cyclic-gmp and Cystic-Fibrosis

The treatment of lung infection in the context of cystic fibrosis (CF) is limited by a biofilm mode of growth of pathogenic organisms. When compared to planktonically grown bacteria, bacterial biofilms can survive extremely high levels of antimicrobials. Within the lung, bacterial biofilms are aggregates of microorganisms suspended in a matrix of self-secreted proteins within the sputum. These structures offer both physical protection from antibiotics as well as a heterogeneous population of metabolically and phenotypically distinct bacteria. The bacteria themselves and the components of the extracellular matrix, in addition to the signaling pathways that direct their behaviour, are all potential targets for therapeutic intervention discussed in this review. This review touches on the successes and failures of current anti-biofilm strategies, before looking at emerging therapies and the mechanisms by which it is hoped they will overcome current limitations.

Topics: Alginates; Anti-Bacterial Agents; Bacteriophages; Biofilms; Cell Communication; Cyclic GMP; Cystic Fibrosis; Humans; Iron; Lung Diseases; Quorum Sensing

The Gram-negative pathogen Pseudomonas aeruginosa is found ubiquitously within the environment and is recognised as an opportunistic human pathogen that commonly infects burn wounds and immunocompromised individuals, or patients suffering from the autosomal recessive disorder cystic fibrosis (CF). During chronic infection, P. aeruginosa is thought to form structured aggregates known as biofilms characterised by a self-produced matrix which encases the bacteria, protecting them from antimicrobial attack and the host immune response. In many cases, antibiotics are ineffective at eradicating P. aeruginosa from chronically infected CF airways. Cyclic-di-GMP has been identified as a key regulator of biofilm formation; however, the way in which its effector proteins elicit a change in biofilm formation remains unclear. Identifying regulators of biofilm formation is a key theme of current research and understanding the factors that activate biofilm formation may help to expose potential new drug targets that slow the onset of chronic infection. This minireview outlines the contribution made by exopolysaccharides to biofilm formation, and describes the current understanding of biofilm regulation in P. aeruginosa with a particular focus on CF airway-associated infections.

Topics: Alginates; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Cystic Fibrosis; Drug Resistance, Bacterial; Escherichia coli Proteins; Glucuronic Acid; Hexuronic Acids; Humans; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common pathogen that colonizes the lungs of patients with cystic fibrosis. Isolates from sputum are typically all derived from the same strain of bacterium but show extensive phenotypic heterogeneity. One of these variants is the so-called small colony variant, which also shows increased ability to form a biofilm and is frequently resistant to multiple antibiotics. The presence of small colony variants in the sputum of patients with cystic fibrosis is associated with a worse clinical condition. The underlying mechanism responsible for generation of the small colony phenotype remains unclear, but a final common pathway would appear to be elevation of intracellular levels of cyclic di-GMP. This phenotypic variant is thus not just a laboratory curiosity, but a significant bacterial adaptation that favors survival within the lung of patients with cystic fibrosis and contributes to the pulmonary damage caused by P. aeruginosa.

Topics: Anti-Bacterial Agents; Biofilms; Chronic Disease; Cyclic GMP; Cystic Fibrosis; Drug Resistance, Bacterial; Humans; Lung; Phenotype; Pseudomonas aeruginosa; Pseudomonas Infections

Heat-stable enterotoxin, produced by Escherichia coli, binds to particulate guanylate cyclase to increase cyclic GMP in intestinal cells. This in turn stimulates the cyclic-GMP- or cyclic-AMP-dependent protein kinase, activating the same chloride channel that is defective in cystic fibrosis. It is possible that the relatively high prevalence of cystic fibrosis in humans results from its protective effect against diarrhea.

Topics: Animals; Bacterial Toxins; Chloride Channels; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Enterotoxins; Escherichia coli Infections; Escherichia coli Proteins; Humans; Ion Transport; Membrane Proteins

There is a time lag between the beginning of sweat secretion and fully effective production of fluid of minimum salt content. It is suggested that changes in permeability to water or electrolytes have to be effected and may account for the time lag. The composition of thermal and pilocarpine sweat indicates that pharmacological stimulation does not wholely reproduce the physiological mechanism: pilocarpine sweat contains more sodium and more cyclic AMP, irrespective of the secretory rate. The sweat obtained from patients with cystic fibrosis has been compared with that from normal children: the concentrations of cyclic AMP and cyclic GMP are the same. Defective sodium reabsorption in the patients is therefore unlikely to be due to inadequate or excessive synthesis of these cyclic nucleotides.

