M2 Receptor Function Research Articles

The effect of allosteric antagonists in modulating muscarinic M2-receptor function in guinea-pig isolated trachea.

1. We have assessed the influence of a range of synthetic cationic polypeptides with putative inhibitory actions at prejunctional muscarinic M2-receptors on electrical field stimulation-induced contraction of guinea-pig isolated tracheal preparations. Electrical field stimulation of epithelium-denuded guinea-pig trachea resulted in frequency-dependent contractile responses. As expected, tracheal smooth muscle sensitivity to electrical field stimulation was increased in tissues pretreated with the muscarinic M2-receptor antagonist, gallamine. In contrast, gallamine did not significantly alter the contractile potency to acetylcholine. 2. Unlike gallamine, the synthetic cationic polypeptides, poly-L-arginine, poly-L-lysine, poly-D-lysine, the cationic dye ruthenium red and the anionic polysaccharide, heparin, failed to increase significantly tracheal smooth muscle sensitivity to electrical field stimulation. 3. Poly-L-arginine, ruthenium red and heparin had no effect on the contractile response to exogenously applied methacholine. 4. These data are consistent with the concept that in guinea-pig tracheal smooth muscle, gallamine is an allosteric antagonist of guinea-pig tracheal muscarinic M2-receptors, whereas the various cationic polypeptides and the polyanion, heparin, are not.

The effect of leukocyte depletion on pulmonary M2 muscarinic receptor function in parainfluenza virus‐infected guinea‐pigs

1. Parainfluenza infections of the airways cause dysfunction of inhibitory M2 muscarinic receptors on the pulmonary parasympathetic nerves. To distinguish the direct effects of virus from the effects of virus-induced airway inflammation on M2 muscarinic receptor function, guinea-pigs were depleted of leukocytes by pretreating with cyclophosphamide (30 mg kg-1, i.p. daily for 7 days) after which they were infected with parainfluenza virus type 1 (Sendai virus). 2. Guinea-pigs were anaesthetized, tracheotomized, and ventilated. The vagus nerves were isolated and cut, and the distal ends were electrically stimulated causing bronchoconstriction. In control animals, pilocarpine (1-100 micrograms kg-1, i.v.) inhibited and gallamine (0.1-10 mg kg-1, i.v.) potentiated vagally-induced bronchoconstriction by stimulating or blocking M2 muscarinic receptors on the vagus. These effects of pilocarpine and gallamine were almost completely lost in virus-infected animals, demonstrating loss of M2 receptor function. 3. Cyclophosphamide depleted peripheral blood leukocytes and inhibited the virus-induced influx of inflammatory cells into the lung. Depletion of leukocytes protected M2 receptor function from viral infection in some, but not all, guinea-pigs tested. 4. Among infected animals that had been depleted of leukocytes, the viral content (expressed as the log of the number of tissue culture infectious doses per g lung tissue) of those that retained normal M2 receptor function was 4.29 +/- 0.05 (mean +/- s.e. mean), while the viral content of those that lost M2 receptor function despite leukocyte depletion was 5.45 +/- 0.20 (P = 0.011). Thus the viral content of the lungs in which M2 receptor function was lost was 16 times greater than that of the lungs in which M2 receptor function was preserved. Viral content correlated with the inhibition of vagally-mediated bronchoconstriction after the maximum dose of pilocarpine (100 Microg kg-1; r2 = 0.81, P =0.0004).5. In antigen-challenged animals, inhibitory M2 muscarinic receptor function is restored when positively charged inflammatory cell proteins are bound and neutralized by heparin. However, heparin(2000 micro kg-1, i.v.) did not reverse virus-induced loss of M2 muscarinic receptor function, even in those guinea-pigs with a lower viral titer.6. Because leukocyte depletion protected M2 muscarinic receptor function only in animals with mild viral infections, it appears that viruses have both an indirect, leukocyte-dependent effect on M2 receptors and, in animals with more severe infections, a leukocyte-independent effect on M2 receptors. The failure of heparin to restore M2 receptor function demonstrates that the leukocyte-dependent loss of M2 receptor function is not mediated by positively charged inflammatory cell proteins.

