PURINERGIC REGULATION OF ANION SECRETION BY CYSTIC-FIBROSIS PANCREATIC DUCT CELLS

The present study explored regulation of anion secretion across cystic fibrosis pancreatic ductal epithelium by extracellular ATP with the s hort-circuit current (I-sc) technique. CFPAC-1 cells grown on Millipor e filters formed polarized monolayers with junctional complexes as rev ealed by light and electron microscopy. The cultured monolayers exhibi ted an increase in I-sc in response to apical application of ATP in a concentration-dependent manner (concentration eliciting 50% of maximal response = 3 mu M). Replacement of Cl- in the bathing solution or tre atment of the cells with a Cl- channel blocker, 4,4'-diisothiocyanosti lbene-2,2'-disulfonic acid (DIDS), markedly reduced I-sc, indicating t hat a substantial portion of ATP-activated I-sc was Cl- dependent. The effects of different adenosine nucleosides and/or nucleotides on I-sc were also studied to identify the type of purinoceptors involved. The order of potency, ATP = UTP > ADP > adenosine, was consistent with th at for P-2 purinoceptors. Reactive blue 2 (100 mu M), a P-2 antagonist , was found to inhibit 86% of ATP-induced I-sc. ATP-induced I-sc was a lso inhibited by pretreatment of the cells with a Ca2+ chelator, ,2-bi s(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (50 mu M) Confocal microscopic study also demonstrated a rise in intr acellular Ca2+ with stimulation by extracellular ATP, indicating a rol e of intracellular Ca2+ in mediating the ATP response. ATP-induced I-s c was observed in monolayers whose basolateral membranes had been perm eabilized by nystatin, which was also sensitive to apical addition of DIDS, suggesting that I-sc was mediated by apical Cl- channels. The re sults of the present study demonstrate the presence of a purinergic re gulatory mechanism involving P-2u receptor and Ca2+ mobilization in pa ncreatic duct anion secretion.