Introduction Lubiprostone stimulates electrogenic Cltransport in human intestinal T84 cells and Clcurrents in HEK293 cells stably transfected with recombinant human ClC-2 (hClC-2) with an EC50 of approximately 20 nM [Cuppoletti et al AJP 287:C1173, 2004]. The purpose of this study was to determine whether lubiprostone (over the concentration range that activates electrogenic Cltransport) causes changes in [Ca]i, cAMP or PKA mediated phosphorylation. Methods T84 cells, HEK293 cells and HEK293 cells stably expressing hClC-2 or mutant hClC-2 were used. T84 cell [Ca]i, was measured using the calcium indicator dye indo-1/AM and intracellular [cAMP] was determined using a commercial ELISA kit. A commercial FLIPER assay was used to measure [Ca]i, and [cAMP] in HEK293 cells. Cl currents were measured by whole cell patch clamp. Results Lubiprostone at 1, 10 or 100 nM did not increase [Ca]i, in T84 cells and only slightly increased [cAMP] from 0.56 ± 0.02 to 4.0 ± 0.3 pmole/105 cells (n=4, P<0.001) at 100 nM (5 times the EC50 for lubiprostone activation of Cl currents). 1 μM PGE1 significantly (P<0.0005) increased [Ca]i from 80.8 ± 2.4 nM (control) to 1175.0 ± 18.7 nM (n=3) and greatly increased [cAMP] to 37.6 ± 0.6 pmole/105 cells (n=4) (P<0.0005). In contrast, 100 nM lubiprostone had no effect on [Ca]i or cAMP in HEK293 cells. Nevertheless hClC-2 in HEK293 cells is activated by lubiprostone, thereby demonstrating that neither [Ca]i or cAMP signaling are involved in lubiprostone activation of hClC-2. To rule out involvement of PKA phosphorylation of hClC-2 in lubiprostone activation of Clcurrents, a mutant hClC-2 Clchannel lacking the two unique PKA consensus phosphorylation sites (RRAT and RGET) essential for PKA activation of ClC-2, was used [Cuppoletti, et al JBC 279:21849, 2004]. In wild-type hClC-2 expressing HEK293 cells, control Clcurrents at -140 mV (n=6) of -27.2 ± 3.5 pA/pF, significantly increased (P<0.0005) to -100.9 ± 6.5 pA/pF with 20 nM lubiprostone and subsequently were reduced with 500 μM CdCl2 to control levels, -29.9 ± 5.6 pA/pF (P<0.0005). Cl currents in mutant hClC-2 expressing HEK293 cells was also significantly (P<0.0005) increased with 20 nM lubiprostone and inhibited by CdCl2 (n=6): control, -27.1 ± 6.9 pA/pF; with lubiprostone, -81.9 ± 5.1 (P<0.0005); after CdCl2, -29.4 ± 3.7 pA/pF (P<0.0005). Conclusions Activation of Clcurrents by lubiprostone is independent of [Ca]i and cAMP signaling and PKA mediated phosphorylation of hClC-2. More direct action of lubiprostone on hClC-2 Clchannels may be involved. Supported by Sucampo Pharmaceuticals, Inc. E001288 Conclusions Activation of Clcurrents by lubiprostone is independent of [Ca]i and cAMP signaling and PKA mediated phosphorylation of hClC-2. More direct action of lubiprostone on hClC-2 Clchannels may be involved.