Contraction of airway smooth muscle is mediated by M3 muscarinic receptors on the airway smooth muscle. However, there is no evidence suggesting that hyperresponsiveness results from any alterations in function of these M3 muscarinic receptors. In contrast, there is clearly increased release of the neurotransmitter acetylcholine in animal models of hyperactivity and in asthma. Release of acetylcholine is controlled by inhibitory M2 muscarinic receptors, and it appears that it is these M2 receptors that are dysfunctional in animal models of hyperresponsiveness. Allergen-induced M2 receptor dysfunction is absolutely dependent upon an influx of eosinophils into the airways. Activated eosinophils release major basic protein, which binds to M2 receptors and prevents binding of acetylcholine. Thus, the normal negative feedback control of acetylcholine release is lost, and acetylcholine release is increased. In conclusion, loss of function of inhibitory M2 muscarinic receptors on the airway parasympathetic nerves causes vagally mediated bronchoconstriction and hyperresponsiveness following antigen challenge. Fryer AD, Jacoby DB. Muscarinic receptors and control of airway smooth muscle.
The parasympathetic nerves provide the dominant autonomic control of airway smooth muscle. They release acetylcholine onto muscarinic receptors, causing contraction and bronchoconstriction (1). The release of acetylcholine from the parasympathetic nerves is controlled by muscarinic autoreceptors located on the nerves. The hyperresponsiveness, that is characteristic of asthma is thought to result from excessive contraction of the airway smooth muscle. However, in the absence of any measurable changes in the function of the muscarinic receptors that mediate contraction of airway smooth muscle, it is possible that excessive release of acetylcholine is the mechanism for hyperresponsiveness.
Muscarinic receptors have been divided into five subtypes, and various lung functions are controlled by distinct muscarinic receptor subtypes. The expression and function of muscarinic receptors may be altered with lung disease, leading to hyperresponsiveness and to the hypersecretion of mucus associated with disease. This review will describe the function of various subtypes of muscarinic receptors controlling smooth muscle contraction within the lungs and the changes in their function with disease.
In 1914 acetylcholine receptors were classified into nicotinic and muscarinic receptors by Sir Henry Dale. Muscarinic receptors were formally divided into two subtypes in 1980, based on high (M1) and low (M2) affinity for the antagonist pirenzepine (2). Eventually, as additional selective antagonists were introduced, M2 muscarinic receptors were further divided into M2, M3, and M4 receptors. At this time there are no selective agonists used to characterize muscarinic receptor subtypes. Thus, all the pharmacology has been carried out using selective antagonists. Five muscarinic receptor genes have been cloned, and they are designated M1–M5 (2).
In the lungs, muscarinic receptors are present on airway smooth muscle and on the nerves that control airway smooth muscle. Muscarinic receptors have also been localized to the epithelium (M1 and M3) (3) and glands (M3) (4).
Quantification of muscarinic receptors within sections of the lung using radioligand binding to slide-mounted tissue sections has demonstrated that muscarinic receptors are present on airway smooth muscle within the trachea and bronchi (5), although muscarinic receptors are more dense in the ganglia and in the glands than in the airway smooth muscle (6). Within the smooth muscle, the greatest density of muscarinic receptors is in the lower trachea. The upper trachea and bronchi contain significantly fewer receptors, and the number decreases from the bronchi with each generation of the airways. In most species the alveoli contain very few muscarinic receptors, while in some species (ferret and guinea pig) the alveoli have no apparent muscarinic receptors (5, 7).
Airway smooth muscle expresses M2 and M3 muscarinic receptors based on binding studies and northern blot analysis (8, 9). In most animal species, there are many more M2 receptors than M3 in the conducting airways. Depending upon the species, 50 to 80% of the receptors can be M2 receptors (2).
There are no studies characterizing the proportion of M2/ M3 muscarinic receptors in human airway smooth muscle. Muscarinic receptors in human trachea are almost certainly either M2 or M3, since they have a low affinity for an M1 antagonist pirenzepine (10). Although muscarinic receptor proteins have not been characterized in human muscle, mRNA for M2 and M3 receptors has been detected in human bronchi (11), supporting the hypothesis that human airways express both M2 and M3 receptors.
While both M2 and M3 muscarinic receptors are present on the airway smooth muscle, functional studies indicate that it is the M3 receptors that mediate smooth muscle contraction in animals (2) and in man (12). Thus, the receptor-mediating contraction of airway smooth muscle is the same as that mediating contraction in ileal, uterine, and bladder smooth muscle (13).
