Stimulation of Na+-K+-2Cl- cotransport by arsenite in ferret erythrocytes.
1. Na+-K+-2Cl- cotransport activity was measured in ferret erythrocytes as the bumetanide-sensitive uptake of 86Rb. 2. The Na+-K+-2Cl- cotransport rate was stimulated by treating erythrocytes with sodium arsenite but not by sodium arsenate (up to 1 mM). Stimulation took an hour to develop fully. Arsenite had no effect on bumetanide-resistant 86Rb uptake. 3. In cells stored for 3 days or less, cotransport stimulation by arsenite could be described by assuming arsenite either acts at a single site (EC50, 60+/-14 microM, mean +/- S.E.M., n = 3) or that it acts at both high- (EC50, 35+/-9 microM, mean +/- S.E.M., n = 3) and low- (EC50 >2 mM) affinity sites. 4. Stimulation by 1 mM arsenite was greatest on the day of cell collection (rate about 3 times that of the control), even exceeding that produced by 20 nM calyculin A, and declined during cell storage. Addition of calyculin A to arsenite-stimulated cells resulted in further stimulation of Na+-K+-2Cl- cotransport, suggesting that arsenite and calyculin act synergistically. This was most apparent in stored cells. 5. Stimulation by 1 mM arsenite was not affected by treating cells with the mitogen-activated protein kinase inhibitors SB203580 (20 microM) and PD98059 (50 microM), but was both prevented and reversed by the kinase inhibitors staurosporine (2 microM), 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1, 50 microM) and genistein (0.3 mM), and with a combination of 10 microM A23187 and 2 mM EDTA (to reduce intracellular Mg2+ concentration). Only treatment with EDTA and A23187 prevented stimulation by the combination of 1 mM arsenite and 20 nM calyculin, whereas no treatment was able to fully reverse this stimulation once elicited. 6. Our data are consistent with arsenite stimulating (perhaps indirectly) a kinase that phosphorylates and activates the Na+-K+-2Cl- cotransporter.
Flatman
P W
PW
Membrane Biology Group, Department of Biomedical Sciences, University Medical School, Teviot Place, Edinburgh EH8 9AG, UK. peter.flatman@ed.ac.uk
Creanor
J
J
D000818:Animals; D018053:Arsenites / Q000494:pharmacology*; D002034:Bumetanide / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002352:Carrier Proteins / Q000097:blood* / Q000187:drug effects; D002712:Chlorides / Q000097:blood; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology*; D005289:Ferrets; D005419:Flavonoids / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006706:Homeostasis; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D008954:Models, Biological; D011188:Potassium / Q000097:blood; D011725:Pyridines / Q000494:pharmacology; D012413:Rubidium / Q000097:blood*; D012414:Rubidium Radioisotopes; D012964:Sodium / Q000097:blood; D028021:Sodium-Potassium-Chloride Symporters; D019311:Staurosporine / Q000494:pharmacology
D018053:Arsenites; D002352:Carrier Proteins; D002712:Chlorides; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D011725:Pyridines; D012414:Rubidium Radioisotopes; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D000001:Calcimycin; D004492:Edetic Acid; D012964:Sodium; D019833:Genistein; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019311:Staurosporine; D012413:Rubidium; C015001:arsenite; C093642:SB 203580; D011188:Potassium; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one