Background & Aims-The effects of trypsin on pancreatic ductal epithelial cells (PDEC) vary among ... more Background & Aims-The effects of trypsin on pancreatic ductal epithelial cells (PDEC) vary among species and depend on localization of proteinase-activated receptor-2 (PAR-2). Bicarbonate secretion is similar in human and guinea pig PDEC; we compared its localization in these cell types and isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on this process.
ABSTRACT Introduction: Pancreatic duct cells secrete an alkaline, HCO3- rich fluid, and this proc... more ABSTRACT 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).
Low doses of chenodeoxycholate (CDC) stimulate apical anion exchange and HCO3(-) secretion in gui... more 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 (pHi), intracellular Ca(2+) concentration ([Ca(2+)]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. 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 pHi and [Ca(2+)]i and apical Cl(-)/HCO3(-) exchange activity. Patch clamp experiments were performed on isolated guinea pig duct cells. Chenodeoxycholate induced a dose-dependent intracellular acidification and a marked increase in [Ca(2+)]i in CFPAC-1 cells. CFTR expression slightly reduced the rate of acidification but did not affect the [Ca(2+)]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. Bile salts modulate pHi, [Ca(2+)]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.
Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF ... more Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl À channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO À 3 -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 HCO À 3 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 À /HCO À 3 and Na þ /H þ exchangers (NHEs). In CFTR-corrected cells, apical Cl À /HCO À 3 exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl À /HCO À 3 exchange was inhibited by dihydro-4,4 0diisothiocyanostilbene-2,2 0 -disulfonic acid (H 2 -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 À and HCO À 3 transport at the apical membrane.
World journal of gastroenterology : WJG, Jan 14, 2006
To characterize H+ and HCO3- transporters in polarized CFPAC-1 human pancreatic duct cells, which... more To 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. CFPAC-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. Our 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 car...
Background & Aims-The effects of trypsin on pancreatic ductal epithelial cells (PDEC) vary among ... more Background & Aims-The effects of trypsin on pancreatic ductal epithelial cells (PDEC) vary among species and depend on localization of proteinase-activated receptor-2 (PAR-2). Bicarbonate secretion is similar in human and guinea pig PDEC; we compared its localization in these cell types and isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on this process.
ABSTRACT Introduction: Pancreatic duct cells secrete an alkaline, HCO3- rich fluid, and this proc... more ABSTRACT 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).
Low doses of chenodeoxycholate (CDC) stimulate apical anion exchange and HCO3(-) secretion in gui... more 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 (pHi), intracellular Ca(2+) concentration ([Ca(2+)]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. 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 pHi and [Ca(2+)]i and apical Cl(-)/HCO3(-) exchange activity. Patch clamp experiments were performed on isolated guinea pig duct cells. Chenodeoxycholate induced a dose-dependent intracellular acidification and a marked increase in [Ca(2+)]i in CFPAC-1 cells. CFTR expression slightly reduced the rate of acidification but did not affect the [Ca(2+)]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. Bile salts modulate pHi, [Ca(2+)]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.
Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF ... more Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl À channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO À 3 -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 HCO À 3 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 À /HCO À 3 and Na þ /H þ exchangers (NHEs). In CFTR-corrected cells, apical Cl À /HCO À 3 exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl À /HCO À 3 exchange was inhibited by dihydro-4,4 0diisothiocyanostilbene-2,2 0 -disulfonic acid (H 2 -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 À and HCO À 3 transport at the apical membrane.
World journal of gastroenterology : WJG, Jan 14, 2006
To characterize H+ and HCO3- transporters in polarized CFPAC-1 human pancreatic duct cells, which... more To 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. CFPAC-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. Our 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 car...
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Papers by M. Gray