Cystic Fibrosis Pancreatic Duct Cells Research Articles

CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector

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.

CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector

CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector

Expression of a Wild-type CFTR Maintains the Integrity of the Biosynthetic/Secretory Pathway in Human Cystic Fibrosis Pancreatic Duct Cells

The structural integrity of the Golgi complex is essential to its functions in the maturation, sorting, and transport of plasma membrane proteins. Previously, we demonstrated that in pancreatic duct CFPAC-1 cells, which express DeltaF508 CFTR (cystic fibrosis transmembrane conductance regulator), the intracellular trafficking of carbonic anhydrase IV (CA IV), a membrane protein involved in HCO(3)(-) secretion, was impaired. To determine whether these abnormalities were related to changes in the Golgi complex, we examined the ultrastructure and distribution of Golgi compartments with regard to the microtubule cytoskeleton in CFPAC-1 cells transfected or not with the wild-type CFTR. Ultrastructural and immunocytochemical analysis showed that in polarized CFPAC-1 cells, Golgi stacks were disconnected from one another and scattered throughout the cytoplasm. The colocalization of CA IV with markers of Golgi compartments indicated the ability of stacks to transfer this enzyme. This Golgi dispersal was associated with abnormal microtubule distribution and multiplicity of the microtubule-organizing centers (MTOCs). In reverted cells, the normalization of Golgi structure, microtubule distribution, and MTOC number was observed. These observations suggest that the entire biosynthetic/secretory pathway is disrupted in CFPAC-1 cells, which might explain the abnormal intracellular transport of CA IV. Taken together, these results point to the fact that the expression of DeltaF508 CFTR affects the integrity of the secretory pathway.

Synergism between a dietary flavonoid and butyric acid derivative in correcting chloride secretory defect in cystic fibrosis pancreatic duct cells

Synergism between a dietary flavonoid and butyric acid derivative in correcting chloride secretory defect in cystic fibrosis pancreatic duct cells

Synergism between a dietary flavonoid and butyric acid derivative in correcting chloride secretory defect in cystic fibrosis pancreatic duct cells

Synergism between a dietary flavonoid and butyric acid derivative in correcting chloride secretory defect in cystic fibrosis pancreatic duct cells

Defective regulation of gap junctional coupling in cystic fibrosis pancreatic duct cells.

The cystic fibrosis (CF) gene encodes a cAMP-gated Cl- channel (cystic fibrosis transmembrane conductance regulator [CFTR]) that mediates fluid transport across the luminal surfaces of a variety of epithelial cells. We have previously shown that gap junctional communication and Cl- secretion were concurrently regulated by cAMP in cells expressing CFTR. To determine whether intercellular communication and CFTR-dependent secretion are related, we have compared gap junctional coupling in a human pancreatic cell line harboring the DeltaF508 mutation in CFTR and in the same cell line in which the defect was corrected by transfection with wild-type CFTR. Both cell lines expressed connexin45 (Cx45), as evidenced by RT-PCR, immunocytochemistry, and dual patch-clamp recording. Exposure to agents that elevate intracellular cAMP or specifically activate protein kinase A evoked Cl- currents and markedly increased junctional conductance of CFTR-expressing pairs, but not in the parental cells. The latter effect, which was caused by an increase in single-channel activity but not in unitary conductance of Cx45 channels, was not prevented by exposing CFTR-expressing cells to a Cl- channel blocker. We conclude that expression of functional CFTR restored the cAMP-dependent regulation of junctional conductance in CF cells. Direct intercellular communication coordinates multicellular activity in tissues that are major targets of CF manifestations. Consequently, defective regulation of gap junction channels may contribute to the altered functions of tissues affected in CF.

Modulation of Ca 2+-dependent anion secretion by protein kinase C in normal and cystic fibrosis pancreatic duct cells

The study investigated the role of protein kinase C (PKC) in the modulation of agonist-induced Ca 2+-dependent anion secretion by pancreatic duct cells. The short-circuit current ( I SC) technique was used to examine the effect of PKC activation and inhibition on subsequent ATP, angiotensin II and ionomycin-activated anion secretion by normal (CAPAN-1) and cystic fibrosis (CFPAC-1) pancreatic duct cells. The I SC responses induced by the Ca 2+-mobilizing agents, which had been previously shown to be attributed to anion secretion, were enhanced in both CAPAN-1 and CFPAC-1 cells by PKC inhibitors, staurosporine, calphostin C or chelerythrine. On the contrary, a PKC activator, phorbol 12-myristate 13-acetate (PMA), was found to suppress the agonist-induced I SC in CFPAC-1 cells and the ionomycin-induced I SC in CAPAN-1 cells. An inactive form of PMA, 4α d-phorbol 12,13-didecanote (4αD), was found to exert insignificant effect on the agonist-induced I SC, indicating a specific effect of PMA. Our data suggest a role of PKC in modulating agonist-induced Ca 2+-dependent anion secretion by pancreatic duct cells. Therapeutic strategy to augment Ca 2+-activated anion secretion by cystic fibrosis pancreatic duct cells may be achieved by inhibition or down-regulation of PKC.

