okadaic-acid and Anemia--Sickle-Cell

Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 +/- 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 +/- 10% per 100 meq/l). Increases in membrane PP2A activity (270 +/- 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 +/- 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes.

Topics: Anemia, Sickle Cell; Biological Transport; Carrier Proteins; Cell Size; Cytosol; Dehydration; Enzyme Inhibitors; Erythrocytes; Humans; Ionophores; K Cl- Cotransporters; Marine Toxins; Membrane Proteins; Nystatin; Okadaic Acid; Osmolar Concentration; Oxazoles; Phosphoprotein Phosphatases; Serine; Symporters; Threonine; Water-Electrolyte Balance

1. The effect of okadaic acid and sodium fluoride on swelling- and N-ethylmaleimide (NEM)-stimulated KCl cotransport was examined in blood cells from homozygote sickle cell anaemia patients. 2. Blood was drawn into heparin or EDTA by vein puncture from sickle cell patients previously diagnosed in the haematology clinics of Princess Badee'a Teaching Hospital. A standard method for measuring flux by using radioactive rubidium was used. 3. Okadaic acid strongly inhibited swelling-stimulated KCl cotransport if added before swelling. Okadaic acid and sodium fluoride added before NEM inhibited the activation of transport by NEM. Okadaic acid added after NEM did not inhibit transport. 4. The inhibition of the effects of NEM by okadaic acid and sodium fluoride indicates that activation of the flux by NEM requires the action of phosphatase.

Topics: Anemia, Sickle Cell; Dose-Response Relationship, Drug; Erythrocytes, Abnormal; Ethylmaleimide; Hemoglobin, Sickle; Homozygote; Humans; Ion Transport; Okadaic Acid; Potassium; Sodium Fluoride

Changes in the degree of sickling in vitro of reticulocytes and nonreticulocytes from patients with sickle cell disease were studied under complete deoxygenation (PO2 = 0 mm Hg) and partial deoxygenation (PO2 = 30 mm Hg, the average PO2 in the venous circulation) conditions at pH 7.4. Degree of sickling was quantitated by image analysis after identification of reticulocytes by acridine orange staining. Sickling in vitro of reticulocytes and nonreticulocytes under complete deoxygenation was similar and relatively unchanged during a 2-hour incubation. In contrast, under partially deoxygenated conditions, at least two populations of reticulocytes were apparent, one more susceptible to sickling than the other; nonreticulocytes were generally less susceptible to sickling. Many of the severely deformed reticulocytes showed formation of long spicules during incubation. These data suggest that a subset of reticulocytes are more susceptible to sickling than nonreticulocytes, and that the degree of reticulocyte sickling in vitro increases dramatically with time even at constant partial oxygen pressures observed in the venous circulation. Since dehydration in sickled reticulocytes seemed to be proceeding, mechanisms of inhibition were also examined. We found that quinine, an inhibitor of the Ca(++)-activated K+ efflux, inhibited part of the reticulocyte sickling while okadaic acid, a K(+)-Cl- co-transport inhibitor, did not inhibit sickling under our experimental conditions. These phenomena observed at pH and oxygen tension similar to physiological venous conditions may be important in understanding the clinical course and pathophysiology of sickle cell disease.

Topics: Anemia, Sickle Cell; Calcium; Carrier Proteins; Erythrocytes, Abnormal; Ethers, Cyclic; Humans; In Vitro Techniques; K Cl- Cotransporters; Okadaic Acid; Oxygen; Potassium; Quinine; Reticulocytes; Symporters; Veins

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