The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl− channel that governs the quantity and composition of epithelial secretions. CFTR function is normally tightly controlled as dysregulation can lead to life-threatening diseases such as secretory diarrhoea and cystic fibrosis. CFTR activity is regulated by phosphorylation of its cytosolic regulatory (R) domain, and ATP binding and hydrolysis at two nucleotide-binding domains (NBDs). Here, we report that CFTR activity is also controlled by extracellular Cl− concentration ([Cl−]o). Patch clamp current recordings show that a rise in [Cl−]o stimulates CFTR channel activity, an effect conferred by a single arginine residue, R899, in extracellular loop 4 of the protein. Using NBD mutants and ATP dose response studies in WT channels, we determined that [Cl−]o sensing was linked to changes in ATP binding energy at NBD1, which likely impacts NBD dimer stability. Biochemical measurements showed that increasing [Cl−]o decreased the intrinsic ATPase activity of CFTR mainly through a reduction in maximal ATP turnover. Our studies indicate that sensing [Cl−]o is a novel mechanism for regulating CFTR activity and suggest that the luminal ionic environment is an important physiological arbiter of CFTR function, which has significant implications for salt and fluid homeostasis in epithelial tissues.

BACKGROUND & AIMS: The effects of trypsin on pancreatic ductal epithelial cells (PDECs) vary among species and depend on the localization of proteinase-activated receptor 2 (PAR-2). We compared PAR-2 localization in human and guinea-pig PDECs, and used isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on bicarbonate secretion. METHODS: PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functionally, guinea pig PDECs were studied by microperfusion of isolated ducts, measurements of intracellular pH and intracellular Ca2+ concentration, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. RESULTS: PAR-2 localized to the apical membrane of human and guinea pig PDECs. Trypsin increased intracellular Ca2+ concentration and intracellular pH and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were markedly reduced. CONCLUSIONS: Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2-dependent inhibition of the apical anion exchanger and the CFTR Cl- channel. This could contribute to the development of chronic pancreatitis by decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts.

Kemény, Lajos V. MD, PhD1; Hegyi, Péter MD, PhD1; Rakonczay, Zoltán Jr MD, PhD1; Borka, Katalin MD, PhD2; Korompay, Anna MD2; Gray, Mike A. PhD3; Argent, Barry E. PhD3; Venglovecz, Viktória PhD4 Author Information

Az akut pankreatitisz egyik legyakoribb oka az epekovesseg. A betegseg kialakulasanak pontos mechanizmusa azonban nem kellőkeppen tisztazott. A projekt soran celul tűztuk ki az epesavak hatasanak vizsgalatat pankreasz duktalis epitel sejtekre (PDEC), illetve azok fő funkciojara, a bikarbonat szekreciora. Azt tapasztaltuk, hogy a luminalisan adott kis dozisu nem konjugalt kenodeoxikolsav (CDC) IP3-medialt kalcium szignalizacion keresztul stimulalja a bikarbonat szekreciot, mig a nagy dozis mind apikalisan, mind a bazalisan gatolta azt. A gatlo hatas hattereben a CDC jelentős mitokondrium karosito hatasa all, mely kovetkezmenyesen ATP deplecioval jar. Kiserleteink soran azt talaltuk, hogy az ATP deplecio onmagaban is kepes gatolni a bikarbonat szekreciot. A projekt masik celja az aktivalodott tripszin hatasanak vizsgalata volt. Kiserleteinkben igazoltuk, hogy a luminalisan adott tripszin, vagy PAR2 aktivalo peptid (AP) gatolja a bikarbonat szekreciot. Kiserleteink alapjan vaoszinű, hogy , hogy a tripszin bikarbonat szekreciot gatlo hatasanak hattereben a luminalis CFTR-ra kifejtett gatlo hatas allhat. Jelen projekt eredmenyei nagy mertekben hozzajarulnak az akut pankreatitisz kialakulasanak pontosabb megertesehez. | Cholelithiasis is one of the most common reasons of acute pancreatitis. However the patomechanism of this disease is not well understood. Our aim was to investigate the effects of bile acids on pancreatic ductal epithelial cells (PDEC). We clearly showed that luminal admnistration of small doses chenodeoxycholate (CDC) stimulate bicarbonate secretion via IP3 mediated calcium signalisation. In addition. high doses of CDC administered either from the basolateral or the luminal membrane, inhibit bicarbonate secretion . CDC induced mitochondrial damage, wich evoked intracellular ATP depletion. We fuond, that ATP depletion by itself can inhibit pancreatic bicarbonate secretion. The other aim of the project was to investigate the effects of activated trypsin. We found, that luminal administration of trypsin, or PAR-2 activating peptide (AP) inhibit pancreatic bicarbonate secretion. Furthermore, patch clamp experiments revealed that the inhibitory effect of trypsin on bicarbonate secretion is caused via the inhibition of CFTR Cl- channel. Our results considerably contibute to the pathogenesis of acute pancreatitis.