Angiotensin II-mediated signal transduction events in cystic fibrosis pancreatic duct cells

Different signal transduction pathways, i.e. Ca 2+- and cAMP-dependent, involved in mediating the effects of angiotensin II (AII) were investigated separately using the short-circuit current ( I sc) technique and radioimmunoassay (RIA) in a cystic fibrosis pancreatic cell line (CFPAC-1) which exhibits defective cAMP-dependent but intact Ca 2+-dependent anion secretion. The AII-induced I sc could be inhibited by the specific antagonist for AT 1, losartan (1 μM), but not the antagonist for AT 2, PD123177 (up to 10 μM). The AII-induced I sc was also reduced by the treatment of the cells with a Ca 2+ chelator, BAPTA-AM (100 μM), indicating a dependence of the AII-induced anion secretion on the intracellular Ca 2+. Treatment of the cells with pertussis toxin (0.1 μg/ml) or a phospholipase C (PLC) inhibitor, U73122 (5 μM), resulted in a substantial reduction in the AII-induced I sc indicating involvement of Gi and PLC in the Ca 2+-dependent anion secretion. RIA measurements showed that AII stimulated an increase in cAMP production which could be reduced by losartan, pertussis toxin and U73122 but not BAPTA-AM. In addition, inhibitors of cyclooxygenase, indomethacin (10 μM) and piroxicam (10 μM), did not have any effect on the AII-induced cAMP production, excluding the involvement of prostaglandins. Our results suggest that both AII-stimulated cAMP and Ca 2+-dependent responses are mediated by the AT 1 receptor and Gi-coupled PLC pathway. However, the AII-stimulated cAMP production in CFPAC-1 cells is not dependent on Ca 2+ or the formation of prostaglandins.

Sulfation of Chondroitin/Dermatan Sulfate by Cystic Fibrosis Pancreatic Duct Cells Is Not Different from Control Cells

Cystic fibrosis is associated with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated plasma membrane chloride channel. Cystic fibrosis patients have been reported to possess elevated sulfation of glycoconjugates, which may contribute to the pathogenesis of the disease. Sulfation of glycosaminoglycans by a cystic fibrosis pancreatic adenocarcinoma cell line homozygous for ΔF508(CFPAC-1), a control pancreatic cell line (PANC-1), two CFPAC-1 cell lines transfected with the gene for CFTR (PLJ-CFTR-4.7, TR20), and a mock-transfected CFPAC-1 control (PLJ-6) was investigated. Cells were radiolabeled with [35S]sulfate and [3H]glucosamine, and glycosaminoglycans secreted into the medium after 24 and 72 h were isolated. Chondroitinase ABC digestion of chondroitin/dermatan sulfate allowed the recovery of disaccharides which were analyzed for their degree of sulfation by strong anion-exchange HPLC. No differences in the extent of sulfation by any of the cell lines were noted. However, glycoaminoglycans synthesized by cystic fibrosis cells consistently exhibited twofold higher [35S]-sulfate:[3H]glucosamine ratios than the controls. We conclude that CFTR plays no role in the sulfation of chondroitin/dermatan sulfate by pancreatic cells and that isotope incorporation ratios alone are insufficient evidence of changes in sulfation levels.

Angiotensin II receptor type I-regulated anion secretion in cystic fibrosis pancreatic duct cells.

The beta-adrenergic (cAMP-dependent) regulation of Cl- conductance is defective in cystic fibrosis (CF). The present study explored alternative regulation of anion secretion in CF pancreatic ductal cells (CFPAC-1) by angiotensin II (AII) using the short-circuit current (ISC) technique. An increase in ISC could be induced in CFPAC-1 cells by basolateral or apical application of AII in a concentration-dependent manner (EC50 at 3 microm and 100 nm, respectively). Angiotensin receptor subtypes were identified using specific antagonists, losartan and PD123177, for AT1 and AT2 receptors, respectively. It was found that losartan (1 microm) could completely inhibit the AII-induced ISC, whereas, PD123177 exerted insignificant effect on the ISC, indicating predominant involvement of AT1 receptors. The presence of AT1 receptors in CFPAC-1 cells was also demonstrated by immunohistochemical studies using specific antibodies against AT1 receptors. Confocal microscopic study demonstrated a rise in intracellular Ca2+ upon stimulation by AII indicating a role of intracellular Ca2+ in mediating the AII response. Depletion of intracellular but not extracellular pool of Ca2+ diminished the AII-induced ISC. Treatment of the monolayers with a Cl- channel blocker, DIDS, markedly reduced the ISC, indicating that a large portion of the AII-activated ISC was Cl--dependent. AII-induced ISC was also observed in monolayers whose basolateral membranes had been permeabilized by nystatin, suggesting that the ISC was mediated by apical Cl- channels. Our study indicates an AT1-mediated Ca2+-dependent regulatory mechanism for anion secretion in CF pancreatic duct cells which may be important for the physiology and pathophysiology of the pancreas.

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 (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.