INTRODUCTION

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It is generally accepted that mast cells are involved in the pathogenesis of obstructive airway disease (1). The mast cells are located in the airway mucosa close to the basement membrane and surface epithelium (2, 4, 5), i.e., placed in a strategically prominent location to become activated by inhaled antigens and other agents. We have recently demonstrated a negative link between airway mucosal mast cells and the cholinergic system in an atropine-sensitive manner (6). Low concentrations of applied acetylcholine inhibited A23187-stimulated and anti-IgE antibody-stimulated histamine release. Because non-neuronal acetylcholine is ubiquitously present within the airway mucosa (7, 8), an inhibitory muscarinic pathway may exist under in vivo conditions to limit mast cell releasability.

Muscarinic receptors represent a heterogeneous population (9). Therefore, one aim of the present study was to characterize the muscarinic receptor subtype by testing subtype-specific antagonists. In the present experiments, affinity constant (pA₂) values were not established, because of the complex pattern of the inhibitory dose-response curve for both agonists, acetylcholine and oxotremorine (6). However, the relative antagonistic potencies of subtype-specific antagonists were evaluated, to obtain more information about the receptor subtype inhibiting histamine release. Pirenzepine binds with the highest affinity to the M1-receptor protein; negative log inhibition constant (K_i) values (corresponding to pA₂) at human cloned M1-, M2-, M3-, and M4-receptors are 8.2, 6.6, 6.8, and 7.4, respectively (10). Para-fluorohexahydrosiladifendiol (pFHHSiD) shows similar affinities (log K_i or pA₂ values) at the human M1-, M3-, and M4-receptor protein (7.6- 7.9, 7.6-7.8, and 7.5, respectively; 10, 11). AF-DX 116 binds preferentially to the M2-receptor protein. The pA₂ values at mammalian, including human, M1-, M2-, and M3-receptors are 6.3, 7.3, and 6.3; a log K_i value of 6.08 has been reported for human M1-receptors (12). Clozapine binds to a number of neurotransmitter receptors, including muscarinic receptors. It has been reported that clozapine acts as agonist at human cloned M4-receptors, but as antagonist at human cloned M1-, M2-, and M3-receptors (15, 16).

The observation of an inhibitory muscarinic link in human airways contrasts with findings of a facilitatory effect of acetylcholine on mast cells in the gastrointestinal tract or glandular tissue (17). This discrepancy can be explained by the heterogeneity of mast cell populations (6, 18), but species differences may also exist. A second aim of the present study was to investigate whether a similar muscarinic pathway inhibits the histamine release in the rat trachea.

	METHODS

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Tissue Preparation

The protocol for obtaining human tissue was approved by the local ethical review board for human studies (Landesärztekammer Rheinland-Pfalz, Germany). Human tissue was obtained at surgery of patients with lung cancer. Bronchi of patients without a history of chronic airway disease were used. Immediately after lobectomy, tumor-free bronchi were dissected down to a diameter of 4 mm, carefully cleaned from adhering parenchyma, and placed in oxygenated, ice-cold salt solution (composition in mmol/L: 125 NaCl, 23.8 NaHCO₃, 5.05 glucose, 2.68 KCl, 1.80 CaCl₂, 1.04 MgCl₂, 0.54 NaH₂PO₄, 0.057 ascorbic acid, and 0.001 choline chloride). Segmental-subsegmental bronchi were washed several times in the oxygenated salt solution, and cut into pieces 1.5 to 2 cm in length weighing 80 to 170 mg. Bronchi were set up vertically in a 2-ml organ bath under a tension of 1 g. Sprague-Dawley rats (200 to 250 g) of either sex were maintained under standard laboratory conditions on 12-h day/night cycles with ad libitum access to food and water. Rats were stunned by a blow to the neck and bled, the tracheae weighing 40 tp 60 mg removed and placed in organ baths as described for human bronchi.

