title: Calcium- and CaMKII-dependent chloride secretion induced by the microsomal Ca(2+)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone in cystic fibrosis pancreatic epithelial cells.
abstract: Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-derived pancreatic epithelial cell line CFPAC-1. DBHQ stimulated 125I efflux and mobilized intracellular free Ca2+ in a dose-dependent manner. The maximal effects were seen at concentrations of 25-50 microM. DBHQ (25 microM) caused a short-term rise in [Ca2+]i in the absence of ambient Ca2+, and a sustained elevation of [Ca2+]i in cell monolayers bathed in the efflux solution (1.2 mM Ca2+), which was largely attenuated by Ni2+ (5 mM). Bath-application of DBHQ induced an outwardly-rectifying whole-cell Cl- current, which was abolished by pipette addition of BAPTA (5 mM) or CaMK [273-302] (20 microM), an inhibitory peptide of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). Pretreatment of monolayers of CFPAC-1 cells with DBHQ for 4-5 min significantly increased the Ca(2+)-independent or autonomous activity of CaMKII assayed in the cell homogenates. Thus, DBHQ appears to enhance Cl- channel activity via a Ca(2+)-dependent mechanism involving CaMKII. Pretreatment of CFPAC-1 cells with up to 50 microM DBHQ for 6 h did not cause any detectable change in cell viability and did not significantly affect the cell proliferation rate. These results suggest that appropriate selective microsomal Ca(2+)-ATPase inhibitors may be therapeutically useful in improving Cl- secretion in CF epithelial cells.
authors.affiliation: Department of Molecular Pharmacology and Medicine, Stanford University School of Medicine, California 94305, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Chao
authors.forename: K
authors.initials: K
authors.lastname: Kouyama
authors.forename: E K
authors.initials: EK
authors.lastname: Heist
authors.forename: Y J
authors.initials: YJ
authors.lastname: Dong
authors.forename: P
authors.initials: P
authors.lastname: Gardner
mesh_terms: D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology*; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism*
chemical_list: D018118:Chloride Channels; D002712:Chlorides; D004791:Enzyme Inhibitors; D006873:Hydroquinones; C019359:2,5-di-tert-butylhydroquinone; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000252:Calcium-Transporting ATPases; D002118:Calcium