Objectives: Low doses of chenodeoxycholate (CDC) stimulate apical anion exchange and HCO3- secretion in guinea pig pancreatic duct cells (Gut. 2008; 57: 1102-1112). We examined the effects of CDC on intracellular pH (pH(i)), intracellular Ca2+ concentration ([Ca2+](i)), and apical Cl-/HCO3- exchange activity in human pancreatic duct cells and determined whether any effects were dependent on cystic fibrosis transmembrane conductance regulator (CFTR) expression and Cl- channel activity.Methods: Polarized CFPAC-1 cells (expressing F508del CFTR) were transduced with Sendai virus constructs containing complementary DNAs for either wild-type CFTR or beta-galactosidase. Microfluorimetry was used to record pH(i) and [Ca2+](i) and apical Cl-/HCO3- exchange activity. Patch clamp experiments were performed on isolated guinea pig duct cells.Results: Chenodeoxycholate induced a dose-dependent intracellular acidification and a marked increase in [Ca2+](i) in CFPAC-1 cells. CFTR expression slightly reduced the rate of acidification but did not affect the [Ca2+](i) changes. Luminal administration of 0.1 mmol/L of CDC significantly elevated apical Cl-/HCO3- exchange activity but only in cells that expressed CFTR. However, CDC did not activate CFTR Cl- conductance.Conclusions: Bile salts modulate pH(i), [Ca2+](i), and apical anion exchange activity in human pancreatic duct cells. The stimulatory effect of CDC on anion exchangers requires CFTR expression but not CFTR channel activity.

Bile reflux into the pancreas is a common causes of acute pancreatitis. Our group has shown that luminal chenodeoxycholate (CDC) at low doses (0.1mM) stimulated HCO3- secretion in intact pancreatic ducts (Venglovecz et al. Gut. 2008). This stimulatory effect of CDC on HCO3- secretion was caused by an IP3-mediated elevation of intracellular calcium concentration and an increase in apical Cl-/HCO3- exchange activity. The aim of this work was to investigate whether CDC could also regulate ion channels in native pancreatic cells. Using standard whole cell current recordings (KCl-rich pipette: NaCl-rich bath solutions) exposure of isolated pancreatic duct cells to CDC (0.1 mM) reversibly increased whole cell currents ∼ 3-fold in 75 % of recordings (15/20 cells), and hyperpolarised membrane potential by ∼ 18 mV. Resting and activated currents showed marked outward rectification, and were moderately voltage-dependent. CDC-induced currents were inhibited by external barium (5 mM, n=6), as well as by the selective high conductance K+ channel blocker, iberiotoxin (100nM, n=7). However, they were not sensitive to TRAM34 (selective blocker of intermediate conductance K+ channels) nor UCL 1684 (selective blocker of small conductance K+ channels). Bile acid induced-activation was abolished when cytosolic Ca2+ buffering was increased with 5.0mM EGTA, and was only moderately reduced by removal of bath Ca2+. Higher concentrations of CDC (>0.5 mM) were deleterious if exposed for prolonged periods. Together these results provide strong evidence that low doses of CDC selectively activate iberiotoxin-sensitive K+ channels through an increase in cytosolic Ca2+, primarily from internal stores. Activation of a K+ conductance would hyperpolarise membrane potential and thereby increase the electrochemical driving force for HCO3- secretion through electrogenic apical anion exchangers. Supported by The Royal Society.