M3 muscarinic receptors in airway smooth muscle are coupled to phospholipase C via a pertussis toxin–insensitive G protein (GTP-binding regulatory protein). Activation of phospholipase C catalyzes the formation of inositol triphosphate and diacylglycerol from the membrane phospholipid phosphatidylinositol 4,5-bisphosphate and phosphorylation of proteins via activation of protein kinases (14, 15). Coupling of M3 receptors to inositol triphosphate has been demonstrated in all species studied, including in human bronchial smooth muscle (14, 15).
Although 50 to 80% of muscarinic receptors on airway smooth muscle are M2 receptors, the function of these receptors is still unclear. Stimulation of M2 muscarinic receptors in airway smooth muscle leads to inhibition of adenylyl cyclase via an inhibitory, pertussis-sensitive G protein (Gi) (16, 17). Thus, although M2 muscarinic receptors do not play a direct role in smooth muscle contraction, they appear to inhibit any relaxation of the smooth muscle that requires activation of adenylate cyclase, for example, β-agonist–induced relaxation (18).
Although patients with asthma are hyperresponsive to cholinergic agonists, there is no evidence that the M3 receptors mediating contraction of the airway smooth muscle have been altered by disease. In vitro, exogenous cholinergic agonists produced no greater contraction of muscle taken from subjects with asthma versus muscle from subjects with no asthma (19, 20). In animal models there is also no evidence for increased expression of M3 receptors, although glucocorticosteroid treatment, which suppresses expression of the M3 receptors in airway smooth muscle, reverses the hyperresponsiveness (21). In animal models of asthma, bronchoconstriction induced in vivo by muscarinic agonists (with the vagus nerves cut to eliminate any reflex) is not different from control subjects (Figure 1B) (22-24), confirming that there are no alterations in the function of the M3 muscarinic receptors on airway smooth muscle that result in hyperresponsiveness.
It has been suggested that the function of the β-adrenoceptors is impaired in asthma, and that the muscle is unable to relax. This effect may be mediated by the M2 muscarinic receptors since activation of these receptors inhibits β-adrenoceptor–mediated relaxation. The airway smooth muscle of Basenji greyhounds, a breed of dogs with inherited nonspecific airway hyperresponsiveness, is hyporesponsive to β-agonists, when compared with mongrel dogs. This inability to relax is associated with an increased ability of muscarinic agonists to inhibit adenylate cyclase and with an increase in the number of M2 muscarinic receptors in this strain of dog (25). While this alteration in M2 receptors on the smooth muscle may explain hyporelaxation, it does not explain hyperresponsiveness.
Animal models of hyperresponsiveness are most certainly characterized by increased release of acetylcholine from the vagus nerves. In animals (including guinea pigs, dogs, mice, and rats) that have been sensitized and challenged with antigen, release of acetylcholine from the vagus nerves and vagally induced bronchoconstriction is enhanced compared with control subjects (Figure 1A) (26-30). Likewise, in animals that are exposed to ozone or acutely infected with parainfluenza virus, vagally induced bronchoconstriction is potentiated (31– 35). In none of these models was there evidence that the M3 muscarinic receptors on airway smooth muscle were altered. Thus hyperresponsiveness is characterized by increased release of acetylcholine from the nerves rather than by increased sensitivity of the M3 muscarinic receptors on airway smooth muscle.
Cholinergic control of the airways is abnormal in patients with asthma (36), and hyperresponsiveness is in part mediated by the release of acetylcholine onto M3 muscarinic receptors in the airways. Anticholinergic drugs, when given intravenously or in high doses, are effective bronchodilators in asthma (37). Since there is no evidence for increased numbers or function of the M3 muscarinic receptors on the airway smooth muscle of patients with asthma, it is possible that hyperresponsiveness in asthma is mediated by increased release of acetylcholine from the vagus nerves. Release of acetylcholine from the vagus nerves is limited by M2 muscarinic receptors on the parasympathetic nerves supplying the airways, and these receptors are altered in asthma and in animal models of disease. Thus, hyperresponsiveness may be mediated by a muscarinic receptor not on the airway smooth muscle.