Superfusion, Incubation, and Stimulation Protocol

Protocol details have been described previously (6). In brief, isolated airways were at first superfused (2 ml/min; 30 min) with the oxygenated salt solution warmed to a constant temperature of 36° C. Thereafter, superfusion was stopped and airways were incubated in 1.1 ml of the salt solution which was exchanged in 5-min periods. The first three incubates were discarded and the subsequent incubates (time zero; Figure 1) collected for measuring histamine content. In each individual experiment 12 samples were collected. The first three samples represent the spontaneous histamine release collected in the absence of muscarinic receptor agonists (acetylcholine, oxotremorine), which were added from the sixteenth minute of incubation and remained. In interaction experiments, the muscarinic receptor antagonists were added from the start of the incubation (time zero) and remained.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Control experiments and the effect of acetylcholine (ACh) alone or in the presence of pirenzepine (PIR) on the histamine release from human, isolated bronchi. Human isolated bronchi were incubated in physiological salt solution and the medium was exchanged in 5-min intervals. The first six samples represent basal histamine release. The calcium ionophore A23187 was added at the 35th minute by a bolus injection given a final bath concentration of 10 µmol/L and washed out 1 min later with the subsequent medium exchange; filled columns indicate the presence of ACh. Histamine release is expressed as percent of the control release (16th to 30th minute; means ± SEM). (A) Histamine release in the absence of test substances (control release: 0.6 ± 0.1 nmol/g × 5 min; stimulated release 270 ± 48%, n = 7). (B) Histamine release in the presence of 100 pmol/L ACh present from the 16th minute of incubation onward (control release: 1.4 ± 0.2 nmol/g × 5 min; stimulated increase above control release: 36 ± 5%, n = 7). (C) Histamine release in the presence of 100 nmol/L PIR (control release: 0.6 ± 0.1 nmol/g × 5 min; stimulated increase above control release: 340 ± 100%, n = 6). (D) Histamine release in the presence of 100 nmol/L PIR present from zero and 100 pmol/L ACh present from the 16th minute of incubation onward (control release: 0.5 ± 0.1 nmol/g × 5 min; stimulated increase above control release: 280 ± 70%, n = 6).

Cells were stimulated by the calcium ionophore A23187 added by a 50-µl bolus injection at the 35th minute of incubation (final bath concentration 10 µmol/L for human bronchi and 1, 3, and 10 µmol/L for rat trachea) and washed out 1 min later with the subsequent bath exchange. At the end of the incubation period bronchi were dried and weighed.

Analytical Procedure, Calculations, and Statistical Analysis

Histamine content of the medium was determined after derivation with o-phthaldialdehyde by high-performance liquid chromatography (HPLC) and fluorometric detection. For a detailed description of the analytical procedure, see Reference 6. Histamine content of the incubation medium was normalized to 1 g airway. The mean histamine content of three prestimulation periods (16th to 30th minute of incubation) was regarded as 100% (individual control release), and histamine release was expressed as percent of this individual control. Stimulated histamine release was calculated by comparing three subsequent samples after mast cell activation (36th to 50th minute of incubation) with three prestimulation samples (16th to 30th minute of incubation; control release). The results are given as means ± SEM. The significance of differences was evaluated by Student's t test; for multiple comparison, the Dunnett test was applied. For statistical analysis the computer program InStat (San Diego, CA) was used; p values less than 0.05 were regarded as significant.

Drugs and Special Chemicals

The following drugs were used: acetylcholine hydrochloride, calcium ionophore A23187, choline chloride, histamine diphosphate, oxotremorine sesquifumarate, o-phthaldialdehyde (all purchased from SigmaChemie, München, Germany); clozapine, para-fluorohexa-hydrosiladifenidol hydrochloride, pirenzepine dihydrochloride (all purchased from RBI Biotrend, Köln, Germany); AF-DX 116 (11[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1, 4]benzodiazepine-6-one; gift from Thomae, Biberach, Germany); and methanol (Roth, Karlsruhe, Germany).

	RESULTS

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Interaction Experiments between Acetylcholine and Pirenzepine in Human Bronchi

Neither of the test substances affected the spontaneous histamine release (see Figures 1 and 2). A 1-min exposure to the calcium ionophore A23187 (10 µmol/L) stimulated the release of histamine (36th to 50th minute) by 270 ± 48% (n = 7) above the prestimulation level (16th to 30th minute; 0.6 ± 0.1 nmol/g/5 min; Figure 1A). When 100 pmol/L acetylcholine was present from 14 min before the application of A23187, the evoked histamine release amounted to 36 ± 5% (n = 7) of the prestimulation level, i.e., evoked histamine release was inhibited by 86% (Figure 1B). When pirenzepine (100 nmol/L) was present from the start of the collection period, A23187 increased histamine release by 340 ± 100% above the prestimulation level (compare with the control; p > 0.05). Pirenzepine, 100 nmol/L, antagonized the inhibitory effect of 100 pmol/L acetylcholine; evoked histamine release amounted to 280 ± 70% of the prestimulation level (Figure 1D; compare with Figure 1B). Almost 10 nmol/L pirenzepine started to antagonize the inhibitory effect of 100 pmol/L acetylcholine (Figure 3; 13 ± 2% versus 54 ± 17% of the respective control release, p < 0.027, two-tailed t test), an observation which supports the involvement of an M1-receptor subtype.

