thapsigargin and Cystic-Fibrosis

Cystic fibrosis (CF), a common lethal inherited disorder defined by ion transport abnormalities, chronic infection, and robust inflammation, is the result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated chloride (Cl-) channel. Macrophages are reported to have impaired activity in CF. Previous studies suggest that Cl- transport is important for macrophage function; therefore, impaired Cl- secretion may underlie CF macrophage dysfunction. To determine whether alterations in Cl- transport exist in CF macrophages, Cl- efflux was measured using N-[ethoxycarbonylmethyl]- 6-methoxy-quinolinium bromide (MQAE), a fluorescent indicator dye. The contribution of CFTR was assessed by calculating Cl- flux in the presence and absence of cftr(inh)-172. The contribution of calcium (Ca(2+))-modulated Cl- pathways was assessed by examining Cl- flux with varied extracellular Ca(2+) concentrations or after treatment with carbachol or thapsigargin, agents that increase intracellular Ca(2+) levels. Our data demonstrate that CFTR contributed to Cl- efflux only in WT macrophages, while Ca(2+)-mediated pathways contributed to Cl- transport in CF and WT macrophages. Furthermore, CF macrophages demonstrated augmented Cl- efflux with increases in extracellular Ca(2+). Taken together, this suggests that Ca(2+)-mediated Cl- pathways are enhanced in CF macrophages compared with WT macrophages.

Topics: Animals; Calcium; Carbachol; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Fluorescent Dyes; Macrophages; Mice; Quinolinium Compounds; Thapsigargin

Cells respond rapidly to endoplasmic reticulum (ER) stress by blocking protein translation, increasing protein folding capacity, and accelerating degradation of unfolded proteins via ubiquitination and ER-associated degradation pathways. The ER resident type 2 deiodinase (D2) is normally ubiquitinated and degraded in the proteasome, a pathway that is accelerated by enzyme catalysis of T(4) to T(3). To test whether D2 is normally processed through ER-associated degradation, ER stress was induced in cells that endogenously express D2 by exposure to thapsigargin or tunicamycin. In all cell models, D2 activity was rapidly lost, to as low as of 30% of control activity, without affecting D2 mRNA levels; loss of about 40% of D2 activity and protein was also seen in human embryonic kidney 293 cells transiently expressing D2. In primary human airway cells with ER stress resulting from cystic fibrosis, D2 activity was absent. The rapid ER stress-induced loss of D2 resulted in decreased intracellular D2-mediated T(3) production. ER stress-induced loss of D2 was prevented in the absence of T(4), by blocking the proteasome with MG-132 or by treatment with chemical chaperones. Notably, ER stress did not alter D2 activity half-life but rather decreased D2 synthesis as assessed by induction of D2 mRNA and by [(35)S]methionine labeling. Remarkably, ER-stress-induced loss in D2 activity is prevented in cells transiently expressing an inactive eukaryotic initiation factor 2, indicating that this pathway mediates the loss of D2 activity. In conclusion, D2 is selectively lost during ER stress due to an eukaryotic initiation factor 2-mediated decrease in D2 synthesis and sustained proteasomal degradation. This explains the lack of D2 activity in primary human airway cells with ER stress resulting from cystic fibrosis.

Topics: Animals; Cell Line; Cystic Fibrosis; Down-Regulation; Endoplasmic Reticulum Stress; Epithelial Cells; Eukaryotic Initiation Factor-2; Gene Expression; Humans; Iodide Peroxidase; Iodothyronine Deiodinase Type II; Mice; Proteasome Endopeptidase Complex; Protein Stability; Proteolysis; Respiratory Mucosa; Signal Transduction; Thapsigargin; Thyroxine; Transcription Factor CHOP; Triiodothyronine; Tunicamycin

Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca(2+) concentration ([Ca](i)) in airway epithelial cell lines JME and Calu-3, but bacteria or flagellin activated NF-kappaB, IL-8 promoter, and IL-8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca(2+) pump, releases endoplasmic reticulum Ca(2+), and triggers Ca(2+) entry through plasma membrane channels) both increased [Ca](i) but hardly stimulated NF-kappaB and IL-8. ATP and thapsigargin elicited larger, synergistic activations of NF-kappaB and IL-8 secretion when combined with flagellin. BAPTA-AM (to buffer [Ca](i)) or Ca(2+)-free solution reduced increases in [Ca](i) due to ATP or thapsigargin and also reduced NF-kappaB activation and IL-8 secretion triggered by flagellin, ATP, thapsigargin, ATP + flagellin, and thapsigargin + flagellin. IL-8 promoter analysis showed that AP-1 and CCAAT/enhancer-binding protein (C/EBP)beta/nuclear factor for IL-6 (NF-IL6) sites were important for IL-8 expression, and the NF-kappaB-binding site was critical for activation by all agonists and for activation by [Ca](i). Thus increased [Ca](i) was not required for P. aeruginosa- or flagellin-activated NF-kappaB and IL-8 expression and secretion, and increased [Ca](i) was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in [Ca](i) synergized with flagellin or P. aeruginosa, and buffering or reducing [Ca](i) reduced these responses. Thus [Ca](i) plays an important regulatory role in P. aeruginosa- or flagellin-activated innate immune responses in airway epithelia. Dose-dependent responses indicated that flagellin-ATP synergism occurred most prominently at ATP concentrations ([ATP]) > 10 microM and [flagellin] >10(-8) g/ml and during steady increases rather than oscillations in [Ca](i).

Topics: Adenosine Triphosphate; Base Sequence; Calcium Signaling; Cell Line; Cystic Fibrosis; DNA Primers; Egtazic Acid; Epithelial Cells; Flagellin; Humans; Interleukin-8; NF-kappa B; Promoter Regions, Genetic; Pseudomonas aeruginosa; Respiratory System; Thapsigargin

Cystic fibrosis (CF) is the most common Caucasian autosomal recessive disease. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, which is a chloride (Cl(-)) channel. The most common mutation leads to a missing phenylalanine at position 508 (DeltaF508). The DeltaF508-CFTR protein is misfolded and retained in the endoplasmic reticulum and may trigger the unfolded protein response (UPR). Furthermore, CF is accompanied by inflammation and infection, which are also involved in the UPR. To date, the UPR transducer ATF6 and ER stress sensor Grp78 have been used as UPR markers. Therefore, our aim was to study the activation of ATF6 and Grp78 in transfected human epithelial cells expressing the DeltaF508-CFTR protein, and we showed that they are activated in these cells. We investigated the effect of exogenous UPR inducers thapsigargin (Tg) and tunicamycin (Tu) on Grp78 and ATF6 expression. Whereas the cells reacted to the UPR induction, we show a difference in the electrophoretic pattern of ATF6. The Grp78/ATF6 complex was previously described, but its stability during UPR is controversial. Using co-immunoprecipitation we show that it is stable in DeltaF508-CFTR-expressing cells and is maintained under UPR conditions. Finally, using siRNA, we show that decreased ATF6 expression induces increased cAMP-dependent halide flux through DeltaF508-CFTR due to its increased membrane localization. Therefore, our results suggest that UPR may be triggered in CF and that ATF6 may be a therapeutic target.

Topics: Activating Transcription Factor 6; Cell Line; Cell Membrane; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Genetic Vectors; Heat-Shock Proteins; Humans; Molecular Chaperones; Protein Binding; Protein Folding; Protein Isoforms; Protein Transport; RNA, Small Interfering; Thapsigargin; Transfection; Tunicamycin

