M2 Receptor Dysfunction Research Articles

Effects of tachykinin NK1 receptor antagonists on vagal hyperreactivity and neuronal M2 muscarinic receptor function in antigen challenged guinea-pigs.

1. The role of tachykinin NK1 receptors in the recruitment of eosinophils to airway nerves, loss of inhibitory neuronal M2 muscarinic receptor function and the development of vagal hyperreactivity was tested in antigen-challenged guinea-pigs. 2. In anaesthetized guinea-pigs, the muscarinic agonist, pilocarpine (1-100 microg kg(-1), i.v.), inhibited vagally induced bronchoconstriction, in control, but not in antigen-challenged guinea-pigs 24 h after antigen challenge. This indicates normal function of neuronal M2 muscarinic receptors in controls and loss of neuronal M2 receptor function in challenged guinea-pigs. Pretreatment of sensitized guinea-pigs with the NK1 receptor antagonists CP99994 (4 mg kg(-1), i.p.), SR140333 (1 mg kg(-1), s.c.) or CP96345 (15 mg kg(-1), i.p.) before antigen challenge, prevented M2 receptor dysfunction. 3. Neither administration of the NK1 antagonists after antigen challenge, nor pretreatment with an NK2 receptor antagonist, MEN10376 (5 micromol kg(-1), i.p.), before antigen challenge, prevented M2 receptor dysfunction. 4. Electrical stimulation of the vagus nerves caused a frequency-dependent (2-15 Hz, 10 V, 0.2 ms for 5 s) bronchoconstriction that was significantly increased following antigen challenge. Pretreatment with the NK1 receptor antagonists CP99994 or SR140333 before challenge prevented this increase. 5. Histamine (1-20 nmol kg(-1), i.v.) caused a dose-dependent bronchoconstriction, which was vagally mediated, and was significantly increased in antigen challenged guinea-pigs compared to controls. Pretreatment of sensitized animals with CP99994 before challenge prevented the increase in histamine-induced reactivity. 6. Bronchoalveolar lavage and histological studies showed that after antigen challenge significant numbers of eosinophils accumulated in the airways and around airway nerves. This eosinophilia was not altered by pretreatment with the NK1 receptor antagonist CP99994. 7. These data indicate that pretreatment of antigen-sensitized guinea-pigs with NK1, but not with NK2 receptor antagonists before antigen challenge prevented the development of hyperreactivity by protecting neuronal M2 receptor function. NK1 receptor antagonists do not inhibit eosinophil accumulation around airway nerves.

Antibody to VLA-4, but not to L-selectin, protects neuronal M2 muscarinic receptors in antigen-challenged guinea pig airways.

Antigen challenge of sensitized guinea pigs decreases the function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lung, potentiating vagally induced bronchoconstriction. Loss of M2 receptor function is associated with the accumulation of eosinophils around airway nerves. To determine whether recruitment of eosinophils via expression of VLA-4 and L-selectin is critical for loss of M2 receptor function, guinea pigs were pretreated with monoclonal antibodies to VLA-4 (HP1/2) or L-selectin (LAM1-116). Guinea pigs were sensitized and challenged with ovalbumin, and M2 receptor function was tested. In controls, blockade of neuronal M2 muscarinic receptors by gallamine potentiated vagally induced bronchoconstriction, while in challenged animals this effect was markedly reduced, confirming M2 receptor dysfunction. Pretreatment with HP1/2, but not with LAM1-116, protected M2 receptor function in the antigen-challenged animals. HP1/2 also inhibited the development of hyperresponsiveness, and selectively inhibited accumulation of eosinophils in the lungs as measured by lavage and histology. Thus, inhibition of eosinophil influx into the lungs protects the function of M2 muscarinic receptors, and in so doing, prevents hyperresponsiveness in antigen-challenged guinea pigs.

Allergen challenge of passively sensitized human bronchi alters M2 and beta2 receptor function.

We investigated whether antigen challenge may alter M2 and beta2 receptor function in isolated passively sensitized human bronchi. Bronchial rings (2-4 mm internal diameter) were obtained from 12 patients. Passive sensitization was induced by serum containing high IgE levels to Dermatophagoides pteronyssinus and Dermatophagoides farinae. Rings from six patients were used to study M2 receptor function by incubating with cumulatively increasing concentrations (10(-8) M to 10(-4) M) of pilocarpine and applying electric field stimulation (EFS) at 24 Hz. The rings from the other six patients were used to study beta2 receptor function by precontracting with carbachol 10(-6) M and adding cumulatively salbutamol (10(-9) M to 10(-4) M). Additional rings from these patients were used to determine whether dysfunction occurred distal to cAMP production by precontracting with carbachol 10(-6) M and adding cumulatively theophylline (10(-9) M to 10(-4) M). The attenuation of EFS-induced force by pilocarpine and the relaxation of carbachol-precontracted rings by salbutamol were less in challenged than in control and sensitized rings. No difference between challenged, sensitized, and control rings was observed with theophylline. We conclude that allergen challenge in passively sensitized isolated human bronchial rings may result in M2 and beta2 receptor dysfunction without involving mechanisms distal to cAMP formation. It appears that products from inflammatory cells recruited from blood are not necessary for this receptor dysfunction.

Pretreatment with an antibody to interleukin-5 prevents loss of pulmonary M2 muscarinic receptor function in antigen-challenged guinea pigs.

