INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Although initially assumed to be a peripheral hormone because of the high concentration found in blood and in the gastrointestinal tract, it has now been established that 5-HT should be considered as a neurotransmitter that produces its physiological effects by activating specific receptors located on cell membranes. The role of 5-HT receptors has been extensively studied in brain tissue (1) as well as in vascular and gastrointestinal smooth muscle contraction (1, 2). Similarly, in respiratory tissues from different species, 5-HT exhibited a wide spectrum of activity through stimulation of a variety of different 5-HT receptor subtypes. In guinea pig airways in vitro, a direct action of 5-HT has been demonstrated on airway smooth muscle, causing contraction at low concentrations and relaxation at high concentrations, both mediated through stimulation of 5-HT₂ receptors (3, 4). 5-HT has also been shown to modulate the noncholinergic nonadrenergic (NANC) contraction in guinea pig airways in vitro. This contraction, mediated by the release of neuropeptides from sensory nerve endings (5), was significantly inhibited by stimulation of prejunctional 5-HT receptors, which were originally described as 5-HT₁-like receptors (6) but fit the pharmacological profile of 5-HT₇ receptors (7). Epinastine and ketotifen, two drugs used in asthma treatment, act as 5-HT agonists and are also able to inhibit the NANC contraction in guinea pig airways in vitro (8, 9). This mechanism of action may partially account for the prophylactic effects of both drugs in obstructive airway disease such as asthma.

5-HT has also been shown to modulate cholinergic neural responses in a number of species by interacting with presynaptic neuronal receptors. However, the type of effects and the 5-HT receptor subtype involved appear to differ according to the nature of the species. In mouse isolated tracheal segments, 5-HT potentiated cholinergic contraction by activating presynaptic 5-HT₁-like receptors (10). In rat bronchi, 5-HT enhanced cholinergic contraction by stimulation of 5-HT₂ receptors (11). Alternatively, in guinea pig airways, several investigators have demonstrated facilitatory effects of 5-HT on postganglionic cholinergic neurotransmission by stimulation of 5-HT₃ receptors (12). In human airways in vitro, stimulatory effects of 5-HT appear to be mediated by both prejunctional 5-HT₃ and 5-HT₄ receptors (13). On cholinergic nerves, the presence of inhibitory 5-HT receptors, which promote relaxation rather than contraction, has also been suggested. In the presence of a 5-HT₂ and 5-HT₃ receptor antagonist, 5-HT relaxed acetylcholine-precontracted guinea pig airways in vitro, possibly through stimulation of 5-HT₁ receptors (14). Intravenous administration of urapidil, a 5-HT_1A agonist, induced significant bronchodilatatory effects in patients with obstructive airway disease (15). Evidence for 5-HT receptor involvement in 5-HT-induced relaxation has also been demonstrated in gastrointestinal preparations (16, 17). 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT agonist which exhibits some selectivity for 5-HT_1A receptors, inhibited electrically evoked neurotransmitter release from the enteric cholinergic neurons of the guinea pig ileum. These effects were significantly inhibited by methiothepin, which suggests the involvement of 5-HT receptors, possibly of the 5-HT_1A subtype (17). We have recently demonstrated prejunctional, inhibitory effects of epinastine on the cholinergic contraction in both guinea pig and human airways in vitro, which could be attenuated by pretreatment with methysergide (18, 19). These findings implicate the presence of inhibitory 5-HT receptors on cholinergic nerves in respiratory tissues. We therefore intended to investigate further this hypothesis using more selective 5-HT agonists and antagonists on the cholinergic contraction in both guinea pig and human airways in vitro.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Tissue Preparation

Dunkin-Hartley guinea pigs of either sex (300 to 600 g) were killed by cervical dislocation. The lungs, with the bronchi and the trachea, were rapidly removed and placed in a carbogenated modified Krebs-Henseleit solution of the following composition (in millimoles per liter): NaCl, 118; MgSO₄, 1.2; KCl, 5.9; CaCl₂, 2.5; NaH₂PO₄, 1.2; NaHCO₃, 25.5; and glucose, 5.05 (pH 7.4). The trachea was carefully stripped of connective tissue, opened longitudinally by cutting through cartilage, and cut in segments containing four to five cartilaginous rings. Silk loops were attached at both ends of the strips and the airway strips were connected to steel hooks both at the bottom of the organ baths and at the force-displacement transducer.

