Figure 6.

Brefeldin A prevented the cell surface expression and stimulation of Na,K-ATPase induced by db-cAMP. Microdissected rat CCDs or mpkCCD_c14 cells were pretreated in absence or presence of 20 μg/ml brefeldin A for 1 h at 30°C and then incubated (15 for CCDs and 30 for mpkCCD_c14 cells) with or without 10⁻³ M db-cAMP at 37°C. Tubules and cells were then biotinylated, lysed, and the labeled proteins were precipitated by streptavidin agarose-beads. The Na,K-ATPase α-subunit was detected by Western blotting as described in Figure 3. (A and D) Representative immunoblots from CCD (A) and mpkCCD_C14 cells (D), respectively. (B and E) Bars represent the densitometric quantification (means ± SE) from four independent experiments. Results (means ± SE) are expressed as percentage of the optical density values from untreated samples (control); ∗, p < 0.05 versus control values. (C) Hydrolytic activity of Na,K-ATPase in rat CCDs treated with brefeldin A and db-cAMP as described above. Values (means ± SE from 7 independent experiments) are percentage of control (370 ± 53 pmol ATP · mm⁻¹ · h⁻¹). ∗, p < 0.05 versus control values.