h-89 and 3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone

Topics: Acrosome Reaction; Alkalies; Benzoates; Cell Movement; Chlorides; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Hydrogen-Ion Concentration; Intracellular Space; Isoquinolines; Membrane Potentials; Models, Biological; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; Sperm Capacitation; Sulfonamides; Thiazolidines

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