Background and aims: Acute pancreatitis is associated with significant morbidity and mortality. Bile reflux into the pancreas is a common cause of acute pancreatitis and, although the bile can reach both acinar and ductal cells, most research to date has focused on the acinar cells. The aim of the present study was to investigate the effects of bile acids on HCO3- secretion from the ductal epithelium.Methods: Isolated guinea pig intralobular/interlobular pancreatic ducts were microperfused and the effects of unconjugated chenodeoxycholate (CDC) and conjugated glycochenodeoxycholate (GCDC) on intracellular calcium concentration ([Ca2+](i)) and pH (pH(i)) were measured using fluorescent dyes. Changes of pH(i) were used to calculate the rates of acid/base transport across the duct cell membranes.Results: Luminal administration of a low dose of CDC (0.1 mM) stimulated ductal HCO3- secretion, which was blocked by luminal H2DIDS (dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). In contrast, both luminal and basolateral administration of a high dose of CDC (1 mM) strongly inhibited HCO3- secretion. Both CDC and GCDC elevated [Ca2+](i), and this effect was blocked by BAPTA-AM (1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid), caffeine, xestospongin C and the phospholipase C inhibitor U73122. BAPTA-AM also inhibited the stimulatory effect of low doses of CDC on HCO3- secretion, but did not modulate the inhibitory effect of high doses of CDC.Conclusions: It is concluded that the HCO3- secretion stimulated by low concentrations of bile acids acts to protect the pancreas against toxic bile, whereas inhibition of HCO3- secretion by high concentrations of bile acids may contribute to the progression of acute pancreatitis.

Hegyi, Péter MD, PhD; Rakonczay, Zoltán Jr MD, PhD; Farkas, Klaudia MD; Venglovecz, Viktória MS; Ózsvari, Béla MS; Seidler, Ursula MD, PhD; Gray, Mike A. PhD; Argent, Barry E. PhD Author Information

Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl- channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO3- -rich fluid. Our aim in this study was to investigate the potential of a recombinant Sendai virus (SeV) vector to introduce normal CFTR into human CF pancreatic duct (CFPAC-1) cells, and to assess the effect of CFTR gene transfer on the key transporters involved in HCO3- transport. Using polarized cultures of homozygous F508del CFPAC-1 cells as a model for the human CF pancreatic ductal epithelium we showed that SeV was an efficient gene transfer agent when applied to the apical membrane. The presence of functional CFTR was confirmed using iodide efflux assay. CFTR expression had no effect on cell growth, monolayer integrity, and mRNA levels for key transporters in the duct cell (pNBC, AE2, NHE2, NHE3, DRA, and PAT-1), but did upregulate the activity of apical Cl-/HCO3- and Na+/H+ exchangers (NHEs). In CFTR-corrected cells, apical Cl-/HCO3- exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl-/HCO3- exchange was inhibited by dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2-DIDS), but not by a specific CFTR inhibitor, CFTR(inh)-172. Our data show that SeV vector is a potential CFTR gene transfer agent for human pancreatic duct cells and that expression of CFTR in CF cells is associated with a restoration of Cl- and HCO3- transport at the apical membrane.

Aims: We have previously shown that luminal administration of low doses (100 l'M) of non-conjugated chenodeoxycholate (CDC) stimulates, whereas, high doses (1000l'M) of this bile acid inhibits HCO3- secretion (administered either from the luminal or basolateral side) in pancreatic ductal epithelial cells (PDEC). The aim of this study was to investigate the intracellular mechanisms by which CDC exerts its effects. Methods: Isolated guinea pig intra/interlobular pancreatic ducts were microperfused from the luminal and basolateral membranes with standard Hepes or HCO3-/CO2 solutions. The effects of CDC on intracellular calcium concentration [Ca2+]i, and intracellular pH (pHi) were estimated using fluorescent dyes (Fura2-AM or BCECF-AM, repectively) by microfluorometry. Results: Basolateral and luminal administration of high doses (1000l'M) of CDC increased the [Ca2+]i. Both the Ca2+-chelator BAPTA-AM and the IP3 inhibitor caffeine blocked the [Ca2+]i elevation. However, the absence of the extracellular Ca2+ did not modify the [Ca2+]i signalling. Furthermore, BAPTA-AM totally blocked the stimulated HCO3- secretion evoked by low doses (100 l'M) of CDC. In contrast, the Ca2+-chelator did not interfere with the inhibitory effect of high doses of CDC. Importantly, low doses of CDC did not increase HCO3- secretion when the anion inhibitor H2DIDS was simultaneously administered into the lumen, suggesting that CDC exerts its stimulatory effect by modulating the anion exchanger located on the luminal membrane of PDEC. Conclusions: Luminal administration of low doses of CDC stimulate the luminal Cl-/HCO3- exchanger via IP3-mediated Ca2+-signaling in guinea pig pancreatic ducts. High doses of CDC inhibit the anion exchanger and induce Ca2+-signaling, however, these effects do not correlate with each other. The inhibition of HCO3- secretion is probably due to the marked acidification caused by this non-conjugated bile acid. This work was supported by OTKA, MTA, OM and Royal Society.

