Stimulation of PI hydrolysis by PAR2-AP via Gα_i and Gα_q proteins.

<p>(<b>A</b>) Concentration-dependent stimulation of PI hydrolysis PAR2-AP. Freshly dispersed muscle cells labelled with myo-2-[³H]inositol were incubated with different concentrations of PAR2-AP for 60 s. Total [³H]inositol phosphates were separated by ion-exchange chromatography. Results are expressed as total [³H]inositol phosphate formation in cpm/mg protein above basal levels (basal levels: 520±66 cpm/mg protein). (<b>B</b>) Selective inhibition of PAR2-AP-stimulated PI hydrolysis by pertussis toxin (PTx). Dispersed muscle cells labelled with myo-2-[³H]inositol were incubated for 10 min with U73122 (10 µM) or with PTx (200 ng/ml) for 60 min, and then PAR2-AP (1 µM) for 60 s. Treatment of cells with PTx (563±102 cpm/mg protein) or U73122 (485±86 cpm/mg protein) alone had no effect on basal PI hydrolysis. (<b>C</b>) Inhibition of PAR2-AP-stimulated PI hydrolysis by PAR2 siRNA. Control muscle cells and cells transfected with PAR2-specific siRNA were labelled with myo-2-[³H]inositol and then treated with PAR2-AP (1 µM) for 60 s. Inset: expression of PAR2 in control cells (lane 1) and cells transfected with PAR2 siRNA (lane 2) was measured by western blot using antibody to PAR2 (1∶1000). (<b>D</b>) Inhibition of PAR2-AP-stimulated PI hydrolysis by Ga_q minigene, Ga_i minigene or both. Cultured gastric muscle cells labelled with myo-2-[³H]inositol and expressing Ga_q minigene, Ga_i minigene, both Gα_q and Gα_i minigenes, or control vector were treated with PAR2-AP (1 µM) for 60 s. In some experiments cells transfected with Ga_q minigene were treated with PTx (200 ng/ml) for 60 min. Inset: expression of Ga_q and Ga_i in control cells (lane 1), cells transfected with Ga_q minigene (lane 2) or Ga_i minigene (lane 3) was measured by western blot using antibody to Ga_q (1: 1000) or a common antibody to Ga_i (1:2000). (<b>E</b>) Regulation of PAR2-AP-stimulated PI hydrolysis by regulator of G protein signaling 4 (RGS4). Cultured gastric muscle cells labelled with myo-2-[³H]inositol and overexpressing wild type RGS4, dominant negative RGS4 (RGS4[N88S]), or vector alone were treated with PAR2-AP (1 µM) for 60 s. Results are expressed as total [³H]inositol phosphate formation in cpm/mg protein. Values are means ± S.E. of four experiments. ** Significant inhibition from control response to PAR2-AP (P<0.001); ## significant increase from control response to PAR2-AP (P<0.01).