The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP4 receptor has been described [2-5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xeno pus oocytes expressing ClC-2, with or without co-expression of the EP4 receptor or p adrenergic receptor (beta AR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either beta AR or the EP4 receptor were also studied. In oocytes co-expressing ClC-2 and beta AR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the beta adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing CIC-2 and EP4 did not respond to the presence of 0.1, 1, or 10 mu M lubiprostone in the superperfusate. Oocytes co-expressing CFTR and beta AR did not respond to hyperpolarization, acidic pH, or 1 mu M lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTRinh 172. Co-expression of CFTR and EP4 resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTRinh 172. The EC50 for lubiprostone mediated CFTR activation was similar to 10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP4 receptor in oocytes. (c) 2012 Elsevier Inc. All rights reserved.