Macroscopically normal human bronchial tissue was obtained from thoracotomy specimens of patients (1 woman, 11 men; mean age 54 ± 15 yr) undergoing surgery for bronchial carcinoma. None of the patients had characteristics of asthma. Immediately after surgical resection, a macroscopically normal part of the lung tissue was immersed in cooled (4° C) and carbogenated (5% CO₂ and 95% O₂) modified Krebs-Henseleit buffer solution. The airways (segmental and subsegmental bronchi) were carefully stripped from surrounding lung tissue and cut into ring segments. Thin silk threads were tied through the bronchial rings and they were connected to steel hooks both at the bottom of the organ baths and at the force-displacement transducer. The tissues remained in fresh carbogenated buffer throughout the dissection procedure and during the experiments and were used between 1 and 18 h after resection.

The guinea pig airway strips or human bronchial preparations were mounted vertically between two platinum wire electrodes in 10-ml organ baths containing Krebs-Henseleit solution, which was maintained at 37° C and continuously bubbled with 5% CO₂ in O₂. The preparations contracted against a load of 1 g for guinea pig tissue and 2 g for human tissue, which have both been shown to produce optimal repeatable responses in preliminary experiments. While being washed with fresh KH solution every 20 min, tissues were allowed to equilibrate under tension for at least 60 min before beginning experimental protocols, during which time a stable baseline tension was achieved. All experiments in guinea pig tracheal strips were performed in the presence of indomethacin (10 µM) to prevent modulation of neural responses by endogenously synthesized prostaglandins (20).

Experimental Protocol

Isometric contractile responses, induced either by electrical field stimulation (EFS) or by adding acetylcholine (ACh), were measured by using a Grass FT 03 force-displacement transducer (STAG Instruments, Ltd., Chalgrove, Oxon, UK). The traces were visualized on a computer screen after digitalization of the signal (Codas, Dataq Instrument, Inc., Akron, OH) and recorded on a personal computer.

Electrical field stimulation. EFS was produced by a Harvard student stimulator (Harvard Apparatus Ltd., Edenbridge, Kent, UK). Biphasic square-wave pulses of a supramaximal voltage of 50 V at source and a pulse duration of 0.5 ms were delivered for 15 s every 4 min at frequencies ranging from 0.5 to 32 Hz in guinea pig tracheal strips and 1 to 32 Hz in human bronchial rings. After the equilibration period a frequency-response curve (FRC) (0.5 to 32 Hz or 1 to 32 Hz) was performed and then discarded. After washing the tissues a control FRC was performed. Eight tissues were simultaneously tested with at least one time control tissue per experiment.

In a first set of experiments in guinea pig tracheal strips, the 5-HT agonists 8-OH-DPAT (1 to 30 µM, 5-HT_1A/7 selective) or 5-carboxamidotryptamine (5-CT, 5-HT_1/2/7 selective) (10 to 100 µM) (21) were added to the organ baths, with only one concentration of drug added per tissue. After an incubation period of 15 min, a third FRC (0.5 to 32 Hz) was obtained.

In a second set of experiments in guinea pig tracheal strips, the effects of 5-HT antagonistsSDZ 216-525 (10 µM, 5-HT_1A selective) (22) and pindobind (10 µM, 5-HT_1A selective) (23), methysergide (1 to 30 µM, 5-HT_1A,1B,1D,2,7 selective), ketanserin (10 µM, 5-HT₂ selective), and tropisetron (1 µM, 5-HT_3-4 selective) (21)were studied on the cholinergic contraction. These antagonists were also studied on the effects of 8-OH-DPAT (10 to 30 µM) on the cholinergic contraction elicited by EFS (0.5 to 32 Hz).

In a third set of experiments, guinea pig airway strips were incubated with capsaicin (10 µM) 1 h before the start of the experiment, which was washed out 30 min later, to deplete the sensory nerves of endogenous tachykinins (24). Subsequently, the effects of 8-OH-DPAT (10 to 30 µM) on the cholinergic contraction were investigated. The same protocol was used as described previously.

In a different set of experiments the effect of 8-OH-DPAT was investigated on the nonadrenergic relaxation, induced by EFS (50 V, 0.5 ms, 1, 2, 4, or 8 Hz for 30 s) in guinea pig tracheal strips in vitro, in the presence of atropine (1 µM) to block cholinergic responses. After the equilibration period 2 control stimuli were applied at 1, 2, 4, or 8 Hz. These stimuli were discarded if they were not consistent (i.e., > 10% variation). Otherwise, 8-OH-DPAT (10 to 30 µM) was applied and after a 15-min incubation period another 2 stimuli were delivered at the same frequency.

In human bronchial rings the effects of 8-OH-DPAT (10 to 30 µM) on the cholinergic contraction elicited by EFS (1 to 32 Hz) were investigated, both in the presence and in the absence of SDZ 216-525 (10 µM) and methysergide (30 µM).

