Muscarinic Receptor Subtypes in Lung Clinical lmplleatlons':? P. A. MINETTE and PETER J. BARNES
Introduction The heterogeneity of muscarinic receptors has been recognized in the last decade with the development of new selective muscarinic antagonists (1-4). The existence of at least three different subtypes is now widely accepted (2-4). Although there is still some confusion over the classification and nomenclature of the different subtypes, it is possible to differentiate receptors with a high affinity for the antagonist pirenzepine (PZ) (called MI receptors), those with a high affinity for AF-DX 116 and methoctramine (called M2 receptors), and those with a high affinity for 4-diphenyl acetoxy N-methyl piperidine (4DAMP) and hexa-hydro siladifenidol (called M3 receptors) (2-4). Further subdivision may be possible in the future, with the development of more selective antagonists. Several muscarinic receptor subtypes have now been identified in airways (5-7) (figure I). Muscarinic receptors are coupled to different postreceptor mechanisms, which include inhibition of adenylate cyclase (AC) and the stimulation of phosphatidylinositol (PI) turnover with formation of inositol triphosphate. There is now increasing evidence that MI and M3 receptors are predominantly linked to PI turnover (8-12),whileM2 receptorsare mainly inhibiting AC (8, 13). Muscarinic receptors have recently been cloned and expressed in human and animal tissues (14, 15). As many as five different receptor proteins have now been described (16), but their precise relationship to pharmacologicallydistinct receptorsis not yetclear (17).
Muscarinic Receptors in Airways Cholinergic nerves are the predominant neural pathway in the airwys (18)and play an important role in airway obstruction. Cholinergic nerve fibers pass down the vagus nerve to synapse in parasympathetic ganglia within the airways. Acetylcholine (ACh) is released from postganglionic fibers and binds to muscarinic receptors located on the target cells. Autoradiographic studies on human lung (19) have shown high densities of muscarinic receptors on central airway smooth muscles, submucosal glands, and parasympathetic ganglia. Low densities of receptors are also present on cholinergic nerves. Identification of receptor subtypes involved in these locations may lead to better understanding of cholinergic control of airway tone and mucus secretion and improve anticholinergic therapy of airway diseases. S162
SUMMARY Several subtypes of muscarinic receptors have been identified in airways and lung parenchyma of different species, including humans. M1 receptors may be present in parasympathetic ganglia where they appear to facilitate ganglionic neurotransmission. M2 receptors seem to be located on cholinergic nerve endings where they function as "autoreceptors," inhibiting acetylcholine release. M3 receptors appear to be located on effectory cells, smooth muscle, and mucus secreting glands. Experimental data in support of this view are presented and discrepant data are discussed. AM REV RESPIR DIS 1990; 141:S162-S165
Smooth Muscle Receptors In vitro, guinea pig (3), bovine (20), and dog (21) airway smooth muscle contractions to cholinergic agonists are blocked with high affinity by 4-DAMP and only with low affinity by PZ (table 1), classifying these receptors as the M3 subtype. Binding studies on bovine trachea (20, 22) and human airways (23) confirm a low affinity of muscarinic receptors for PZ and high affinity for 4-DAMP. According to these functional and binding data, a recent autoradiographic mapping of muscarinic receptors in guinea pig and human airways showed consistent M3 receptors on smooth muscle in all airways from trachea to peripheral bronchioles (24). By contrast, Roffel and coworkers (20) found heterogeneous binding with selective M2 antagonists AF-DX 116 and methoctramine in bovine trachea, suggesting