Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:A cystic fibrosis pancreatic adenocarcinoma cell line. 169 30

Cystic fibrosis (CF), the most common lethal genetic disease in Caucasians, is characterized by defective electrolyte transport in several epithelia. In sweat duct, pancreatic, intestinal, and airway epithelia, abnormalities in transepithelial ion transport may account for the manifestations of the disease. A Cl- impermeable apical cell membrane is a common feature in these CF epithelia. The rate of transepithelial Cl- transport is controlled in part by hormonally regulated apical membrane Cl- channels; in CF epithelia, Cl- channels are present but their regulation is defective. Most regulation studies have focused on an outwardly rectifying Cl- channel, although other channels may be involved in Cl- secretion. Phosphorylation of Cl- channels or associated regulatory proteins by cAMP-dependent protein kinase or by protein kinase C (at a low internal [Ca2+]) in excised patches of membrane activates Cl- channels in normal cells but not in CF cells. Phosphorylation with protein kinase C at a high internal [Ca2+] in excised patches of membrane inactivates the channel; such inactivation is normal in CF cells. Cl- channels can also be activated by other maneuvers including an increase in the cytosolic [Ca2+], sustained membrane depolarization, an increase in temperature, proteolysis, and changes in osmolarity; the response to such maneuvers is not defective in CF. In addition to the Cl- channel abnormalities, Na+ absorption is increased in CF epithelia. It is not certain whether the increased rate of Na+ absorption results from an increase in the number of cation channels or an alteration of their kinetics. The relation of these ion channel abnormalities to the CF gene product is unknown, but an understanding of the function of the protein product and its defective function in CF should yield important new insights into the pathogenesis and potential therapy of this disease.
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PMID:Abnormal regulation of ion channels in cystic fibrosis epithelia. 169 93

Apical membrane Cl- channels control the rate of transepithelial Cl- secretion in airway epithelia. cAMP-dependent protein kinase and protein kinase C regulate Cl- channels by phosphorylation; in cystic fibrosis cells, phosphorylation-dependent activation of Cl- channels is defective. Another important signaling system involves arachidonic acid, which is released from cell membranes during receptor-mediated stimulation. Here we report that arachidonic acid reversibly inhibited apical membrane Cl- channels in cell-free patches of membrane. Arachidonic acid itself inhibited the channel and not a cyclooxygenase or lipoxygenase metabolite because (i) inhibitors of these enzymes did not block the response, (ii) fatty acids that are not substrates for the enzymes had the same effect as arachidonic acid, and (iii) metabolites of arachidonic acid did not inhibit the channel. Inhibition occurred only when fatty acids were added to the cytosolic surface of the membrane patch. Unsaturated fatty acids were more potent than saturated fatty acids. Arachidonic acid inhibited Cl- channels from both normal and cystic fibrosis cells. These results suggest that fatty acids directly inhibit apical membrane Cl- channels in airway epithelial cells.
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PMID:Fatty acids inhibit apical membrane chloride channels in airway epithelia. 169 96

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.
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PMID:Expression and characterization of the cystic fibrosis transmembrane conductance regulator. 169 61

Cystic fibrosis transmembrane conductance regulator (CFTR) generates cAMP-regulated Cl- channels; mutations in CFTR cause defective Cl- channel function in cystic fibrosis epithelia. We used the patch-clamp technique to determine the single channel properties of Cl- channels in cell expressing recombinant CFTR. In cell-attached patches, an increase in cellular cAMP reversibly activated low conductance Cl- channels. cAMP-dependent regulation is due to phosphorylation, because the catalytic subunit of cAMP-dependent protein kinase plus ATP reversibly activated the channel in excised, cell-free patches of membrane. In symmetrical Cl- solutions, the channel had a channel conductance of 10.4 +/- 0.2 (n = 7) pS and a linear current-voltage relation. The channel was more permeable to Cl- than to I- and showed no appreciable time-dependent voltage effects. These biophysical properties are consistent with macroscopic studies of Cl- channels in single cells expressing CFTR and in the apical membrane of secretory epithelia. Identification of the single channel characteristics of CFTR-generated channels allows further studies of their regulation and the mechanism of ion permeation.
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PMID:Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel. 171 15

Patch-clamp studies have identified a cAMP-dependent Cl- conductance in lymphocytes that is defectively regulated in cystic fibrosis. In this study we used 125I efflux and whole-cell patch-clamp studies to investigate whether prostaglandin E1 (PGE1), an agonist that generates intracellular cAMP in Jurkat T lymphocytes, activates a Cl- conductance. Stimulation of T cells by externally applied PGE1 stimulated 125I efflux and activated a slowly developing membrane current. When external and internal Cl- were about equal, the current reversed at about zero mV, but when external Cl- was lowered from 157 to 7 mM the reversal potential shifted 75 mV in the positive direction, demonstrating that the current carrier was Cl-. In addition, the current was blocked by 10 microM 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB), a potent Cl- channel blocker. A membrane-permeable cAMP analog mimicked the effect of PGE1, whereas intracellular application of a cAMP antagonist Rp-cAMP blocked the effect of PGE1. Addition of purified catalytic subunit of cAMP-dependent protein kinase (PKA) plus ATP to the recording pipette also activated a similar current, whereas internally applied Walsh inhibitor, the synthetic peptide inhibitor of PKA, blocked the PGE1 effect. These results suggest that PGE1, acting through PKA, activates a Cl- current in Jurkat T cells.
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PMID:Prostaglandin E1 activates a chloride current in Jurkat T lymphocytes via cAMP-dependent protein kinase. 172 93