View larger version (41K): [in this window] [in a new window]   Figure 2.   Antagonistic potencies of pirenzepine, pFHHSiD, AF-DX 116, and clozapine on the inhibitory effect of 100 pmol/L acetylcholine on A23187-evoked histamine release from human isolated bronchi. The experimental protocol is described in Figure 1. Shown is the A23187-evoked histamine release (percent of the respective control value) in the presence of 100 pmol/L acetylcholine either alone (C) or in combination with the muscarinic receptor antagonists. Means ± SEM of 5 to 9 experiments are given. Significance of differences from C: *p < 0.05, **p < 0.001; significance of difference of the individual antagonist concentrations in relation to the lowest concentration applied: p < 0.05; p < 0.001.

View larger version (26K): [in this window] [in a new window]   Figure 3.   Control experiments and the effect of oxotremorine on the spontaneous and A23187-evoked histamine release from rat isolated trachea. The experimental protocol corresponds to that shown in Figure 1. Histamine release is expressed as percent of the control release (16th to 30th minute; means ± SEM). (A) Histamine release in the absence of test substances (control release: 0.9 ± 0.2 nmol/g × 5 min; stimulated release 145 ± 50%, n = 6). (B, C, D) Histamine release in the presence of 1, 100, and 10,000 nmol/L oxotremorine present from the sixteenth minute of incubation onward, respectively. The values for the control and stimulated histamine release are shown in Table 2. Means ± SEM of 4 to 6 experiments are shown.

Interaction Experiments between Acetylcholine and Further Muscarinic Receptor Antagonists in Human Bronchi

Figure 2 summarizes the antagonistic potencies of pirenzepine, pFHHSiD, AF-DX 116, and clozapine to prevent the inhibitory effect of 100 pmol/L acetylcholine on evoked histamine release. AF-DX 116 and clozapine significantly attenuated the inhibitory effect of acetylcholine only at a concentration of 1 µmol/L. pFHHSiD, which shows similar affinities at the human cloned M1-, M3-, and M4-subtype (10), antagonized the inhibitory effect of 100 pmol/L acetylcholine with a similar potency as pirenzepine (Figure 3).

Effects of Muscarinic Receptor Antagonists Alone on Evoked Histamine Release from Human Bronchi

The A23187-evoked release of histamine under control condition (absence of muscarinic receptor agonists or antagonists) was normalized to 100% to compare with the release obtained in the presence of the muscarinic receptor antagonists alone (Table 1). Obviously, in all series carried out with the antagonists, evoked histamine release was increased but the differences were not statistically significant (significance levels are indicated in Table 1).

Effect of Acetylcholine and Oxotremorine on Evoked Histamine Release from the Isolated Rat Trachea

A23187-evoked histamine release from the isolated rat trachea was measured. The experimental protocol was identical to that applied for human bronchi. Rat tracheal mast cells appeared somewhat more sensitive to the calcium ionophore than human airways. One-minute exposure of 10 µmol/L A23187 increased histamine release to 1,480 ± 400 above the prestimulation level (Table 2; compare with human bronchi 270 ± 48%). A concentration of 3 µM A23187 which increased histamine release by 145 ± 50% above the prestimulation level was used, to test the effects of acetylcholine or oxotremorine, a stable muscarinic receptor agonist. Neither acetylcholine (100 nmol/L) nor oxotremorine (1 and 100 nmol/L), concentrations effectively inhibiting histamine release in human airways (6), significantly inhibited evoked histamine release in the rat trachea. In contrast, 10,000 nmol/L oxotremorine appeared to facilitate evoked histamine release in the rat trachea (Figure 3, Table 2).

	DISCUSSION

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The present experiments demonstrate two findings. First, in human airways the release of histamine is inhibited by muscarinic receptors most likely of the M1 subtype. Second, a species difference was found. In the rat trachea a comparable inhibitory link between muscarinic receptors and histamine release has not been detected.

The calcium ionophore A23187 has been used in the present experiments to stimulate histamine release. This trigger does not correspond to the physiological activation pathway. However, it should be considered that histamine release from human airways was also inhibited by acetylcholine with a similar potency, when anti-IgE was used as release stimulus (6). Thus, the inhibitory control is also operating with the physiological stimulation mode. The results with anti-IgE (6) suggest that the inhibitory pathway directly targets the mast cells.

A23187 activates not only mast cells but more or less every cell. Nevertheless, in the present experiments mucosal mast cells can be identified as the source for the stimulated histamine release for the following two reasons: first, removal of the surface epithelium abolished the A23187-induced histamine release (6); second, the preparations were isolated from noninflammatory lung tissue, i.e., a significant contribution of basophils can be excluded.

