Abstract
Purpose: :
To characterize in FHL124 cells the response to hyposmotic challenge of molecularly identified CCCs, especially Na–K–2Cl cotransport (NKCC) and K–Cl cotransport (KCC) and their volume set point.
Methods: :
Reverse transcriptase polymerase chain reaction (RTPCR), Western blots and immunofluorescence were used to identify the NKCC and KCC isoforms in FHL124 cells (kindly provided by Dr. John Reddan, Oakland University). K–influx was measured with Rb (as K congener) in 300 to 150 mOsM Cl or sulfamate media and 0.1 mM ouabain ± 5 µM bumetanide, and various ion channel inhibitors.
Results: :
1) RT–PCR revealed the expected sizes for NKCC1 (336bp) and KCC1 (233bp), KCC3a (250bp), KCC3b (409bp) and KCC4 (556bp) isoforms. NKCC1 and KCC1,3&4 isoform expression was verified by Western blots and immunocytohistochemistry with primary rabbit anti–NKCC1, anti–rtKCC1, anti–hmKCC3 and anti–msKCC4 antibodies, and secondary horseradish peroxidase– and Cy3–labeled donkey anti–rabbit IgG, respectively. 2) In 300 mOsM Na–media, the Na/K pump, NKCC, and KCC were ∼35, 50, and <10% of total Rb influx, the remainder due to channels. 3) Lowering the osmolarity to 150 mOsM failed to inhibit NKCC and activate KCC, and reduced intracellular K content by 40–50% independently of the chosen anion. 4) At maximum concentrations, niflumic acid, 9–AC, loop diuretics, DIDS, and tamoxifen, inhibitors of anion channels, and of cation channels such as 4–aminopyridine, TEA, Gd, and octanol, respectively, failed to prevent hyposmotically–induced K loss.
Conclusions: :
Like SV40–large T antigen–containing human lens epithelial B3 cells, primary FHL124 cells show most K influx activity through the Na/K pump, NKCC1, and KCC (1, 3 and/or 4 isoform). The hyposmotically–induced cell K and volume reduction likely involves swelling–activated non–selective anion and cation channels, apparently neither related to typical volume–responsive anion channels (i.e. VRAC) nor to cation–non–selective connexins, which only when inhibited would permit volume set point determination. However, additional inhibitor studies are pending. Hence, due to presence of swelling–activated channel, NKCC remains activated and KCC inhibited. In contrast, thiol–modification, volume–independently, inhibits NKCC and activates KCC, respectively, through their regulatory machineries, as shown in B3 cells (Exp. Eye Res. 81: 2005).