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 short-circuit current (I-sc) technique. CFPAC-1 cells grown on Millipore filters formed polarized monolayers with junctional complexes as revealed by light and electron microscopy. The cultured monolayers exhibited 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 treatment of the cells with a Cl- channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), markedly reduced I-sc, indicating that 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 that 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 also inhibited by pretreatment of the cells with a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (50 mu M) Confocal microscopic study also demonstrated a rise in intracellular Ca2+ with stimulation by extracellular ATP, indicating a role of intracellular Ca2+ in mediating the ATP response. ATP-induced I-sc was observed in monolayers whose basolateral membranes had been permeabilized by nystatin, which was also sensitive to apical addition of DIDS, suggesting that I-sc was mediated by apical Cl- channels. The results of the present study demonstrate the presence of a purinergic regulatory mechanism involving P-2u receptor and Ca2+ mobilization in pancreatic duct anion secretion.