the existence of both M2 and M3 receptors in bovine tracheal membranes. Functional studies, however, showed low affinity of tracheal smooth muscle for both M2 antagonists and high affinity for M3 antagonists. The significance of these discrepancies is still unclear. Peripheral Lung Membranes Binding studies on peripheral lung membranes in rabbit (25), rat (26),guinea pig (27), and humans (27-29) show a high proportion (25 to 70070) of receptors with high affinity for PZ (table 2). These results are difficult to interpret since membranes included in such homogenates belong to many different cell types (smooth muscles, mucus glands, ganglia, and pulmonary vessels). Therefore, it is not possible to conclude which cell membranes bear MI receptors. There is functional evidence that muscarinic receptors in parasympathetic ganglia belong to the MI subtype (30-32). Binding studies on isolated submucosal glands of swine trachea (33) showed that Ml receptors made up 27% of total muscarinic receptors. It is, however, unlikely that gland and ganglion receptors could account for as much as 50% of total lung muscarinic receptors. Recent autoradiographic mapping of muscarinic receptor subtypes in human airways
(24) showed MI receptors to be localized to submucosal glands and alveolar wallsbut not to airway smooth muscle. The role of MI receptors on alveoli is as yet unclear. Airway Submucosal Glands Little is known about the subtype of receptor involved in airway mucous and serous glands. Muscarinic receptors of salivary glands (2-4), lachrymal glands (3), and pancreatic acini (34) have been shown to belong to the M3 subtype (2). Binding studies on swine tracheal submucosal glands (33) have shown 27% of muscarinic receptors with a high affinity for PZ (MI), the remaining receptors having an affinity for PZ consistent with M3 receptors. In bovine tracheal mucosa, Madison and colleagues (22) found only receptors with a low affinity for PZ. In this study, no further characterization into M2 or M3 subtype wasmade. Autoradiographic studies have demonstrated the presence of Ml and M3 receptors on human bronchial submucosal glands (24), MI receptors accounting for approximately 30% of the receptors. Functional studies with selective antagonists in isolated cat trachea suggest a response intermediate between Ml and M3 receptors (35), supporting the idea that both receptor subtypes are involved in mucus secretion. Muscarinic Receptors in Parasympathetic Ganglia Historically, MI receptors were first demonstrated in brain and sympathetic ganglia (1-4). Pirenzepine is used clinically to reduce gastric acid secretion and it has been shown to act predominantly on parasympathetic ganI From the Department of Thoracic Medicine, National Heart and Lung Institute, London, United Kingdom. 2 Supported by Boehringer IngeIheim(Germany). J Correspondence and requests for reprints should be addressed to Prof. P. J. Barnes, Department of Thoracic Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom.
8163
MUSCARINIC RECEPTOR SUBTYPES
TABLE 2 MUSCARINIC RECEPTORS ON PERIPHERAL LUNG MEMBRANES PZ+
(0/0) Rabbit (23) Rat (24) Guinea-pig (25) Human (26) (27) (25)
Ganglion Cholinergic nerve
AGONIST
McN-A-343
ANTAGONIST
Airway Smooth muscle
Pilocarpine
Plrenzeplne
Gallamine
4-DAMP
AF-DX 116
Hexahydroslla-dlfenldol
"1ethoctramlne
78 50 25 70 62 43
PZ(%) 22 50 75 (= M3)* 30 38 (= M3)* 57 (= M3)*
Note: Binding studies on peripheral lung homogenates show a high proportion of receptors with high affinity for pirenzepine. Definitionof abbreviations: PZ + = receptors with high affinity for pirenzepine; PZ - = receptors with low affinity for pirenzepine; M3 = M3 SUbtype muscarinic receptor. • Use of 4-DAMP allowed classification of the receptors with low affinity for PZ as M3 receptors.
Fig. 1. Muscarinic receptor subtypes in airways. From Barnes and colleagues (5) with permission.
