Fig. 5. Lysosome exocytosis upregulates P2X₄ receptors at the plasma membrane. (A) NRK cells transfected with vector encoding P2X₄ (AU5) receptors were incubated with 5 µM ionomycin for 10 minutes in normal extracellular solution (NES) at 37°C. Next, cells were washed and returned to NES lacking ionomycin for a further 45 minutes at 37°C, and the solution was then assayed for -Hex activity. The experiment was repeated three times and the data shown are from one representative experiment. Shown are means+s.d., n=3 (^***=P<0.001). Alongside are shown confocal images of cells incubated with ionomycin for 10 minutes in NES at 37°C and then surface labelled with antibodies against AU5 or LAMP-1 (LYC16 clone) at 12°C for 30 minutes. Bars, 10 µm. Between 50 and 70 cells were analysed for each condition. The experiment was repeated three times and the data shown are from one representative experiment. Shown are means+s.e.m., n=3 (^***=P<0.001). (B) A similar experiment was carried out with cultured rat brain microglia, but surface expression of endogenous P2X₄ was measured by biotinylation at 12°C followed by immunoblotting with antibody against P2X₄. `Total' gels show dilutions of total cell lysates. (C) Time course of lysosomal enzyme release induced by 50 mM methylamine (MA) in peritoneal macrophages and HEK 293 cells. Cells were incubated in NES, and -Hex activity was measured at different time points (mean±s.d., n=3). (D) Surface expression of P2X₄ and LAMP-1 in peritoneal macrophages following 15 minutes incubation with 50 mM MA. Surface receptors were detected by biotinylation at 12°C followed by immunoblotting. Samples from the total cell lysates show that the overall expression of P2X₄ and LAMP-1 was unchanged by treatment with MA.