Fig. 2.

Ouabain (Ouab) increases the effect of carbachol (Carb) on the phosphorylation of proteins in the extracellular signal-regulated kinase-1/2 (ERK1/2) signaling cascade but blocks AMP-activated protein kinase (AMPK) phosphorylation. A: parotid acinar cells were treated with 10⁻⁶ M Carb for 2 min after a 1-min exposure to DMSO (−) or 1 mM Ouab and to 10⁻⁵ M Carb after a 1-min exposure to DMSO. Cells in basal (−) condition were exposed for Ouab or DMSO for 3 min. Lysates were subjected to immunoblot analysis as indicated. Representative Western blot analysis of replicate samples from one experiment. Ouab increases the phosphorylation of RAF-S³³⁸, mitogen-activated protein kinase kinase (MEK), and ERK1/2 in cells treated with 10⁻⁶ M Carb. B: quantification of changes in phosphorylation of ERK1/2, MEK, and RAF. For each condition, the level of phosphoprotein was quantified and normalized to the total individual protein (ERK2, MEK, RAF), and values are presented relative to basal (no Ouab). Number of individual experiments is indicated below bars. *P < 0.02 vs. basal (no Ouab). #P < 0.01 vs. 10⁻⁶ M Carb (no Ouab). ##P ≤ 0.05 vs. 10⁻⁶ M Carb (no Ouab). All other conditions are P ≤ 0.01 vs. basal (no ouabain). C: parotid acinar cells were treated with 10⁻⁶ and 10⁻⁵ M Carb for 2 min after a 1-min exposure to DMSO (−) or 1 mM Ouab. Cells in basal (−) condition were exposed for Ouab or DMSO for 3 min. Lysates were subjected to immunoblot analysis using a P-AMPK antibody. Representative Western blot analysis of replicate samples from one experiment. Ouab reduces/blocks the phosphorylation of AMPK. D: quantification of AMPK phosphorylation. For each condition, the phosphorylation of AMPK was quantified and normalized to total AMPK (or ERK2) as a loading control, and values are shown relative to basal (no Ouab). Number of individual experiments is indicated at base of bars. *P < 0.01; **P < 0.05. P values were calculated based on matched pairs from same experiment.