3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone and fenamic-acid

Sputum symptoms are commonly seen in the elderly. This study aimed to identify an efficacious expectorant treatment stratagem through evaluating the secretion-promoting activation and cystic fibrosis transmembrane conductance regulator (CFTR) expression of the bioactive herbal monomer naringenin.. Vectorial Cl- transport was determined by measuring short-circuit current (ISC) in rat airway epithelium. cAMP content was measured by ELISA in primary cultured epithelial cells and Calu-3 cells. CFTR expression in Calu-3 cells was determined by qPCR.. Addition of naringenin to the basolateral side of the rat airway led to a concentration-dependent sustained increase in ISC. The current was suppressed when exposed to Cl--free solution or by bumetanide, BaCl2, and DPC but not by DIDS and IBMX. Forskolin-induced ISC increase and CFTRinh-172/MDL-12330A-induced ISC inhibition were not altered by naringenin. Intracellular cAMP content was significantly increased by naringenin. With lipopolysaccharide stimulation, CFTR expression was significantly reduced, and naringenin dose-dependently enhanced CFTR mRNA expression.. These results demonstrate that naringenin has the ability to stimulate Cl- secretion, which is mediated by CFTR through a signaling pathway by increasing cAMP content. Moreover, naringenin can increase CFTR expression when organism CFTR expression is seriously hampered. Our data suggest a potentially effective treatment strategy for sputum.

Topics: Animals; Barium Compounds; Benzoates; Cells, Cultured; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Female; Flavanones; Humans; Imines; Ion Transport; Male; Microscopy, Fluorescence; ortho-Aminobenzoates; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Thiazolidines; Trachea

Mammalian sperm must undergo a maturational process, named capacitation, in the female reproductive tract to fertilize the egg. Sperm capacitation is regulated by a cAMP/protein kinase A (PKA) pathway and involves increases in intracellular Ca(2+), pH, Cl(-), protein tyrosine phosphorylation, and in mouse and some other mammals a membrane potential hyperpolarization. The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel modulated by cAMP/PKA and ATP, was detected in mammalian sperm and proposed to modulate capacitation. Our whole-cell patch-clamp recordings from testicular mouse sperm now reveal a Cl(-) selective component to membrane current that is ATP-dependent, stimulated by cAMP, cGMP, and genistein (a CFTR agonist, at low concentrations), and inhibited by DPC and CFTR(inh) -172, two well-known CFTR antagonists. Furthermore, the Cl(-) current component activated by cAMP and inhibited by CFTR(inh) -172 is absent in recordings on testicular sperm from mice possessing the CFTR ΔF508 loss-of-function mutation, indicating that CFTR is responsible for this component. A Cl(-) selective like current component displaying CFTR characteristics was also found in wild type epididymal sperm bearing the cytoplasmatic droplet. Capacitated sperm treated with CFTR(inh) -172 undergo a shape change, suggesting that CFTR is involved in cell volume regulation. These findings indicate that functional CFTR channels are present in mouse sperm and their biophysical properties are consistent with their proposed participation in capacitation.

Topics: Animals; Benzoates; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiological Phenomena; Female; Genistein; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Inbred CFTR; Mutant Proteins; ortho-Aminobenzoates; Patch-Clamp Techniques; Sperm Capacitation; Spermatozoa; Thiazolidines

Virulence of the intracellular pathogen Listeria monocytogenes (Listeria) requires escape from the phagosome into the host cytosol, where the bacteria replicate. Phagosomal escape is a multistep process characterized by perforation, which is dependent on the pore-forming toxin listeriolysin O (LLO), followed by rupture. The contribution of host factors to Listeria phagosomal escape is incompletely defined. Here we show that the cystic fibrosis transmembrane conductance regulator (CFTR) facilitates Listeria cytosolic entry. CFTR inhibition or mutation suppressed Listeria vacuolar escape in culture, and inhibition of CFTR in wild-type mice before oral inoculation of Listeria markedly decreased systemic infection. We provide evidence that high chloride concentrations may facilitate Listeria vacuolar escape by enhancing LLO oligomerization and lytic activity. We propose that CFTR transiently increases phagosomal chloride concentration after infection, potentiating LLO pore formation and vacuole lysis. Our studies suggest that Listeria exploits mechanisms of cellular ion homeostasis to escape the phagosome and emphasize host ion-channel function as a key parameter of bacterial virulence.

