Figure 4. Effects of ionophores on spermine transport in Drosophila S2 cells.

(A, C) [¹⁴C]Spermine (500 nM) was added to cell suspensions either immediately (black bars) or 5 min (grey bars) after the addition of ionophores. Transport was calculated after 5 min of incubation with the radiolabelled substrate as described in the Materials and methods section. (A) Transport activity after addition of 500 nM valinomycin, 500 nM CCCP or 500 μM KCN. The control group shows transport in the absence of any ionophore. We observed a significant reduction in transport in the absence of pre-incubation using CCCP compared with the control (†, P<0.00001, ANOVA with Tukey's post-hoc test), but not with valinomycin or KCN (not significant, N.S.). Using a 5 min pre-incubation, transport was significantly reduced using all three agents compared with the control (‡, P<0.00001). Post-hoc comparison between values obtained for valinomycin and KCN using 0 min compared with 5 min pre-incubated cells also showed a significant difference (P<0.001, not indicated on the Figure). A smaller reduction was seen for CCCP using 0 min compared with 5 min pre-incubated cells (P<0.05, not indicated on the Figure). (B) Addition of valinomycin (represented by a black box) in K⁺-free medium will clamp the cells at the equilibrium potential of K⁺, by allowing K⁺ ions to flow down their electrochemical gradient (i). If nigericin is added (grey box), H⁺ is pumped out as the K⁺ cycles in and out of the cell (ii). (C) Transport activity of [¹⁴C]spermine after addition of 500 nM nigericin or 500 nM nigericin+500 nM valinomycin. We detected a significant difference between ionophore treatment and control at 0 min pre-incubation time for both nigericin and nigericin+valinomycin (†, P<0.00001, ANOVA with Tukey's post-hoc test). In addition, we observed significant differences between nigericin compared with nigericin+valinomycin at 0 min pre-incubation time (‡, P<0.00001). Transport using 0 min compared with 5 min pre-incubation also differed for both nigericin and for nigericin+valinomycin (P<0.05, not indicated on the Figure).