Cl- channels in the apical membranes of salt-secreting epithelia are activated by both cAMP and Ca2+ second-messenger systems, and dysfunctions in their hormonal regulation have been demonstrated in patients with cystic fibrosis. We have transfected the epithelial cell line T84 with an expression vector containing a mutant form of the regulatory subunit of the cAMP-dependent protein kinase. Stable transformants that express this construct have reduced basal cAMP-dependent protein kinase activity and do not increase kinase activity beyond the basal level of control cells in response to cAMP. Forskolin, vasoactive intestinal peptide, and prostaglandin E2 each stimulate intracellular cAMP accumulation in both mutant and control clones; however, the activation of Cl- channels in response to elevated cAMP is blocked in mutant clones, indicating direct involvement of the cAMP-dependent protein kinase. In contrast, Ca2+ ionophores retain their ability to activate the Cl- channel in T84 cells expressing the mutant regulatory subunit, suggesting that activation of the channel by means of Ca2+ does not require the participation of cAMP-dependent protein kinase activity. These clones will be useful for further studies of the interactions between the cAMP- and Ca2(+)-dependent regulatory pathways in salt-secreting epithelial cells. They can also be used to identify the mediators of Ca2(+)-dependent Cl- channel activation in isolation from interactions with the cAMP second-messenger pathway.
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PMID:Regulation of Cl- transport in T84 cell clones expressing a mutant regulatory subunit of cAMP-dependent protein kinase. 217 70

Chloride impermeability of epithelial cells can account for many of the experimental and clinical manifestations of cystic fibrosis (CF). Activation of apical-membrane Cl- channels by cyclic AMP-mediated stimuli is defective in CF airway epithelial cells, despite normal agonist-induced increases in cellular cAMP levels. This defect in Cl- channel regulation has been localized to the apical membrane by exposing the cytoplasmic surface of excised membrane patches to the catalytic subunit (C subunit) of cAMP-dependent protein kinase and ATP. In membranes from normal cells, C-subunit activated Cl- channels with properties identical to those stimulated by cAMP-dependent agonists during cell-attached recording. Activation by the C subunit was not observed in CF membranes, but the presence of Cl- channels was verified by voltage-induced activation. The failure of the C subunit to activate the Cl- channels of CF membranes indicates that the block in their cAMP-mediated activation lies distal to induction of cAMP-dependent protein kinase activity and focuses our attention on the Cl- channel and its membrane-associated regulatory proteins as the probable site of the CF defect.
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PMID:Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells. 244 2

Chloride (Cl-) secretion by the airway epithelium regulates, in part, the quantity and composition of the respiratory tract fluid, thereby facilitating mucociliary clearance. The rate of Cl- secretion is controlled by apical membrane Cl- channels. Apical Cl- channels are opened and Cl- secretion is stimulated by a variety of hormones and neurotransmitters that increase intracellular levels of cyclic AMP (cAMP). In cystic fibrosis (CF), a common lethal genetic disease of Caucasians, airway, sweat-gland duct, secretory-coil and possibly other epithelia are anion impermeable. This abnormality may explain several of the clinical manifestations of the disease. The Cl- impermeability in CF-airway epithelia has been localized to the apical cell membrane, where regulation of Cl- channels is abnormal: hormonal secretagogues stimulate cAMP accumulation appropriately but Cl- channels fail to open. Here we report that the purified catalytic subunit of cAMP-dependent protein kinase plus ATP opens Cl- channels in excised, cell-free patches of membrane from normal cells, but fails to open Cl- channels in CF cells. These results indicate that in normal cells, the cAMP-dependent protein kinase phosphorylates the Cl- channel or an associated regulatory protein, causing the channel to open. The failure of CF Cl- channels to open suggests a defect either in the channel or in such an associated regulatory protein.
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PMID:Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium. 244 45

1. Recent discoveries have implicated regulation of an apical membrane chloride channel as site of a defect in cystic fibrosis (CF). The channel fails to respond to stimuli that elevate intracellular cAMP. 2. This paper describes properties of reversible cycles of protein phosphorylation and considers substrate specificity, reactions with model peptides, and space-filling structural models. 3. Mutation of a channel regulatory protein is proposed to involve either: (a) change of phosphorylated serine residue to an unreactive residue, (b) change in a nearby residue that does not affect phosphorylation by cAMP-dependent kinase, but results in dephosphorylation by a different phosphatase, or (c) change in a nearby residue that produces a structure unreactive with cAMP-dependent protein kinase. 4. Perhaps in CF sidechains with branched structures at the beta carbons occur on either side of the phosphorylated serine, like in glycogen phosphorylase, and prohibit reaction of a regulatory protein with cAMP-dependent protein kinase.
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PMID:Molecular defects in ion channel regulation in cystic fibrosis predicted from analysis of protein phosphorylation/dephosphorylation. 245 79


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