glia (36) by inhibiting neurally mediated gastric secretion. Since the innervation of the airways is derived embryologically from that of the gut, it seemslikelythat Ml receptors might exist in airway parasympathetic ganglia. Pirenzepine blocks bronchoconstriction due to vagus nerve stimulation in rabbit (30) and in dog (37)at much lowerdoses than those required to inhibit vagally induced decrease in heart rate, suggesting the existence of Ml receptors mediating bronchoconstriction. Since muscarinic receptors on airway smooth muscle have a low affinity for PZ (3, 20, 21), excitatory MI receptors must be located on the vagal pathway controlling airway caliber, most likely on parasympathetic ganglia. In support of this hypothesis, PZ has been shown to be more effectivein blocking pre- than postganglionic vagal nerve stimulation in rabbit airways in vitro (31). There is some evidence that excitatory Ml receptors are also present on human vagal pathways. Pirenzepine is a bronchodilator when given intravenously (38), but at doses that were not shown to be selective for MI receptors. Recently (32), PZ given by inhalation to atopic volunteers was shown to be as effective as inhaled ipratropium bromide in blocking the cholinergic reflex bronchocon-
striction triggered by exposure to sulfur dioxide. The same doses of PZ failed to displace the dose-response curves to inhaled methacholine in the same subjects, demonstrating that PZ inhibits selectively the cholinergicreflex mechanism, presumably at ganglionic level. The physiologic role of Ml receptors in autonomic ganglia remains speculative because ganglionic transmission is classically due to activation of nicotinic receptors. It is possible that MI receptors close potassium channels in ganglionic neurons, inducing slow depolarization and facilitating rapid firing on nicotinic activation. Confirmation of this hypothesis needs further investigation. Prejunctional Inhibitory Muscarinic Receptors Prejunctional muscarinic receptors, which inhibit the release of ACh from cholinergic nerves (autoreceptors), have been shown in airways in guinea pig (39) and cat (40) in vivo and in guinea pig (41) and dog (42) in vitro. Selective inhibition of these receptors by gallamine potentiates neurally mediated smooth muscle contractions (38- 41). Autoreceptors have also been demonstrated in human air-
TABLE 1 MUSCARINIC RECEPTORS ON AIRWAY SMOOTH MUSCLE CELLS Binding Studies
Functional Studies
Pirenzepine
4-DAMP
Type
pA2*
Canine bronchi Bovine trachea Guinea pig trachea Guinea pig trachea
6.8 6.9 7.1 6.7
Bovine trachea Guinea pig trachea Guinea pig trachea
9.0 9.0 8.7
Type
pKit
Reference
19 18 3 Personal communication
Bovine trachea Bovine trachea Human bronchi
6.3 6.4 5.8
19:1: 18 22:1:
18 3 Personal communication
Bovine trachea
8.0
18
Reference
Note: Functional and binding studies on airway smooth muscle show a low affinity of the receptors for pirenzepine and a high affinity for 4-DAM?, consistent with an "13 receptor. * pA2 .. -logarithm of the concentration of antagonist that inhibits 50% of the effect of the agonist. t pKi = -logarithm of the dissociation constant for the antagonist-receptor complex. pKi was not given by the authors but calculated from the Ki: pKj = -log (Kil.
*
ways in vitro (43) using pilocarpine as an autoreceptor-agonist to inhibit bronchial ring contractions induced by electric field stimulation. This effect was inhibited by gallamine in a dose-related fashion (figure 2), suggesting a muscarinic receptor-mediated mechanism. In guinea pig tracheal strips in vitro, contractions induced by electric field stimulation were found to be potentiated by AFOX 116 at doses that are selective for M2 receptors; no such potentiation was found with MI antagonist PZ (personal unpublished data, figure 3). Pilocarpine, which stimulates autoreceptors in vitro (39, 43), has an inhibitory effect on cholinergic reflex bronchoconstriction induced by inhalation of sulfur dioxide in healthy atopic volunteers (44), suggesting the existence of autoreceptors in vivo. These receptors presumably serveto limit neurally mediated bronchoconstriction. In asthmatic subjects, pilocarpine has no such inhibitory action, suggesting a possible dysfunction of the autoreceptor, which would result in exaggerated