Acids; Phosphatidylinositols; Phosphoinositide Phospholipase C; Phospholipids; Phosphoric Diester Hydrolases; Phosphorus Radioisotopes

For 25 subjects with sickle cell anemia the mean red cell life span measured with Di-isopropylfluorophosphite-32P (DF32P) was 17.32 +/- 4.51 days. Performed simultaneously, the half life (T1/2 of radioactively-labelled chromium 51Cr) was 10.11 +/- 2.82 days (14 subjects). Eight additional subjects, or more than 30 per cent of those studied using both red cell tags, had 51Cr red cell survival curves better described by two exponents than by one, apparently due to two different rates of 51Cr elution from the red cells. This finding limits the value of quantitative data obtained by this procedure. A negative correlation was found between the mean red cell life span measured with DF32P-tagged cells and the proportion of irreversibly sickled cells in venous or capillary blood. A similar negative correlation was found between the red cell half survival time measured with 51Cr-tagged cells and the proportion of irreversibly sickled cells. These data are compatible with the view that repeated sickling and, in particular, the formation of irreversibly sickled cells play a distinct role in the pathogenesis of hemolysis in sickle cell anemia.

Topics: Anemia, Sickle Cell; Blood Cell Count; Chromium Radioisotopes; Erythrocyte Aging; Half-Life; Hemolysis; Humans; Isoflurophate; Phosphorus Radioisotopes; Reticulocytes

Red cell survival was measured in ten subjects with S-C disease and one with S-O Arab (alpha 2 beta 2-121 glu yields lys) disease using both DF32p and 51Cr as tags. Red cell volume was slightly reduced in most patients (87% plus or minus 20% of predicted normal). In nine SC patients, mean red cell life (DF32p) was 28.9 plus or minus 4.0 days. For one SC subject it was significantly longer (47.9 days), as it was for the one with S-O Arab. The S-O Arab subject had irreversibly sickled cells in the peripheral blood, shereas those with SC had few (less than 1/1000 red cells) or none. The S-O Arab hemolysate gelled at a hemmoglobin concentration (16.2 g/100ml) near that for sickle cell anemia hemolysates (15.9 plus or minus 1.0 g/100 ml; n equals 8) but significantly lower than that for SC hemolysates (21.6 plus or minus 1.9 g/100 ml; n equals 5). It seems likely that properties of S-C red cells other than their relative ease of sickling contribute significantly to their rate of hemolysis.

Topics: Adult; Anemia, Sickle Cell; Cell Survival; Chromium Radioisotopes; Erythrocytes, Abnormal; Female; Gels; Hemoglobin C Disease; Humans; Phosphorus Radioisotopes

Topics: Adult; Aged; Anemia, Pernicious; Anemia, Sickle Cell; Cell Survival; Chromium Radioisotopes; Erythrocytes; Female; Half-Life; Hemoglobins, Abnormal; Humans; Isoflurophate; Male; Middle Aged; Phosphorus Radioisotopes

Topics: Adult; Anemia, Sickle Cell; Blood Protein Electrophoresis; Chromium Radioisotopes; Diagnosis, Differential; Erythrocyte Count; Erythrocytes; Female; Hematocrit; Hemoglobin, Sickle; Hemoglobinopathies; Hemoglobins, Abnormal; Humans; Male; Oxygen; Phosphorus Radioisotopes; Physical Exertion; Reticulocytes; Syndrome

Topics: Adenosine Triphosphate; Anemia, Hemolytic; Anemia, Sickle Cell; Autoradiography; Avian Myeloblastosis Virus; Cell-Free System; Centrifugation, Density Gradient; Chromatography, Affinity; Chromatography, DEAE-Cellulose; DNA; DNA Nucleotidyltransferases; Electrophoresis, Disc; Escherichia coli; Globins; Hemoglobin H; Hemoglobinopathies; Hemolysis; Humans; Nucleic Acid Hybridization; Oligonucleotides; Pancreas; Phosphorus Radioisotopes; Poly U; Reticulocytes; Ribonucleases; RNA, Messenger; Thalassemia; Ultracentrifugation

Topics: Alkaline Phosphatase; Anemia, Sickle Cell; Animals; Avian Myeloblastosis Virus; Base Sequence; Centrifugation, Density Gradient; Chromatography, Gel; Chromatography, Thin Layer; DNA; DNA-Directed RNA Polymerases; Ducks; Escherichia coli; Globins; Humans; Iodine Radioisotopes; Mice; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Protein Biosynthesis; Rabbits; Ribonucleases; Ribonucleotides; RNA, Messenger; Species Specificity; Transcription, Genetic; Tritium

By using three isotopes of diisopropyl-phosphofluoridate ([(3)H]-, [(14)C]-, and [(32)P]DFP) simultaneously, the life span of red cells from 20 patients with sickle cell anemia (Hb SS) has been studied after varying degrees of carbamylation in vitro with cyanate (NCO) and carbamyl phosphate (CP). The results are expressed in terms of the red cell mean life span (MLS). The MLS of red cells in the patients studied averaged 15.2+/-6.3 (SD) days. After carbamylation the increase in red cell life span was linearly proportional to the concentration of cyanate used, so that at 0.01. 0.02, and 0.3 M NCO (approximately 1, 1.6, and 2 mol NCO/mol Hb) the average increase in MLS was 8.14+/-4.9 days, 14.7+/-4.1 days, and 18.4+/-8.8 days, respectively. Analysis of survival curves and the results of an experiment using a population of Hb SS cells separated by centrifugation indicated that carbamylation had a disproportionate effect on the survival of the youngest cells in the population. Improvement in MLS correlated with the reticulocyte count of the cells carbamylated. This finding is explained on the hypothesis that the life span of irreversibly sickled and other damaged cells is not improved by carbamylation but that carbamylation greatly improves the life span of the young, and as yet undamaged, cells. For this reason extracorporeal carbamylation is not favored as a form of therapy. At the level of carbamylation attainable by oral therapy, however, it would appear likely that only a modest increase in red cell life span will be achieved.

Topics: Adolescent; Adult; Anemia, Sickle Cell; Carbamates; Carbon Radioisotopes; Erythrocyte Aging; Erythrocytes; Female; Humans; Isoflurophate; Male; Phosphorus Radioisotopes; Tritium

Topics: Anemia; Anemia, Sickle Cell; Humans; In Vitro Techniques; Phosphorus; Phosphorus Radioisotopes; Radioactivity; Sickle Cell Trait