In 1984 Fryer and Maclagan (38) demonstrated that functional M2 muscarinic receptors were present in the postganglionic parasympathetic nerves supplying the lung. These muscarinic receptors provide a negative feedback mechanism whereby acetylcholine released from the vagus nerve stimulates the muscarinic receptors and inhibits further release of acetylcholine (Figure 2). These receptors were demonstrated in vivo using the muscarinic agonist pilocarpine, which by stimulating these neuronal receptors inhibits vagally induced bronchoconstriction. In contrast, gallamine, a selective M 2 antagonist, potentiated bronchoconstriction induced by electrical stimulation of the vagus nerves by blocking these receptors (38). Inhibitory neuronal M2 receptors were initially described in the guinea pig (38) and have now been described in the parasympathetic nerves supplying the lungs of all species studied thus far, including man (26, 37-44).
The neuronal M2 muscarinic receptors function under physiologic conditions and exert a profound inhibitory influence on acetylcholine release. Stimulation of the receptors with muscarinic agonists inhibits vagally induced bronchoconstriction in the guinea pig as much as 80% (Figure 3A) (38). In human bronchi contracted by electrical field stimulation, the muscarinic agonist pilocarpine inhibits the contraction by 96%, demonstrating that the M2 muscarinic receptors also exert a marked degree of inhibition in human lung (41). Conversely, blockade of the M2 receptors with antagonists produces an increase in acetylcholine release and a 10-fold increase in vagally induced bronchoconstriction (38, 45). Thus, the neuronal M2 receptors play a critical role in maintaining normal autonomic control of the lung.
The inhibitory M2 muscarinic receptors on the pulmonary parasympathetic nerves are dysfunctional in three different animal models of asthma: acute infection with parainfluenza virus (34, 35), sensitization and challenge with antigen (26, 29, 37), and acute exposure to ozone (22). In all of these models the agonist pilocarpine no longer inhibits vagally induced bronchoconstriction, demonstrating that the M2 receptors cannot be stimulated with exogenous agonists (Figure 3B). Furthermore, the potentiation of vagally induced bronchoconstriction by gallamine is greatly inhibited, indicating that endogenous acetylcholine is also not effectively stimulating the receptors. Thus, in multiple animal models of hyperresponsiveness, the neuronal M2 receptors are dysfunctional. Decreased neuronal M2 receptor function increases release of acetylcholine from the nerves. Since the vagus nerves control airway tone and mediate reflex bronchoconstriction, loss of neuronal M2 receptor function results in increased tone and increased reflex bronchoconstriction, thus causing hyperresponsiveness.
Loss of neuronal M2 receptor function is therefore a mechanism for airway hyperresponsiveness. Alone, loss of M2 receptor function causes hyperresponsiveness via increased release of acetylcholine and increased contraction of airway smooth muscle. An additional side effect of increased acetylcholine that should be considered is that it may further enhance M2-mediated inhibition of adenylyl cyclase in the smooth muscle. This effect can be further augmented since chronic stimulation of the M3 muscarinic receptors (as may be seen with neuronal M2 dysfunction) decreases the responsiveness of adenylyl cyclase (46). In animal models of asthma, prevention of neuronal M2 receptor dysfunction or acute restoration of M2 receptor function (see below) prevents or reverses hyperresponsiveness to vagal stimulation (23, 24, 47).
M2 muscarinic autoreceptors are present and functional on parasympathetic nerves in human airways (41). The parasympathetic nerves of patients with asthma are hyperresponsive compared with control subjects since in vitro there is increased release of acetylcholine in response to electrical field stimulation (48). As in animal models of asthma, there is evidence that the neuronal M2 muscarinic receptors are not functioning properly in humans with asthma. Muscarinic agonists inhibit reflex-induced bronchoconstriction in subjects with no asthma but do not inhibit bronchoconstriction in patients with mild asthma (Figure 4) (49, 50), demonstrating loss of M2 receptor function. Thus, M2 muscarinic autoreceptors are present in human lung, have an important role in controlling acetylcholine release from the vagus nerves, and are dysfunctional in patients with asthma.
Recently it has been demonstrated that release of acetylcholine from the parasympathetic nerves supplying the mucus glands is also controlled by inhibitory M2 muscarinic receptors (51). Since neuronal M2 receptors on the nerves supplying the smooth muscle are dysfunctional in asthma, they may also be dysfunctional on the nerves supplying the glands, increasing acetylcholine release and thus increasing mucous secretion in asthma.
