The cystic fibrosis transmembrane conductance regulator (CFTR) protein forms a tightly regulated channel that mediates the passive diffusion of chloride ions. [1] However, transepithelial chloride efflux, long a hallmark of a functioning CFTR protein, has yet to be linked to any of the pathologies associated with cystic fibrosis lung disease. Research studies showing the permeability of the CFTR to the anionic tripeptide glutathione (GSH) provide the link that explains both the increased inflammatory reaction, and the lack of antimicrobial function observed in CF lung tissues. [2] Moreover, the resultant lack of formation of extracellular cysteinyl peptide-nitric oxide adducts, such as thionein-NO, and S-nitrosoglutathione in the cystic fibrosis (CF) lung provides the key to understanding both of these pathologies as well as the affinity for and virulence of microbes such as Stapholoccus aureas and Pseudomonas aeruginosa in the CF lung.
Thiols such as GSH are potentially powerful antioxidants since they contain a sulfhydryl (-SH) group in their structure and are readily oxidised by giving up their hydrogen atom to form stable disulphide bonds. Large amounts of the sulfhydryl-containing antioxidant GSH have been measured in the epithelial lining fluid (ELF) of the lower respiratory tract in normal individuals. Cantin et al calculated total GSH of normal ELF to be 140-fold higher than that of GSH in the plasma of the same individuals; and 96% of the GSH to be in reduced form. [19] Cell types which are known to be present in the lower respiratory tract, such as mononuclear phagocytes, lymphocytes, and fibroblasts, are also known to export GSH extracellularly, and GSH is found in high concentrations in the extracellular fluid of other organs as well. [20-22, 90] Moreover, because the process of moving products out of the environment has been shown to be rather slow, it is probable that GSH remains for some time in the lower respiratory tract. [23, 24]
Roum et al observed a systemic deficiency of GSH in patients with cystic fibrosis. [3] Additionally, Gao et al found that GSH content in the apical fluid of CF cell cultures was significantly lower than in those cultures having normal CFTR function. [72] GSH is probably removed from the extracellular space by uptake and subsequent catabolism by cells which, in the lung, is likely accomplished by virtue of the plasma membrane-bound y-glutamyl transpeptidase (GGT), an enzyme that cleaves the y-glutamyl bond of GSH during the uptake process. [91-93] It has been demonstrated that the plasma concentrations of GSH are markedly elevated in hereditary deficiencies of GGT, whereas extracellular concentrations of GGT in the lung of CF patients have been shown to be significantly increased. [94, 95] Lack of extracellular GSH in the ELF of CF patients plays a major role in the increased inflammatory response seen in this disease.
Protease/antiprotease imbalance is well documented in CF lung disease. [107] Oxidants have been shown to inactivate both alpha 1-antitrypsin (a1-AT), the major antiprotease of the lower respiratory tract and secretory leukoprotease inhibitor (SLPI), the major antiprotease of the upper respiratory tract. [105, 106] In the lung, GSH is responsible for the scavenging of both intracellular and extracellular toxic oxygen radicals, which are known to damage lung tissue. However, proteases, while known to damage lung tissue, can also inactivate antioxidants. Although CF ELF is known to contain all of the elements necessary for the GSH redox cycle, a lack of transport of the tripeptide to the extracellular environment by the CFTR protein would serve to explain both the increase in oxidant-mediated damage demonstrated in this disease as well as the protease/antiprotease imbalance.
Increased lipid peroxidation has been demonstrated in children with CF. [59] Phosphatidylcholine, which is the main component of lung surfactant, is synthesized within the lamellar bodies of type 2 alveolar epithelial cells. [110, 111] Peroxynitrite, via damage to both its lipid and protein components, may inactivate pulmonary surfactant. [60] As well, the perilamellar membrane contains enzymes which are required for phosphatidylcholine synthesis and oxidative damage to this membrane have been associated with decreased synthesis of phosphatidylcholine. Although ascorbic acid has been shown to have a protective effect on the levels of phosphatidylcholine in adult mice, GSH levels are important to the maintenance of ascorbic acid in the reduced state, and a deficiency in these levels have been shown to be responsible for the reduction of ascorbic acid levels in tissues of newborn rats. [112, 113] It is likely that unopposed oxidant radical formation, due to deficiency in extracellular GSH levels in CF lungs, and the resultant inability to maintain necessary ascorbic acid levels is responsible for the abnormalities seen in lung surfactant in CF tissues.