Topics: Absorption; Adolescent; Child; Cyclic AMP; Cyclic GMP; Cystic Fibrosis; Humans; Pilocarpine; Potassium; Secretory Rate; Sodium; Sweat; Sweat Glands

A subpopulation of small-colony variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolates obtained from colonized cystic fibrosis lungs. Since most SCVs have until now been isolated from clinical samples, it remains unclear how widespread the ability of P. aeruginosa strains to develop this phenotype is and what the genetic mechanism(s) behind the emergence of SCVs are according to the origin of the isolate. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to spontaneously adopt an SCV-like smaller alternative morphotype distinguishable from that of the ancestral parent strain under laboratory culture conditions. We found that all the P. aeruginosa strains tested could adopt an SCV phenotype, regardless of their origin. Whole-genome sequencing of SCVs obtained from clinical and environmental sources revealed single mutations exclusively in two distinct c-di-GMP signaling pathways, the Wsp and YfiBNR pathways. We conclude that the ability to switch to an SCV phenotype is a conserved feature of P. aeruginosa and results from the acquisition of a stable genetic mutation, regardless of the origin of the strain.

Topics: Cyclic GMP; Cystic Fibrosis; Humans; Mutation; Phenotype; Pseudomonas aeruginosa; Pseudomonas Infections

Topics: Amino Acid Sequence; Amino Acids; Arginine; Bacterial Proteins; Biofilms; Burkholderia cenocepacia; Burkholderia Infections; Cloning, Molecular; Cyclic GMP; Cystic Fibrosis; Escherichia coli Proteins; Flagellin; Gene Expression Regulation, Bacterial; Glutamic Acid; Locomotion; Lung; Mutagenesis, Insertional; Peptide Hydrolases; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Sequence Alignment; Sputum

How diversity evolves and persists in biofilms is essential for understanding much of microbial life, including the uncertain dynamics of chronic infections. We developed a biofilm model enabling long-term selection for daily adherence to and dispersal from a plastic bead in a test tube. Focusing on a pathogen of the cystic fibrosis lung, Burkholderia cenocepacia, we sequenced clones and metagenomes to unravel the mutations and evolutionary forces responsible for adaptation and diversification of a single biofilm community during 1,050 generations of selection. The mutational patterns revealed recurrent evolution of biofilm specialists from generalist types and multiple adaptive alleles at relatively few loci. Fitness assays also demonstrated strong interference competition among contending mutants that preserved genetic diversity. Metagenomes from five other independently evolved biofilm lineages revealed extraordinary mutational parallelism that outlined common routes of adaptation, a subset of which was found, surprisingly, in a planktonic population. These mutations in turn were surprisingly well represented among mutations that evolved in cystic fibrosis isolates of both Burkholderia and Pseudomonas. These convergent pathways included altered metabolism of cyclic diguanosine monophosphate, polysaccharide production, tricarboxylic acid cycle enzymes, global transcription, and iron scavenging. Evolution in chronic infections therefore may be driven by mutations in relatively few pathways also favored during laboratory selection, creating hope that experimental evolution may illuminate the ecology and selective dynamics of chronic infections and improve treatment strategies.

Topics: Bacterial Adhesion; Base Sequence; Biofilms; Burkholderia cenocepacia; Burkholderia Infections; Chronic Disease; Cyclic GMP; Cystic Fibrosis; Directed Molecular Evolution; DNA, Bacterial; Ecosystem; Genome, Bacterial; Humans; Lung Diseases; Mannose; Metagenome; Mutation; Opportunistic Infections; Phylogeny; Selection, Genetic

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Down-Regulation; Enterotoxins; Escherichia coli Proteins; Female; Gastrointestinal Hormones; Genes, Modifier; HEK293 Cells; Heterozygote; Humans; Intestinal Mucosa; Intestinal Obstruction; Male; Meconium; Mice; Molecular Sequence Data; Mutation; Natriuretic Peptides; Pedigree; Phenotype; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide

The airways of patients with cystic fibrosis (CF) exhibit decreased nitric oxide (NO) concentrations, which might affect airway function. The aim of this study was to determine the effects of NO on ion transport in human airway epithelia. Primary cultures of non-CF and CF bronchial and bronchiolar epithelial cells were exposed to the NO donor sodium nitroprusside (SNP), and bioelectric variables were measured in Ussing chambers. Amiloride was added to inhibit the Na(+) channel ENaC, and forskolin and ATP were added successively to stimulate cAMP- and Ca(2+)-dependent Cl(-) secretions, respectively. The involvement of cGMP was assessed by measuring the intracellular cGMP concentration in bronchial cells exposed to SNP and the ion transports in cultures exposed to 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (ODQ), or to 8Z, a cocktail of 8-bromo-cGMP and zaprinast (phosphodiesterase 5 inhibitor). SNP decreased the baseline short-circuit current (I(sc)) and the changes in I(sc) induced by amiloride, forskolin, and ATP in non-CF bronchial and bronchiolar cultures. The mechanism of this inhibition was studied in bronchial cells. SNP increased the intracellular cGMP concentration ([cGMP](i)). The inhibitory effect of SNP was abolished by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger (PTIO) and ODQ and was partly mimicked by increasing [cGMP](i). In CF cultures, SNP did not significantly modify ion transport; in CF bronchial cells, 8Z had no effect; however, SNP increased the [cGMP](i). In conclusion, exogenous NO may reduce transepithelial Na(+) absorption and Cl(-) secretion in human non-CF airway epithelia through a cGMP-dependent pathway. In CF airways, the NO/cGMP pathway appears to exert no effect on transepithelial ion transport.

Topics: Adenosine Triphosphate; Adult; Aged; Amiloride; Bronchi; Chloride Channels; Colforsin; Cyclic GMP; Cyclic N-Oxides; Cystic Fibrosis; Epithelial Sodium Channel Blockers; Epithelial Sodium Channels; Free Radical Scavengers; Guanylate Cyclase; Humans; Imidazoles; Middle Aged; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxadiazoles; Purinones; Quinoxalines; Young Adult

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.

Topics: Bacterial Proteins; Cell Line, Tumor; Chromatography, Thin Layer; Cyclic GMP; Cystic Fibrosis; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Humans; Lung; Oligonucleotide Array Sequence Analysis; Polysaccharides, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction

Topics: Animals; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Exocrine Glands; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucus; Signal Transduction

The CFTR gene encodes a chloride channel with pleiotropic effects on cell physiology and metabolism. Here, we show that increasing cGMP levels to inhibit epithelial Na(+) channel in cystic fibrosis (CF) respiratory epithelial cells corrects several aspects of the downstream pathology in CF. Cell culture models, using a range of CF cell lines and primary cells, showed that complementary pharmacological approaches to increasing intracellular cGMP, by elevating guanyl cyclase activity though reduced nitric oxide, addition of cell-permeable cGMP analogs, or inhibition of phosphodiesterase 5 corrected multiple aspects of the CF pathological cascade. These included correction of defective protein glycosylation, bacterial adherence, and proinflammatory responses. Furthermore, pharmacological inhibition of phosphodiesterase 5 in tissues ex vivo or in animal models improved transepithelial currents across nasal mucosae from transgenic F508del Cftr(tm1Eur) mice and reduced neutrophil infiltration on bacterial aerosol challenge in Pseudomonas aeruginosa-susceptible DBA/2 mice. Our findings define phosphodiesterase 5 as a specific target for correcting a number of previously disconnected defects in the CF respiratory tract, now linked through this study. Our study suggests that phosphodiesterase 5 inhibition provides an opportunity for simultaneous and concerted correction of seemingly disparate complications in CF.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cell Line; Cells, Cultured; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelial Sodium Channels; Guanylate Cyclase; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Ion Transport; Mice; Mice, Transgenic; Nitric Oxide; Nitric Oxide Synthase Type II; Piperazines; Purines; Respiratory System; Signal Transduction; Sildenafil Citrate; Sodium; Sulfones; trans-Golgi Network