Cumulative concentration-response curve to acetylcholine. To determine whether the effects of 8-OH-DPAT on the cholinergic contraction were due to activation of pre- or postjunctional receptors, the effects of a 15-min incubation period with 8-OH-DPAT (30 µM) were studied on the cumulative-concentration relationship to exogenously applied ACh in guinea pig and human airways in vitro. Concentration-effect curves to ACh were performed in a cumulative manner, by adding incremental concentrations, spaced at half log ₁₀ intervals (10 nM to 30 mM). The results were expressed as a percentage of the maximal contraction to ACh (10 mM), which was determined at the beginning of the experiment.

Drugs

Drugs used in these experiments were obtained from the following sources: 8-OH-DPAT, ketanserin, capsaicin, hexamethonium, tetrodotoxin (Biomol; Sanver Tech, Boechout, Belgium); SDZ 216-525, methysergide, tropisetron (ICS205-930) (a kind gift from Sandoz- Novartis Pharma, Basel, Switzerland); pindobind (RBI; Sanver Tech, Boechout, Belgium); ACh, indomethacin (Sigma Chemical Co., Eupen, Belgium). SDZ 216-525 and tropisetron were dissolved in dimethylsulfoxide; capsaicin was dissolved in ethanol; pindobind was dissolved in methanol. All other drugs were dissolved in distilled water. Control tissues were treated with an equivalent amount of the appropriate solvent. Fresh drug solutions were made up daily. Drug additions did not exceed 1% (vol/vol) of the organ bath volume. All concentrations refer to the final organ bath concentration.

Analysis of Results

Results are expressed as means ± SEM. All contractile or relaxing responses were measured as the difference between peak tension and resting tension. The effects of a single concentration of 8-OH-DPAT or 5-CT with or without antagonist were expressed as a percentage inhibition, comparing the contractile or relaxing responses at each stimulation frequency after pretreatment with the contraction or relaxation at the same frequency in the control responses. Because each tissue acted as its own control, analysis of data was possible using a Student's t test for paired data. Significance between tissues treated with 8-OH-DPAT with or without antagonists was assessed using a Student's t test for unpaired data. The same test was used to assess the effects of 8-OH-DPAT versus control on the cumulative concentration-response curve to exogenous ACh. Probability values of < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Electrical Field Stimulation

Effects of 8-OH-DPAT on the cholinergic contraction in guinea pig tracheal strips in vitro. In guinea pig tracheal strips, EFS (50 V, 0.5 ms, 0.5 to 32 Hz for 15 s every 4 min) caused a rapid and transient contraction that was abolished by pretreatment of the tissues with atropine (1 µM), confirming that the contractile responses were caused by the release of ACh. Hexamethonium (10 µM), a ganglion blocker, had no effect on the cholinergic contraction elicited by EFS, which confirms that the contractile responses were mediated by the release of ACh from postganglionic cholinergic nerves. The responses to EFS were completely blocked by tetrodotoxin (1 µM) confirming their neuronal origin (Figure 1A).

View larger version (16K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   Figure 1.   (A) Trace showing the effect of hexamethonium (10 µM), atropine (1 µM), and tetrodotoxin (1 µM) on the cholinergic contraction, elicited by EFS (closed squares: 50 V at source, 0.5 ms, 32 Hz for 15 s) in guinea pig tracheal strips in vitro. Hexamethonium had no effect on the cholinergic contraction. The responses to EFS were completely blocked by tetrodotoxin and atropine. (B) Trace showing the effect of 8-OH-DPAT (30 µM) on the cholinergic contraction, elicited by EFS (closed squares: 50 V at source, 0.5 ms, 0.5 to 32 Hz for 15 s every 4 min) in guinea pig tracheal strips in vitro. 8-OH-DPAT produced a potent and frequency-dependent inhibition of the cholinergic contraction.

EFS resulted in a cholinergic contraction whose amplitude increased with increasing frequencies of stimulation and ranged from 0.4 ± 0.1 g tension at 0.5 Hz to 0.9 ± 0.1 g tension at 32 Hz. Tissues that exhibited a potent noncholinergic nonadrenergic contraction in addition to the cholinergic contraction were discarded. A typical trace of the frequency-response curve is shown in Figure 1B, which also demonstrates the effects of 8-OH-DPAT (30 µM) on the cholinergic responses. 8-OH-DPAT (30 µM) inhibited the cholinergic contraction at all frequencies of stimulation. Moreover, when contractile responses were expressed as percentage inhibition, comparing the contractile responses at each stimulation frequency after pretreatment with the contraction at the same frequency in the control responses, it was clear that the effect on the cholinergic contraction by 8-OH-DPAT was more pronounced at lower frequencies of stimulation. Preliminary experiments involving a time course of the inhibitory effects of 8-OH-DPAT demonstrated no further inhibition of the cholinergic contraction with longer incubation time than 15 min.