cholinergic reflex bronchoconstriction (44). Pulmonary Arteries Muscarinic receptors are also present on pulmonary arteries where they mediate vasodilatation, secondary to release of endothelial cell-derived relaxant factor (45). In vitro relaxation of precontracted rat pulmonry arteries by ACh is inhibited with high affinity by 4-0AMP and with low affinity by methoctramine and PZ, classifying these receptors as M3 (26). Clinical Relevance and Conclusions From available experimental data, it is possible to speculate about the role of each muscarinic receptor subtype (figure 3). Ml and M2 receptors appear mainly to control parasympathetic neurotransmission, Ml receptors facilitating ganglionic transmission and M2 receptors acting as negative feedback mechanisms at postganglionic level,while M3 receptors appear to activate target cells like
5164
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Introduction The heterogeneity of muscarinic receptors has been recognized in the last decade with the development of new selective muscarinic antagonists (1-4). The existence of at least three different subtypes is now widely accepted (2-4). Although there is still some confusion over the classification and nomenclature of the different subtypes, it is possible to differentiate receptors with a high affinity for the antagonist pirenzepine (PZ) (called MI receptors), those with a high affinity for AF-DX 116 and methoctramine (called M2 receptors), and those with a high affinity for 4-diphenyl acetoxy N-methyl piperidine (4DAMP) and hexa-hydro siladifenidol (called M3 receptors) (2-4). Further subdivision may be possible in the future, with the development of more selective antagonists. Several muscarinic receptor subtypes have now been identified in airways (5-7) (figure I). Muscarinic receptors are coupled to different postreceptor mechanisms, which include inhibition of adenylate cyclase (AC) and the stimulation of phosphatidylinositol (PI) turnover with formation of inositol triphosphate. There is now increasing evidence that MI and M3 receptors are predominantly linked to PI turnover (8-12),whileM2 receptorsare mainly inhibiting AC (8, 13). Muscarinic receptors have recently been cloned and expressed in human and animal tissues (14, 15). As many as five different receptor proteins have now been described (16), but their precise relationship to pharmacologicallydistinct receptorsis not yetclear (17).
Muscarinic Receptors in Airways Cholinergic nerves are the predominant neural pathway in the airwys (18)and play an important role in airway obstruction. Cholinergic nerve fibers pass down the vagus nerve to synapse in parasympathetic ganglia within the airways. Acetylcholine (ACh) is released from postganglionic fibers and binds to muscarinic receptors located on the target cells. Autoradiographic studies on human lung (19) have shown high densities of muscarinic receptors on central airway smooth muscles, submucosal glands, and parasympathetic ganglia. Low densities of receptors are also present on cholinergic nerves. Identification of receptor subtypes involved in these locations may lead to better understanding of cholinergic control of airway tone and mucus secretion and improve anticholinergic therapy of airway diseases. S162
SUMMARY Several subtypes of muscarinic receptors have been identified in airways and lung parenchyma of different species, including humans. M1 receptors may be present in parasympathetic ganglia where they appear to facilitate ganglionic neurotransmission. M2 receptors seem to be located on cholinergic nerve endings where they function as "autoreceptors," inhibiting acetylcholine release. M3 receptors appear to be located on effectory cells, smooth muscle, and mucus secreting glands. Experimental data in support of this view are presented and discrepant data are discussed. AM REV RESPIR DIS 1990; 141:S162-S165
