Inhibition of the PI3Kβ isoform produces ligand-independent [3H]Eb receptor upregulation and enhances upregulation produced by choline and nicotine.
A-C) The results from measurements of stably transfected 293 cells that were either not treated (C) or treated with the PI3K inhibitors LY294002 (LY, broad range), PIK75 (PI3Kα), PI828 (PI3Kβ), or AS60524 (PI3Kγ) as in (A), or in the presence of (B) choline or (C) nicotine as normalized to the control in (A). The specific [3H]Eb binding to membranes from these cells was measured for 3 independent experiments, the results normalized to the average no-treatment control value of 1.0 and all values from each experiment were then summed. D) Experiments similar to those in Panels A-C tested the impact to combining LY294002 and PI828 on upregulation of [3H]Eb binding or enhancement of upregulation produced by either choline or nicotine as indicated by + signs indicating addition of the agent. Upregulation relative to the control for each group is in light grey and enhancement of choline or nicotine upregulation is in dark grey. Error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. E) An example of a representative western blot showing the relative expression of α4 or β2 subunits in crude membrane fractions of cells after receiving the treatment as labeled. Nic, nicotine; Ch, choline. The ratio of the band density for each subunit was measured, normalized to the control (1.0) and then plotted for each treatment. Above each lane is the change in the β2/α4 ratio calculated after normalization.
