Fig. 5.

Rho-GTPase activity is correlated with LPA- but not carbachol-induced ATP release. A and B: 1321N1 cells were treated with 300 μM βγ-meATP for 15 min. A sample of the reaction media was taken for a baseline [ATP] measurement (open bars), and then cells were stimulated with 100 μM carbachol or 10 μM LPA for 15 min before removing second sample of the reaction media (solid bars). Extracellular ATP concentration was measured via an off-line luciferin-luciferase assay as described in methods. Data represent means ± SE of 4 independent experiments each performed in triplicate. *P < 0.01 vs. LPA-treated control. #P < 0.05 vs. LPA-treated control. C: suspended 1321N1 cells were loaded with fura 2-AM and stimulated with 100 μM carbachol or 10 μM LPA to determine that ToxB loading had no observable effect on elevations in cytosolic [Ca²⁺]. This experiment was performed once. D: stimulation with 10 μM LPA for 2 min induced RhoA-GTP accumulation, whereas stimulation with 100 μM carbachol does not. Aliquots of lysate were subjected to Rhotekin (TRBD)-RhoA-GTP pull-down assays as described in methods and WB were done using anti-RhoA antibody. The data are representative of two separate experiments.