Developmental expression of muscarinic acetylcholine receptors in chick retina: selective induction of M2 muscarinic receptor expression in ovo by a factor secreted by muller glial cells.

Muscarinic acetylcholine receptors (mAChRs) play an important role in signal processing in the retina. We have used subtype-specific antibodies to identify the changes in the localization of mAChR expression during embryonic development of the retina in vivo and their relationship to the changes in mAChRs in retinal cells in culture. We have demonstrated previously that treatment of fresh retinal cultures with conditioned media from mature retinal cultures specifically induces expression of the M(2) mAChR (McKinnon et al., 1998). We show that the M(2)-inducing activity, which we tentatively have called MARIA (muscarinic acetylcholine receptor-inducing activity) is produced by Müller glial cells in culture, because significant activity can be found in media conditioned by essentially neuron-free cultures of Müller glia, as well as by a Müller glial cell line but not several neuroblastoma cell lines. We also demonstrate that the appearance of the M(2) receptor in vivo occurs concomitantly with the appearance of significant numbers of Müller glial cells in the developing retina. Furthermore, the administration of crude or partially purified preparations of MARIA to developing chick embryos in ovo induces precocious expression of M(2) mAChRs in the appropriate cell types in the retina. These results show that a factor secreted by cultured retinal Müller glia can regulate M(2) mAChR expression in vivo and in vitro and suggest that the secretion of MARIA by Müller glia in vivo may be responsible for the normal induction of M(2) mAChR expression during embryonic development.

Substance P-induced airway hyperreactivity is mediated by neuronal M(2) receptor dysfunction.

Neuronal muscarinic (M(2)) receptors inhibit release of acetylcholine from the vagus nerves. Hyperreactivity in antigen-challenged guinea pigs is due to blockade of these M(2) autoreceptors by eosinophil major basic protein (MBP) increasing the release of acetylcholine. In vivo, substance P-induced hyperactivity is vagally mediated. Because substance P induces eosinophil degranulation, we tested whether substance P-induced hyperreactivity is mediated by release of MBP and neuronal M(2) receptor dysfunction. Pathogen-free guinea pigs were anesthetized and ventilated. Thirty minutes after intravenous administration of [Sar(9),Met(O(2))(11)]- substance P, guinea pigs were hyperreactive to vagal stimulation and M(2) receptors were dysfunctional. The depletion of inflammatory cells with cyclophosphamide or the administration of an MBP antibody or a neurokinin-1 (NK(1)) receptor antagonist (SR-140333) all prevented substance P-induced M(2) dysfunction and hyperreactivity. Intravenous heparin acutely reversed M(2) receptor dysfunction and hyperreactivity. Thus substance P releases MBP from eosinophils resident in the lungs by stimulating NK(1) receptors. Substance P-induced hyperreactivity is mediated by blockade of inhibitory neuronal M(2) receptors by MBP, resulting in increased release of acetylcholine.

Antigen-induced hyperreactivity to histamine: role of the vagus nerves and eosinophils.

M2 muscarinic receptors limit acetylcholine release from the pulmonary parasympathetic nerves. M2 receptors are dysfunctional in antigen-challenged guinea pigs, causing increased vagally mediated bronchoconstriction. Dysfunction of these M2 receptors is due to eosinophil major basic protein, which is an antagonist for M2 receptors. Histamine-induced bronchoconstriction is composed of a vagal reflex in addition to its direct effect on airway smooth muscle. Because hyperreactivity to histamine is seen in antigen-challenged animals, we hypothesized that hyperreactivity to histamine may be due to increased vagally mediated bronchoconstriction caused by dysfunction of M2 receptors. In anesthetized, antigen-challenged guinea pigs, histamine-induced bronchoconstriction was greater than that in control guinea pigs. After vagotomy or atropine treatment, the response to histamine in antigen-challenged animals was the same as that in control animals. In antigen-challenged animals, blockade of eosinophil influx into the airways or neutralization of eosinophil major basic protein prevented the development of hyperreactivity to histamine. Thus hyperreactivity to histamine in antigen-challenged guinea pigs is vagally mediated and dependent on eosinophil major basic protein.

