Effect of NYD-SP27 down-regulation on ATP-induced Ca2+-dependent pancreatic duct anion secretion in cystic fibrosis cells

Our previous study demonstrated that NYD-SP27 is a novel inhibitory PLC isoform, expressed endogenously in human pancreas and upregulated in CFPAC-1 cells. The present study investigated the effect of NYD-SP27 down-regulation on the ATP-stimulated and Ca2+-dependent pancreatic anion secretion by CFPAC-1 cell line using short-circuit current (I-SC) recording. NYD-SP27 anti sense-transfected CFPAC-1 (AT-CF) cells exhibited a significantly higher basal transmembrane potential difference and current than those of empty vector-transfected CFPAC-1 (VT-CF) cells. Cl- channel blocker, DPC or Glibenclamide (1 mM), and inhibitor of Na+-K+-Cl- cotransporter, bumetanide (100 mu M) significantly inhibited the basal current in AT-CF cells. The inhibitor of adenylate cyclase, MDL12330A (20 mu M), and Ca2+-dependent Cl- channel (CaCC) blocker, DIDS (100 mu M) also significantly reduced the basal current in AT-CF. Apical application of ATP (10 mu M) stimulated a fast transient I-SC increase in VT-CF cells, but a more sustained rise with slower decline in AT-CF cells. Pretreatment with BAPTA-AM (50 mu M) reduced the ATP-induced I-SC response in AT-CF cells by 77.9%. PMA (1 mu M), a PKC activator, inhibited the ATP-stimulated current increase (the transient peak) in VT-CF cells, but had no effect on the AT-CF cells. However, PKC inhibitor, staurosporine (40 mu M) could inhibit the ATP-induced I-SC response in AT-CF cells. The present results confirm the previously proposed inhibitory role of NYD-SP27 in the PLC pathway and demonstrate that the suppression of its expression could result in an enhancement of ATP-stimulated Ca2+ dependent pancreatic anion secretion. (C) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.