3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone and 2-aminoethoxydiphenyl-borate

Under conditions of high dietary salt intake, prostaglandin E2 (PGE2) production is increased in the collecting duct and promotes urinary sodium chloride (NaCl) excretion; however, the molecular mechanisms by which PGE2 increases NaCl excretion in this context have not been clearly defined. We used the mouse inner medullary collecting duct (mIMCD)-K2 cell line to characterize mechanisms underlying PGE2-regulated NaCl transport. When epithelial Na(+) channels were inhibited, PGE2 exclusively stimulated basolateral EP4 receptors to increase short-circuit current (Isc(PGE2)). We found that Isc(PGE2) was sensitive to inhibition by H-89 and CFTR-172, indicating that EP4 receptors signal through protein kinase A to induce Cl(-) secretion via cystic fibrosis transmembrane conductance regulator (CFTR). Unexpectedly, we also found that Isc(PGE2) was sensitive to inhibition by BAPTA-AM (Ca(2+) chelator), 2-aminoethoxydiphenyl borate (2-APB) (inositol triphosphate receptor blocker), and flufenamic acid (FFA) [Ca(2+)-activated Cl(-) channel (CACC) inhibitor], suggesting that EP4 receptors also signal through Ca(2+) to induce Cl(-) secretion via CACC. Additionally, we observed that PGE2 stimulated an increase in Isc through crosstalk between cAMP and Ca(2+) signaling; BAPTA-AM or 2-APB inhibited a component of Isc(PGE2) that was sensitive to CFTR-172 inhibition; H-89 inhibited a component of Isc(PGE2) that was sensitive to FFA inhibition. Together, our findings indicate that PGE2 activates basolateral EP4 receptors and signals through both cAMP and Ca(2+) to stimulate Cl(-) secretion in IMCD-K2 cells. We propose that these signaling pathways, and the crosstalk between them, may provide a concerted mechanism for enhancing urinary NaCl excretion under conditions of high dietary NaCl intake.

Topics: Animals; Benzoates; Boron Compounds; Calcium; Calcium Signaling; Cell Line; Chloride Channels; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Dinoprostone; Egtazic Acid; Flufenamic Acid; Inositol 1,4,5-Trisphosphate Receptors; Ion Transport; Isoquinolines; Kidney Medulla; Kidney Tubules, Collecting; Mice; Patch-Clamp Techniques; Receptors, Prostaglandin E, EP4 Subtype; Sodium Channel Blockers; Sodium Channels; Sodium Chloride; Sulfonamides; Thiazolidines

Retention of F508del-CFTR proteins in the endoplasmic reticulum (ER) is dependent upon chaperone proteins, many of which require Ca(2+) for optimal activity. Here, we show in human tracheal gland CF-KM4 cells, that after correction of F508del-CFTR trafficking by miglustat (N-butyldeoxynojirimycin) or low temperature (27 degrees C), the Ca(2+) mobilization is decreased compared to uncorrected cells and becomes identical to the Ca(2+) response observed in non-CF MM39 cells. In CF-KM4 and human nasal epithelial CF15 cells, we also show that inhibiting vesicular trafficking by nocodazole prevents not only the rescue of F508del-CFTR but also the Ca(2+) mobilization decrease. Finally, experiments using the CFTR inhibitor CFTR(inh)-172 showed that the presence but not the channel activity of F508del-CFTR at the plasma membrane is required to decrease the Ca(2+) mobilization in corrected CF cells. These findings show that correction of the abnormal trafficking of F508del-CFTR proteins might have profound consequences on cellular homeostasis such as the control of intracellular Ca(2+) level.

Topics: 1-Deoxynojirimycin; Adenosine Triphosphate; Benzoates; Boron Compounds; Calcium Signaling; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; Epithelial Cells; Histamine; Humans; Inositol 1,4,5-Trisphosphate Receptors; Nocodazole; Protein Transport; Respiratory Mucosa; Sequence Deletion; Temperature; Thiazolidines