Inhibitory M1-receptors in Human Airways

Acetylcholine in a rather low concentration of 100 pmol/L inhibited the A23187-evoked histamine release from human bronchi. All muscarinic receptor antagonists (pirenzepine, pFHHSiD, AF-DX 116, clozapine) applied in the present experiments attenuated the inhibitory action of acetylcholine. In addition, we have recently reported that atropine also prevented the inhibitory action of acetylcholine (6). All these observations indicate that the inhibitory effect of acetylcholine is mediated via stimulation of muscarinic receptors.

Muscarinic receptor antagonists are applied in the treatment of chronic airway diseases to reduce the smooth muscle tone. However, the simultaneous blockade of the inhibitory muscarinic receptors controlling mucosal mast cell function may induce undesired side effects by increasing the exocytotic release activity. Therefore, the muscarinic receptor subtype involved in the control of histamine release should be characterized in order to optimize drug therapy. The following reasons support the view that the receptors mediating inhibition of histamine release in human airways belong to the M1 subtype:

1. Pirenzepine, 100 nmol/L, antagonized the inhibitiory effect of acetylcholine by 83%. This high potency corresponds with its binding at the M1-receptor protein, as previously discussed. In binding experiments with human lung membranes a log pK_i of 7.8 was found indicating the existence of M1-receptors in this tissue (19).
2. The antagonistic potency of pFHHSiD was similar to that obtained for pirenzepine. This observation agrees with the binding parameters reported for the human cloned M1-receptor (log K_i values 8.2 and 7.65 for pirenzepine and pFHHSiD, respectively) (11) as well as with data (pA₂ value 7.9) from functional experiments measuring phosphatidylinositol hydrolysis triggered by human M1-receptors (12).
3. The low potency of AF-DX 116 excludes a M2-receptor subtype, but corresponds with its affinity at the human M1-receptor protein (pA₂ value 6.3; log K_i 6.08; see References 12-14).
4. Clozapine has been reported to act as antagonist at the human cloned M1-, M2-, and M3-receptors but as agonist at M4-receptors (15, 16). The antagonistic potency of clozapine in the present experiments favors a receptor population other than the M4 subtype. In this context, it should be considered that in the same tissue an example for an agonistic activity of clozapine has also been reported. Clozapine inhibited the release of newly synthesized [³H]acetylcholine in human bronchi by stimulating prejunctional muscarinic receptors (20). In contrast, in the present study clozapine did not reduce evoked histamine release alone, but antagonized the effect of acetylcholine.

Although pA₂ values were not determined, the present results support the view that muscarinic receptors of the M1 subtype are involved in the inhibition of histamine release. Whether this muscarinic brake is operating under in vivo conditions is difficult to determine from the present in vitro experiments. However, there is some indication that an inhibitory muscarinic tone may exist. For example, we reported that atropine increased the kinetics of histamine release significantly in human airways (6). In the present experiments all muscarinic receptor antagonists tended to increase the evoked histamine release. A more significant observation has been made under in vivo conditions. Atropine increased histamine release after nasal antigen challenge in humans (21). For inhalational drug therapy selective antagonists at M3-receptors should be developed to maintain the inhibitory muscarinic control of histamine release.

Species Differences

The present experiments confirm the existence of an inhibitory link between muscarinic receptors and mucosal mast cells in human airways. However, an opposite regulation has been repeatedly reported in other species and tissues. For example, histamine release from rat isolated peritoneal mast cells is facilitated by acetylcholine (17). A facilitatory effect of cholinergic agonists was found on mast cells originating from the peritoneal cavity, lung parenchyma, glandular tissue, and nasal polyps (17, 22, 23). The existence of different mast cell populations may explain the different results. However, species differences also should be considered.

In the present experiments, histamine release from the rat trachea was investigated to test whether rat airway might be a suitable model for studies of muscarinic receptor modulation of histamine release. Oxotremorine, at concentrations suppressing the histamine release from human bronchi (6), did not inhibit the A23187-evoked histamine release from the rat trachea. In contrast, the highest concentration of oxotre-morine tended to increase histamine release. Similarly, 100 nmol/L acetylcholine which mediated the maximal inhibitory effect in human bronchi (6) did not reduce the histamine release in the rat trachea. Thus, histamine release from trachea is not controlled by inhibitory muscarinic receptors.

In conclusion, acetylcholine may play an important role in limiting histamine release in human airways via stimulation of muscarinic receptors, most likely of the M1 subtype. To optimize drug therapy, inhalational muscarinic receptor antagonists should act exclusively at M3-receptors.