Defective antigen presentation by lavage cells from terminal patients with CF has been demonstrated. [96] GSH levels in antigen presenting cells such as macrophages have also been shown to affect the development of either Th-1 or Th2 immune responses in AIDS patients. [62] However, although defective antigen presentation has been correlated with a low level of intracellular GSH in AIDS patients, these patients have also been shown to have subnormal GSH levels in lung epithelial lining fluid. [108] As well, although levels of intracellular GSH have been shown to be increased in CF epithelial cells, extracellular GSH has been shown to be well below normal levels. [72, 97, 98]. This information argues for an extracellular mechanism by which GSH metabolism affects antigen presentation and, indeed, deleterious effects of H2O2 have been implicated in the impairment of antigen presentation in murine epidermal-derived dendritic cell lines. [99]
The glutathione cycle is a sequence of reactions by which the degradation of H2O2 is coupled to the increased activity of the hexose monophosphate shunt (HMS). Two enzymes of the HMS, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, reduce the oxidized form of nicotinamide-adenine dinucleotide phophate (NADP+) to the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), and the continued activity of the shunt depends on the reoxidation of NADPH. The latter can be accomplished by the glutathione cycle, which is initiated by the oxidation of GSH by H2O2 catalyzed by glutahtione peroxidase. The oxidized glutathione (GSSG) formed is reduced by NADPH in the presence of glutathione reductase with the formation of NADP+. Thus, the HMS is a sink for H2O2.
Although a study by Worlitzsch et al shows that, despite the increased inflammation and infection in CF subjects, the H2O2 levels in breath condensates were comparable to healthy controls, an inverse correlation between myeloperoxidase (MPO), a polymorphonuclear-derived enzyme which reacts with H2O2 to form HOCl, and CF lung function has been reported. [25-28] Both CF homozygotes and heterozygotes have been shown to produce larger amounts of intracellular MPO-dependent oxidants than controls. [51] Respectively, hydrogen peroxide is degraded by two enzymes: glutathione peroxidase and catalase, the first of which provides a relatively high affinity, low capacity degradative system, and is thus the first line of defense against H2O2 toxicity, and the second of which represents a low affinity, high capacity system, and thus is the second line of defense. [69, 70] Glutathione peroxidase, in a reaction that is coupled to the oxidation of two molecules of glutathione, can reduce H2O2 to water. Thus, a systemic deficiency of GSH, as has been reported in CF, can lead to an increase in the level of hydroperoxides and subsequent burden of oxidant stress. A compensatory reaction to this increased burden is evidenced by an increase in G6PD activity in erythrocytes of CF patients. [159]
Further, the balance between oxidants and antioxidants influences critical steps in the signal transduction cascade. Binding of transcription factors to consensus sites on DNA are driven by physiological oxidant-antioxidant homeostasis, especially by the thiol-disulfide balance. Endogenous glutathione and thioredoxin systems are considered to be an effective regulator of redox-sensitive gene expression.
Accordingly, disturbances of the GSH cycle have been linked to redox regulation of the induction of transforming growth factor beta 1 (TGF beta 1) expression. [39]. TGF beta 1 is a 25kDa homodimeric peptide belonging to a family of structurally related, multifunctional cytokines which are involved in cellular morphogenesis, differentiation, and fibrosis. [57, 58] Nath et al demonstrated that deprivation of antioxidants can directly induce TGF beta 1 mRNA in vitro. [39] Finally, Corrin et al demonstrated that the intra-alveolar buds of granulation tissue (Masson bodies), in the organizing pneumonia seen in cystic fibrosis, reacted strongly with antibody to the extracellular form of TGF-beta 1, and that the hyperplastic epithelium covering these connective tissue polyps reacted with the antibody to the intracellular form. [68]
As well, TGFb1 has been linked to interference with the expression of inducible nitric oxide synthase (iNOS), a calcium dependent enzyme which is expressed during inflammation and is responsible for large amounts of nitric oxide (NO) production, as well as being associated with protective cellular responses to invading pathogens. [14]. Produced in immune cells by the induction of NOS via stimulation of cytokines or lipopolysacchride, NO has cytotoxic or cytostatic effects on invading microorganisms, as well as being involved in many signalling pathways. It has been shown that, at concentrations normally found in the upper airways, NO is bacteriostatic to Staphylococcus aureus, which is one of the primary respiratory pathogens of cystic fibrosis. [6] Indeed, host defence in response to a number of pathogens has been proven to be severely impaired in iNOS-deficient mice. [16] In a study by Kelly et al, TGF beta 1 expression was shown to be increased in the airways of CF mice, even in the absence of infection; and the cytokine altered NO production by 72%, in response to lipopolysaccharide, a major component of the cell wall of gram-negative bacteria. [50]