Smooth Muscle Receptors In vitro, guinea pig (3), bovine (20), and dog (21) airway smooth muscle contractions to cholinergic agonists are blocked with high affinity by 4-DAMP and only with low affinity by PZ (table 1), classifying these receptors as the M3 subtype. Binding studies on bovine trachea (20, 22) and human airways (23) confirm a low affinity of muscarinic receptors for PZ and high affinity for 4-DAMP. According to these functional and binding data, a recent autoradiographic mapping of muscarinic receptors in guinea pig and human airways showed consistent M3 receptors on smooth muscle in all airways from trachea to peripheral bronchioles (24). By contrast, Roffel and coworkers (20) found heterogeneous binding with selective M2 antagonists AF-DX 116 and methoctramine in bovine trachea, suggesting the existence of both M2 and M3 receptors in bovine tracheal membranes. Functional studies, however, showed low affinity of tracheal smooth muscle for both M2 antagonists and high affinity for M3 antagonists. The significance of these discrepancies is still unclear. Peripheral Lung Membranes Binding studies on peripheral lung membranes in rabbit (25), rat (26),guinea pig (27), and humans (27-29) show a high proportion (25 to 70070) of receptors with high affinity for PZ (table 2). These results are difficult to interpret since membranes included in such homogenates belong to many different cell types (smooth muscles, mucus glands, ganglia, and pulmonary vessels). Therefore, it is not possible to conclude which cell membranes bear MI receptors. There is functional evidence that muscarinic receptors in parasympathetic ganglia belong to the MI subtype (30-32). Binding studies on isolated submucosal glands of swine trachea (33) showed that Ml receptors made up 27% of total muscarinic receptors. It is, however, unlikely that gland and ganglion receptors could account for as much as 50% of total lung muscarinic receptors. Recent autoradiographic mapping of muscarinic receptor subtypes in human airways
(24) showed MI receptors to be localized to submucosal glands and alveolar wallsbut not to airway smooth muscle. The role of MI receptors on alveoli is as yet unclear. Airway Submucosal Glands Little is known about the subtype of receptor involved in airway mucous and serous glands. Muscarinic receptors of salivary glands (2-4), lachrymal glands (3), and pancreatic acini (34) have been shown to belong to the M3 subtype (2). Binding studies on swine tracheal submucosal glands (33) have shown 27% of muscarinic receptors with a high affinity for PZ (MI), the remaining receptors having an affinity for PZ consistent with M3 receptors. In bovine tracheal mucosa, Madison and colleagues (22) found only receptors with a low affinity for PZ. In this study, no further characterization into M2 or M3 subtype wasmade. Autoradiographic studies have demonstrated the presence of Ml and M3 receptors on human bronchial submucosal glands (24), MI receptors accounting for approximately 30% of the receptors. Functional studies with selective antagonists in isolated cat trachea suggest a response intermediate between Ml and M3 receptors (35), supporting the idea that both receptor subtypes are involved in mucus secretion. Muscarinic Receptors in Parasympathetic Ganglia Historically, MI receptors were first demonstrated in brain and sympathetic ganglia (1-4). Pirenzepine is used clinically to reduce gastric acid secretion and it has been shown to act predominantly on parasympathetic ganI From the Department of Thoracic Medicine, National Heart and Lung Institute, London, United Kingdom. 2 Supported by Boehringer IngeIheim(Germany). J Correspondence and requests for reprints should be addressed to Prof. P. J. Barnes, Department of Thoracic Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom.
8163
MUSCARINIC RECEPTOR SUBTYPES
TABLE 2 MUSCARINIC RECEPTORS ON PERIPHERAL LUNG MEMBRANES PZ+
(0/0) Rabbit (23) Rat (24) Guinea-pig (25) Human (26) (27) (25)
Ganglion Cholinergic nerve
AGONIST
McN-A-343
ANTAGONIST
Airway Smooth muscle
Pilocarpine
Plrenzeplne
Gallamine
4-DAMP
AF-DX 116
Hexahydroslla-dlfenldol
"1ethoctramlne
78 50 25 70 62 43
PZ(%) 22 50 75 (= M3)* 30 38 (= M3)* 57 (= M3)*
Note: Binding studies on peripheral lung homogenates show a high proportion of receptors with high affinity for pirenzepine. Definitionof abbreviations: PZ + = receptors with high affinity for pirenzepine; PZ - = receptors with low affinity for pirenzepine; M3 = M3 SUbtype muscarinic receptor. • Use of 4-DAMP allowed classification of the receptors with low affinity for PZ as M3 receptors.
Fig. 1. Muscarinic receptor subtypes in airways. From Barnes and colleagues (5) with permission.