Endosomal hyperacidification in cystic fibrosis (CF) respiratory epithelial cells is secondary to a loss of sodium transport control owing to a defective form of the CF transmembrane conductance regulator CFTR. Here, we show that endosomal hyperacidification can be corrected by activating the signalling cascade controlling sodium channels through cyclic GMP. Nitric oxide (NO) donors corrected the endosomal hyperacidification in CF cells. Stimulation of CF cells with guanylate cyclase agonists corrected the pH in endosomes. Exposure of CF cells to an inhibitor of cGMP-specific phosphodiesterase PDE5, Sildenafil, normalized the endosomal pH. Treatment with Sildenafil reduced secretion by CF cells of the proinflammatory chemokine interleukin 8 following stimulation with Pseudomonas aeruginosa products. Thus, the endosomal hyperacidification and excessive proinflammatory response in CF are in part due to deficiencies in NO- and cGMP-regulated processes and can be pharmacologically reversed using PDE5 inhibitors.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Cells, Cultured; Cyclic GMP; Cystic Fibrosis; Endosomes; Humans; Hydrogen-Ion Concentration; Nitric Oxide; Piperazines; Purines; Respiratory Mucosa; Signal Transduction; Sildenafil Citrate; Sulfones

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is an important pathway for duodenal mucosal bicarbonate secretion. Duodenal biopsies from CF patients secrete bicarbonate in response to heat-stable enterotoxin from Escherichia coli (STa) but not cAMP. To explore the mechanism of STa-induced bicarbonate secretion in CF more fully, we examined the role of CFTR in STa-stimulated duodenal bicarbonate secretion in mice. In vivo, the duodenum of CFTR (-/-) or control mice was perfused with forskolin (10(-4) M), STa (10(-7) M), uroguanylin (10(-7) M), 8-bromoguanosine 3',5'-cGMP (8-Br-cGMP) (10(-3) M), genistein (10(-6) M) plus STa, or herbimycin A (10(-6) M) plus STa. In vitro, duodenal mucosae were voltage-clamped in Ussing chambers, and bicarbonate secretion was measured by pH-stat. The effect of genistein, DIDS (10(-4) M), and chloride removal was also studied in vitro. Control, but not CF, mice produced a significant increase in duodenal bicarbonate secretion after perfusion with forskolin, uroguanylin, or 8-Br-cGMP. However, both control and CF animals responded to STa with significant increases in bicarbonate output. Genistein and herbimycin A abolished this response in CF mice but not in controls. In vitro, STa-stimulated bicarbonate secretion in CF tissues was inhibited by genistein, DIDS, and chloride-free conditions, whereas bicarbonate secretion persisted in control mice. In the CF duodenum, STa can stimulate bicarbonate secretion via tyrosine kinase activity resulting in apical Cl(-)/HCO(3)(-) exchange. Further studies elucidating the intracellular mechanisms responsible for such non-CFTR mediated bicarbonate secretion may lead to important therapies for CF.

Topics: Animals; Bacterial Toxins; Benzoquinones; Bicarbonates; Cell Membrane; Chloride-Bicarbonate Antiporters; Colforsin; Cyclic GMP; Cystic Fibrosis; Duodenum; Enterotoxins; Enzyme Inhibitors; Escherichia coli Proteins; Genistein; In Vitro Techniques; Lactams, Macrocyclic; Mice; Natriuretic Peptides; Peptides; Protein-Tyrosine Kinases; Quinones; Rifabutin

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.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Calcium Channel Blockers; Cell Line; Charybdotoxin; Cyclic AMP; Cyclic GMP; Cystic Fibrosis; Diltiazem; Enzyme Inhibitors; Epithelial Cells; Glutathione; Humans; Membrane Potentials; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Oxadiazoles; Pancreas; Patch-Clamp Techniques; Penicillamine; Quinoxalines; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione

