Figure 1.
Temperature-dependence of 86Rb+ uptake.
86Rb+ uptake in the absence or presence of 10 µM bumetanide was assessed following a 60 min pre-incubation in isotonic basic (open bars) or hypotonic, low [Cl−] (filled bars) medium at either RT (A) or 37°C (B). Bumetanide-sensitive (B-S) 86Rb+ uptakes are shown as mean ± s.e.m. (n = 9–14). In experiments designed specifically to assess the effects of increasing the temperature from RT to 37°C, 86Rb+ uptake increased by: fNKCC2A, 343±13% (n = 4); fNKCC1, 509±22% (n = 3); and HEK-293, 150±49% (n = 3) under basal conditions; and by: 236±36% (n = 4), 106±17% (n = 5), and 145±31% (n = 3) following incubation in a hypotonic, low [Cl−] medium. Bumetanide-resistant 86Rb+ uptakes at 37°C in basic medium were (nmol mg protein−1 min−1): fNKCC2A, 1.86±0.77 (n = 10); fNKCC1, 1.95±0.37 (n = 10); and HEK-293, 1.04±0.39 (n = 8) and did not change substantially under any condition tested.
Figure 2.
Effect of ouabain on 86Rb+ uptake and phosphorylation of fNKCC2A and fNKCC1.
(A) time-dependent activation of 86Rb+ uptake by ouabain. Bumetanide-sensitive (B-S) 86Rb+ uptakes were measured at 37°C following pre-incubation of cells in an isotonic medium containing 0.1 mM ouabain for the times indicated. Values are means ± s.e.m. (n = 4–7). Circles represent fNKCC1; squares, fNKCC2A; and triangles, control HEK-293 cells. Lines, fitted to the data by non-linear regression analysis, are of the form: y = R+S(1−exp(−kt)), where R is the initial, and R+S the maximum uptake, k is the rate constant and t, the time in min. (B) Effect of omitting Ca2+ from the medium during exposure to ouabain. Cells were exposed to 0.1 mM ouabain and 86Rb+ uptake was measured in the presence (open symbols) or absence (filled symbols) of 1 mM Ca2+ in the medium. Ca2+-chelators were not used. (C) Lysates from cells exposed to ouabain (in the presence of Ca2+) for 30 min were immunoblotted and probed with antibodies against phospho-NKCC1 and 2 (NKCC-P, R5), NKCC2 (anti-NKCC2), NKCC1 and 2 (T4) and NKCC1 (N1). An indication of molecular mass is shown to the right of each panel. Similar results were obtained when the experiment was repeated.
Table 1.
Effects of inhibitors and changes to the ionic environment on cotransporter phosphorylation.
Figure 3.
86Rb+ uptake after pre-incubation in Na+-free medium.
Bumetanide-sensitive (B-S) 86Rb+ uptake was measured at 37°C in confluent HEK-293 cells (triangles), fNKCC2A cells (squares) or fNKCC1 cells (circles). Cells were pre-incubated in a Na+-free (NMDG) medium for the times indicated with 0.1 mM ouabain present for the last 5 min. 86Rb+ uptake was then measured in the presence of Na+. Points represent means ± s.e.m. (n = 5–11).
Figure 4.
Effects of phosphatase and kinase inhibitors on 86Rb+ uptake.
Cells were pre-incubated at 37°C in basic medium for 30 min followed by 30 min in basic medium in the absence (control) or presence of: 1% DMSO (vehicle), 0.25 µM calyculin (A), 1 mM orthovanadate for the entire 60 min (B), 50 µM PP1 (C) or 120 µM genistein (D) before measuring 86Rb+ uptake in the presence of the drugs. Bumetanide-sensitive (B-S) 86Rb+ uptakes are shown as mean ± s.e.m. (n = 3–7, values for HEK-293 cells in B and C are means of triplicates in a single experiment). Using paired, normalised comparisons * signifies P<0.05. 86Rb+ uptakes in the presence of DMSO were (% control): fNKCC2A, 91.7±2.4 (n = 6); fNKCC1, 93.0±5.2 (n = 6); and HEK-293, 101±5.7 (n = 3), none significantly different from controls.
Figure 5.
Effects of phosphatase and kinase inhibitors on phosphorylation of fNKCC2A and fNKCC1.
Experiments were carried out as described in Figure 4, but without the addition of 86Rb+. Cell lysates were subjected to SDS-PAGE, immunoblotted, and probed with antibodies against phospho-NKCC1 and 2 (NKCC-P, R5), NKCC2 (anti-NKCC2), NKCC1 and 2 (T4) and NKCC1 (N1). Representative blots are shown with control and drug-treated samples from the same blot. Estimated molecular mass is indicated to the right of each panel. (A) Effect of 0.25 µM calyculin A on monomeric NKCC. An approximate 20 kDa band-shift in calyculin-treated cells is only observed with the phospho-specific antibody R5 (arrow) but not with NKCC-P. (B) High molecular mass bands in calyculin A treated fNKCC2A cells. (C) Example of 20 kDa band-shift (arrow) in HEK-293 cells. Cells had been pre-incubated in basic medium, a Na+-free medium or with calyculin. (D) Effect of 50 µM PP1 and 120 µM genistein on monomeric NKCC. (E) High molecular mass bands in PP1 or genistein treated fNKCC2A cells.