glia (36) by inhibiting neurally mediated gastric secretion. Since the innervation of the airways is derived embryologically from that of the gut, it seemslikelythat Ml receptors might exist in airway parasympathetic ganglia. Pirenzepine blocks bronchoconstriction due to vagus nerve stimulation in rabbit (30) and in dog (37)at much lowerdoses than those required to inhibit vagally induced decrease in heart rate, suggesting the existence of Ml receptors mediating bronchoconstriction. Since muscarinic receptors on airway smooth muscle have a low affinity for PZ (3, 20, 21), excitatory MI receptors must be located on the vagal pathway controlling airway caliber, most likely on parasympathetic ganglia. In support of this hypothesis, PZ has been shown to be more effectivein blocking pre- than postganglionic vagal nerve stimulation in rabbit airways in vitro (31). There is some evidence that excitatory Ml receptors are also present on human vagal pathways. Pirenzepine is a bronchodilator when given intravenously (38), but at doses that were not shown to be selective for MI receptors. Recently (32), PZ given by inhalation to atopic volunteers was shown to be as effective as inhaled ipratropium bromide in blocking the cholinergic reflex bronchocon-
striction triggered by exposure to sulfur dioxide. The same doses of PZ failed to displace the dose-response curves to inhaled methacholine in the same subjects, demonstrating that PZ inhibits selectively the cholinergicreflex mechanism, presumably at ganglionic level. The physiologic role of Ml receptors in autonomic ganglia remains speculative because ganglionic transmission is classically due to activation of nicotinic receptors. It is possible that MI receptors close potassium channels in ganglionic neurons, inducing slow depolarization and facilitating rapid firing on nicotinic activation. Confirmation of this hypothesis needs further investigation. Prejunctional Inhibitory Muscarinic Receptors Prejunctional muscarinic receptors, which inhibit the release of ACh from cholinergic nerves (autoreceptors), have been shown in airways in guinea pig (39) and cat (40) in vivo and in guinea pig (41) and dog (42) in vitro. Selective inhibition of these receptors by gallamine potentiates neurally mediated smooth muscle contractions (38- 41). Autoreceptors have also been demonstrated in human air-
TABLE 1 MUSCARINIC RECEPTORS ON AIRWAY SMOOTH MUSCLE CELLS Binding Studies
Functional Studies
Pirenzepine
4-DAMP
Type
pA2*
Canine bronchi Bovine trachea Guinea pig trachea Guinea pig trachea
6.8 6.9 7.1 6.7
Bovine trachea Guinea pig trachea Guinea pig trachea
9.0 9.0 8.7
Type
pKit
Reference
19 18 3 Personal communication
Bovine trachea Bovine trachea Human bronchi
6.3 6.4 5.8
19:1: 18 22:1:
18 3 Personal communication
Bovine trachea
8.0
18
Reference
Note: Functional and binding studies on airway smooth muscle show a low affinity of the receptors for pirenzepine and a high affinity for 4-DAM?, consistent with an "13 receptor. * pA2 .. -logarithm of the concentration of antagonist that inhibits 50% of the effect of the agonist. t pKi = -logarithm of the dissociation constant for the antagonist-receptor complex. pKi was not given by the authors but calculated from the Ki: pKj = -log (Kil.
*
ways in vitro (43) using pilocarpine as an autoreceptor-agonist to inhibit bronchial ring contractions induced by electric field stimulation. This effect was inhibited by gallamine in a dose-related fashion (figure 2), suggesting a muscarinic receptor-mediated mechanism. In guinea pig tracheal strips in vitro, contractions induced by electric field stimulation were found to be potentiated by AFOX 116 at doses that are selective for M2 receptors; no such potentiation was found with MI antagonist PZ (personal unpublished data, figure 3). Pilocarpine, which stimulates autoreceptors in vitro (39, 43), has an inhibitory effect on cholinergic reflex bronchoconstriction induced by inhalation of sulfur dioxide in healthy atopic volunteers (44), suggesting the existence of autoreceptors in vivo. These receptors presumably serveto limit neurally mediated bronchoconstriction. In asthmatic subjects, pilocarpine has no such inhibitory action, suggesting a possible dysfunction of the autoreceptor, which would result in exaggerated cholinergic reflex bronchoconstriction (44). Pulmonary Arteries Muscarinic receptors are also present on pulmonary arteries where they mediate vasodilatation, secondary to release of endothelial cell-derived relaxant factor (45). In vitro relaxation of precontracted rat pulmonry arteries by ACh is inhibited with high affinity by 4-0AMP and with low affinity by methoctramine and PZ, classifying these receptors as M3 (26). Clinical Relevance and Conclusions From available experimental data, it is possible to speculate about the role of each muscarinic receptor subtype (figure 3). Ml and M2 receptors appear mainly to control parasympathetic neurotransmission, Ml receptors facilitating ganglionic transmission and M2 receptors acting as negative feedback mechanisms at postganglionic level,while M3 receptors appear to activate target cells like
5164
MINETTE AND BARNES
110
100
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