Figure 1: Effect of isoform-selective PI3K inhibitors on catecholamine release from chromaffin cells.

Cultured bovine chromaffin cells were briefly washed with buffer A and incubated with increasing concentrations of (a) TGX-221 (b) IC87114 or (c) AS252424 before being further stimulated using barium chloride (2 mM) for 2 min in the continuing presence of inhibitors. (d) Chromaffin cells were treated with IC87114 (1 μM) for the indicated time period before being stimulated using barium chloride (2 mM) for 2 min. (e) Chromaffin cells were incubated with or without IC87114 for 20 min before being stimulated using barium chloride (2 mM) for 2 min. (f) Chromaffin cells were treated with the indicated concentrations of IC87114 for 5 min before stimulation using digitonin (20 μM) permeabilization in the presence of 10 μM Ca²⁺ and 2 mM MgATP in KGEP buffer. (g) Chromaffin cells were pre-incubated with or without VO-OHpic (100 nM) in buffer A before the addition of IC87114 (1 μM). Cells were then stimulated as described above. (h) Chromaffin cells were pre-treated with VO-OHpic (100 nM) before being stimulated using barium chloride (2 mM) for 2 min. Aliquots of the supernatant were removed and assayed fluorimetrically for catecholamine content. Data are expressed as mean±s.e.m. n=8, **Student's t-test, P<0.01, representative experiments carried out at least 4 times in octuplicate. (i) Subcellular fractions were prepared from bovine adrenal medulla (12KP and 12KSN=pellet and supernatant from 12,000g_av spin; 100KP and 100KSN=pellet and supernatant from the 100,000g_av spin). The chromaffin granule-containing 12KP fraction was loaded on a linear sucrose gradient (1M–2.2M) and fractions collected from the top (1–12). 50 μg protein from each fraction were separated and analysed by SDS–PAGE and western blotting using the indicated antibodies. The position of the early endosome, plasma membrane and secretory granules containing fractions was detected using anti-EEA1, anti-SNAP-25 and anti-VAMP2 antibodies, respectively.