Pathway Map Details
NO-dependent CFTR activation (normal and CF)
Object list (links open in MetaCore):
norB (P. aeruginosa), CFTR, NifD (Burkholderia sp.), Guanylate cyclase alpha-2/beta-1, NO, beta-ENaC, eNOS, ARG1, ENaC, Cl(-) cytosol, GMP,
NO-dependent CFTR activation (normal and CF)
Cystic fibrosis (CF) is a multisystem disease associated with mutations in the gene encoding the CF transmembrane conductance regulatory ( CFTR ) protein . The most common mutation associated with CF results in deletion of a single amino acid, phenylalanine, at position 508 in CFTR protein (mutant deltaF508 CFTR protein) , .
Exhaled nitric oxide ( NO ), elevated in most inflammatory lung diseases, is decreased in CF. According to some studies, impaired NO formation in lower airways correlates with more progressive lung disease .
NO is produced by a family of NO synthases (NOS) by transformation of L-arginine to L-citrulline and NO. In human, there are three isoforms of NOS: neuronal NOS ( nNOS ), endothelial NOS ( eNOS ) and inducible NOS ( iNOS ). nNOS and eNOS are constitutively expressed and produce small amounts of NO, whereas iNOS expression is mainly induced by inflammatory stimuli. Induced iNOS synthesizes relatively large quantities of NO. All three isoforms are expressed in human airways .
In the case of CF, airway epithelial cells are more susceptible to bacterial and viral infection due to impairment of the host NO defense pathway. Polymorphisms of constitutive NOS ( nNOS and eNOS ) and reduced iNOS expression contributes to decreased NO production along with bacterial consumption , , , , . Alternatively, lower concentration of airway NO in CF could be caused by lack of substrate, L-arginine. . L-arginine is a common substrate for both NOSs (e.g., iNOS ) and arginase ( ARG1 ). ARG1 activity is increased in CF patients. Increased ARG1 activity can result in L-arginine deficiency and thereby contribute to low airway NO formation and impaired pulmonary function , .
NO can also be released from S-Nitrosoglutathione, an endogenous signaling molecule that at low micromolar concentrations increases expression, maturation, and function of both wild-type and deltaF508 CFTR in epithelial cells , , . S-Nitrosoglutathione mainly acts independently of the classic NO radical/cyclic GMP pathway . A number of enzymes can catabolize S-Nitrosoglutathione to form free NO radicals, including Cu/Zn superoxide dismutase ( SOD1 ) and thioredoxin reductases ( TXNRD1, TXNRD2 and TXNRD3 ) , , , .
NO mediates many effects through production of cyclic GMP ( cGMP ) by NO-sensitive guanylate cyclase. cGMP activates type II isoform of cGMP dependent protein kinase G ( Protein kinase G 2 ). The latter phosphorylates CFTR and increases its activity. NO-sensitive Guanylate cyclase alpha-2/beta-1 isoform is localized in the plasma membrane where it mediates stimulatory effect of NO on CFTR activity much more efficiently than other NO-dependent guanylate cyclase isoforms . The selective membrane effects of the Guanylate cyclase alpha-2/beta-1 isoform in this signaling relay are mediated by a compartmentalized pool of cGMP that is resistant to degradation by cellular phosphodiesterases, such as phosphodiesterase 5A ( PDE5A ) , , .
CFTR is an apical membrane Cl - channel in epithelial cells. CFTR negatively regulates the amiloride-sensitive epithelial sodium channel ( ENaC ). ENaC inhibition by CFTR is documented in CF patients which suffer from airway obstruction and chronic infection that results from decreased mucociliary clearance secondary to missing CFTR and high ENaC activity .
CFTR and ENaC are the principal rate-limiting steps for Cl - secretion and Na + absorption by ciliated airway epithelia. Mutations in the CFTR gene lead to hyperabsorption of sodium chloride and a reduction in the periciliary salt and water content which leads to impaired mucociliary clearance , , .
There is also a unique relationship in the CF airway between prokaryotic and eukaryotic cells. The airway colonization with denitrifying bacteria, such as Pseudomonas aeruginosa and Burkholderia species, alters nitrogen balance in the CF airway. CF lung infection is associated with decreased concentrations of oxidized forms of nitrogen (such as NO ) and increased concentrations of reduced forms of nitrogen (such as nitrous oxide ( N(2)O ) and ammonium ( NH(3) )) in the CF airways .
The reduction of NO to N(2)O can be catalyzed by enzymes like Nitric oxide reductase ( NOR (P. aeruginosa) ) in the P. aeruginosa denitrification pathway , , . NOR (P. aeruginosa) can also catalyze the reduction of N(2)O to molecular nitrogen ( N2 ) , . One of the most prevalent species of the Burkholderia cepacia complex found in CF patients, Burkholderia vietnamiensis, can produce a Nitrogenase complex that catalyzes the reduction of N(2) to NH(3) .
In epithelial cells, NH(3) inhibits chloride transport (via CFTR channel), and NO inhibits amiloride-sensitive sodium transport (via ENaC channel) and augments chloride transport , , . Thereby, a shift from oxidized to reduced forms of nitrogen can increase epithelial salt transport abnormalities in CF airways and adversely affect CF disease.
Mucoid, mucA mutant P. aeruginosa bacteria cause chronic lung infections in CF patients and are refractory to phagocytosis and antibiotics. As chronic CF lung disease progresses, mucoid, alginate-overproducing strains emerge and become the predominant form .
P. aeruginosa is capable of anaerobic growth by respiration using nitrite ( NO(2)(-) ) as terminal electron acceptor. The reduction of ( NO(2)(-) ) to NO is catalyzed by P. aeruginosa enzyme Nitric reductase ( NirS (P. aeruginosa) ) .
Mucoid, mucA mutant P. aeruginosa has the inherently low NirS and NOR activity and, thereby, has limited capacity for NO(2)(-) and NO removal. Treatment of mucoid, mucA mutant bacteria with NO(2)(-) (15 mM) at pH 6.5 under anaerobic conditions, similar to conditions within mucopurulent secretions in the airways of CF patients, leads to the death of these bacteria. Thereby, the treatment (e.g. aerosolization) of CF patients with NO(2)(-) and NO can exert antimicrobial effect on mucoid P. aeruginosa .
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