PERSPECTIVE
Nucleosides and Nucleotides in the Lung
Role in Asthma

Jeffrey S. Fedan

Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia

Adenine nucleosides and nucleotides have multiple effects as extracellular mediators in every organ system (1) and initiate or modulate cellular responses via cell surface receptors. Current evidence indicates the existence of four receptors for adenosine: A₁, A_2A, A_2B, and A₃ (2). These G protein-coupled receptors transduce activation or inhibition of adenylate cyclase and phospholipase C. Reasonably selective antagonists are available for some adenosine receptor subtypes. The receptors for adenine nucleotides, such as adenosine triphosphate (ATP), now encompass seven distinct P2X class receptors (P2X₁ through P2X₇) (3) and ten P2Y subfamily receptors: P2Y₁ through P2Y₁₁ (the former P2Y₇-receptor is no longer included as a subtype) (4). Five of these receptors are mammalian. Despite its structure, uridine triphosphate (UTP) is a potent ligand at several P2Y-receptors. Responses to P2X-receptor stimulation result from activation of nonselective cation channels in the cell membrane. P2Y-receptor activation stimulates signaling mediated via phospholipase C. There is a paucity of specific antagonists for P2X- and P2Y-receptors, and their characteristics have been defined through the use of relative agonist potencies. Additional information about the molecular characteristics of these receptors, their pharmacologic properties, and associated signaling pathways can be found in several recent compendia and reviews (1, 5).

Nucleosides and nucleotides have several important actions in the lung (9), among which are contractile and relaxant effects on airway smooth muscle, stimulation of mucus and surfactant secretion, and stimulation of ciliary-beating activity. Nucleosides and nucleotides activate or modulate a number of inflammatory cells that are involved in lung disease, including mast cells, neutrophils, macrophages, and eosinophils (1, 10, 11). Two particular aspects of nucleosides and nucleotides have direct applicability to lung disease and its treatment. First, ATP and UTP stimulate with comparable affinity epithelial alternative Cl channels both in normal epithelium and in epithelium from cystic fibrosis (CF) patients, who lack a normally functioning CF-transmembrane conductance regulator Cl channel (12, 13). Promoting Cl secretion with these P2Y₂-receptor-activating ligands offers an opportunity in CF patients for hydrating the hypophase (13).

The second significant aspect of nucleoside action in pulmonary disease involves the observation by Cushley and colleagues (14) that inhalation of aerosols of 5'-adenosine monophosphate (5'-AMP) or adenosine, its breakdown product, provokes bronchoconstriction in atopic and nonatopic patients with asthma but not in normal subjects (9, 15). Although the precise mechanism(s) have not been fully defined, adenosine-induced bronchoconstriction involves histamine released from mast cells and the subsequent contraction of airway smooth muscle, inasmuch as the response is abrogated by H₁-histamine receptor blockers and mast cell "stabilizing" drugs such as sodium cromoglycate. The bronchoconstriction is inhibited by cyclooxygenase inhibitors, such as indomethacin, and by the muscarinic receptor antagonists, atropine and ipratropium, implying that prostanoid release and cholinergic neural pathways are activated or facilitated by adenosine (18). Adenosine-induced bronchoconstriction is also inhibited by theophylline, a finding that led to the proposal that the methylxanthine is an adenosine receptor antagonist. The precise therapeutic mechanism(s) of action of theophylline in asthma remain clouded to this day, as the drug's ability to block adenosine receptors or inhibit cyclic AMP phosphodiesterase are not manifest at therapeutic blood levels. Nevertheless, it has been reliably demonstrated in in vitro studies that, while it has little effect by itself, adenosine potentiates the release of histamine and leukotrienes stimulated by other agents, such as antigen. Mast cell activation by adenosine is currently postulated to be mediated by activation of A_2B- and A₃-receptors.

The article by Michoud and associates in this issue of the American Journal of Respiratory Cell and Molecular Biology, the second on this topic from this laboratory (19), examines another mechanism by which inhaled adenosine could cause bronchoconstriction; namely, its direct effects on airway smooth muscle. In normal guinea pig airway smooth muscle in vitro adenosine elicits contraction and relaxation (20). A₃-receptors mediate the contraction, and these responses are heightened in airways isolated from guinea pigs sensitized with ovalbumin. These same airways also display bronchoconstriction to inhaled adenosine (21, 22). Similar changes occur in sensitized rabbits but appear to involve A₁-receptors (23). These findings correlate well with the observations (24) that isolated bronchi from patients with asthma are more reactive to the contractile effect of adenosine, but not to histamine or leukotriene C₄, and that histamine and leukotrienes mediate the responses.

