1a, b and d). After the

current reached its maximal activation (i.e. at steady state; hypo ss), 10 μM curcumin (Fig. 1a) or 0.1% DMSO (Fig. 1b) was added to the extracellular hypotonic solution for 10 min. However, no significant difference in IClswell was detected in cells exposed to either 10 μM curcumin or 0.1% DMSO (paired Student’s t-test). Fig. 1c shows the current density-to-time relation in hypotonic solution and in the presence of curcumin or DMSO; accordingly, no effect of curcumin was detected (paired Student’s t-test). Similar results were obtained after the addition CH5424802 in vitro of curcumin to cells kept in extracellular isotonic solution, demonstrating that curcumin is unable to directly stimulate IClswell in HEK293 Phoenix cells under these conditions (paired Student’s t-test, data not shown). The same experimental design described above was employed with 50 μM curcumin or 0.5% DMSO (vehicle). Current density-to-voltage relations show that a 10 min extracellular exposure to neither curcumin (Fig. 2a) nor DMSO (Fig. 2b) following hypotonic shock had an effect on IClswell (paired Student’s t-test). Fig. 2c shows the time course of the current elicited in hypotonic solution in the presence of curcumin or DMSO; accordingly, no effect of curcumin or DMSO was detected (paired Student’s t-test). Similar experiments were performed after adding

50 μM curcumin or 0.5% DMSO to the pipette filling solution ( Fig. 2d); after establishing the whole cell configuration, the substances dissolved in the pipette filling solution have access to the intracellular space. The current density-to-voltage learn more relations were measured in hypertonic

extracellular solution and after 10 min of hypotonic shock; no differences were detected between IClswell measured in the presence of intracellular 50 μM curcumin or 0.5% DMSO (F test). Fig. 3a–k show the results of patch clamp experiments obtained from HEK293 Phoenix cells after a long-term exposure (15–23 h in the medium used for cell growth) to 0.1–10 μM curcumin or 0.05% DMSO (vehicle). In contrast to the experiments described above, curcumin or DMSO were not present in the extracellular solutions during current recordings. After establishing the seal, IClswell was activated as mentioned above. The current density-to-voltage relations (Fig. Vorinostat datasheet 3a, c, e, g and i) were determined in extracellular hypertonic solution and every 10 min for 30 min in extracellular hypotonic solution. Long-term exposure to 0.1 μM curcumin (Fig. 3a) did not affect IClswell (F test); in contrast, 0.5, 1.0 and 5.0 μM curcumin ( Fig. 3c, e and g) significantly up-regulated IClswell (curcumin vs DMSO: p = 0.0001, p < 0.0001 and p < 0.0001 respectively, F test). Surprisingly, a further increase in curcumin concentration led to the opposite effect. As shown in Fig. 3i, long-term exposure to 10 μM curcumin significantly impaired IClswell activation with respect to DMSO (p < 0.0001, F test).