Regulation of potassium transport in human lens epithelial cells.
The major K influx pathways and their response to thiol modification by N-ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors were characterized in human lens epithelial B3 (HLE-B3) cells with Rb as K congener. Ouabain (0.1 mM) and bumetanide (5 microM) discriminated between the Na/K pump ( approximately 35% of total Rb influx) and Na-K-2Cl cotransport (NKCC) ( approximately 50%). Cl-replacement with nitrate or sulfamate revealed <10% residual [ouabain+bumetanide]-insensitive K-Cl cotransport (KCC). At 0.3-0.5 mM, NEM stimulated the Na/K pump by 2-fold independent of external Na, KCC between 2 and 4-fold, and abolished approximately 90% of NKCC. Calyculin-A, a serine/threonine protein phosphatase-1 inhibitor, did not affect NKCC but inhibited KCC, whereas 10 microM staurosporine, a serine/threonine kinase inhibitor, abolished NKCC, and stimulated KCC only when followed by NEM treatment. The tyrosine-kinase inhibitor genistein, at concentrations >100 microM, activated the Na/K pump and abolished NKCC but did not affect KCC. The data suggest at least partial inverse regulation of KCC and NKCC in HLE-B3 cells by signaling cascades involving serine, threonine and tyrosine phosphorylation/dephosphorylation equilibria.
Cell Biophysics Group, Wright State University School of Medicine, Dayton, OH 45435, USA. peter.lauf@wright.edu
Peter K
PK
Lauf
Ronald
R
Warwar
Thomas L
TL
Brown
Norma C
NC
Adragna
D002034:Bumetanide / Q000494:pharmacology; D002460:Cell Line; D004232:Diuretics / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005665:Furosemide / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D007908:Lens, Crystalline / Q000378:metabolism*; D008387:Marine Toxins; D010042:Ouabain / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011188:Potassium / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D054645:Protein Phosphatase 1; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012413:Rubidium / Q000493:pharmacokinetics; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D019311:Staurosporine / Q000494:pharmacology; D027981:Symporters / Q000378:metabolism; D000096922:K Cl- Cotransporters
D002034:Bumetanide; D004232:Diuretics; D004791:Enzyme Inhibitors; D005033:Ethylmaleimide; D005665:Furosemide; D019833:Genistein; D008387:Marine Toxins; D010042:Ouabain; D010080:Oxazoles; D010749:Phosphoprotein Phosphatases; D011188:Potassium; D011493:Protein Kinase C; D054645:Protein Phosphatase 1; D011505:Protein-Tyrosine Kinases; D012413:Rubidium; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase; D019311:Staurosporine; D027981:Symporters; D000096922:K Cl- Cotransporters; C059041:calyculin A