The effect of Lipin-2 on cellular ionic currents generated by ATP. RAW264.7 cells treated with control siRNA (siRNACtrl.), siRNA against lipin-2 (siRNALpin2; A, left), or BMDMs from WT and Lpin2^−/− mice (A, right) were stimulated with 100 ng/ml LPS for 4 h, 2 mM ATP or 10 µM nigericin for 40 min, 200 µg/ml monosodium urate (MSU) or 150 µg/ml alum for 6 h, or LPS for 4 h, followed by ATP or nigericin for 40 min, MSU, or alum for 6 h, as indicated. IL-1β present in cellular supernatants was analyzed by specific ELISA (A). (B) Representative traces obtained with whole-cell recordings using a 1-s ramp protocol in peritoneal macrophages from WT and Lpin2^−/− animals stimulated with 2 mM ATP in control solution (140 mM NaCl) or in a solution where NaCl has been substituted for 140 mM N-methyl-d-glucamine (NMDG), as indicated. (top) Current density versus voltage plots; (bottom) time course of the current density at −120 and +100 mV. (C) Mean current density from 25–39 macrophages analyzed is shown. RAW264.7 cells were also evaluated as in B and mean current density from 18 cells is shown (D). Time course of the current amplitude obtained at −120 mV and +100 mV in a peritoneal macrophage from WT mice upon exposure to 2 mM ATP during the indicated time, to illustrate the calculations of t_on (time from opening to maximal stimulation) and t_off (time from maximal opening to closure; E, left). (right) Mean t_on and t_off for −120 mV and +100 mV from WT and Lpin2^−/− mice peritoneal macrophages (14–40 cells) stimulated with 2 mM ATP (E). Data from A are shown as mean ± SD, and experiments shown are representative of at least three independent ones made in triplicate. *, P < 0.05, Student’s t test. Dada from C, D, and E, are shown as means ± SEM *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA, followed by Tukey's test.