Inhalational challenge with antigen decreases the function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lung, increasing the release of acetylcholine from the vagus nerves and potentiating vagally induced bronchoconstriction. It is possible that eosinophils cause M2 receptor dysfunction, perhaps by releasing positively charged proteins that are M2 receptor antagonists. Because of the probable role of interleukin-5 in initiating and maintaining the eosinophil infiltration, we tested the function of neuronal M2 receptors in antigen-challenged guinea pigs after pretreatment with a monoclonal antibody to interleukin-5 (TRFK-5). Ovalbumin was given intraperitoneally to sensitize the animals. Three weeks later, the animals were injected intraperitoneally with either TRFK-5 (240 micrograms/kg i.p.) or saline. Beginning three days later, they were challenged with an ovalbumin aerosol for 5 min on each of four consecutive days. M2 receptor function was tested 24 h after the last antigen challenge. Electrical stimulation of both vagi caused bronchoconstriction and bradycardia. In control animals, pilocarpine attenuated, and gallamine potentiated, vagally induced bronchoconstriction by stimulating and blocking neuronal M2 muscarinic receptors, respectively. In challenged animals that did not receive TRFK-5, these effects were markedly reduced, confirming M2 receptor dysfunction. In TRFK-5-treated guinea pigs, the effects of both pilocarpine and gallamine were the same as those in control animals, demonstrating normal M2 receptor function. Pretreatment with TRFK-5 selectively inhibited the migration of eosinophils into the lungs as measured by lung lavage. Thus the function of M2 muscarinic receptors in antigen-challenged guinea pigs can be protected by inhibiting eosinophil influx into the lungs.

ＯＡ感作ぜん息モルモットにおけるムスカリン性アセチルコリン受容体Ｓｕｂｔｙｐｅの検討

We examined the correlation between airway hyperreactivity and the density of muscarinic acetylcholine receptor (mACh-R), and reported that the density of mACh-R in OA-sensitized guinea pig was higher than that in a control group. We also examined the characteristics of the M1, M2 and M3 receptors in guinea pig pulmonary tissue, and the following results were obtained.1) For the M1 and M2 receptors, no difference was observed between the density and affinity of OA-sensitized lungs and those of the control lungs.2) The density of M3 receptors in the lung tissue of OA-sensitized guinea pigs washigher than that in the control group. No difference was observed between the affinity of M3 receptors in the OA-sensitized lungs and that in the control lungs.3) Pilocarpine, which is an M2 receptor agonist, increased the density of M3 receptors in the lung tissue of guinea pigs, and the rate of the increase in the density of M3 receptors in OA-sensitized guinea pigs was less than that in the control group. On the other hand, methoctramine, which is an M2 receptor antagonist, decreased the density of M3 receptors in the lung tissue of guinea pigs, and the rate of this decrease was as same as that in the control group.These results suggest that the increase in M3 receptors in the lung tissue of OA-sensitized guinea pigs is related to airway hyperreactivity and that there is a dysfunction of M2 receptors in OA-sensitized guinea pigs.

Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.

The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of MBP with the M2 muscarinic receptor is allosteric. This effect of MBP suggests that it may function as an endogenous allosteric inhibitor of agonist binding to the M2 muscarinic receptor. Inhibition of [3H]NMS binding by MBP was reversible by treatment with heparin, which binds and neutralizes MBP. Eosinophil peroxidase (EPO) also inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors and inhibited atropine-induced dissociation of [3H]NMS-receptor complexes. On a molar basis, EPO is less potent than MBP. Neither eosinophil cationic protein nor eosinophil-derived neurotoxin affected binding of [3H]NMS to M2 receptors. Thus both MBP and EPO are selective allosteric antagonists at M2 receptors. The effects of these proteins may be important causes of M2 receptor dysfunction and enhanced vagally mediated bronchoconstriction in asthma.

Function of pulmonary M2 muscarinic receptors in antigen-challenged guinea pigs is restored by heparin and poly-L-glutamate.

The effect of heparin and poly-L-glutamate on the function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lung was tested in antigen-challenged guinea pigs. After antigen challenge, M2 receptor function is decreased, thus increasing release of acetylcholine from the vagus and potentiating vagally induced bronchoconstriction. Guinea pigs were anesthetized, tracheostomized, vagotomized, paralyzed, and ventilated. Electrical stimulation of the vagi caused bronchoconstriction and bradycardia. In controls, pilocarpine attenuated vagally induced bronchoconstriction by stimulating neuronal M2 muscarinic receptors. Conversely, blocking these autoreceptors with gallamine potentiated vagally induced bronchoconstriction. In challenged animals the effects of both drugs were markedly reduced, confirming M2 receptor dysfunction. 20 min after heparin or poly-L-glutamate, the effects of both pilocarpine and gallamine on vagally induced bronchoconstriction were restored, demonstrating recovery of M2 receptor function. Neither heparin nor poly-L-glutamate affected vagally induced responses in control animals. Thus antigen-induced dysfunction of M2 receptors can be reversed by polyanionic polysaccharides (heparin) or polyanionic peptides (poly-L-glutamate). This suggests that a polycationic substance such as eosinophil major basic protein, cationic protein, or peroxidase may be responsible for antigen-induced pulmonary M2 receptor dysfunction.