The role of specific inflammatory cells in loss of M2 receptor function was examined since depletion of inflammatory cells with cyclophosphamide prior to exposure to ozone prevented loss of M2 receptor function (52). The role of eosinophils was examined because eosinophils are found in asthmatic airways (53), and appear to be selectively recruited to the airway nerves in sections taken from patients who have died from acute asthma (54). In addition, eosinophils are also present in guinea pig lungs following acute ozone exposure or antigen challenge of sensitized animals (54). Finally, inhibition of eosinophil migration into the lungs inhibits hyperresponsiveness (55, 56).
Major basic protein and M2 receptor function. Eosinophils contain charged proteins such as major basic protein, eosinophil cationic protein, and eosinophil peroxidase (57). This is important since M2 muscarinic receptors are particularly prone to blockade by positively charged proteins, including poly-l-arginine, poly-l-lysine, basic histone, and protamine. These proteins bind to an allosteric site on the M2 receptor and inhibit agonist binding (58). In contrast, M3 muscarinic receptors are not affected by positively charged proteins. Thus, charged proteins from inflammatory cells may inhibit agonist binding to neuronal M2 muscarinic receptors without altering the M3 muscarinic receptors on airway smooth muscle.
In guinea pig lungs and in lungs from patients who have died from asthma, there is a selective recruitment of eosinophils to the airway nerves. Eosinophils are present along and within the nerve bundles, ganglia, and along the nerve fibers in the smooth muscle (Figure 5) (54). Thus, recruitment of eosinophils into the lungs and nerves brings charged proteins into the lungs and directly to the nerves and M2 muscarinic receptors.
Eosinophil major basic protein is an allosteric antagonist for M2 muscarinic receptors. It selectively displaces labeled antagonist from M2 muscarinic receptors in a dose related fashion with a Ki of 1.5 × 10−5 M. Since major basic protein is found in the sputum of patients with asthma in micromolar concentrations, it is likely that relevant concentrations are present around the M2 receptors in vivo (see Reference 57). Major basic protein did not bind to M3 muscarinic receptors in vitro, and thus would not be expected to affect agonist binding to M3 receptors on airway smooth muscle. Although our initial hypothesis was that major basic protein interacts with M2 muscarinic receptors on the basis of charge, the positively charged eosinophil cationic protein did not bind to the M2 receptors. Thus, the interaction between M2 receptors and major basic protein must be more complex.
Eosinophils, airway hyperresponsiveness, and M2 function. The role of eosinophils and of eosinophil major basic protein on loss of M2 receptor function and hyperresponsiveness has since been confirmed in vivo. The initial studies took advantage of the fact that heparin and poly-l-glutamic acid bind and neutralize positively charged eosinophil proteins (47). After a stable, reproducible bronchoconstrictor response to vagal stimulation was obtained in antigen-challenged guinea pigs, either heparin, desulfated heparin (which has no anticoagulating properties), or poly-l-glutamic acid was given and vagally induced bronchoconstriction measured every minute thereafter. The response to vagal stimulation was unchanged for 5 min, after which a progressive decrease in vagally mediated bronchoconstriction was observed over 20 min, reaching a plateau at approximately 50% of the bronchoconstriction before administration of the heparins or poly-l-glutamic acid (47, 59) (Figure 6). Neither heparin, desulfated heparin, nor poly-l-glutamic acid inhibited bronchoconstriction induced by intravenous acetylcholine, demonstrating that the M3 receptors on the airway smooth muscle were not affected. In addition, none of the polyanionic substances affected vagally induced or intravenous acetylcholine-induced bronchoconstriction in control (nonantigen-challenged) animals (47, 59). Thus, heparin, desulfated heparin, and poly-l-glutamic acid can acutely reverse hyperresponsiveness in antigen-challenged guinea pigs. While the use of heparin in the treatment of hyperresponsiveness is problematic due to its anticoagulating properties, desulfated heparin, which is not an anticoagulant, may be useful.