Topics: Animals; Bacterial Toxins; Benzoates; Calcium Channel Blockers; Cell Line; Cells, Cultured; Chlorides; Cystic Fibrosis Transmembrane Conductance Regulator; Cytosol; Female; Glycine; Heat-Shock Proteins; Hemolysin Proteins; Host-Pathogen Interactions; Hydrazines; Hydrogen-Ion Concentration; Listeria monocytogenes; Listeriosis; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; ortho-Aminobenzoates; Phagosomes; Protein Multimerization; Thiazolidines; Vacuoles

The aim of this study was to investigate the functional expression of cystic fibrosis transmembrane conductance regulator (CFTR) with electrophysiological and molecular technique in rat oviduct epithelium. In whole-cell patch clamp, oviduct epithelial cells responded to 100 microM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) with a rise in inward current in Gap-free mode, which was inhibited successively by 5 microM CFTR(inh)-172, a CFTR specific inhibitor, and 1 mM diphenylamine-2-carboxylate (DPC), the Cl- channel blocker. The cAMP-activated current exhibited a linear current-voltage (I-V) relationship and time- and voltage-independent characteristics. The reversal potentials of the cAMP-activated currents in symmetrical Cl- solutions were close to the Cl- equilibrium, 0.5+/-0.2 mV (n=4). When Cl- concentration in the bath solution was changed from 140 mM to 70 mM and a pipette solution containing 140 mM Cl- was used, the reversal potential shifted to a value close to the new equilibrium for Cl-, 20+/-0.6 mV (n=4), as compared with the theoretic value of 18.7 mV. In addition, mRNA expression of CFTR was also detected in rat oviduct epithelium. Western blot analysis showed that CFTR protein is found in the oviduct throughout the cycle with maximal expression at estrus, and immunofluorescence and immunohistochemistry analysis revealed that CFTR is located at the apical membrane of the epithelial cells. These results showed that the cAMP-activated Cl- current in the oviduct epithelium was characteristic of CFTR, which provided direct evidence for the functional expression of CFTR in the rat oviduct epithelium. CFTR may play a role in modulating fluid transport in the oviduct.

Topics: Animals; Benzoates; Calcium Channel Blockers; Cells, Cultured; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; Epithelial Cells; Epithelium; Estrous Cycle; Female; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Immunohistochemistry; ortho-Aminobenzoates; Oviducts; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thiazolidines

Chloride (Cl(-)) channels expressed in vascular smooth muscle cells (VSMC) are important to control membrane potential equilibrium, intracellular pH, cell volume maintenance, contraction, relaxation and proliferation. The present study was designed to compare the expression, regulation and function of CFTR Cl(-) channels in aortic VSMC from Cftr(+/+) and Cftr(-)(/)(-) mice. Using an iodide efflux assay we demonstrated stimulation of CFTR by VIP, isoproterenol, cAMP agonists and other pharmacological activators in cultured VSMC from Cftr(+/+). On the contrary, in cultured VSMC from Cftr(-)(/)(-) mice these agonists have no effect, showing that CFTR is the dominant Cl(-) channel involved in the response to cAMP mediators. Angiotensin II and the calcium ionophore A23187 stimulated Ca(2)(+)-dependent Cl(-) channels in VSMCs from both genotypes. CFTR was activated in myocytes maintained in medium containing either high potassium or 5-hydroxytryptamine (5-HT) and was inhibited by CFTR(inh)-172, glibenclamide and diphenylamine-2,2'-dicarboxylic acid (DPC). We also examined the mechanical properties of aortas. Arteries with or without endothelium from Cftr(-/-) mice became significantly more constricted (approximately 2-fold) than that of Cftr(+/+) mice in response to vasoactive agents. Moreover, in precontracted arteries of Cftr(+/+) mice, VIP and CFTR activators induced vasorelaxation that was altered in Cftr(-/-) mice. Our findings suggest a novel mechanism for regulation of the vascular tone by cAMP-dependent CFTR chloride channels in VSMC. To our knowledge this study is the first to report the phenotypic consequences of the loss of a Cl(-) channel on vascular reactivity.

Topics: Adrenergic beta-Agonists; Angiotensin II; Animals; Aorta, Thoracic; Benzoates; Cells, Cultured; Chlorides; Colforsin; Cystic Fibrosis Transmembrane Conductance Regulator; Genistein; Glyburide; In Vitro Techniques; Isoproterenol; Mice; Mice, Inbred CFTR; Muscle, Smooth, Vascular; ortho-Aminobenzoates; Quinolizines; Serotonin; Thiazoles; Thiazolidines; Vasoactive Intestinal Peptide; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents