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.

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

1. The novel thromboxane (TX) antagonist, BAY u3405, has been evaluated against bronchoconstriction induced by the TXA2 mimetic U-46619, prostaglandin D2 (PGD2), 5-hydroxytryptamine (5-HT), leukotriene D4 (LTD4) and histamine in the guinea-pig in vivo by use of a modification of the model described by Konzett & Rossler. 2. When given intravenously (i.v.) at 30 or 100 micrograms kg-1, U-46619 caused 80% maximal bronchoconstriction in most animals. In contrast, PGD2 caused a smaller 40%-50% maximal bronchoconstriction at the highest dose tested (300 micrograms kg-1, i.v.). 3. BAY u3405, given intravenously, orally (p.o.) or by aerosol antagonized U-46619-induced bronchoconstriction in a dose-related manner. The approximate ID50 values were 600 micrograms kg-1, i.v., 1.7 mg kg-1 p.o. and 0.1% w/v 20 breaths by aerosol. 4. BAY u3405 had similar inhibitory activities against U-46619-induced bronchoconstriction and hypertension suggesting that it had no preferential activity on the airways. 5. When given intravenously BAY u3405 antagonized the bronchoconstrictor effect of intravenous PGD2 with ID50 values between 30-100 micrograms kg-1. 6. The action of BAY u3405 (10 mg kg-1, p.o.) was long lasting, causing significant inhibition of U-46619-induced bronchoconstriction 7 h after dosing. 7. At 1 mg kg-1, i.v., a dose that abolished the response to U-46619 and PGD2, BAY u3405 had no effect on histamine-, 5-HT- or LTD4-induced bronchoconstriction. 8. BAY u3405 potently and selectively antagonized U-46619- or PGD2-induced bronchoconstriction in the Konzett-Rossler model of guinea-pig lung function. It should therefore prove to be a useful tool for defining the role of TXA2- and PGD2 in airway diseases such as asthma.

BAY u3405 an antagonist of thromboxane A2- and prostaglandin D2-induced bronchoconstriction in the guinea-pig.

Passive lung anaphylaxis (PLA) was investigated in rats sensitized by the intravenous injection of high titre reaginic antiserum prepared in rats. 2 The effect of various pharmacological antagonists on anaphylactic bronchoconstriction in vivo were examined. An antihistamine (mepyramine), a kallikrein inactivator (aprotinin) or a prostaglandin synthesis inhibitor (aspirin) did not inhibit PLA, whereas an anti-5-hydroxytryptamine agent (methysergide) and an anti-slow reacting substance-A agent (FPL 55712) significantly reduced the response. 3 Isolated perfused lungs taken from sensitized rats released, on challenge with the sensitizing antigen, histamine, 5-hydroxytryptamine, slow reacting substance of anaphylaxis (SRS-A) and prostaglandins, but no rabbit aorta contracting substance (RCS). 4 Disodium cromoglycate inhibited both anaphylactic bronchoconstriction in vivo and the anaphylactic release of mediators in vitro. Inhibition in vivo was dose-related. 5 Mediators from the intestine, the primary shock organ of anaphylaxis in the rat, did not contribute to the lung response. 6 Vagal reflex pathways were found not to be important in PLA in vivo. 7 The relationship between the mediators released following antigen challenge of passively sensitized rat lung in vitro and passive lung anaphylaxis in vivo is discussed.

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Mediators of passive lung anaphylaxis in the rat

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.

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

1. The guinea-pig trachea was isolated with its extrinsic innervation intact and placed in a water-jacketed dissecting dish containing warmed, oxygenated Krebs solution. The trachea was not separated from the oesophagus. Two adjacent cartilage rings of the rostral portion of the trachea were cut open opposite the trachealis and prepared for isometric tension measurements. 2. Following the addition of atropine and contraction of the trachealis with prostaglandin F2 alpha (PGF2 alpha), stimulation of the cervical sympathetic trunks elicited relaxations that were abolished by propranolol or hexamethonium. Stimulation of the vagus nerves caudal to the nodose ganglia also elicited relaxations. These vagally mediated relaxations were unaffected by propranolol but were abolished by hexamethonium or by cutting the recurrent laryngeal nerves. 3. After cutting the vagi caudal to the nodose ganglia, stimulation of the vagi rostral to the nodose ganglia elicited relaxations of the trachealis that were not significantly affected by either propranolol or hexamethonium but were abolished by cutting the superior laryngeal nerves. Stimulation of right vagi which had undergone supranodose vagotomy 14 days prior to experimentation was without effect on the smooth muscle of the guinea-pig trachea while the response to stimulation of the left vagus was unchanged. 4. Acute capsaicin desensitization abolished relaxations of the guinea-pig trachealis elicited by stimulation of the vagal fibres carried by the superior laryngeal nerves. In contrast, capsaicin desensitization only modestly inhibited relaxations elicited by stimulation of the preganglionic parasympathetic fibres carried by the recurrent laryngeal nerves and had no effect on sympathetic nerve-induced relaxations. 5. Removing the oesophagus selectively abolished relaxations elicited by stimulation of both vagal pathways of non-adrenergic relaxant innervation. Non-adrenergic relaxations of the trachealis elicited by electrical field stimulation were unaffected by removing the oesophagus. Oesophagus removal also had no effect on the parasympathetic-cholinergic contractile innervation or the sympathetic relaxant innervation of the trachealis. 6. The results indicate that the guinea-pig trachealis receives non-adrenergic relaxant innervation from both parasympathetic and capsaicin-sensitive vagal pathways. The results also suggest that the neurones mediating non-adrenergic relaxations of the trachea are sensitive to oesophagus removal. The observation that oesophagus removal abolishes parasympathetic relaxations of the trachealis while having no effect on parasympathetic contractions supports the hypothesis that the guinea-pig trachealis receives excitatory and inhibitory innervation from distinct vagal parasympathetic pathways.

Relaxant innervation of the guinea-pig trachealis: demonstration of capsaicin-sensitive and -insensitive vagal pathways.

Summary

1
Propranolol augmented the bronchoconstrictor response to methacholine or histamine, recorded by air overflow in the anaesthetized, vagotomized guinea-pig

2
After adrenalectomy, propranolol was still active, though less so than before

3
In the pithed guinea-pig, there was no augmentation of the effect of bronchoconstrictors on air overflow The action of propranolol could thus be due to the β-adrenoceptor blockade of compensatory sympathetic bronchodilator activity, as concluded by McCulloch, Proctor & Rand (1967)

4
Electrical stimulation of the thoracic region of the spinal cord of the pithed guinea-pig reduced the effect of bronchoconstrictors on air overflow This reduction could be blocked by propranolol; practolol was much less effective

M. W. Parkes

Papers

Effect of β-adrenoceptive blocking agents on the response to bronchoconstrictor drugs in the guinea-pig air overflow preparation