Purinergic regulation of anion secretion by cystic fibrosis pancreatic duct cells

Abstract

The present study explored regulation of anion secretion across cystic fibrosis pancreatic ductal epithelium by extracellular ATP with the short-circuit current (Isc) 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 Isc in response to apical application of ATP in a concentration-dependent manner (concentration eliciting 50% of maximal response = 3 microM). 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 Isc, indicating that a substantial portion of ATP-activated Isc was Cl- dependent. The effects of different adenosine nucleosides and/or nucleotides on Isc were also studied to identify the type of purinoceptors involved. The order of potency, ATP = UTP > ADP > adenosine, was consistent with that for P2 purinoceptors. Reactive blue 2 (100 microM), a P2 antagonist, was found to inhibit 86% of ATP-induced Isc. ATP-induced Isc 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 microM). 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 Isc was observed in monolayers whose basolateral membranes had been permeabilized by nystatin, which was also sensitive to apical addition of DIDS, suggesting that Isc was mediated by apical Cl- channels. The results of the present study demonstrate the presence of a purinergic regulatory mechanism involving P2U receptor and Ca2+ mobilization in pancreatic duct anion secretion.

Purinergic receptors in the endocrine and exocrine pancreas

“…UTP≈ATP>ADP≫α,β-methylene ATP, β,γ-methylene ATP, 2-methylene ATP [124,127]. These data indicate the presence of UTP-preferring receptors, such as P2Y 2 or perhaps P2Y 4 [13].…”

“…Usually, the function of P2X 4 receptors has been difficult to separate from P2X 7 (Fig. 3c) [122][123][124][125][126]. In addition, ATP/UTP also stimulated HCO À 3 efflux, associated with Cl À HCO À 3 exchange and monitored by intracellular pH measurements [125,126].…”

“…In most studies on cultured cells, cells were grown on coverslips and nucleotides were added to the apical side. However, in a few studies where nucleotides could be added to either the apical/luminal side as opposed to the basolateral/serosal side, they had a more pronounced effect from the apical side [102,124,126]. In two studies nucleotides had the following order of efficacy on stimulation of Cl − transport:…”

Purinergic signalling in the pancreas in health and disease

“…The following information is available. Purinergic regulation of Cl K and ion transport and Ca 2C signalling by CFPAC-1 cells via P2U (P2Y 2 or P2Y 4 ) receptors has been described (Chan et al 1996, Cheung et al 1998, O'Reilly et al 1998, Zsembery et al 2000. CFPAC-1 cells express mRNA and proteins for P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11-14 and P2X 1-7 .…”

The Epac1 Signaling Pathway Regulates Cl− Secretion via Modulation of Apical KCNN4c Channels in Diarrhea

“…The complete inhibition of FSK-stimulated Isc by clotrimazole and TRAM-34 compared with the lack of effect of apamin, iberiotoxin, or zinc suggests a possible role for apical KCNN4 channels but not BK, SK, or KCNQ1/KCNE3 channels in cAMP-stimulated Cl Ϫ secretion. To further show that TRAM-34 inhibits FSK-stimulated Cl Ϫ secretion via blockage of the apical KCNN4 channel, we examined I K(ap) by applying a basolateral to apical K ϩ gradient with K ϩ as the sole permeant ion as described under "Materials and Methods" (39). After nystatin permeabilization of the basolateral membrane and in the presence of this K ϩ gradient, FSK elicited a rise in I K(ap) that was significantly (32 Ϯ 5 versus 8 Ϯ 2 A/cm 2 , p Ͻ 0.01) inhibited by 10 M TRAM-34 (Fig.…”