In support of these findings, low concentrations of NO have been measured in the upper airways of cystic fibrosis patients by Balfour-Lynn et al. [29] These low levels have been found despite high levels of pro-inflammatory cytokines, such as Interleukin-1 beta, Interleukin-6, and tumor necrosis factor alpha, which have been detected in bronchoalveolar lavage fluids and the supernatant fluid of CF patients, and which would be expected to increase iNOS transcriptional rates. [17, 30-32] Indeed, iNOS expression has been shown to be reduced or absent in CF tissues [18] And, although it has been shown that the stable NO oxidation products nitrite and nitrate are elevated in CF sputum during exacerbations, the source of NO for these metabolites is suggested to be a product of the constitutively expressed NOS, instead of the inducible form of the enzyme. [17, 18]
Witko-Sarsat et al reported that NADPH oxidase activity of opsonized zymosan-stimulated neutrophils is increased in both CF heterozygoes as well as homozygotes. [51] While this indicates that there is no defect, in terms of initiation of the respiratory burst in CF neutrophils, it has been reported that neutrophils derived from CF patients are defective in their response, in terms of beta-glucuronidase exocytosis, to the chemotactic peptide formyl-met-leu-phe (fMLP). [UI91369791-55] Beta-glucuronidase, a lysosomal enzyme of the hydrolase class, can have an effect on invading pathogens in a number of different ways: (1) by a direct lethal effect through its catalytic activity; (2) by a mechanism independent of enzymatic action; (3) in conjunction with other antimicrobial agents; or (4) by aiding in the digestion of a cell already killed by another agent (or agents). Defective beta-glucuronidase exocytosis, and a concommitant downregulation of these functions, in CF, is likely to increase the incidence of colonization with Psuedomonas aeruginosa as well as other pathogens. NO has been shown to be a key physiological mediator, in terms of beta-glucuronidase exocytosis. [56] VanUffeleu et al showed that exogenous NO could enhance the release of both lysozyme and beta-glucuronidase induced by fMLP. [56]
While it has been demonstrated that there exists no defect in CF neutrophils, regarding activation of the respiratory burst, it has been shown that there is an alteration in terms of an increase in oxidative burst. Neutrophil oxidative burst has been shown to be increased in both CF heterozygotes and homozygotes. [51] Since superoxide anion has been shown to react to form nitrate and nitrites, this would more likely explain the increase, during acute pulmonary infection in CF, in total sputum nitrate and nitrite, than any increased metabolism of products of constitutive NOS. [ 17, 87] The ensuing imbalance, in terms of NO and superoxide, thus, can explain much of the oxidative damage that is seen in CF lung tissues. When NO is in excess of superoxide, oxidative damage is limited. [10] Nathan et al, in studies involving influenza virus pneumonitis in mice, showed that inflammatory tissue damage is increased in the absence of iNOS. [15] Studies performed by Gutierrez et al on cultured pulmonary epitheilial cells and lung tissue in vivo, suggest that NO plays a protective role via its inhibitioni of O2 and hydrogen peroxide-dependent toxicity, when these tissues were subject to inflammatory mediators or acute oxidative stress. [10] As well, NO might have protective effects in terms of decreasing neutrophil accumulation in the lung. [9] Finally, Grosemann et al found a positive correlation between NO and forced vital capacity, which indicates that lower levels of NO are a marker for more severe lung disease in CF. [37]
As well, NO has been found to function as a signaling agent that acts through the activation of soluble guanylate cyclase (GC-S), thus stimulating the production of cyclic guanosine monophosphate (cGMP). [34] In turn, an increase in the production of cGMP has been demonstrated in several systems to effectively downregulate amiloride sensitive sodium absorption. [35, 36] As well, NO has been shown to activate chloride currents in human lung epithelial cells via a regulatory effect involving this same mechanism. [88] Kelley et al found that inhibition of iNOS both significantly increased the transepithelial potential difference in mice nasal epithelia and reduced the ability, in excised mouse lungs, to prevent the growth of Pseudomonas aeruginosa. [33]
S-nitrosoglutathione (GSNO), a stable derivative of NO, is formed in the extracellular millieu; its formation from GSH and NO is an oxygen dependent process. [4] Studies suggest that the NO adduct with GSH is one of the metabolic intermediaries that exist in the lung which modulate the bioactivity and toxicity of NO. Moreover, Gaston et al reported that GSNO, the predominant nitrosothiol found in the lungs is present at nano-to macromolar concentrations in the airways. [5] GSH efflux by the CFTR protein, and the tripeptides subsequent affiliation with NO, to form GSNO in the extracellular space, is particularly significant in terms of the nitrosothiols role in the transport and packaging of NO in biological systems, as well as in terms of the preservation of NO bioactivity and the limitation of its potential O2-dependent toxicity. However, there is a distinct lack of correlation between S-nitrosothiols bioactivity and their rate of spontaneous NO release. [120, 121] Indeed, the lack of formation of GSNO in the extracellular environment represents the key element which explains both the inflammatory component and the lack of antimicrobial function seen in the CF lung environment.