Aims: Exposure of the pancreas to bile acids is considered to be one of the possible causes of acute pancreatitis. However, no information is available on the effects of bile acids on intact pancreatic epithelia. Methods: Isolated guinea pig intra/interlobular pancreatic ducts were perfused from the luminal and basolateral membranes with standard Hepes or HCO3-/CO2 solutions. The effects of chenodeoxycholate (non-conjugated bile acid, CDC) and glycochenodeoxycholate (conjugated bile acid, GCDC) on intracellular pH (pHi) of pancreatic ductal epithelial cells (PDEC) were measured using the fluorescent dye BCECF and microfluorometry. Results: Basolateral or luminal administration of the membrane permeable CDC dose dependently decreased the pHi of PDEC. However, GCDC decreased the pHi mostly from the basolateral side, suggesting that functionally active bile acid transporters are expressed in PDEC. Moreover, luminal administration of low doses (100µM) of CDC significantly stimulated the HCO3- secretion of PDEC, most likely via the luminal anion exchanger. Neither basolateral exposure of low doses of CDC nor low or high doses (100µM and 1000µM, respectively) of GCDC (administered either from the basolateral or luminal side) had an effect on ductal HCO3- secretion. Interestingly, basolateral or luminal administration of high doses (1000µM) of CDC inhibited HCO3- secretion. Low doses of bile acids had no effect on the activities of the Na+/HCO3- cotransporter and the Na+/H+ exchanger. On the other hand, high doses (1000µM) of CDC inhibited the above mentioned acid/base transporters localised on the basolateral membrane of PDEC. Conclusion: These results suggest the presence of specific bile acid transporters in PDEC. Low doses of CDC stimulate pancreatic HCO3- secretion which may serve as a defence mechanism during acute pancreatitis. The inhibition of acid/base transporters by high doses of CDC is probably due to the marked acidification (toxic effect) caused by this non-conjugated bile acid. This work was supported by OTKA, MTA, OM and Royal Society.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (CftrG551D and Cftrtm1UncTgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Introduction: Pancreatic duct cells secrete an alkaline, HCO3- rich fluid, and this process is markedly reduced in cystic fibrosis (CF). Investigations of the H+ and HCO3- transporters involved in ductal HCO3- secretion have been performed mainly on animal tissue. The aim of this study was to characterize the HCO3-/CO2 permeabilities and acid/base transporters in polarised CFPAC-1 human pancreatic duct cells (derived from a CF patient). Methods: CFPAC-1 cells grown on Transwells were loaded with the pH sensitive fluorescent dye BCECF, and mounted into a perfusion chamber, which allowed the simultaneous perfusion of different solutions to the basolateral and apical membranes. Transmembrane acid/base flux was calculated from changes in intracellular pH and the buffering capacity of the cells. Results: The apical membrane of the cells was freely permeable to CO2, but did not allow HCO3- influx from the lumen. In contrast, the basolateral membrane had transporters that promoted rapid HCO3- entry into the cells. Furthermore, our data suggest the functional presence of Na+/H+ exchangers (NHE) on both the apical and basolateral membranes, and Cl-/HCO3- exchanger (AE) and Na+/HCO3- M amiloride µco-transporter (pNBC) activities on the basolateral membrane. 300 completely blocked NHEs on both membranes of the cells. Basolateral HCO3- uptake was sensitive to variations of extracellular K+ concentration and blocked by the anion transport inhibitor H2-DIDS. The application of the membrane permeable carbonic anhydrase inhibitor, acetazolamide, partially decreased the rate of apical and basolateral acid flux and base flux, respectively. RT-PCR revealed the expression of pNBC1, AE2 and NHE1 mRNA. Conclusions: These data suggest that polarised CFPAC-1 human pancreatic duct cells exhibit a differential permeability to HCO3-/CO2at the apical and basolateral membranes and will be a useful model for studying acid/base transporters in CF. Supported by The Wellcome Trust, OTKA (PF63951), ETT (517/2006) and MTA (BO 00218/06).

