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Results for “PANCREATIC CYSTIC FIBROSIS”

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At least 37 records · Page 2Linked to original sources
PubMed1992

cAMP-activated Cl channels in CFTR-transfected cystic fibrosis pancreatic epithelial cells.

Retrovirus-mediated transfection of cDNA for the cystic fibrosis (CF) gene into the CF pancreatic cell line, CFPAC-1, confers adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of Cl conductance. We used patch-clamp techniques to identify the single-channel basis of this conductance pathway and to study its properties. Forskolin or cAMP activated Cl channels with a conductance of 9 +/- 1 pS in 26 of 62 cell-attached patches of cystic fibrosis transmembrane conductance regulator (CFTR)-transfected CFPAC-1 cells. The current-voltage (I-V) relation showed slight outward rectification (chord conductance of 10 +/- 2 pS at +80 mV vs. 7 +/- 1 pS at -80mV) with high Cl concentrations (170 mM) in the pipette solution. Channel kinetics were voltage sensitive, with longer openings at positive clamp voltages. Channel properties were unaffected by the substitution of N-methyl-D-glucamine for pipette Na or by the addition of disulfonic stilbenes (100 microM DNDS or DIDS) to the pipette. The channels usually inactivated within seconds of patch excision, but in three of nine patches, activity could be maintained by addition of the catalytic subunit of protein kinase A and ATP. With equal Cl concentrations on both membrane surfaces, the single-channel I-V relation was linear, suggesting that the outward rectification of the cell-attached channel is due to a pipette-to-cell Cl gradient. Anion substitution on the extracellular side of the membrane indicates a halide permselectivity of Br approximately Cl greater than I.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line, Transformed↗
PubMed1999

Modulation of Ca2+-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 Ca2+-dependent anion secretion by pancreatic duct cells. The short-circuit current (ISC) 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 ISC responses induced by the Ca2+-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 ISC in CFPAC-1 cells and the ionomycin-induced ISC in CAPAN-1 cells. An inactive form of PMA, 4alphad-phorbol 12, 13-didecanote (4alphaD), was found to exert insignificant effect on the agonist-induced ISC, indicating a specific effect of PMA. Our data suggest a role of PKC in modulating agonist-induced Ca2+-dependent anion secretion by pancreatic duct cells. Therapeutic strategy to augment Ca2+-activated anion secretion by cystic fibrosis pancreatic duct cells may be achieved by inhibition or down-regulation of PKC.

Adenosine Triphosphate↗
PubMedDate not supplied

Stimulation of HCO3- secretion across cystic fibrosis pancreatic duct cells by extracellular ATP.

Previous studies have demonstrated that extracellular ATP is able to activate Ca2+-dependent Cl- channels which may be important in circumventing the defective cAMP-dependent pancreatic ductal HCO3- secretion in cystic fibrosis (CF). The present study further investigated the effect of extracellular ATP on the stimulation of HCO3- secretion across CF pancreatic duct cells (CFPAC-1). Cells were grown in culture plate inserts which enabled the access of a pH microelectrode to the apical compartment. Changes in apical pH upon stimulation by extracellular ATP, as an indication of HCO3- secretion, were measured. ATP induced a rapid increase in apical pH, which reached a plateau with an averaged pH unit of 0.3 higher than that measured in unstimulated cells. The effect of ATP was concentration dependent. The ATP-induced change in pH could be blocked by apical addition of Cl- channel blockers, indicating that activation of apical Cl- channel is vital for HCO3- secretion by the pancreatic duct cells. Together with the previous finding, the present study suggests that HCO3- secretion may be stimulated in CF pancreatic duct cells by extracellular ATP via a cAMP-independent pathway.

Adenosine Triphosphate↗
PubMed1990

A cystic fibrosis pancreatic adenocarcinoma cell line.

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.

Adenocarcinoma↗
PubMed1990

Genetic complementation in cystic fibrosis pancreatic cells by somatic cell fusion.

The biochemical defect that underlies the genetic disorder cystic fibrosis (CF) has been proposed to involve an altered regulation of epithelial Cl- permeability by agents such as adenosine 3',5'-cyclic monophosphate (cAMP). We report here the successful complementation of this functional defect achieved by using the technique of somatic cell fusion to introduce the normal CF allele into mutant cells. CF epithelial cells were fused with transfectant mouse fibroblasts that contain the normal human gene. The resulting heterokaryons were examined for restoration of cAMP-activated Cl- transport using an optical assay of Cl- permeability. Our results provide direct evidence for the involvement of the protein product of the normal CF allele in modulating epithelial Cl- permeability.

Animals↗
PubMed1983

Pancreatic polypeptide secretion. A marker for disturbed pancreatic function in cystic fibrosis.

