CALCIUM-DEPENDENT AND CAMKII-DEPENDENT CHLORIDE SECRETION INDUCED BY THE MICROSOMAL CA2+-ATPASE INHIBITOR 2,5-DI-(TERT-BUTYL)-1,4-HYDROQUINONE IN CYSTIC-FIBROSIS PANCREATIC EPITHELIAL-CELLS

Microsomal Ca2+-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 Ca2+-dependent pathway, in the cystic fibrosis (CF)-derived pancreatic epithelial cell line CFPAC-1, DBHQ stimulated I-125 efflux and mobilized intracellular free Ca2+ in a dose-dependent manner, The maximal effects were seen at concentrations of 25-50 mu M. DBHQ (25 mu M) 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 mu M), 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 Ca2+-independent or autonomous activity of CaMKII assayed in the cell homogenates. Thus, DBHQ appears to enhance Cl- channel activity via a Ca2+-dependent mechanism involving CaMKII. Pretreatment of CFPAC-1 cells with up to 50 mu M 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 Ca2+-ATPase inhibitors may be therapeutically useful in improving Cl- secretion in CF epithelial cells.