Journal
EXPERIMENTAL EYE RESEARCH
Volume 82, Issue 1, Pages 55-64Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.exer.2005.05.002
Keywords
human lens epithelial cells; cation-chloride cotransporters; Na/K pump; K-Cl cotransport (KCC); Na/K-Cl cotransport (NKCC); N-ethylmaleirnide; protein kinases; protein phosphatases; loop diuretics; cataract
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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 mu M) discriminated between the Na/K pump (similar to 35% of total Rb influx) and Na-K-2Cl cotransport (NKCC) (similar to 50%). Cl-replacement with nitrate or sulfamate revealed < 10% residual [ouabain+bumetanide]-inseiisitive 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 similar to 90% of NKCC. Calyculin-A, a serine/threonine protein phosphatase-1 inhibitor, did not affect NKCC but inhibited KCC, whereas 10 mu M 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 mu M, 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. (C) 2005 Elsevier Ltd. All rights reserved.
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