Deletion of phenylalanine 508 (deltaF508) accounts for nearly 70% of all mutations that occur in the cystic fibrosis transmembrane conductance regulator (CFTR). The deltaF508 mutation is a class II processing mutation that results in very little or no mature CFTR protein reaching the apical membrane and thus no cAMP-mediated Cl- conductance. Therapeutic strategies have been developed to enhance processing of the defective deltaF508 CFTR molecule so that a functional cAMP-regulated Cl- channel targets to the apical membrane. Sarcoplasmic/endoplasmic reticulum calcium (SERCA) inhibitors, curcumin and thapsigargin, have been reported to effectively correct the CF ion transport defects observed in the deltaF508 CF mice. We investigated the effect of these compounds in human airway epithelial cells to determine if they could induce deltaF508 CFTR maturation, and Cl- secretion. We also used Baby Hamster Kidney cells, heterologously expressing deltaF508 CFTR, to determine if SERCA inhibitors could interfere with the interaction between calnexin and CFTR and thereby correct the deltaF508 CFTR misfolding defect. Finally, at the whole animal level, we tested the ability of curcumin and thapsigargin to (1) induce Cl- secretion and reduce hyperabsorption of Na+ in the nasal epithelia of the deltaF508 mouse in vivo, and (2) induce Cl- secretion in intestine (jejunum and distal colon) and the gallbladder of the deltaF508 CF mouse. We conclude that curcumin and thapsigargin failed to induce maturation of deltaF508 CFTR, or induce Cl- secretion, as measured by biochemical and electrophysiologic techniques in a variety of model systems ranging from cultured cells to in vivo studies.

Topics: Animals; Bronchi; Calcium-Transporting ATPases; Calnexin; Cells, Cultured; Chlorides; Cricetinae; Curcumin; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gallbladder; Humans; Intestines; Ion Transport; Mice; Mice, Mutant Strains; Mutation; Protein Folding; Respiratory Mucosa; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium; Thapsigargin

Cystic fibrosis (CF) is caused by genetic mutations that lead to dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The most common mutation, DeltaF508, causes inefficient trafficking of mutant CFTR protein from the endoplasmic reticulum to the cell membrane. Therapeutic efforts have been aimed at increasing the level of DeltaF508-CFTR protein in the membrane using agents such as sodium butyrate. In this study, we investigated the effects of culturing a human airway epithelial cell line, Calu-3, in the presence of 5 mM sodium butyrate. Within 24 h, butyrate exposure caused a significant decrease in the basal, as well as Ca(2+)-activated, anion secretion by Calu-3 cell monolayers, determined by the change in transepithelial short-circuit current in response to the Ca(2+)-elevating agent thapsigargin. The secretory response to 1-ethyl-2-benzimidazolinone, an activator of the basolateral Ca(2+)-activated K(+) channel KCNN4, was similarly reduced by butyrate treatment. Quantitative PCR revealed that these functional effects were associated with dramatic decreases in mRNA for both KCNN4 and CFTR. Furthermore, the KCNQ1 K(+) channel was upregulated after butyrate treatment. We suggest that prolonged exposure to sodium butyrate downregulates the expression of both KCNN4 and CFTR, leading to a functional loss of Ca(2+)-activated anion secretion. Thus, butyrate may inhibit, rather than stimulate, the anion secretory capacity of human epithelial cells that express wild-type CFTR, particularly in tissues that normally exhibit robust Ca(2+)-activated secretion.

Topics: Benzimidazoles; Butyric Acid; Calcium; Calcium Channel Agonists; Cell Line; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Enzyme Inhibitors; Epithelial Cells; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Lung; Thapsigargin