Pancreatic polypeptide, a 36-amino peptide, is released from the pancreas by a variety of stimuli, including intravenous Boots secretin. Studies in a generalized destructive and inflammatory process such as chronic pancreatitis have revealed a markedly diminished response to stimulation. To assess whether pancreatic polypeptide release in response to Boots secretin provides a useful measure of pancreatic destruction in cystic fibrosis, 41 patients with proven cystic fibrosis, aged 14 months-23 years, and seven control subjects, aged 18-24 years were studied. Serum pancreatic polypeptide, measured by radioimmunoassay, rose from a basal of 18.5 +/- 2.7 pmol/liter to a peak of 35.6 +/- 4.3 pmol/liter at 5 min in cystic fibrosis, and from a basal of 10.8 +/- 2.8 pmol/liter to a 5-min peak of 109 +/- 27.7 pmol/liter in control subjects. The basal levels of both groups were similar but the cystic fibrosis patients had a significantly lower peak response than controls (P less than 0.05). The peak over basal pancreatic polypeptide ratio was calculated and was less than five in 93% of cystic fibrosis patients, whereas all control subjects had a ratio greater than five. Pancreatic polypeptide measurements in response to secretin may be a convenient and useful means of following the course of pancreatic disease in a chronic illness such as cystic fibrosis.

Adolescent↗
PubMed1997

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Nitric oxide inhibits whole-cell current in cystic fibrosis pancreatic epithelial cells.

We characterized the effects of nitric oxide (NO) on whole-cell current in pancreatic epithelial cell lines from control (PANC-1) and cystic fibrosis patients (CFPAC-1). The nitric oxide donor S-nitrosoglutathione (GSNO) significantly reduced whole-cell current in CFPAC-1 cells but had no effect in PANC-1 cells. This inhibitory effect of NO could be eliminated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or charybdotoxin, suggesting the involvement of DIDS-sensitive Cl- channels and charybdotoxin-sensitive K+ channels. Pretreatment of cells with a selective inhibitor of soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3,1]quinoxalin-1-one (ODQ, 10 microM), eliminated the inhibitory effect of NO, but not 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP; 1 mM), indicating that NO acts via a cGMP-dependent pathway. There was a striking difference in cGMP production in response to GSNO in CFPAC-1 cells as compared with PANC-1 cells. GSNO induced a 90-fold increase in cGMP level in CFPAC-1 cells, compared with a threefold increase in PANC-1. Similarly, CFPAC-1 cells showed elevated levels of sGC and constitutive nitric oxide synthase activity as compared with PANC-1 cells. Therefore excessive production of NO, as is seen in inflammatory states, may contribute to the CF phenotype by inhibiting transepithelial ion movement and preventing secretion of digestive enzymes produced by the pancreas.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

New modalities in the treatment of exocrine pancreatic insufficiency in cystic fibrosis.

Pancreatic insufficiency is the second most important pathophysiological expression of cystic fibrosis (CF) and occurs in the majority of patients. It leads to fat malabsorption and high energy losses in the stools and is one of the major causes of malnutrition often seen in CF. Although the development of enteric-coated enzyme preparations offers a dramatic improvement in therapy, it is still difficult to achieve complete correction of fat malabsorption. The cause for this treatment failure is the relative acidic environment in the duodenum induced by a decreased pancreatic bicarbonate output. To improve the efficacy of enteric-coated preparations the dissolution of these preparations in the duodenum must be optimised in order to achieve a high intraduodenal enzyme concentration. With the aim to increase intraduodenal pH, additional therapy with H2-antagonists and oral prostaglandins has been tested without unequivocal success. Omeprazole, a gastric acid inhibitor with more potency and duration of action compared to H2-antagonists, improves the efficacy of enteric-coated capsules of pancreatin dramatically. With a daily dose of 20 mg in addition to Pancrease (3 x 4 capsules) near normalization of faecal fat excretion will be reached in most CF patients with persistent steatorrhoea.

Cystic Fibrosis↗
PubMed2005

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.

Carbonic Anhydrase IV↗
PubMed1995

Calcium- and CaMKII-dependent chloride secretion induced by the microsomal Ca(2+)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone in cystic fibrosis pancreatic epithelial cells.

Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-derived pancreatic epithelial cell line CFPAC-1. DBHQ stimulated 125I efflux and mobilized intracellular free Ca2+ in a dose-dependent manner. The maximal effects were seen at concentrations of 25-50 microM. DBHQ (25 microM) caused a short-term rise in [Ca2+]i in the absence of ambient Ca2+, and a sustained elevation of [Ca2+]i in cell monolayers bathed in the efflux solution (1.2 mM Ca2+), which was largely attenuated by Ni2+ (5 mM). Bath-application of DBHQ induced an outwardly-rectifying whole-cell Cl- current, which was abolished by pipette addition of BAPTA (5 mM) or CaMK [273-302] (20 microM), an inhibitory peptide of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). Pretreatment of monolayers of CFPAC-1 cells with DBHQ for 4-5 min significantly increased the Ca(2+)-independent or autonomous activity of CaMKII assayed in the cell homogenates. Thus, DBHQ appears to enhance Cl- channel activity via a Ca(2+)-dependent mechanism involving CaMKII. Pretreatment of CFPAC-1 cells with up to 50 microM DBHQ for 6 h did not cause any detectable change in cell viability and did not significantly affect the cell proliferation rate. These results suggest that appropriate selective microsomal Ca(2+)-ATPase inhibitors may be therapeutically useful in improving Cl- secretion in CF epithelial cells.

Calcium↗