Glutaminase and the Control of Airway pH
Yet Another Problem for the Asthmatic Lung?

Owen W. Griffith, Ph.D.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin

	ARTICLE

TOP ARTICLE REFERENCES

Studies completed over the past decade have significantly advanced our understanding of the molecular basis of the pathology of asthma, particularly with respect to inflammatory mediators and their influence on immune response and on the normal physiological functions of lung. Among the more important and instructive of those recent findings is the appreciation that an exuberant and chronic innate immune response contributes significantly to the pathology of asthma. Thus exhaled nitric oxide (eNO) levels are high in patients with asthma, and much, but perhaps not all (1), of the excessive NO synthesis is due directly or indirectly to the expression of the inducible isoform of nitric oxide synthase (iNOS) (2, 3). Such expression, presumably in response to inflammatory mediators or inhaled irritants, is characteristic of innate immunity (4). Spontaneous reaction of NO with O₂ or superoxide forms highly reactive and toxic nitrosative stress species that contribute to airway damage. Although some NO is converted to S-nitrosothiols having strong and potentially beneficial relaxant effects on bronchial smooth muscle, such compounds are rapidly degraded in asthmatic, but not in normal, lung by mechanisms not yet fully elucidated (5). Interestingly, accelerated S-nitrosothiol breakdown was not observed in glucocorticoid-treated patients with asthma (6).

Acidification of the extracellular environment limits bacterial growth and is another characteristic of innate immunity. Last year, Hunt and coworkers reported that expired breath condensate (EBC) collected from patients with asthma is substantially more acidic than EBC from healthy control subjects (3). They suggested that dysregulation of airway pH control is a previously unsuspected aspect of the pathology of asthma. In the current issue of the American Journal of Respiratory and Critical Care Medicine (pp. 101-107), Hunt and coworkers extend those findings by showing that exposure to acidic media causes lung epithelial and Type II alveolar cells in culture to express phosphate-dependent glutaminase, generate substantial ammonia* from glutamine, and alkalinize their culture medium (7). They suggest that glutamine-dependent, epithelial cell-mediated ammonia production plays an important role in limiting lung acidity. Consistent with this view and their earlier finding that patients with asthma produce acidic EBC (3), Hunt and coworkers show further that glutaminase expression and ammonia levels are diminished in asthmatic lung and EBC, respectively (7). Mechanistic insight is provided by the observation that interferon (IFN)- and tumor necrosis factor (TNF)-, inflammatory cytokines known to be elevated in asthma, downregulate glutaminase expression and ammonia production in tissue culture. Conversely, corticosteroid administration, which suppresses inflammatory cytokine levels, increased glutaminase expression to above normal levels in a single corticosteroid-treated individual without asthma; a similar effect is anticipated but not yet demonstrated in patients with asthma (7). These results offer additional and important insight into the metabolic derangements affecting the asthmatic lung. As Hunt and coworkers point out, failure of the asthmatic lung to limit the acidification of its lining fluid diminishes ciliary action and increases both mucus viscosity and the reactivity and potential cytotoxicity of nitrosative stress species such as peroxynitrite (7). In the context of a chronic inflammatory disease such as asthma, those changes are likely to contribute to both mucus plugging and epithelial cell damage.