Increases in superoxide anion release by activated neutrophils have been demonstrated in CF tissues. [51] As well, NO has been shown to inhibit superoxide anion release in neutrophils. [115-117] Clancy et al showed that synthetic preparations of GSNO also had the capacity to inhibit superoxide anion production and activate the HMS. [80] Accordingly, GSNO has been demonstrated to be a NADPH oxidizing substrate for human or calf thymus thioredoxin reductase. In studies by Nikitovic et al, physiological concentrations of GSNO displayed catalytic activity in the thioredoxin system, which resulted in liberation of NO and GSH and which was coupled to the oxidation of NADPH. GSNO was shown to be both a substrate and to inhibit the mammalian thioredoxin system. [114] As well, the nitrosothiols function as the bioactive intermediary by which the HMS is activated in neutrophils has significance in terms of NO redox signaling responses in neutrophils. Indeed, GSNO has been found to inhibit human GSH reductase, which is the central enzyme of GSH redox metabolism. [122] Moreover, it is likely that decomposition of GSNO, and its action as a donor of NO+, NO, and NO-, depending on the ambient redox millieu, is the method by which the function of NO, as a redox signaling agent, is accomplished.
Glutathione peroxidase, which is known to catalyze the detoxifying reduction of H2O2 via oxidation of GSH, has also been shown to be involved in the catalysis of S-nitrosothiol breakdown. [118] In fact, studies have shown that GSNO afforded significant protection during hydrogen peroxide treatment of cells. [119] Additionally, Gaston et al demonstrated that nitrosothiols might limit the putative toxicity of NO in the O2-rich environment of the lung. [83] Indeed, glutathione has been reported to scavenge the product of superoxide and NO, peroxynitrite, leading to the formation of GSNO. [101] This finding is particularly significant in terms of the functioning of macrophages and neutrophils, which utilize NO for microbial killing, since it is necessary for these cells to be resistant to attack by NO and its products. [81, 82] Finally, via signaling pathways involving H2O2, TGF beta 1 is known to induce the expresssion of macrophage colony stimulating growth factor (M-CSF), which has been implicated in the pathenogenesis of CF and other fibrotic lung diseases. [142] Hong et al demonstrated that the activation of NF-kappa-b was inhibited by GSNO; as well as the repression of 85% of M-CSF promoter activity with the addition of GSNO and catalase to the cell medium. [144]
Additionally, it has been shown that oxidants such as superoxide anion react to form nitrate and nitrites. [123] These products of NO degradation have also been shown to be increased during CF exacerbations, probably due in part to the increased respiratory burst in response to NADPH oxidase, which has been displayed by both CF heterozygotes and homozygotes, as well as the lack of extracellular GSH necessary to the formation of extracellular GSNO, which lack would contribute to the breakdown of NO, from both constitutive and inducible sources, into nitrates and nitrites. The lack of extracellular formation of GSNO, which is known to downregulate the action of NADPH oxidase in terms of the respiratory burst in neutrophils thus becomes significant in terms of Pseudomonas aeruginosa (PA) colonization in CF airways. Although PA has been extensively studied as an aerobic pathogen, Neut et al has shown that, in the presence of nitrite, PA displays dense growth under strict anaerobic conditions. [124] Indeed, nitrate reducing bacteria frequently displays a higher pathogenic potential in terms of the enzyme products from gram-negative bacteria, such as lipopolysaccharidase, than non-nitrite reducing bacteria. [125, 126, 127]