Several lines of evidence indicate, therefore, that adenosine acts indirectly via released mediators to cause bronchoconstriction. Michoud and colleagues designed experiments to clarify the direct effects of adenosine on airway smooth muscle, as a modulator (as opposed to an initiator) of contractile responses. These investigators employed primary cultures of rat tracheal smooth muscle, which are devoid of mast cells and their mediators. In their earlier investigation (19) using Fura-2 to measure increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i), Michoud and coworkers observed that adenosine itself had no significant effect on [Ca²⁺]_i, but it potentiated the [Ca²⁺]_i elevation in response to ATP; the potentiating effect was sensitive to inhibition of phospholipase C. On the basis of the effects of selective antagonists, the potentiating effect of adenosine was attributed to A₃-receptors. In the current article Michoud and associates report that the A₃-selective agonist 1-deoxy-1-[6-[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl--D-ribofuranuronamide mimics adenosine's potentiating effect on ATP-induced [Ca²⁺]_i elevation. They also determined that the potentiating effect of adenosine is not specific for ATP because responses to 5-hydroxytryptamine (5-HT) were also potentiated. An inhibitor of phospholipase A₂ abolished the potentiating effect of adenosine, whereas inositol phosphate levels were unaffected by adenosine. In toto, this work establishes the existence and function of A₃-receptors, which modulate reactivity of airway smooth muscle, and it provides a paradigm for investigation of possible changes in this system in the airways of the patient with asthma.

Are the agonists studied by Michoud and coworkers relevant to the understanding of adenosine-induced bronchoconstriction in asthma? In all likelihood they are. In the rat 5-HT performs many of the functions of histamine in other species, and it has been demonstrated in dog isolated bronchi that adenosine potentiates histamine-induced contractions (18). Mast cells release ATP upon degranulation, and it has been demonstrated that during antigen stimulation the released ATP may serve as an intercellular mediator causing nearby mast cells to trigger a histamine secretory response via the P2X₇-receptors (10, 25). An amplification scenario can be envisaged in which the released ATP, in addition to causing contraction of the airway smooth muscle directly (9) and indirectly through the released histamine, would be degraded progressively via ectonucleotidases to give rise to adenosine, which would intensify the response to ATP and other contractile mediators.

A central question remains unanswered by the studies of Michoud and fellow investigators: If the potentiation of responses by adenosine in smooth muscle from nonsensitized rats is of (patho)physiologic relevance, why doesn't adenosine cause obstruction in nonasthmatic patients and nonsensitized laboratory animals instead of bronchodilation or no effect? Mast cell numbers are increased in the airways of the patient with asthma (26), and allergen challenge of sensitized subjects elicits adenosine release (17). Discounting for a moment the possibility that A₃-receptors could be upregulated, as has been reported for A₁-receptors in sensitized rabbits (23), it is tempting to speculate that the availability of a larger pool of mediators intensifies the effect of adenosine both on the mast cells and on the smooth muscle. It must now be determined whether the potentiating effect of adenosine occurs in human airway smooth muscle, and whether it is exacerbated in tissues from patients with asthma or sensitized animals.

	Footnotes

Address correspondence to: Jeffrey S. Fedan, PPRB, HELD, NIOSH, L-2015, 1095 Willowdale Rd., Morgantown, WV 26505. E-mail: jsf2{at}cdc.gov

(Received in original form May 11, 1999).

	References

1. Dubyak, G. R., and J. S. Fedan, editors. 1990. Biological Actions of Extracellular ATP. The New York Academy of Sciences, New York.

2. Fredholm, B. B., A. P. Ijzerman, K. A. Jacobson, J. Linden, U. Schwab, and G. R. Stiles. 1998. Adenosine receptors. In The IUPHAR Compendium of Receptor Characterization and Classification. IUPHAR Media Ltd., Cambridge. 48-57.

3. Humphrey, P. P. A., B. S. Khakh, C. Kennedy, B. F. King, and G. Burnstock. 1998. P2X receptors. 1998. In The IUPHAR Compendium of Receptor Characterization and Classification. IUPHAR Media Ltd., Cambridge. 195-208.

4. Harden, T. K., E. A. Barnard, H.-M. Boeynaems, G. Burnstock, G. Dubyak, S. M. O. Hourani, and P. A. Insel. 1998. P2Y receptors. In The IUPHAR Compendium of Receptor Characterization and Classification. IUPHAR Media Ltd., Cambridge. 209-218.

5. Harden, T. K., J. Boyer, and R. A. Nicholas. 1995. P₂-purinergic receptors: subtype-associated signaling responses and structure. Annu. Rev. Pharmacol. Toxicol. 35: 541-579 [Medline].

6. Palmer, T. M., and G. R. Stiles. 1997. Structure-function analysis of inhibitory adenosine receptor regulation. Neuropharmacol. 36: 1141-1147 [Medline].