Using the whole-cell patch-clamp technique, we identified an amiloride (AMI)-sensitive Na(+) current in cystic fibrosis cells, JME/CF15, growing in standard medium. The reversal potential of this current depended on Na(+) concentrations and the cation selectivity was much higher for Na(+) than for K(+), indicating that the current is through ENaC channels. In contrast, cells from EGF-containing medium lacked AMI-sensitive Na(+) currents. In permeabilized cells growing in EGF-containing medium, alphaENaC was mainly detected in a perinuclear region, while in cells from standard medium it was distributed over the cell body. Western-blot analysis showed that in standard medium cells expressed fast-migrating EndoH-insensitive and slow-migrating EndoH-sensitive alphaENaC fractions, while in cells growing in the presence of EGF, alphaENaC was only detected as the fast-migrating EndoH-insensitive fraction. Long-term incubation of cells with EGF resulted in an increased basal Ca(2+) level, [Ca(2+)](i). A similar increase of [Ca(2+)](i) was also observed in the presence of 2muM thapsigargin, resulting in inhibition of ENaC function. Thus, in JME/CF15 cells inhibition of the ENaC function by chronic incubation with EGF is a Ca(2+)-mediated process that affects trafficking and surface expression of ENaC channels.

Topics: Calcium; Cells, Cultured; Cystic Fibrosis; Electric Conductivity; Epidermal Growth Factor; Epithelial Sodium Channels; Glycosylation; Humans; Ion Channel Gating; Ion Transport; Patch-Clamp Techniques; Protein Transport; Sodium; Sodium Channels; Thapsigargin; Time Factors

Airway submucosal glands have been proposed as a primary site for initiating and sustaining airway disease in cystic fibrosis (CF). However, it has been difficult to define the role of CFTR in gland fluid secretion because of concerns in interpreting experiments on diseased CF human airways subjected to chronic infection and inflammation. Here, we test the role of CFTR in gland fluid secretion by using a selective CFTR inhibitor (CFTRinh-172) in pig and human airways. Measurements of single-gland fluid secretion rates showed inhibition of both cholinergic and cAMP-stimulated fluid secretion by CFTRinh-172. Secreted fluid [Na+] and [Cl-] measured by fluorescence ratio imaging were 101 and 116 mM, respectively, and not significantly altered by secretory agonists or CFTR inhibition. Gland fluid pH was 7.1 and reduced by 0.4 units after CFTR inhibition. Gland fluid viscosity, determined by photobleaching of FITC-dextran, was threefold increased in pilocarpine-stimulated gland fluid after CFTR inhibition, and protein concentration was increased from 12 to 20 mg/ml. Our data provide strong evidence that gland fluid secretion is CFTR-dependent. The relatively hyper-viscous and acidic fluid secretions produced by acute CFTR inhibition support a role for defective gland function in CF lung disease and provide a rational basis for pharmacological creation of a large animal model of CF.

Topics: Animals; Benzoates; Body Fluids; Bronchi; Cells, Cultured; Chlorides; Cholinergic Agents; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Exocrine Glands; Humans; Hydrogen-Ion Concentration; Pilocarpine; Second Messenger Systems; Sodium; Swine; Thapsigargin; Thiazoles; Thiazolidines; Viscosity

The most common mutation in cystic fibrosis, Delta F508, results in a cystic fibrosis transmembrane conductance regulator (CFTR) protein that is retained in the endoplasmic reticulum (ER). Retention is dependent upon chaperone proteins, many of which require Ca(++) for optimal activity. Interfering with chaperone activity by depleting ER Ca(++) stores might allow functional Delta F508-CFTR to reach the cell surface. We exposed several cystic fibrosis cell lines to the ER Ca(++) pump inhibitor thapsigargin and evaluated surface expression of Delta F508-CFTR. Treatment released ER-retained Delta F508-CFTR to the plasma membrane, where it functioned effectively as a Cl(-) channel. Treatment with aerosolized calcium-pump inhibitors reversed the nasal epithelial potential defect observed in a mouse model of Delta F508-CFTR expression. Thus, ER calcium-pump inhibitors represent a potential target for correcting the cystic fibrosis defect.