Moreover, PA has long been known to have a denitrification system by which it processes the breakdown products of NO. [128, 129] This ability is particular relevant to the adaptive responses this pathogen is known to display in terms of aminoglycoside resistance. Although PA is commonly classified as a strict aerobe, this is misleading since PA is well adapted to conditions of oxygen limitation, given the presence of a suitable terminal electron acceptor molecule such as nitrite or one of its derivatives. [130] Nitrite reductase (denA) and its regulatory protein, anr, which is a redox regulator for expression of denitrification genes, mRNA levels have both been shown to be increased in gentamicin-resistant PA. [131] As well, denA has been shown to increase increase levels of interleukin-8 and cause dense neutrophil infiltrations in airways of patients with chronic airway diseases, such as CF. [132] Thus, an increased O2 depletion characteristic of chronic airway disease, such as in the case of CF, leads to an increase in the adaptive anaerobic metabolism of pathogens such as PA, in terms of nitrites, and an increase in the pathogenic potential of the microbe, along with an increase in resistance. The pathways of this adaptive response, in terms of GSNO, would also explain the more severe pulmonary phenotypes found in glutathione S-transferase M1 (GSTM1) deficient CF populations, since GSNO formation has been found to be catalyzed from organic nitrites by microsomal GSTs. [133-135]
In addition, GSNO decomposition is known to be mediated by GSH and copper-zinc superoxide dismutase (Cu-Zn SOD). [146] Alterations in zinc turnover have been documented, as well as have reduced copper enzyme activities, such as Cu-Zn SOD, in neutrophils and erythrocytes of children with CF. [147, 148] These anomalies would be expected in a system with high intracellular levels of GSH, such as seen in the case of CF, not only via the demonstrated attenuation of SOD-dependent release of NO from GSNO in the case of increased intracellular levels of GSH, but also because of the deficiency of GSNO in the extracellular millieu. [149]
Additionally, by a mechanism that is independent of the spontaneous cleavage of the nitrosothiol to yield NO, nitrosothiols have been found to inhibit bacterial growth. [85] Morris et al found that the antimicrobial action of nitrosothiols results from covalent modification of bacterial membrane sulfhydryl groups. [86] In fact, direct inhibition of bacterial membrane sulfhydryl groups, such as glyceraldehyde phosphate dehydrogenase, has been attributed to nitrosothiols generally, independent of their size, shape, charge, hydrophobicity or membrane permeability. [85, 138, 139] Thus, NO transfer, via nitrosothiols, to critical membrane and cellular thiols in bacteria, and the concomitant formation of protein nitrosothiols and subsequent evolution to disulfide, disrupts vital microbial functions.
Goldman et al found that the human beta-defensin-1 (HBD-1), which is a salt-sensitive antibiotic peptide in the lung, is inactivated in CF because of the high sodium chloride concentration in the epithelial fluid lining the lungs, but that adenoviral vectors of the normal CFTR gene, which restored less than 15% of CFTR expression, reactivated HBD-1 and its antimicrobial function. [89] GSNO has been shown to activate chloride currents in human lung epithelial cells, as well as inhibiting lung sodium transport through a cGMP- mediated inhibition of epithelial cation channels. [150, 151] Although other mechanisms, including the CFTR protein itself, may be implicated in restoration of the antimicrobial function of HBD-1 and other beta-defensins necessary to lung defence, it is likely that alternative chloride channels and regulation of sodium channels make a significant contribution to the pathology associated with CF, and that GSNO and its effects, given the necessity of a restoration of only 15% of the function of the CFTR protein, on these regulatory functions could act to restore lung defence in terms of human beta defensin peptides.
