calyculin-a and Anemia--Sickle-Cell

In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb.

Topics: Anemia, Sickle Cell; Antisickling Agents; Dimethyl Adipimidate; Enzyme Inhibitors; Erythrocytes; Erythrocytes, Abnormal; Ethylmaleimide; Humans; K Cl- Cotransporters; Magnesium; Manometry; Marine Toxins; Oxazoles; Oxygen; Symporters

In response to hypoxia, sickle red blood cells (SS RBC) and leukocytes exhibit increased adherence to the vascular endothelium, while diapedesis of leukocytes through the blood vessel increases. However, the cellular signaling pathway(s) caused by hypoxia is poorly understood. We utilized CoCl2 as a mimetic molecule for hypoxia to study cellular signaling pathways. We found that in human umbilical vein endothelial cells (HUVEC), CoCl2 at 2 mM concentration induced the surface expression of a subset of CAMs (VCAM-1) and activation of transcription factor NF-kappaB in the nuclear extracts of HUVEC. Furthermore, CoCl2 also caused time-dependent tyrosine phosphorylation of mitogen-activated protein (MAP) kinase isoform ERK2 without significantly affecting ERK1, indicating ERK2 is the preferred substrate for upstream kinase of the MAPK pathway. Inhibitors of MAP kinase (PD98059) or platelet-activating factor (PAF)- receptor antagonist (CV3988) inhibited the CoCl2-induced NF-kappaB activation and VCAM-1 expression. Augmented expression of VCAM-1 led to increased SS RBC adhesion, inhibitable by a VCAM-1 antibody. Additionally, CoCl2 caused a two- to threefold increase in the rate of transendothelial migration of monocyte-like HL-60 cells and a twentyfold increase in phosphorylation of platelet endothelial cell adhesion molecules (PECAM-1). The transendothelial migration of monocytes was inhibited by an antibody to PECAM-1. Both phosphorylation of PECAM-1 and transendothelial migration of monocytes in response to CoCl2 were inhibited by protein kinase inhibitor (GF109203X) and augmented by protein phosphatase inhibitor (Calyculin A). Our data suggests that CoCl2-induced cellular signals directing increased expression of VCAM-1 in HUVEC involve downstream activation of MAP kinase and NF-kappaB, while the phosphorylation of PECAM-1 occurs as a result of activation of PKC. We conclude that PAF-receptor antagonist inhibits the CoCl2- or hypoxia-induced increase in the adhesion of SS RBC, PECAM-1 phosphorylation, and the concomitant transendothelial migration of monocytes.

Topics: Adult; Anemia, Sickle Cell; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion; Cell Movement; Cells, Cultured; Cobalt; E-Selectin; Endothelium, Vascular; Enzyme Inhibitors; Erythrocytes, Abnormal; Flavonoids; HL-60 Cells; Humans; Hypoxia; Indoles; Intercellular Adhesion Molecule-1; Maleimides; Marine Toxins; Mitogen-Activated Protein Kinase 1; Molecular Sequence Data; NF-kappa B; Oxazoles; Phospholipid Ethers; Phosphoprotein Phosphatases; Phosphorylation; Platelet Endothelial Cell Adhesion Molecule-1; Platelet Membrane Glycoproteins; Protein Processing, Post-Translational; Receptors, Cell Surface; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Transcription Factor AP-1; Umbilical Veins; Vascular Cell Adhesion Molecule-1

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. K+ influx and efflux were measured in normal (HbA) and sickle (HbS) red blood cells to investigate the interaction of swelling, H+ ions and urea with O2 (0 to 150 mmHg O2) in the presence of ouabain and bumetanide (both 100 microM). 2. In HbA cells, K+-C1- cotransport was O2 dependent. At low oxygen tensions (PO2s) the transporter was inactive and refractory to low pH, swelling or urea. 3. C1--independent K+ influxes in sickle cells were elevated at low PO2s, as previously reported. C1--dependent K+ influxes were large at both high and low PO2s, whether stimulated by swelling, H+ ions or urea. In the absence of O2, C1--dependent K+ influxes were similar in magnitude to those measured at high PO2s. The minimum for C1--dependent K+ influx was observed at PO2s of about 40-70 mmHg. 4. K+ efflux from HbS cells was stimulated by the addition of urea (500 mM). The rate constants were of similar magnitude whether measured at high PO2 or in the absence of O2, and were predominantly C1- dependent under both conditions. 5. In HbS red blood cells, reduction of extracellular Ca2+, addition of 1 mM Mg2+ or nitrendipine (10 microM) to the saline had no effect. Inhibitors of K+-C1- cotransport, [(dihydroindenyl)oxy] alkanoic acid (DIOA; 100 microM) or calyculin A (0.1 microM), inhibited influxes by a similar magnitude to C1- substitution. 6. Results are significant for the pathophysiology of sickle cell disease. Low pH and urea are able to stimulate KC1 loss from sickle cells, leading to cellular dehydration, even in regions of low PO2.

Topics: Anemia, Sickle Cell; Bumetanide; Carboxylic Acids; Carrier Proteins; Chlorides; Egtazic Acid; Enzyme Inhibitors; Erythrocytes; Hemoglobin A; Hemoglobin, Sickle; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Indenes; Magnesium; Marine Toxins; Ouabain; Oxazoles; Oxygen; Potassium; Reference Values; Sodium-Potassium-Chloride Symporters; Urea