In the context of diagnosing and differentiating various forms of asthma, the report by Hunt and coworkers adds ammonia to a growing list of putative metabolic and inflammatory markers that can be noninvasively determined by collecting and analyzing EBC; others include pH, hydrogen peroxide, nitrosothiols, nitrite, leukotrienes, and 8-isoprostanes (8, 9). As pointed out by others (9), each of these markers needs to be rigorously correlated with more invasive but better established measures of lung pathology (e.g., biopsy histology, bronchoalveolar lavage (BAL) analysis, and methacholine challenge). Each also needs to be validated as to its reproducibility and ability to accurately reflect the state of the lower airways. In this regard, Effros has earlier suggested that EBC is derived primarily from condensed water vapor rather than solute-containing droplets and has raised concerns about using filtered EBC to determine epithelial cell lining fluid pH (10). Additional studies addressing lung acidity in asthma are eagerly awaited. With respect to ammonia, urease-containing bacteria in the mouth are capable of producing high levels of ammonia (11), and trapping of gaseous NH₃ by acidic EBC in either the mouth or the collection apparatus is therefore a potential concern. Fortunately, Hunt and coworkers find ammonia levels are generally lowest in the more acidic EBC samples (see Figure 6B in Hunt and coworkers [7]), a result arguing against significant NH₃ trapping. In addition, the in vivo EBC measurements showing diminished ammonia in asthma are strongly supported by the in vitro studies showing that cytokines known to be elevated in asthma diminish the ability of lung epithelial cells to form ammonia. Although a recent abstract reports that exhaled ammonia is increased in patients with mild or moderate to severe steroid-treated asthma (12), details of those studies are not yet available. As was previously observed with S-nitrosothiol levels (6), it may be that steroid treatment reverses the metabolic defect in ammonia production.

Quite apart from its implications for asthma pathology, the report by Hunt and coworkers opens an interesting and as yet unfinished new chapter in the increasingly complicated story of lung glutamine metabolism. Thus it is well-established that glutaminase activity by itself can have no effect on pH as the products, NH₄⁺ and glutamate, neither provide nor consume protons. Because ~ 1% of NH₄⁺ is dissociated into NH₃ and H⁺ at neutral pH, it is indeed likely that some NH₃ will diffuse from glutaminase-containing epithelial cells and be trapped by an external pool of acidic alveolar lining fluid as proposed by Hunt and coworkers (7). However, the H⁺ left behind would quickly acidify the epithelial cell cytosol in the absence of other metabolic processes to neutralize it. Because lung epithelial cells in culture clearly release ammonia and alkalinize their medium (7), further metabolism of the glutamate coproduct must be occurring. In fact, complete catabolism of glutamate as a metabolic fuel yields an additional NH₄⁺ in addition to 3CO₂ and 2HCO₃. It is presumably the formation of HCO₃ that serves to neutralize the cell culture medium in vitro or the alveolar lining fluid in vivo. Diffusion of NH₃ may have some role in "alkali transport," but it is likely that HCO₃ is the key species transported.

We note that it is not yet established that glutamate is fully catabolized by lung epithelial cells and that use of alternative pathways could be of considerable interest. Transamination of glutamate with pyruvate and metabolism of the resulting -ketoglutarate to regenerate pyruvate yields one equivalent of HCO₃ (glutamate  alanine + CO₂ + HCO₃). This pathway is consistent with the observation by Hunt and coworkers that growth of epithelial cells on glutamate rather than glutamine produced little ammonia, although it would predict an equal effect on medium pH. Alternatively, glutamate may be metabolized to some extent through glyconeogenic or lipogenic pathways to yield ¹/₂ glucose equivalent, NH₄⁺ and 2 HCO₃ or acetyl-CoA, NH₄⁺, CO₂, and 2HCO₃, respectively. In all cases, net synthesis of alkali (i.e., HCO₃) occurs. Use of glutamine to support surfactant lipogenesis has been shown in lung Type II cells (13) and that pathway is of particular interest because the step catalyzed by malic enzyme produces NADPH needed for both lipogenesis and defense against oxidative stress. It will be of considerable interest to determine which, if any, of these pathways is occurring in lung epithelial cells.

In the larger context of lung glutamine metabolism, several studies now show that the lung as a whole can be a significant net exporter of glutamine, although there is marked variation among species in the magnitude of its role (large in rats, lower in humans) (14). Net glutamine synthesis is attributed to lung endothelial cells and is apparently fueled by glutamate and ammonia extracted from the plasma. The studies by Hunt and coworkers (7) suggest that intralung trafficking in glutamate, glutamine, and ammonia should now also be considered, particularly because glucocorticoids are reported to upregulate glutamine synthesis (14). In addition, because glutamine has an inhibitory effect on NO synthesis from arginine (17), interactions between glutamine and arginine metabolism also warrant further investigation in the lung. Hunt and coworkers have not only provided new insight into the molecular basis of asthma pathology but have also further opened the door to an interesting world of lung intermediary metabolism.

