Figure 8.

db-cAMP–induced cell surface expression and stimulation of Na, K-ATPase were dependent on intracellular Ca²⁺. Microdissected rat CCDs or mpkCCD_c14 cells were preincubated without or with 10 μM BAPTA-AM at room temperature (CCDs) or at 37°C (mpkCCD_c14 cells) for 1 h, and then incubated C (15 min for CCDs and 30 min for mpkCCD_c14 cells) in the absence or presence of 10⁻³ M db-cAMP at 37°. Samples were then biotinylated, lysed, and 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 three and four independent experiments in rat CCDs and mpkCCD_c14 cells, respectively. 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 BAPTA-AM 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.