Fig. 3.

Activators of heterotrimeric G proteins and Gαo₂ inhibit uptake of [³H]serotonin by permeabilized BON cells.A, SLO-treated and washed cells (see Materials and Methods) were resuspended in KG buffer containing 2 mmMg-ATP with additions as indicated and incubated for 25 min at 36°C. The reserpine-sensitive serotonin uptake was inhibited by addition of the GTP analogs GMppNp and GTPγS each at a final concentration of 50 μm. Their effects were statistically significant, calculated by Students’ t test (*,**p < 0.003). Preincubating the cells with pertussis toxin (100 ng/ml) prevented the inhibition of serotonin uptake by GTP analogs. Values (n = 3 ± SD) represent the reserpine-sensitive uptake. Unspecific accumulation (picomoles per milligram of protein) in the presence of 2 μm reserpine was 0.93 ± 0.05 and 0.82 ± 0.08 for untreated and pertussis toxin-treated samples, respectively.B, Permeabilized cells were incubated as given inA with purified Gαo₁ (20 nm), Gαo₂ (10 nm), or Gαo₂ that had been denatured by heating to 100°C. Only effects of Gαo₂ were statistically significant (*p < 0.05). Unspecific accumulation in the presence of reserpine was 0.83 ± 0.1 pmol/mg of protein.C, Permeabilized BON cells were preincubated for 20 min with KG buffer in the absence or presence of TeNt/LC (200 nm final concentration) before uptake was started by addition of fresh KG buffer plus Mg-ATP supplemented with 10 nm AlF^4₋-activated or heat-denaturated Gαo₂. Only effects of Gαo₂ were statistically significant (*p < 0.02). Values (n = 3 ± SD) represent reserpine-sensitive uptake. Uptake in the presence of reserpine (2 μm) was 0.28 ± 0.2 pmol/mg of protein. D, Under the experimental conditions given in C, addition of TeNt/LC between 200 nm and 1 μm completely cleaved synaptobrevin without affecting synaptophysin analyzed for comparison.n.p., Nonpermeabilized.