Topics: Amiloride; Calcium Channel Blockers; Cell Membrane; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Expression Regulation; Humans; Isoproterenol; Patch-Clamp Techniques; Sequence Deletion; Thapsigargin

Much of the pulmonary disease in cystic fibrosis is associated with polymorphonuclear leukocyte-dominated airway inflammation caused by bacterial infection. Respiratory epithelial cells express the polymorphonuclear chemokine interleukin-8 (IL-8) in response to ligation of asialylated glycolipid receptors, which are increased on damaged or regenerating cells and those with cystic fibrosis transmembrane conductance regulator mutations. Because both Pseudomonas aeruginosa and Staphylococcus aureus, the most common pathogens in cystic fibrosis, bind asialylated glycolipid receptors such as asialoGM1, we postulated that diverse bacteria can activate a common epithelial signaling pathway to elicit IL-8 expression. P. aeruginosa PAO1 but not pil mutants and S. aureus RN6390 but not the agr mutant RN6911 stimulated increases in [Ca(2+)](i) in 1HAEo- airway epithelial cells. This response stimulated p38 and ERK1/2 mitogen-activated protein kinase (MAPK) signaling cascades resulting in NF-kappaB activation and IL-8 expression. Ligation of the asialoGM1 receptor or thapsigargin-elicited Ca(2+) release activated this pathway, whereas P. aeruginosa lipopolysaccharide did not. The rapid kinetics of epithelial activation precluded bacterial invasion of the epithelium. Recognition of asialylated glycolipid receptors on airway epithelial cells provides a common pathway for Gram-positive and Gram-negative organisms to initiate an epithelial inflammatory response.

Topics: Adhesins, Bacterial; Blotting, Western; Calcium; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Egtazic Acid; Enzyme Activation; Epithelial Cells; G(M1) Ganglioside; Genes, Reporter; Humans; Inflammation; Interleukin-8; Kinetics; Lipopolysaccharides; Luciferases; Lung; MAP Kinase Signaling System; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutation; NF-kappa B; Pseudomonas aeruginosa; Receptors, Cell Surface; Signal Transduction; Spectrophotometry; Staphylococcus aureus; Thapsigargin; Time Factors; Trachea

Lysozyme is secreted in large quantities in human airways (10-20 mg/day), where it helps to defend against bacterial and fungal infection. Lysozyme expression is restricted to the serous cells of the submucosal glands, which also express high levels of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. It is often assumed that mucus secretion in human airways is coupled to anion secretion through CFTR Cl(-) channels located in the apical membrane. Therefore, a defect in CFTR function could cause abnormal mucus secretion leading to persistent bacterial infection and inflammation of the airways. In this study we measured simultaneous secretion of lysozyme and Cl(-) from human airway epithelial serous cells. Secretion of lysozyme was measured by a turbidimetric assay that relies on the ability of lysozyme to disrupt the wall of the bacterium Micrococcus lysodeikticus, thus causing a fall in the optical density of the sample. Secretion of Cl(-) was measured as short-circuit current in a modified Ussing chamber. Activation of Cl(-) secretion by stimulation of cAMP- or Ca(2+)-dependent pathways caused comparable increases in lysozyme secretion. Similarly, blockers of Cl(-) secretion, such as diphenylamine-2-carboxylate (DPC), also reduced lysozyme secretion. However, while treatment of airway submucosal gland cells with antisense oligonucleotides directed against CFTR reduced Cl(-) secretion, it had no significant effect on the total amount of lysozyme secretion. These results suggest a role for functional CFTR in regulation of lysozyme secretion in human airways.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Biological Transport; Cell Line; Chlorides; Colforsin; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Inhibitors; Humans; Muramidase; Nitric Oxide Donors; Oligonucleotides, Antisense; Respiratory Mucosa; S-Nitrosoglutathione; Thapsigargin