Neuronal M2Muscarinic Receptor Function in Guinea-Pig Lungs Is Inhibited by Indomethacin

The function of M2 muscarinic autoreceptors on pulmonary parasympathetic nerves was investigated in the absence and presence of cyclooxygenase inhibitors in vivo. Guinea pigs were anesthetized, paralyzed, and artificially ventilated. Pulmonary inflation pressure, heart rate, and blood pressure were recorded. Electrical stimulation of vagus nerves produced bronchoconstriction (measured as an increase in pulmonary inflation pressure) and bradycardia. In control guinea pigs, pilocarpine (1 to 100 micrograms/kg) given intravenously stimulated inhibitory M2 muscarinic receptors on pulmonary parasympathetic nerves, thus attenuating vagally induced bronchoconstriction. Conversely, blockade of these autoreceptors by the selective M2 antagonist gallamine (0.1 to 10 mg/kg given intravenously) potentiated vagally induced bronchoconstriction. Separate groups of animals were given either indomethacin or naproxen. These cyclooxygenase inhibitors potentiated vagally induced bronchoconstriction. Furthermore, in those animals pretreated with either indomethacin or [+] naproxen, pilocarpine did not inhibit and gallamine did not potentiate vagally induced bronchoconstriction. In the heart, the effects of pilocarpine and gallamine on M2 muscarinic receptors were not altered by either cyclooxygenase inhibitor. Neither intravenously administered indomethacin (1 mg/kg) nor [+] naproxen (5 mg/kg) altered baseline pulmonary inflation pressure or baseline heart rate in the treated guinea pigs. These studies demonstrate that inhibitory M2 muscarinic receptors on pulmonary parasympathetic nerves do not function in the presence of cyclooxygenase inhibitors. Loss of M2 receptor function may contribute to aspirin-induced airway hyperresponsiveness.

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.

Differential effects of amitriptyline on sudomotor, cardiovagal, and adrenergic function in human subjects.

Tricyclic antidepressants, especially amitriptyline (AMI), are widely used by patients who require tests of autonomic function. AMI effects autonomic function, but its quantitative and differential effects are not known. We prospectively evaluated the effect of AMI on sudomotor (M3 receptor), cardiovagal (M2) and adrenergic functions in 6 subjects, aged 20-40 years before, during, and 48 hours after AMI 75 mg/d. M3 receptor function was evaluated using QSART (quantitative sudomotor axon reflex test) recordings from four sites, M2 receptor function from cardiovagal studies [HR response to deep breathing (HRDB) and the Valsalva ratio (VR)], and adrenergic function from an analysis of beat-to-beat BP responses to tilt and the Valsalva maneuver. Plasma AMI was determined from blood samples obtained within 2 hours of autonomic testing. QSART volume was reduced by 47% by AMI (P = 0.01), and recovered to 81% of baseline following a 48-hour washout. HRDB was unaffected by AMI (P > 0.05). VR was increased by 10% (NS) with AMI and by 14% (P < 0.05) with washout. The percent change in hemodynamic parameters by AMI were: orthostatic reduction in SBP, 62% (NS); orthostatic HR increment, 66%, (P = 0.03); and phase IIe SBP decrement, 665%, (P = 0.02). We conclude that AMI in the moderate doses used, resulted in greater inhibition of M3 than M2 receptors. The BP, HR, and beat-to-beat BP alterations likely reflect adrenergic inhibition resulting in a reduction in effective plasma volume. A washout of 48 hours is adequate for muscarinic but not adrenoreceptors.

Parainfluenza virus infection damages inhibitory M2 muscarinic receptors on pulmonary parasympathetic nerves in the guinea-pig.

