thapsigargin and fenamic-acid

The effect of genistein on anion secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in cultured rat cauda epididymal epithelia was studied by short-circuit current (Isc) technique. Genistein added apically stimulated a concentration-dependent rise in Isc due to Cl(-) and HCO(3)(-) secretion. The genistein-induced Isc was observed in basolaterally permeabilized monolayers, suggesting that the Isc response was mediated by the apical anion channel. The response could be blocked by the nonspecific Cl(-) channel blocker, diphenylamine-2-carboxylate (DPC), but not by the Ca(2+)-activated Cl(-) channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Genistein did not increase intracellular cAMP, but H-89, a protein kinase A inhibitor, completely abolished the Isc response to genistein. Moreover, pretreatment of the tissues with MDL-12330A, an adenylate cyclase inhibitor, markedly attenuated the Isc response to genistein, but the response was restored upon the addition of exogenous cAMP. Ca(2+), protein kinase C, tyrosine kinase, and protein phosphatase signalling pathways were not involved in the action of genistein. It is speculated that genistein stimulates anion secretion by direct interaction with CFTR. This requires a low level of phosphorylation of CFTR by basal protein kinase A activity. It is suggested that genistein may provide therapeutic benefit to male infertility associated with cystic fibrosis.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenylyl Cyclase Inhibitors; Animals; Bicarbonates; Cells, Cultured; Chloride Channels; Chlorides; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Enzyme Inhibitors; Epididymis; Epithelium; Genistein; Male; ortho-Aminobenzoates; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Thapsigargin

This study was designed to investigate the involvement of myosin light-chain kinase (MLCK) in bradykinin- and thapsigargin-induced changes in intracellular Cl- and Ca2+ concentrations ([Cl-]i; [Ca2+]i) in porcine aortic endothelial cells.. Using the fluorescent probes N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE) and fura-2/AM, the effects of different MLCK inhibitors on bradykinin- and thapsigargin-induced changes in [Cl-]i and [Ca2+]i were assessed.. Bradykinin and thapsigargin significantly decreased the MQAE fluorescence intensity, which indicates increased [Cl-]i; these changes were reversed by removal of extracellular chloride (Cl-o) and were significantly inhibited by Cl(-)-channel inhibitor N-phenylanthranilic acid but not by Na(+)-K(+)-Cl- cotransport inhibitor furosemide. Pretreatment with ML-9 and wortmannin, two different selective inhibitors of MLCK, significantly reduced these changes in a dose-dependent manner. The inhibitory effects of ML-9 and wortmannin on the Cl- responses were not significantly different and were not additive. Bradykinin and thapsigargin provoked large increases in [Ca2+]i, which were significantly diminished by removal of Cl-o and by pretreatment with the Cl(-)-channel inhibitor N-phenylanthranilic acid.. The study shows that an increase in [Cl-]i may be involved in the Ca2+ influx in response to bradykinin and thapsigargin and that MLCK might be involved in the Cl- response. We suggest that MLCK might be involved in the Cl(-)-sensitive endothelial Ca2+ responses to bradykinin and thapsigargin.

Topics: Androstadienes; Animals; Aorta; Azepines; Bradykinin; Calcium; Calcium Channel Blockers; Carrier Proteins; Cells, Cultured; Chlorides; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Furosemide; Myosin-Light-Chain Kinase; ortho-Aminobenzoates; Sodium-Potassium-Chloride Symporters; Swine; Thapsigargin; Wortmannin