C-type natriuretic peptide (CNP), a hormone which stimulates particulate guanylate cyclase activity, was studied for its ability to stimulate chloride permeability through the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. Two cell lines, Calu-3 and CF-T43, were used as models of normal and cystic fibrosis (CF) airway epithelial cells, respectively. Calu-3 cells, derived from a lung carcinoma, express relatively high levels of wild-type CFTR. CF-T43 is a transformed line derived from a nasal polyp and expresses the mutant CFTR, deltaF508. Calu-3 cells exposed to the nucleotide guanosine-3',5'-monophosphate (cGMP) analogue 8-Br-cGMP exhibit increased 36Cl- efflux, demonstrating that cGMP can mediate changes in chloride permeability. CNP induces a bumetanide-sensitive short circuit current across Calu-3 monolayers. Whole-cell currents stimulated by CNP display linear current-voltage relationships and have inhibitor pharmacology and ion selectivity consistent with CFTR channel activity. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, and CNP both increase cGMP levels and short circuit current in Calu-3 cells. In contrast, exposure of CF-T43 cells to CNP resulted in an increased 36Cl- efflux rate only when combined with the adenylate cyclase agonist isoproterenol and the response was sensitive to kinase inhibitors. CF-T43 cells exposed to isoproterenol and SNP showed no increase in chloride efflux. Together, these data indicate that CNP can activate wild-type and mutant CFTR through a cAMP-dependent protein kinase pathway and that the sensitivity of Calu-3 cells for this stimulation is greater than that of the CF-T43 cells.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Cell Line; Cell Membrane Permeability; Chlorides; Cyclic GMP; Cystic Fibrosis; Guanylate Cyclase; Ion Transport; Natriuretic Peptide, C-Type; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Proteins; Trachea

Inhibitors of guanosine 3',5'-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl- transport across the nasal epithelia of cystic fibrosis mice carrying the delta F508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (delta F/delta F)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and delta F508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(-/-)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 +/- 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3',5'-cyclic monophosphate (6.2 +/- 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl- secretion across the nasal epithelium of CFTR(delta F/delta F) mice. No effect on TEPD was seen with this combination when used on CFTR(-/-) mice, implying that the CNP-induced change in TEPD in CFTR(delta F/delta F) mice is CFTR-dependent.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Anti-Inflammatory Agents, Non-Steroidal; Chlorides; Colforsin; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Mice; Mice, Knockout; Mice, Mutant Strains; Models, Biological; Nasal Mucosa; Natriuretic Peptide, C-Type; Nitroprusside; ortho-Aminobenzoates; Proteins; Trachea

Nitric oxide, which is produced by cytokine-activated mononuclear cells, is thought to play an important role in inflammation and immunity. While the function of nitric oxide as a direct cytotoxic effector molecule is well established, its function as a transducer molecule in immune cells is not. By use of whole-cell patch clamp recordings, we show that nitric oxide activates cystic fibrosis transmembrane conductance regulator CI- currents in normal human cloned T cells by a cGMP-dependent mechanism. This pathway is defective in cystic fibrosis-derived human cloned T cells. These findings not only delineate a novel transduction mechanism for nitric oxide but also support the hypothesis that an intrinsic immune defect may exist in cystic fibrosis.

Topics: Aminoquinolines; Arginine; CD4-Positive T-Lymphocytes; Chloride Channels; Clone Cells; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Guanylate Cyclase; Humans; Membrane Proteins; Nitric Oxide; Patch-Clamp Techniques; Penicillamine; S-Nitroso-N-Acetylpenicillamine; Signal Transduction

Intestinal chloride (Cl-) secretion can be induced by the heat-stable enterotoxin (STa) from Escherichia coli via generation of cGMP. We investigated the regulatory pathway responsible for cGMP-mediated Cl- secretion in the human colonic carcinoma cell line Caco-2 using whole-cell voltage clamp techniques. Cyclic GMP or cAMP induced a 5-fold increase in Cl- conductance (gCl) in the presence of intracellular ATP and 3-isobutyl-1-methylxanthine. Current activation by cGMP persisted in the presence of the type I cGMP-dependent protein kinase (PKG) inhibitor, KT5823, but was inhibited by the specific peptide inhibitor of the cAMP-dependent protein kinase A (PKA), PKI5-24. The stimulatory effects of cGMP and cAMP on gCl were not additive. The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by intracellular ATP and by cAMP-dependent phosphorylation. In order to determine whether CFTR was involved in the cGMP-dependent increase in gCl, we tested the effect of intracellularly injected anti-CFTR505-511 antibodies previously shown to inhibit CFTR function. Antibodies introduced into individual cells via the patch pipette completely inhibited cGMP-dependent current activation. Cyclic GMP also failed to activate gCl in cystic fibrosis cells. Taken together, these studies demonstrate that activation of the CFTR via PKA-dependent phosphorylation accounts for the cGMP-mediated increase in Cl- secretion in Caco-2 cells.

