PAR1 agonist-induced Gq but not Gi signaling is impaired in NCI-H28 cells.
A, thrombin-induced intracellular Ca2+ mobilization in HMEC-1, Met-5A, and NCI-H28 cells. B, selective-PAR1-AP-induced intracellular Ca2+ mobilization in Met-5A and NCI-H28 cells. Serum and growth factor starved cells were loaded with Fluo-3AM to measure [Ca2+]i variations as indicated by changes in fluorescence intensity. Fluorescence was recorded before agonist addition (F0) and then every 3 seconds after thrombin (10 nM) or PAR1-AP (10 µM) addition for another 120 seconds. Data shown are mean ± SEM of a single experiment done in triplicate. Experiments were repeated two additional times with similar results. The results are reported as relative fluorescence (RF = F/F0 where F0 is basal fluorescence and F is fluorescence recorded after cell stimulation with the agonist). C, inhibition of isoproterenol stimulated cAMP production in Met-5A and NCI-H28 cells by different concentrations of thrombin in the presence and absence of 100 nM SCH 79797. D, no effect of the selective PAR1-AP on isoproterenol stimulated cAMP production in Met-5A and NCI-H28 cells. Serum and growth factor starved cells were exposed to different agonist concentrations. Assays were initiated by the addition of 1 µM isoproterenol. Production of cAMP was measured using a competition binding assay which includes the bovine adrenal cAMP binding protein and [3H]cAMP. Data shown are mean ± SEM of three independent experiments performed in triplicate. The differences between thrombin- and thrombin plus SCH 79797-treated cells were significant (**P≤0.01, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).