The effect of heparin, desulfated heparin, and poly-l-glutamic acid on neuronal M2 muscarinic receptor function was tested by measuring the ability of pilocarpine to suppress, and of gallamine to potentiate, vagally induced bronchoconstriction after administration of heparin and poly-l-glutamic acid. Treatment with either negatively charged substance acutely restored the effects of pilocarpine and of the M2 antagonist gallamine on vagally induced bronchoconstriction (47, 59). Thus the decrease in vagally mediated bronchoconstriction after the heparins or poly-l-glutamate is accompanied by, and probably due to, a restoration of function of the M2 receptors. The effect of these three substances on vagally induced bronchoconstriction in antigen-challenged animals is consistent with positively charged proteins being responsible for the loss of M2 receptor function.
The specific role of eosinophils in loss of M2 receptor function and subsequent hyperresponsiveness has also been demonstrated. Depletion of eosinophils prior to antigen challenge by pretreatment with an antibody to interleukin-5 protected the function of the neuronal M2 muscarinic receptors from antigen challenge (60). In addition, blockade of eosinophil migration into the lungs with an antibody to the adhesion molecule, very late activation antigen-4, also protected neuronal M2 muscarinic receptor function from antigen challenge (23). Thus, the presence of eosinophils is required for loss of neuronal M2 muscarinic receptor function.
The role of eosinophil major basic protein in loss of M2 receptor function was demonstrated using an antibody to major basic protein. Administration of anti-major basic protein prior to antigen challenge protected the function of the neuronal M2 muscarinic receptors and prevented hyperresponsiveness. However, anti-major basic protein did not inhibit eosinophil migration into the lungs or to the nerves within the lungs. Thus, even in the presence of eosinophils, specific blockade of major basic protein protected neuronal M2 muscarinic receptor function (24), conclusively demonstrating that loss of neuronal M2 muscarinic receptor function is due to release of major basic protein from eosinophils.
Finally, it is major basic protein–induced loss of M2 receptor function that causes hyperresponsiveness. Antigen-challenged guinea pigs are hyperresponsive to electrical stimulation of the vagus nerves. Protection of the neuronal M2 receptor function with either the antibody to very late activation antigen-4, or with the antibody to major basic protein, prevented development of hyperresponsiveness (23, 24, 55) (Figure 1A). These data agree with the experiments that demonstrated that removal of positively charged proteins with heparin and other anionic substances restored M2 receptor function in vivo and acutely reversed airway hyperresponsiveness. Thus, loss of neuronal M2 muscarinic receptor function via release of eosinophil major basic protein is a mechanism for hyperresponsiveness in antigen-challenged animals.
In summary, eosinophils are recruited to the airway nerves in antigen-challenged guinea pigs, where they release major basic protein. Major basic protein is a selective, endogenous antagonist for the neuronal M2 receptors. Blockade of the neuronal receptors results in increased release of acetylcholine from the parasympathetic nerves (Figure 7). Thus, loss of M2 function results in increased airway tone due to increased tonic release of acetylcholine and potentiation of vagally mediated reflex bronchoconstriction. Since M2 receptors are not functional in some humans with asthma, and asthma is also characterized by an influx of eosinophils, this may be a mechanism for the hyperresponsiveness associated with asthma. There are other potential mechanisms for altering the function of the neuronal M2 muscarinic receptors; thus, whether major basic protein is also the mechanism for hyperresponsiveness in ozone-exposed and virus-infected animals remains to be tested.
Although eosinophils are the mechanism for loss of M2 function in antigen-challenged guinea pigs, they are not involved in M2 receptor dysfunction and hyperresponsiveness after viral infection (61). It may be that with viral infection there are other mechanisms for loss of neuronal M2 receptor function. We have demonstrated that deglycosylation of the M2 receptors (as with viral neuraminidase) decreases agonist affinity for M2 receptors (62), which would compromise the negative feedback control of acetylcholine release. Neuraminidase is also present endogenously in inflammatory cells such as macrophages, neutrophils, and lymphocytes, and thus may be an additional mechanism whereby inflammatory cells may affect M2 receptor function. Furthermore, both parainfluenza virus and interferon-γ substantially decrease the expression of the gene encoding the M2 receptor in airway parasympathetic nerves (63). Thus, decreased synthesis of M2 receptors may also be a mechanism for decreased function of the neuronal M2 muscarinic receptors.
Loss of neuronal M2 muscarinic receptor function, and the resulting increase in release of acetylcholine from the parasympathetic nerves, is the mechanism of hyperresponsiveness in antigen-challenged guinea pigs. Neuronal M2 receptor dysfunction has been demonstrated in humans with asthma, but whether this contributes to hyperresponsiveness associated with this disease remains to be tested.
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