3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone and 5-nitro-2-(3-phenylpropylamino)benzoic-acid

Besides being a Ca²-regulating hormone, parathyroid hormone (PTH) has also been shown to regulate epithelial transport of certain ions, such as Cl, HCO₃, and Na, particularly in the kidney. Although the intestinal epithelium also expressed PTH receptors, little was known regarding its mechanism in the regulation of intestinal ion transport. We investigated the ion regulatory role of PTH in intestinal epithelium-like Caco-2 monolayer by Ussing chamber technique and alternating current impedance spectroscopy. It was found that Caco-2 cells rapidly responded to PTH within 1 min by increasing apical HCO₃- secretion. CFTR served as the principal route for PTH-stimulated apical HCOV efflux, which was abolished by various CFTR inhibitors, namely, NPPB, glycine hydrazide-101 (GlyH-101), and CFTRinh-172, as well as by small interfering RNA against CFTR. Concurrently, the plasma membrane resistance was decreased with no changes in the plasma membrane capacitance or paracellular permeability. HCOV was probably supplied by basolateral uptake via the electrogenic Na⁺-HCO₃⁻ cotransporter and by methazolamide-sensitive carbonic anhydrase, while the resulting intracellular H⁺ might be extruded by both apical and basolateral Na/H exchangers. Furthermore, the PTH-stimulated HCO₃-secretion was markedly reduced by protein kinase A (PKA) inhibitor (PKI 14-22 amide) and phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY-294002), but not by intracellular Ca²⁺ chelator (BAPTA-AM) or protein kinase C inhibitor (GF-109203X). In conclusion, the present study provided evidence that PTH directly and rapidly stimulated apical HCO₃- secretion through CFTR in PKA- and PI3K-dependent manner, which was a novel noncalciotropic, ion regulatory action of PTH in the intestinal epithelium.

Topics: Benzoates; Bicarbonates; Caco-2 Cells; Cells, Cultured; Cystic Fibrosis Transmembrane Conductance Regulator; Dielectric Spectroscopy; Glycine; Humans; Intestinal Mucosa; Ion Transport; Nitrobenzoates; Parathyroid Hormone; Polymerase Chain Reaction; RNA, Small Interfering; Thiazolidines

In autosomal-dominant polycystic kidney disease (ADPKD), cAMP-stimulated cell proliferation and Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel drive the enlargement of fluid-filled epithelial cysts. To investigate how CFTR blockers inhibit cyst growth, we studied cAMP-dependent Cl- secretion, cell proliferation, and cyst growth using type I Madin Darby canine kidney (MDCK) cells as a model of renal cyst development and growth.. We grew MDCK cysts in collagen gels in the presence of the cAMP agonist forskolin, measured Cl- secretion with the Ussing chamber technique, and assayed cell proliferation using nonpolarized and polarized MDCK cells. To inhibit CFTR, we used glibenclamide, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), genistein, and the specific CFTR inhibitor CFTRinh-172. As controls, we tested the effects of blockers of other types of apical membrane Cl- channels and inhibitors of basolateral membrane ion channels and transporters.. In the absence of inhibitors of transepithelial ion transport, forskolin stimulated dramatic cyst growth. CFTR blockers and inhibitors of basolateral membrane ion channels and transporters retarded cyst growth. In contrast, blockers of other types of apical membrane Cl- channels, which were without effect on CFTR, failed to inhibit cyst growth. Inhibition of cyst growth by CFTR blockers was correlated with inhibition of cAMP-stimulated Cl- current (correlation coefficient = 0.81; P < 0.05), but not cell proliferation (correlation coefficient = 0.50; P > 0.05).. Our data suggest that CFTR blockers might retard cyst growth predominantly by inhibiting fluid accumulation within the cyst lumen.

Topics: Animals; Benzoates; Cell Division; Cell Line; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Dogs; Electric Conductivity; Epithelium; Genistein; Glyburide; Ion Transport; Kidney Diseases, Cystic; Nitrobenzoates; Thiazoles; Thiazolidines