As well, the increased lipid peroxidation which has been demonstrated in children with CF, besides being a function of the reduced amount of GSH in the extracellular space, maybe also be exacerbated by a lack of NO. [59] Indeed, NO has been shown to be cytoprotective to the endothelium in the presence of perioxidants through its reduction of lipid peroxidation. [61] And, although peroxynitrite has been shown to inactivate pulmonary surfactant by damaging both its lipid and protein components, xanthine oxidase-dependent liposomal oxidation has also been demonstrated by Rubbo et al. Significantly, while NO and GSH alone have been shown to have no effect on lipid peroxidation by these radical products, NO donors, such as GSNO, have been shown to completely inhibit their effects. [145]
Finally, studies have shown that nitrosothiols such as GSNO exist in the airways in concentrations that are potentially sufficient to influence airway tone and that they possess potent bronchodilator activity. [102, 103] Indeed, studies have shown that although asthmatics have an increase in NO gas concentration, mean concentrations of S-nitrosothiols are lower than normals in these patients. [152, 153] Moreover, in a study by Bannenberg et al, GSNO, along with other S-nitrosothiols, was found to induce a sustained reversal of bronchoconstriction induced by methacholine, with a rapid onset of action in common with salbutamol. [154] Lack of formation of S-nitrosothiols, subsequent to depletion of GSH and NO, whose reaction to form the NO adduct of GSH, GSNO, is catalyzed by O2 in the extracellular space, could explain the 25% incidence of asthma associated with CF.
Lellemand et al proposed that the MRP and MDR proteins, which are, respectively, 50% and 30% homologous to the CFTR protein, and transport both glutathione adducts as well as metallothione adducts, such as zinc and copper, both of which are necessary to proper immune function, were responsible for the "remission effect" of chemotherapy drug-elicited expression of these proteins in CF patients who had undergone chemotherapy. Trials with colchicine are now being conducted with CF patients, in an attempt to induce MRP and MDR protein synthesis.
The liver, however, which is largely responsible for heavy metal metabolism, expresses these proteins even in CF patients. The macrolide drug, azithromycin, which has a heavy metal component (zinc) has been shown to effect an increase in pulmonary function scores in CF children over the long term (six months). As well, an anti-inflammatory mechanism has been proposed as an explanation for azithromycin's effect in these patients. This is likely the case, if azithromycin is inducing an increased expression of MT in the liver of these patients. Since liver cells, even those of CF patients, express the MRP protein, it is likely that metallothionein (MT) is transported to the extracellular milieu in the liver and transported via erythrocytes to the lungs.
Moreover, thionein, the non-apoform of metallothionein, which is transiently present in tissues, after GSH removes the zinc from metallothionein, contains twenty times the cysteinyl residues, which NO can bond to, as glutathione. In a pathology which presents with a 72% reduction in exhaled nitric oxide, such as CF, compounds which can transport NO in a non-toxic manner, such as GNSO and thionein-NO become extremely relevant.
It is likely that the defect evidenced in the inflammatory cascade begins very early in the course of CF lung disease. Goldberg et al found evidence for Th-2 like immune activity in bronchoalveolar lavage fluid taken from infants with CF. [63] Indeed, it has been demonstrated that the chronic imbalance of the neutrophil elastase/anti-elastase protective screen that characterizes CF respiratory epithelial surface is likely well established by 1 year of age, with resultant potential for lung damage. [64] Accordingly, studies have shown that bronchial surfactant is also altered in infancy in patients with CF. [67] Inflammation has been shown to be present as early as 4 weeks in the course of CF lung disease, before colonization and infection of the lungs with potentially pathogenic bacteria occurs. [65, 66]
While these findings, and the related discovery that GSH is effluxed by the CFTR would seem to argue for supplementation of GSH, via aerosolization into the epithelial extracellular space in CF, aerosolized GSH has only a very short half-life time (<2 hours), and antioxidants similiar to GSH, such as Nacystelyn and N-acetylcysteine, have been shown to have a greater effect in terms of their abrogation of the inflammatory effects of H2O2, when delivered exogenously. [75, 76] In addition, in a study of aerosolized GSH use in asthma patients, bronchoconstriction has been noted as a side effect. This effect was repeatedly observed and was not related to osmolarity or pH of the GSH solution. [155]
