Lithium fluxes indicate presence of Na-Cl cotransport (NCC) in human lens epithelial cells.
During regulatory volume decrease (RVD) of human lens epithelial cells (hLECs) by clotrimazole (CTZ)-sensitive K fluxes, Na-K-2Cl cotransport (NKCC) remains active and K-Cl cotransport (KCC) inactive. To determine whether such an abnormal behavior was caused by RVD-induced cell shrinkage, NKCC was measured in the presence of either CTZ or in high K media to prevent RVD. NKCC transports RbCl + NaCl, and LiCl + KCl; thus ouabain-insensitive, bumetanide-sensitive (BS) or Cl-dependent (ClD) Rb and Li fluxes were determined in hyposmotic high NaCl media with CTZ, or in high KCl media alone, or with sulfamate (Sf) or nitrate as Cl replacement at varying Rb, Li or Cl mol fractions (MF). Unexpectedly, NKCC was inhibited by 80% with CTZ (IC(50) = 31 microM). In isosmotic (300 mOsM) K, Li influx was approximately 1/3 of Rb influx in Na, 50% lower in Sf, and bumetanide-insensitive (BI). In hypotonic (200 mOsM) K, only the ClD but not BS Li fluxes were detected. At Li MFs from 0.1-1, Li fluxes fitted a bell-shaped curve maxing at approximately 0.6 Li MF, with the BS fluxes equaling approximately 1/4 of the ClD-Li influx. The difference, i.e. the BI/ClD Li influx, saturated with increasing Li and Cl MFs, with K(ms) for Li of 11 with, and 7 mM without K, and of approximately 46 mM for Cl. Inhibition of this K-independent Li influx by thiazides was weak whilst furosemide (<100 microM) was ineffective. Reverse transcription polymerase chain reaction and Western blots verified presence of both NKCC1 and Na-Cl cotransport (NCC). In conclusion, in hyposmotic high K media, which prevents CTZ-sensitive K flux-mediated RVD in hLECs, NKCC1, though molecularly expressed, was functionally silent. However, a K-independent and moderately thiazide-sensitive ClD-Li flux, i.e. LiCC, likely occurring through NCC was detected operationally and molecularly.
Lauf
Peter K
PK
Cell Biophysics Group, Department of Pathology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA. Peter.Lauf@wright.edu
Chimote
Ameet A
AA
Adragna
Norma C
NC
D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D008094:Lithium / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D049971:Thiazides / Q000494:pharmacology
D016160:Retinoblastoma Protein; D028021:Sodium-Potassium-Chloride Symporters; D049971:Thiazides; D008094:Lithium