Role of insulin in antigen-induced airway eosinophilia and neuronal M2 muscarinic receptor dysfunction.

In the lungs, neuronal M2 muscarinic receptors limit ACh release from parasympathetic nerves. In antigen-challenged animals, eosinophil proteins block these receptors, resulting in increased ACh release and vagally mediated hyperresponsiveness. In contrast, diabetic rats are hyporesponsive and have increased M2 receptor function. Because there is a low incidence of asthma among diabetic patients, we investigated whether diabetes protects neuronal M2 receptor function in antigen-challenged rats. Antigen challenge of sensitized rats decreased M2 receptor function, increased vagally mediated hyperreactivity by 75%, and caused a 10-fold increase in eosinophil accumulation around airway nerves. In antigen-challenged diabetic rats, neuronal M2 receptor function was preserved and there was no eosinophil accumulation around airway nerves. Insulin treatment of diabetic rats completely restored loss of M2 receptor function, vagally mediated hyperresponsiveness, and eosinophilia after antigen challenge. These data demonstrate that insulin is required for development of airway inflammation, loss of neuronal M2 muscarinic receptor function, and subsequent hyperresponsiveness in antigen-challenged rats and may explain decreased incidence of asthma among diabetic humans.

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.

Increased function of inhibitory neuronal M2 muscarinic receptors in diabetic rat lungs.

1. The function of inhibitory neuronal M2 muscarinic receptors in diabetic rat lungs was investigated. 2. Neuronal M2 muscarinic receptors inhibit acetylcholine release from parasympathetic nerves. Thus, stimulation of neuronal M2 muscarinic receptors with muscarinic agonists, such as pilocarpine, inhibits acetylcholine release and vagally induced bronchoconstriction. In contrast, blockade of neuronal M2 muscarinic receptors with selective M2 muscarinic antagonists, such as AF-DX 116, potentiates acetylcholine release and vagally induced bronchoconstriction. 3. Rats were made diabetic by streptozotocin (65 mg kg (-1), i.v.). After 7 14 days the rats were anaesthetized with urethane (1.5 g kg (-1), i.p.), tracheostomized, vagotomized, ventilated and paralysed with suxamethonium (30 mg kg (-1), i.v.). Some 7 day diabetic rats were treated with low doses of long acting (NPH) insulin (2 units day (-1), s.c.) for 7 days before experimentation. This dose of insulin was not sufficient to restore normoglycaemia in diabetic rats. Thus, insulin-treated diabetic rats remained hyperglycaemic. 4. Distal electrical stimulation (5 70 Hz, 6 s, 40 V, 0.4 ms) of the vagi caused bronchoconstriction, measured as an increase in inflation pressure and bradycardia. In diabetic rats, vagally induced bronchoconstriction was significantly depressed vs controls. In contrast, bronchoconstriction caused by i.v. acetylcholine was similar in diabetic and control animals. 5. The function of neuronal M2 muscarinic receptors was tested with the muscarinic agonist pilocarpine (0.001-100.0 microg kg (-1), i.v.) and the antagonist AF-DX 116 (0.01-3.0 mg kg (-1), i.v.). Pilocarpine inhibited vagally-induced bronchoconstriction (30 Hz, 20-40 V, 0.4 ms at 6 s) and AF-DX 116 potentiated vagally-induced bronchoconstriction (20 Hz, 20-40 V, 0.4 ms at 6 s) to a significantly greater degree in diabetic rats compared to controls. 6. Both frequency-dependent vagally-induced bronchoconstriction and M2 muscarinic receptor function could be restored to nearly control values in diabetic rats treated with low doses of insulin. 7. Displacement of [3H]QNB (1 nM) with the agonist carbachol (10.0 nM-10.0 mM) from diabetic cardiac M2 muscarinic receptors revealed a half log increase in agonist binding affinity at both the high and low affinity binding sites vs controls. In contrast, M2 receptors from insulin-treated diabetic rat hearts showed no significant difference in binding affinity vs controls. 8. These data show that neuronal M2 muscarinic receptors in the lungs have increased function in diabetic rats, suggesting that insulin modulates M2 muscarinic receptor function.