Indeed, a deficiency of S-nitrosothiols in the extracellular environment has been noted in patients who suffer asthmatic respiratory failure. [157] Although the formation of sulfites has been suggested as a likely source of the bronchoconstriction in studies of asthmatics and inhaled GSH, GSH has been shown to decompose GSNO in a concentration-dependent manner in vitro, and since the rate of decomposition was decreased by a factor of 2 in the absence of O2, it is likely that the decomposition of GSNO is increased in the O2-rich extracellular environment of the lung. [158] A depletion of extracellular GSNOis also more likely in CF patients since there is an increased amount of the glutathione rate-limiting enzyme gamma-glutamyl transpeptidase, which is a membrane-bound enzyme, of which GSNO has recently been shown to be a substrate. [72, 104]
Since GSNO is known to be taken up by cells, and has been suggested by several investigators to be the method by which the physiological role of NO is mediated, a more efficacious therapy, and one which would take into account both the deficiency of GSH and the lack of transcription of iNOS that has been demonstrated in CF lung tissue, is the delivery of S-nitrosoglutathione (GSNO), which has a half-life of 5.5 hours, via aerosolization into the extracellular space of CF lung tissue. [77, 78, 79] Nitrosothiols such as GSNO present a viable therapeutic alternative to existing NO donor drugs since they do not appear to undergo tolerance or generate potentially toxic metabolites. Indeed, s-nitrosothiols (general formula, R-SNO, where R- is one of a range of chemical moieties) decompose spontaneously in solution to the relevant disulfide and NO. In addition, they exhibit tissue-specificity not seen with existing donor drugs. NO adducts such as S-nitrosoglutathione, as compared to NO itself, are also relatively resistant to inactivation at physiological O2 concentrations.
The fact that RS-NO form upon inhalation of NO gas indicates that these compounds form indirectly through reaction with O2 in the extracellular airway environment, which provides a selective mechanism by which they act as pulmonary vasodilators. [100] Indeed, in cases such as CF, where the effects of the increased inflammatory cascade, and theoretically, the concomitant reduction in the expression of iNOS, as indicated by the presentation of Th-2 like immune response, can be detected as early as four weeks of age, exogenous delivery of GSNO to the extracellular millieu represents a much safer and more efficacious alternative to the exogenous delivery of GSH. In addition, it might be possible to cause the formation of extracellular GSNO by aerosolizing, separately, both GSH and L-arginine, which acts to increase the production of NO via the constitutive nitric oxide synthases. Since nitrosothiols have been known to form upon inhalation of NO gas, it is theoretically possible to cause their formation in CF lung extracellular space via both the exogenous delivery of GSH, and the delivery of L-arginine, according to the method used by Solomon. [156]
As well, metallothionein, probably in the zinc form, and working in conjunction with both copper, zinc, and glutathione, would probably be of great benefit to CF patients. It might be possible to provide enough metallothione to the diet, which could be transported by erythrocytes to the lungs and other epithelial organs, such as the kidney, liver, and pancreas. And, it would probably be more efficacious to aerosolize a form of MT to the lungs, and thus resolve any problems associated with transport to the other epithelial, exocrine organs.
To conclude, the lack of transport of GSH to the extracellular millieu leads to the lack of formation of extracellular GSNO. The lack of this messenger molecule, as in the case of CF, acts to perturb the normal functioning of the glutathione cycle in the lung. This perturbance, in turn, causes an increase in the transcriptional rate of TGF beta 1, which interferes with the expression of iNOS. Lack of transport of GSH to the extracellular lung environment by the CFTR leads to both an increase in oxidative stress in these tissues and a lack of transcription of iNOS which, in turn, leads to an increased susceptibility to infection and increased susceptibility to oxidative stress. Exogenous aerosol delivery of GSNO represents both a safe and efficacious method by which to remediate the effects of the basic defect involved in CF lung disease. As well, an increase, either through diet, or through aerosolization to the lungs, of MT, would restore both the antimicrobial function of zinc and copper to these patients, as well as increase the amount of cysteinyl NO adducts.
S-nitrosothiol adducts are among the primary bioorganic products of the reaction of cellular constituents with the intermediates of NO/O2 reaction. As well, competition studies have shown that cysteine residues have an affinity for NO metabolites that is 3 orders of magnitude greater than that of the nonsulfhydryl amino acids. [136, 137] Moreover, since it has been suggested that the multi-drug resistance associated protein (MRP), which has been shown to be 50% homologous to the CFTR protein, is also responsible for the transport of other cysteinyl compounds such as leukotrienne C4, further research is needed to ascertain as to whether additional cysteinyl compounds, capable of carrying NO, are implicated in the normal functioning of the epithelial lining fluid, in terms of both inflammation and antimicrobial function.
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