FIG. 2.

Thrombin-induced AMPK activation is mediated via PAR-1, G_q coupling, and phospholipase C activation. A. HUVEC were stimulated with the PAR-1 agonist peptide TFLLR (10 μM) for the indicated times. B. HUVEC were pretreated with pertussis toxin (PTX) (500 ng/ml, 3 h) or with G_q-protein antagonist 2A (GPA-2A) (5 μM, 10-min preincubation in transiently permeabilized cells) and subsequently stimulated with thrombin (1 U/ml, 1 min). C. HUVEC were pretreated with U-73122 (10 μM, 30 min) and stimulated with thrombin (1 U/ml, 1 min) or alternatively treated with the phospholipase C activator m-3M3FBS (100 μM, 2 min). For panels A to C, cells were lysed and subjected to immunoblotting using antibodies against phosphorylated AMPKα (threonine 172) and total AMPKα for counterstaining. Representative figures and densitometry data (means ± SEMs) of three independent experiments for each treatment are shown. Phosphospecific signals from TFLLR-stimulated and nonstimulated cells (A) or from cells stimulated with thrombin in the absence or presence of inhibitors (B and C) were compared. , P < 0.05.