7. Jacobson, K. A., and M. F. Jarvis, editors. 1997. Purinergic Approaches in Experimental Therapeutics. Wiley-Liss, New York.

8. Turner, J. T., G. A. Weisman, and J. S. Fedan, editors. 1998. The P2 Nucleotide Receptors: The Receptors. Humana Press. Totowa, NJ.

9. Fedan, J. S. Nucleotides and nucleosides in pulmonary function. 1997. In Purinergic Approaches in Experimental Therapeutics. K. A. Jacobson and M. F. Jarvis, editors. Wiley-Liss, Inc., New York.

10. Cockcroft, S., and B. D. Gomperts. 1979. Activation and inhibition of calcium-dependent histamine secretion by ATP ions applied to rat mast cells. J. Physiol. 296: 229-243 [Abstract/Free Full Text].

11. Walker, B. A., M. A. Jacobson, D. A. Knight, C. A. Salvatore, T. Weir, D. Zhou, and T. R. Bai. 1997. Adenosine A3 receptor expression and function in eosinophils. Am. J. Respir. Cell Mol. Biol. 16: 531-537 [Abstract].

12. Mason, S. J., A. M. Paradiso, and R. C. Boucher. 1991. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium. Br. J. Pharmacol. 103: 1649-1656 [Medline].

13. Knowles, M. R., L. L. Clarke, and R. C. Boucher. 1991. Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. New Eng. J. Med. 325: 533-538 [Abstract].

14. Cushley, M. J., A. E. Tattersfield, and S. T. Holgate. 1983. Inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. Br. J. Clin. Pharmacol. 15: 161-165 [Medline].

15. Jacobson, M. L., and T. R. Bai. 1997. The role of adenosine in asthma. In Purinergic Approaches in Experimental Therapeutics. K. A. Jacobson and M. F. Jarvis, editors. Wiley-Liss, Inc., New York. 315-331.

16. Polosa, R., and S. T. Holgate. 1997. Adenosine bronchoprovocation: a promising marker of allergic inflammation in asthma? Thorax 52: 919-923 [Medline].

17. Marquardt, D. 1997. Adenosine. In Asthma. P. J. Barnes, M. M. Grunstein, A. R. Leff, and A. J. Woolcock, editors. Lippincott-Raven Publishers, Philadelphia. 585-591.

18. Sakai, N., J. Tamaoki, K. Kobayashi, M. Katayama, and T. Takizawa. 1989. Adenosine potentiates neurally- and histamine-induced contraction of canine airway smooth muscle. Int. Arch. Allergy Appl. Immunol. 90: 280-284 [Medline].

19. Michoud, M. C., B. Tollocsko, and J. G. Martin. 1997. Effects of purine nucleotides and nucleosides on cytosolic calcium levels in rat trachea smooth muscle. Am. J. Respir. Crit. Care Med. 16: 199-205 .

20. Farmer, S. G., B. J. Canning, and D. E. Wilkins. 1988. Adenosine receptor-mediated contraction and relaxation of guinea pig isolated tracheal smooth muscle: effect of adenosine antagonists. Br. J. Pharmacol. 95: 371-378 [Medline].

21. Thorne, J. R., and K. J. Broadley. 1994. Adenosine-induced bronchoconstriction in conscious hyperresponsive and sensitized guinea pigs. Am. J. Respir. Crit. Care Med. 149: 392-399 [Abstract].

22. Thorne, J. R., H. Danahay, and K. J. Broadley. 1996. Analysis of the bronchoconstrictor responses to adenosine receptor agonists in sensitized guinea-pig lungs and trachea. Eur. J. Pharmacol. 316: 263-271 [Medline].

23. Ali, S., S. J. Mustafa, and W. J. Metzger. 1994. Adenosine receptor-mediated bronchoconstriction and contraction of airway smooth muscle from allergic rabbits with late phase obstruction: evidence for an inducible adenosine A1 receptor. J. Pharmacol. Exp. Ther. 268: 1328-1334 [Abstract/Free Full Text].

24. Bjorck, T., L. E. Gustafsson, and S. E. Dahlen. 1992. Isolated bronchi from asthmatics are hyperresponsive to adenosine, which apparently acts indirectly by liberation of leukotrienes and histamine. Am. Rev. Respir. Dis. 145: 1087-1091 [Medline].

25. Osipchuk, Y., and M. Cahalan. 1992. Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells. Nature 359: 241-244 [Medline].

26. Shimizu, Y., and L. B. Schwartz. 1997. Mast cell involvement in asthma. In Asthma. P. J. Barnes, M. M. Grunstein, A. R. Leff, and A. J. Woolcock, editors. Lippincott-Raven, Philadelphia. 353-365.