	Footnotes

	References

TOP ARTICLE REFERENCES

1. Wechsler ME, Grasemann H, Deykin A, Silverman EK, Yandava CN, Israel E, Wand M, Drazen JM. Exhaled nitric oxide in patients with asthma. Am J Respir Crit Care Med 2000; 162: 2043-2047 [Abstract/Free Full Text].

2. Yates DH. The role of exhaled nitric oxide in asthma. Immunol Cell Biol 2001; 79: 178-190 [Medline].

3. Hunt JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TAE, Gaston B. Endogenous airway acidification: implications for asthma pathology. Am J Respir Crit Care Med 2000; 161: 694-699 [Abstract/Free Full Text].

4. Nathan C. Natural resistance and nitric oxide. Cell 1995; 82: 873-876 [Medline].

5. Gaston B, Sears S, Woods J, Hunt J, Ponaman M, McMahon T, Stamler JS. Bronchodilator S-nitrosothiol deficiency in asthmatic respiratory failure. Lancet 1998; 351: 1317-1319 [Medline].

6. Corradi M, Montuschi P, Donnelly LE, Pesci A, Kharitonov SA, Barnes PJ. Increased nitrosothiols in exhaled breath condensate in inflammatory airway disease. Am J Respir Crit Care Med 2001; 163: 854-858 [Abstract/Free Full Text].

7. Hunt JF, Erwin E, Palmer L, Vaughan J, Malhotra N, Platts-Mills TAE, Gaston B. Expression and activity of pH-regulatory glutaminase in human airway epithelium. Am J Respir Crit Care Med 2002; 165: 101-107 [Abstract/Free Full Text].

8. Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crit Care Med 2001; 163: 1693-1722 [Free Full Text].

9. Mutlu GM, Garey KW, Robbins RA, Danziger LH, Rubinstein I. Collection and analysis of exhaled breath condensate in humans. Am J Respir Crit Care Med 2001; 164: 731-737 [Free Full Text].

10. Effros RM. Endogenous airway acidification: implications for asthma pathology (Letter). Am J Respir Crit Care Med 2001; 163: 293 [Free Full Text].

11. Huizenga JR, Gips CH. Determination of ammonia in saliva using indophenol, an ammonium electrode and an enzymatic method: a comparative investigation. J Clin Chem Clin Biochem 1982; 20: 571-574 [Medline].

12. Kharitonov SA, Barnes PJ. Exhaled ammonia in asthma, cystic fibrosis and upper respiratory tract infection [abstract]. Am J Respir Crit Care Med 2000; 161: A307 .

13. Fox RE, Hopkins IB, Cabacungan ET, Tildon JT. The role of glutamine and other alternate substrates as energy sources in the fetal rat lung type II cell. Pediatr Res 1996; 40: 135-141 [Medline].

14. Abcouwer SF, Lukaszewicz GC, Souba WW. Glucocorticoids regulate glutamine synthetase expression in lung epithelial cells. Am J Physiol 1996; 270: L141-L151 [Abstract/Free Full Text].

15. Herskowitz K, Plumley DA, Martin TD, Hautamaki RD, Copeland EM, Souba WW. Lung glutamine flux following open heart surgery. J Surg Res 1991; 51: 82-86 [Medline].

16. Souba WW, Herskowitz K, Plumley DA. Lung glutamine metabolism. JPEN 1990; 14: 68S-70S .

17. Wu G, Haynes TE, Li H, Yan W, Meininger CJ. Glutamine metabolism to glucosamine is necessary for glutamine inhibition of endothelial nitric oxide synthesis. Biochem J 2001; 353: 245-252 [Medline].