The inhibitory control of pancreatic ductal HCO 3 − secretion may be physiologically important in terms of limiting the hydrostatic pressure developed within the ducts and in terms of switching off pancreatic secretion after a meal. Substance P (SP) inhibits secretin-stimulated HCO 3 − secretion by modulating a Cl − -dependent HCO 3 − efflux step at the apical membrane of the duct cell (Hegyi P, Gray MA, and Argent BE. Am J Physiol Cell Physiol 285: C268–C276, 2003). In the present study, we have shown that SP is present in periductal nerves within the guinea pig pancreas, that PKC mediates the effect of SP, and that SP inhibits an anion exchanger on the luminal membrane of the duct cell. Secretin (10 nM) stimulated HCO 3 − secretion by sealed, nonperfused, ducts about threefold, and this effect was totally inhibited by SP (20 nM). Phorbol 12,13-dibutyrate (PDBu; 100 nM), an activator of PKC, reduced basal HCO 3 − secretion by ∼40% and totally blocked secretin-stimulated secretion. In addition, bisindolylmaleimide I (1 nM to 1 μM), an inhibitor of PKC, relieved the inhibitory effect of SP on secretin-stimulated HCO 3 − secretion and also reversed the inhibitory effect of PDBu. Western blot analysis revealed that guinea pig pancreatic ducts express the α-, β I -, δ-, ε-, η-, θ-, ζ-, and μ-isoforms of PKC. In microperfused ducts, luminal H 2 DIDS (0.5 mM) caused intracellular pH to alkalinize and, like SP, inhibited basal and secretin-stimulated HCO 3 − secretion. SP did not inhibit secretion further when H 2 DIDS was present in the lumen, suggesting that SP and H 2 DIDS both inhibit the activity of an anion exchanger on the luminal membrane of the duct cell.

AIMTo characterize H+ and HCO3- transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the DeltaF508 CFTR mutation.METHODSCFPAC-1 cells were seeded at high density onto permeable supports and grown to confluence. The cells were loaded with the pH-sensitive fluorescent dye BCECF, and mounted into a perfusion chamber, which allowed the simultaneous perfusion of the basolateral and apical membranes. Transmembrane base flux was calculated from the changes in intracellular pH and the buffering capacity of the cells.RESULTSOur results showed differential permeability to HCO3-/CO2 at the apical and basolateral membranes of CFPAC-1 cells. Na+/ HCO3- co-transporters (NBCs) and Cl-/ HCO3- exchangers (AEs) were present on the basolateral membrane, and Na+/H+ exchangers (NHEs) on both the apical and basolateral membranes of the cells. Basolateral HCO3- uptake was sensitive to variations of extracellular K+ concentration, the membrane permeable carbonic anhydrase (CA) inhibitors acetazolamide (100 micromol/L) and ethoxyzolamide (100 micromol/L), and was partially inhibited by H2-DIDS (600 micromol/L). The membrane-impermeable CA inhibitor 1-N-(4-sulfamoylphenylethyl)-2,4,6-trimethylpyridine perchlorate did not have any effect on HCO3- uptake. The basolateral AE had a much higher activity than that in the apical membrane, whereas there was no such difference with the NHE under resting conditions. Also, 10 micromol/L forskolin did not significantly influence Cl-/ HCO3- exchange on the apical and basolateral membranes. The administration of 250 micromol/L H2-DIDS significantly inhibited the basolateral AE. Amiloride (300 micromol/L) completely inhibited NHEs on both membranes of the cells. RT-PCR revealed the expression of pNBC1, AE2, and NHE1 mRNA.CONCLUSIONThese data suggest that apart from the lack of CFTR and apical Cl-/ HCO3- exchanger activity, CFPAC-1 cells express similar H+ and HCO3- transporters to those observed in native animal tissue.