Topics: Chloride Channels; Colonic Neoplasms; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Membrane Proteins; Protein Kinases; Tumor Cells, Cultured

Calcium- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretions in the human colon are abnormal in cystic fibrosis, but the effect of guanosine 3',5'-cyclic monophosphate (cGMP) is unknown. This study examined the effects of the cGMP activator Escherichia coli heat-stable enterotoxin (STa) on rectal ion transport of controls and subjects with cystic fibrosis.. In vivo rectal potential difference (PD) was measured in response to 10(-7) mol/L STa in adult cystic fibrosis (n = 6) and control subjects (n = 7). Cl- transport was also evaluated in 24-hour primary cultures of human colonocytes using 6-methoxy-quinolyl-acetoethyl ester in response to STa (1 mumol/L) and 8-bromo-cGMP (100 mumol/L) with or without Cl- transport inhibitors.. Whereas STa increased rectal potential difference in controls, there was no effect in cystic fibrosis subjects. STa stimulated the cGMP concentration in rectal biopsy specimens from both control and cystic fibrosis subjects approximately twofold. In vitro Cl- transport in non-cystic fibrosis colonocytes increased threefold and fivefold with STa and 8-bromo-cGMP, respectively. These transport increases were inhibited by furosemide and the Cl- channel blocker diphenylamine-2-carboxylate.. Human colonocytes secrete Cl- in response to STa and cGMP in normal subjects, but this response is absent in cystic fibrosis.

Topics: Adult; Bacterial Toxins; Cell Membrane Permeability; Cell Separation; Chlorides; Colon; Cyclic GMP; Cystic Fibrosis; Enterotoxins; Escherichia coli Proteins; Female; Humans; Intestinal Mucosa; Male; Rectum; Reference Values

Cystic fibrosis transmembrane conductance regulator (CFTR) is a regulated Cl- channel; in secretory epithelia, it is located in the apical membrane where it regulates transepithelial Cl- secretion. Previous studies have shown that cAMP-dependent protein kinase (PKA) can phosphorylate and activate CFTR Cl- channels. We asked whether other kinases would phosphorylate CFTR in vitro and activate CFTR Cl- channels in excised, inside-out patches of membrane from NIH 3T3 fibroblasts stably expressing recombinant CFTR. We found that both Ca(2+)-independent and Ca(2+)-dependent isoforms of protein kinase C (PKC) activated the CFTR Cl- channel. Consistent with this finding, PKC also phosphorylated CFTR in vitro. In contrast, the multifunctional Ca2+/calmodulin-dependent protein kinase failed to either activate or to phosphorylate CFTR Cl- channels, suggesting that this enzyme has no direct effect on CFTR. We found that cGMP-dependent protein kinase (cGK) (purified from bovine lung) phosphorylated CFTR in vitro. However, cGMP failed to increase the apical membrane Cl- permeability in human airway epithelia, and addition of cGMP, ATP, and cGK failed to activate CFTR Cl- channels. These results suggest that if cGK phosphorylates CFTR in vivo, it does so at sites not involved in CFTR Cl- channel activation. Because cAMP-dependent activation of CFTR Cl- channels and Cl- secretion in intact cells is reversible, we asked whether specific phosphatases can dephosphorylate and inactivate CFTR Cl- channels. Addition of protein phosphatase 2A (PP2A) decreased PKA-activated current by 67% within 10 min. The phosphatase inhibitor calyculin-A blocked the effect of PP2A. In contrast, neither protein phosphatases 1, 2B, nor two preparations of alkaline phosphatase inactivated PKA-phosphorylated CFTR Cl- channels. The effects of protein phosphatases on CFTR function were paralleled by their ability to dephosphorylate CFTR in vitro. Our data indicate that CFTR Cl- channels can be phosphorylated and activated by PKA as well as by Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC and can be dephosphorylated and thus inactivated by PP2A.

Topics: 3T3 Cells; Animals; Calcium; Calmodulin; Cell Membrane; Chloride Channels; Chlorides; Cyclic AMP; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Isoenzymes; Membrane Proteins; Mice; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Protein Kinases; Protein Phosphatase 2; Recombinant Proteins

Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.