The regulation of chloride efflux from cystic fibrosis pancreatic adenocarcinoma cells (CFPAC-1) and wild-type CFTR-transfected CFPAC-1 cells (TPAC) was compared. Forskolin (10 microM) stimulated chloride efflux from the corrected TPAC cells but not from CFPAC-1 cells. Chloride efflux from both cell types was activated by thapsigargin (0.5 microM). The nucleotides ATP and UTP and the non-hydrolyzable ATP analogue, adenosine 5'-O-(3-thio) triphosphate (ATPgammaS), stimulated chloride efflux from both cell types. None of the other P2 purinoceptor agonists investigated elicited a response. The order of potency was ATP > or = UTP > or = ATPgammaS. Adenosine (10-100 microM) activated choride efflux from the TPAC but not the CFPAC cell line with no increase in intracellular cyclic AMP. Small but statistically significant inhibitions of the adenosine-(50 microM)-stimulated increase in chloride efflux were elicited by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 100 nM) and the A2 receptor antagonist 3,7-dimethyl-1-propylargylxanthine (DMPX, 10 microM). The A2A receptor antagonist 8-(3-chlorostyryl)caffeine (CSC, 100 nM) had no significant effect. These results provide evidence for the regulation of chloride efflux by P2Y2 purinoceptors in genetically-corrected and CF pancreatic cell lines. Studies with adenosine receptor antagonists indicate some possible involvement of A1 and A2 (but not A2A) receptors in the adenosine stimulation of chloride efflux, but the relatively small effects of the inhibitors coupled with lack of increase in cyclic AMP and a response only in the CFTR-transfected cells also suggests a possible direct effect of adenosine on CFTR.

Topics: Adenosine Triphosphate; Cell Line; Chloride Channels; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Inhibitors; Humans; Pancreas; Purinergic Agonists; Receptors, Purinergic; Thapsigargin; Transfection; Uridine Triphosphate

In human airway epithelial cell lines 9HTEo- and CFNPE9o, histamine causes a transient elevation of intracellular free calcium concentration ([Ca2+]i) detected by fura 2 fluorescence, which is due to both release from intracellular stores and extracellular Ca2+ entry. The effect of histamine is abolished by the Ca(2+)-ATPase inhibitor thapsigargin. Histamine also stimulates inositol phosphate accumulation. Changes in [Ca2+]i and inositol phosphate production exhibit a similar dose-response relationship for histamine (maximal effect at 10(-4) M), with both phenomena being blocked by the H1 antagonist mepyramine and being insensitive to pertussis toxin treatment. The effects of histamine on phosphoinositide metabolism and [Ca2+]i are abolished by a short-term preincubation with phorbol ester, and this effect is reversed by staurosporine and calphostin C, suggesting a feedback regulation by protein kinase C. The results indicate that human airway epithelial cells contain H1 receptors coupled to phospholipase C through a pertussis toxin-insensitive G protein.

Topics: Calcium; Calcium-Transporting ATPases; Cell Line; Cimetidine; Cystic Fibrosis; Enzyme Activation; Enzyme Inhibitors; Epithelium; GTP-Binding Proteins; Histamine; Histamine H1 Antagonists; Histamine H2 Antagonists; Humans; Inositol Phosphates; Pertussis Toxin; Protein Kinase C; Pyrilamine; Receptors, Histamine H1; Signal Transduction; Thapsigargin; Trachea; Type C Phospholipases; Virulence Factors, Bordetella

Activation of Cl- and K+ conductances by nucleotide receptor-operated mobilization of intracellular Ca2+ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltage-clamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl- ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1 +/- 0.3 min. Its amplitude was about 2.7 nA at -100 mV with 100 mumol/l of either ATP or UTP. The second current was carried by K+ ions and was blocked by Cs+. This current peaked more slowly and had a mean duration of 4.6 +/- 0.7 min. Its amplitude was 0.9 nA and 0.5 nA at -20 mV with 100 mumol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca2+ concentration ([Ca2+]i) that was similar in the presence or absence of extracellular Ca2+. The ED50 for UTP was 24 mumol/l and that for ATP was 94 mumol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store by thapsigargin prevented both the nucleotide-induced [Ca2+]i increase and the activation of membrane currents. Addition of 2 mmol/l Ca2+ to thapsigargin-treated cells produced a sustained increase of Cl- and K+ currents, which was reversed by Ca2+ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca2+]i that stimulates Ca(2+)-dependent Cl- and K+ currents.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cells, Cultured; Chloride Channels; Cystic Fibrosis; Electrophysiology; Extracellular Space; Fura-2; Humans; Membrane Potentials; Pancreatic Ducts; Potassium Channels; Terpenes; Thapsigargin; Uridine Triphosphate

