8-bromocyclic-gmp and Cystic-Fibrosis

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

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