49 EFFECT OF PKC STIMULATION ON SODIUM CHLORIDE COTRANSPORTER FUNCTION AND SURFACE EXPRESSION IN MAMMALIAN CELLS

The sodium chloride cotransporter (NCC) belongs to the family of electroneutral cation-chloride transporters (SLC12). It is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. NCC is the principal salt-absorptive pathway in the mammalian distal convoluted tubule, but its regulatory mechanisms have been principally studied in the Xenopus oocyte expression system and have not been well defined in mammalian models. Therefore, we have developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse distal convoluted (mDCT) cell line. We now use this system to assess the effects of activators of PKA (forskolin), PKC (TPA), or neither (4α-PDD, a non-PKC-stimulating phorbol ester) on function and surface expression of NCC. This is the first reported correlation of NCC function (22Na+ uptakes) with surface expression in mammalian cells. For this, mDCT cells were grown to 90% confluence in 12-well plates and then placed in Cl−-free media for 30 minutes. The cells were then incubated in medium-containing vehicle (DMSO), TPA, forskolin, or 4α-PDD. For functional assessment, cells were then incubated in a 22Na+-containing medium with or without thiazides and then washed with ice-cold wash buffer and lysed. Radioactivity was counted, protein concentrations of the lysates were determined, and uptakes were normalized to nmol/mg. Thiazide-sensitive uptake was given by the difference of the uptakes with and without thiazide. For assessment of surface expression, the cells were washed with ice-cold PBS and incubated with NHS-SS-biotin at 40°C for 30 minutes. The cells were lysed, and labeled protein was isolated. The biotinylated protein was eluted in SDS sample buffer with DTT. Protein concentration was measured using the lysate from each group. Biotinylated surface protein and the corresponding amount of total NCC in the cell lysates were analyzed by Western blotting and were quantified by densitometric analysis. TPA completely suppressed NCC function, essentially eliminating thiazide-sensitive uptake (statistically no different from zero for TPA vs 43.4 ± 8.68 nmol/mg for vehicle, p < .01 by ANOVA). Stimulation of PKA did not significantly alter NCC function compared with vehicle or 4α-PDD. For the biotinylation study, the fraction of cell surface NCC over total NCC in the cell lysate was calculated for each group. Cell surface NCC protein represented 48.4 ± 8.9%