Figure 3.

PD 102807-induced p-AMPK is independent of calcium mobilization.A and B, cells were stimulated with PD 102807 (10 and 100 μM) or MCh (100 μM), and calcium mobilization was measured using fluo-4 AM calcium indicator and Flexstation III as described in Experimental procedures. C, cells were permeabilized using β-escin (100 nM) then treated with increasing concentrations of CaCl₂ (0, 0.2, 2, and 20 mM) and allowed to equilibrate for 5 min. Cells were then stimulated with PD 102807 (100 μM; t = 20 min) or MCh (100 μM; t = 5 min). Cells were lysed, and phosphorylation status of AMPK was determined by immunoblotting. Representative blots are shown. Loading was corrected for β-actin. Data are means ± SD. ∗p < 0.05, ∗∗∗p < 0.001 versus vehicle stimulation; one-way ANOVA followed by Bonferroni multiple comparison test. AMPK, AMP-activated protein kinase; MCh, methacholine; p-AMPK, phospho-AMPK.