The effect of purinergic compounds on [Ca2+]i and membrane currents of cell lines derived from the airway epithelium of normal and cystic fibrosis individuals has been investigated. 2-Chloroadenosine (2-CADO), as well as other agonists of the A1 adenosine receptors, causes a transient elevation of cytosolic [Ca2+] that is antagonized by the A1 adenosine receptor antagonist 8-cyclopentyl-1,3 dipropylxanthine (DPCPX). ATP is also effective, but at a lower extent. The [Ca2+]i increase induced by 2-CADO and ATP is abolished by preincubation with phorbol 12-myristate 13-acetate and the Ca(2+)-ATPase inhibitor thapsigargin. This latter result suggests that purinergic agonists mobilize Ca2+ from inositol 1,4,5-trisphosphate-sensitive stores. Pertussis toxin completely inhibits the effect of 2-CADO, whereas only it partially affects that of ATP, suggesting the involvement of different types of G proteins. Perforated patch clamp experiments carried out in both current clamp and voltage clamp modes show that 2-CADO and ATP activate K(+)- and Cl(-)-selective membrane currents, with a mechanism inhibited by preincubation with DPCPX and thapsigargin. These data indicate that activation of adenosine A1 receptor, in a similar way to ATP receptor, causes [Ca2+]i increase and ion channels activation through a transduction mechanism that is not impaired in cystic fibrosis airway epithelial cells.

Topics: 2-Chloroadenosine; Adenosine Triphosphate; Adult; Calcium; Cells, Cultured; Chloride Channels; Cystic Fibrosis; Humans; Membrane Potentials; Potassium Channels; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Terpenes; Thapsigargin; Trachea; Xanthines

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

Topics: Calcimycin; Calcium; Carcinoma; Cations, Divalent; Colonic Neoplasms; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Half-Life; Humans; Membrane Proteins; RNA, Messenger; Terpenes; Thapsigargin; Transcription, Genetic; Tumor Cells, Cultured

1. Cultured epithelia derived from whole human sweat glands, isolated secretory coils, isolated reabsorptive ducts and whole glands from cystic fibrosis (CF) subjects have been used to examine drug sensitivity by use of short circuit current recording. 2. Short circuit current increases were observed with lysylbradykinin, carbachol and histamine in epithelia of different origins. All responses were due to stimulation of electrogenic sodium absorption, evidenced by the inhibition of these responses by amiloride. The latter also abolished the basal current. The terpenes, thapsigargin and forskolin had no effect on transport. 3. The stimulation of a sodium current by agonists was dependent upon calcium, responses being inhibited by lanthanum ions and EGTA. Further A23187 induced a sodium current. 4. Pronounced oscillations in the sodium currents were a feature of the responses, implying synchronous, regulated cell activity. 5. Forskolin produced a ten fold increase in adenylate cyclase activity. All agonists listed in 2 except forskolin caused an increase in intracellular calcium [Ca]i, [Ca]i responses in CF cells were not different from those of normal cells, except with thapsigargin where the responses were smaller. 6. It is concluded that in culture, cells develop ductal characteristics, whether derived from normal or CF glands, coils or ducts. An increase in [Ca]i followed by activation of calcium-sensitive potassium channels and apical membrane hyperpolarization may be the major mechanism for increasing sodium influx.

Topics: Adenylyl Cyclases; Amiloride; Barium; Calcium; Carbachol; Cells, Cultured; Colforsin; Cyclic AMP; Cystic Fibrosis; Epithelium; Humans; Kallidin; Lanthanum; Sodium Channels; Sweat Glands; Terpenes; Thapsigargin