Topics: Amino Acid Sequence; Cell Line; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophoresis, Gel, Two-Dimensional; Membrane Proteins; Molecular Sequence Data; Phosphorylation; Protein Kinase C; Protein Kinases; Substrate Specificity

Adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels were measured in labial gland slices from controls and patients with cystic fibrosis (CF). Incubation in vitro with 10 microM epinephrine, 50 microM isoproterenol, or 10 microM carbachol increased cAMP levels by 2.3-fold, 3.1-fold, and 1.8-fold, respectively, in control glands and by similar amounts in CF glands. The only statistically significant CF-related difference was a decreased response to isoproterenol. Addition of MIX (3-isobutyl-1-methylxanthine) increased cAMP levels in control and CF glands by an order of magnitude under all conditions but did not eliminate the CF-related decrease in cAMP level obtained with isoproterenol. cGMP levels were measured only in the presence of MIX. Incubation with carbachol nearly doubled cGMP levels in control and CF glands but only the control gland response approached statistical significance (P = 0.06). cGMP levels in CF glands were nearly threefold greater than those in control glands, and disease-related differences obtained in the presence of carbachol and isoproterenol were statistically significant.

Topics: 1-Methyl-3-isobutylxanthine; Adolescent; Adult; Cyclic AMP; Cyclic GMP; Cystic Fibrosis; Female; Humans; Lip; Male; Nucleotides, Cyclic; Reference Values; Salivary Glands; Stimulation, Chemical

Topics: Animals; Calcium; Chloride Channels; Chlorides; Cyclic AMP; Cyclic GMP; Cystic Fibrosis; Electrophysiology; Humans; Ion Channel Gating; Ion Channels; Membrane Proteins; Models, Biological

Topics: Adenylyl Cyclases; Animals; Cholera; Cholera Toxin; Cyclic AMP; Cyclic GMP; Cystic Fibrosis; Humans; Intestinal Mucosa; Rabbits

Urinary adenosine 3':5' (cyclic) monophosphate and guanosine 3':5' (cyclic)-monophosphate excretion are significantly elevated in adult male cystic fibrosis patients compared to normal men when values are expressed in ways which consider the smaller size of the cystic fibrosis subjects of the same age. Cystic fibrosis patients excreted 3.22 +/- 1.16 nmol adenosine 3':5' (cyclic)monophosphate/g creatinine per 24 h vs. 1.97 +/- 0.43 for normal men (p less than 0.02); and cystic fibrosis patients excreted 0.50 +/- 0.16 nmol guanosine 3':k' (cyclic)monophosphate/g creatinine per 24 h vs. 0.30 +/- 0.07 for normal men (p less than 0.05). Subjects were all adults who had completed linear growth and sexual development, thus eliminating any possible effects of slower maturation of cystic fibrosis patients, and all subjects were male, thus avoiding the fluctuation of urinary cyclic nucleotides with the menstrual cycle, problems which had complicated interpretation of previous studies.

Topics: Adult; Body Surface Area; Creatinine; Cyclic AMP; Cyclic GMP; Cystic Fibrosis; Humans; Male; Middle Aged; Reference Values

The urinary excretion of adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP) was examined in 98 normal children and 46 children with cystic fibrosis between the ages of 9 months and 18 yr. Diurnal variations in cyclic AMP and cyclic GMP excretion were observed in subjects from either group, and peak levels of cyclic nucleotide excretion were generally observed during the period of 0700 to 2100 h. Excretion rates (mumol/day) of cyclic AMP and cyclic GMP increased significantly with age. When cyclic AMP and cyclic GMP excretion rates were normalized for urinary creatine, or body weight, the values declined significantly with age in both groups of patients. Cyclic GMP excretion normalized for body surface area also decreased with age, while the value for cyclic AMP (2.86 plus or minus 0.08 mumol/day/m2, mean plus or minus SE) was constant with age in both normals and cystic fibrosis children. With some comparisons of age groups there were significant differences in cyclic nucleotide excretion between normal subjects and children with cystic fibrosis. The differences noted were dependent upon the methods used to normalize excretion rates (urinary creatine, body weight, surface area, and the ratio of cyclic AMP to cyclic GMP excreated). In general patients with cystic fibrosis excreted greater amounts of cyclic GMP than did normals. The most striking comparison was the ratio of cyclic AMP to cyclic GMP excreted which was 9.09 plus or minus 0.50 in all normal children and 4.41 plus or minus 0.32 in children with cystic fibrosis (P smaller than 0.001).

Topics: Adolescent; Age Factors; Body Surface Area; Body Weight; Child; Child, Preschool; Circadian Rhythm; Creatinine; Cyclic AMP; Cyclic GMP; Cystic Fibrosis; Female; Humans; Male