1. The effect of viral infection on the function of neuronal M2 muscarinic autoreceptors in the lungs was studied in anaesthetized guinea-pigs. 2. Guinea-pigs were inoculated intranasally with either parainfluenza type 3 or with a vehicle control. Four days later the animals were anaesthetized, paralysed and artificially ventilated. Pulmonary inflation pressure, tidal volume, blood pressure, and heart rate were recorded. Both vagus nerves were cut and electrical stimulation of the distal portions caused bronchoconstriction (measured as an increase in pulmonary inflation pressure) and bradycardia. 3. In control animals, pilocarpine (1-100 micrograms kg-1, i.v.) attenuated vagally-induced bronchoconstriction by stimulating inhibitory M2 muscarinic receptors on parasympathetic nerves in the lungs. Conversely, blockade of these receptors with the antagonist gallamine (0.1-10 mg kg-1, i.v.) produced a marked potentiation of vagally-induced bronchoconstriction. These results confirm previous findings. 4. In guinea-pigs infected with parainfluenza virus, pilocarpine did not inhibit vagally-induced bronchoconstriction. Furthermore, gallamine did not potentiate vagally-induced bronchoconstriction to the same degree as in uninfected controls. 5. There was no increase in baseline pulmonary inflation pressure in the infected animals over the controls. Receptors on airway smooth muscle were unchanged by viral infection since large doses of pilocarpine caused equivalent bronchoconstriction in both groups of animals. Gallamine inhibited the vagally-induced fall in heart rate equally in both groups of animals indicating that virus-induced changes in M2 receptor function on pulmonary parasympathetic nerves are not part of a generalized decrease in M2 receptor function. 6. These results demonstrate that the M2 muscarinic receptor-mediated inhibition of acetylcholine release from parasympathetic nerves in the lungs is decreased in animals infected with parainfluenza virus. Loss of this inhibition would result in increased release of acetylcholine from the parasympathetic nerves and may explain virus-induced airway hyperresponsiveness.

Chronic β1‐adrenoceptor antagonist treatment sensitizes β2‐adrenoceptors, but desensitizes M2‐muscarinic receptors in the human right atrium

1. In 64 patients undergoing coronary artery bypass grafting the effects of chronic beta 1-adrenoceptor antagonist (metoprolol, atenolol, bisoprolol) treatment on right atrial beta-adrenoceptor and muscarinic M2-receptor number and functional responsiveness were investigated. 2. The beta 1-adrenoceptor antagonists increased right atrial beta 1-adrenoceptor number, did not affect beta 2-adrenoceptor number, and decreased muscarinic M2-receptor number. 3. Concomitantly, activation of right atrial adenylate cyclase by 10 microM GTP, 10 microM isoprenaline and 1 microM forskolin was enhanced and inhibition by 100 microM carbachol was diminished. 4. On isolated, electrically driven right atria the beta 1-adrenoceptor-mediated positive inotropic effect of noradrenaline was - even with beta 1-adrenoceptor number increased - not altered, while the beta 2-adrenoceptor-mediated effect of procaterol was markedly enhanced. However, the carbachol-induced negative inotropic effect was decreased. 5. It is concluded that chronic beta 1-adrenoceptor antagonist treatment increases beta 1-adrenoceptor number and concomitantly sensitizes beta 2-adrenoceptor function, but desensitizes muscarinic M2-receptor function in the human heart.

Differential regulation of human cardiac beta‐adrenergic and muscarinic receptors by chronic beta‐adrenoceptor antagonist treatment.

In patients undergoing coronary artery bypass grafting chronic beta 1-adrenoceptor antagonist treatment increased right atrial beta 1-adrenoceptor number, did not affect beta 2-adrenoceptor number and decreased muscarinic M2-receptor number. Concomitantly, the M2-receptor-mediated negative inotropic effect of carbachol was reduced, while the beta 1-adrenoceptor-mediated positive inotropic effect of noradrenaline was not altered. The beta 2- adrenoceptor mediated positive inotropic effect of procaterol, however, was markedly enhanced. We conclude that chronic beta 1-adrenoceptor antagonist treatment increases beta 1-adrenoceptor number, sensitizes beta 2-adrenoceptor function and desensitizes M2-receptor function in the human heart.