8-OH-DPAT (1 µM to 30 µM) produced a concentration-dependent inhibition of the cholinergic contraction in guinea pig tracheal strips with a maximal inhibition of 75.8% ± 4.7% at 0.5 Hz (n  5, p < 0.01 compared with control) (Figure 2). The responses to EFS in vehicle-treated tissues remained stable throughout the period of the experiments.

View larger version (20K): [in this window] [in a new window]   Figure 2.   The effect of 8-OH-DPAT (1 to 30 µM) on the cholinergic contraction, elicited by EFS (50 V at source, 0.5 ms, 0.5 to 32 Hz for 15 s every 4 min) in guinea pig tracheal strips in vitro. 8-OH-DPAT produced a concentration-dependent inhibition of the cholinergic contraction. Curves are shown for 8-OH-DPAT 30 µM (closed circles), 8-OH-DPAT 10 µM (closed squares), 8-OH-DPAT 3 µM (closed triangles), 8-OH-DPAT 1 µM (inverted, closed triangles). Points represent mean ± SEM of at least n = 5 observations. Significance of inhibition: *p < 0.05, **p < 0.01, ***p < 0.001, compared with control.

Effects of 5-HT antagonists on the 8-OH-DPAT-inhibition of the cholinergic contraction in guinea pig tracheal strips in vitro. Addition of methysergide (1 to 30 µM, a 5-HT_1,2,7 antagonist) had no effect on the cholinergic contraction on its own (Figure 3). However, methysergide (1 to 30 µM) significantly and concentration-dependently attenuated the 8-OH-DPAT- (30 µM) induced inhibition of the cholinergic contraction at all stimulation frequencies (n  5, p < 0.01 compared with 8-OH-DPAT alone) (Figure 3).

View larger version (20K): [in this window] [in a new window]   Figure 3.   Inhibitory effect of 8-OH-DPAT 30 µM (closed circles) on the cholinergic contraction, elicited by EFS (50 V at source, 0.5 ms, 0.5 to 32 Hz for 15 s every 4 min) in guinea pig tracheal strips in vitro, and attenuation of this inhibition by methysergide 1 µM (open squares), methysergide 10 µM (inverted, open triangles), and methysergide 30 µM (open triangles). Methysergide 30 µM alone (single stars) had no effect on the cholinergic contraction in guinea pig airways in vitro. Points represent mean ± SEM of at least n = 5 observations. Significance of inhibition: **p < 0.01, ***p < 0.001, compared with 8-OH-DPAT 30 µM.

Ketanserin (10 µM, a 5-HT₂ antagonist) and tropisetron (1 µM, a 5-HT_3/4 antagonist) had themselves no effect on the cholinergic contraction. They also had no effect on the inhibitory effects of 8-OH-DPAT (10 to 30 µM) on the cholinergic contraction (n = 5, p = NS compared with 8-OH-DPAT alone) (Figure 4).

View larger version (17K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 4.   Inhibitory effect of 8-OH-DPAT 30 µM (closed circles) and 8-OH-DPAT 10 µM (closed squares) on the cholinergic contraction, elicited by EFS (50 V at source, 0.5 ms, 0.5 to 32 Hz for 15 s every 4 min) in guinea pig tracheal strips in vitro. Ketanserin 10 µM (inverted, open triangles) and tropisetron 1 µM alone (open triangles) did not modulate the cholinergic contraction in guinea pig airways in vitro. (A) Pretreatment with ketanserin 10 µM had no significant effect on the inhibition of the cholinergic contraction by 8-OH-DPAT 30 µM (open circles) and 8-OH-DPAT 10 µM (open squares). (B) Pretreatment with tropisetron (1 µM) also failed to prevent the inhibition of the cholinergic contraction by 8-OH-DPAT 30 µM (open circles) and 8-OH-DPAT 10 µM (open squares). Points represent mean ± SEM of at least n = 5 observations.

SDZ 216-525 (10 µM, a 5-HT_1A antagonist) did not affect the cholinergic contraction produced by EFS (Figure 5). Moreover, this antagonist failed to prevent the inhibition of the cholinergic contraction by 8-OH-DPAT (10 to 30 µM) (n  5, p = NS compared with 8-OH-DPAT alone) (Figure 5). Pindobind (10 µM, a 5-HT_1A antagonist) also failed to modulate the cholinergic contraction and the inhibition of the cholinergic contraction by 8-OH-DPAT (30 µM) (n = 5, p = NS compared with 8-OH-DPAT alone) (data not shown). The responses to EFS in vehicle-treated tissues remained stable throughout the period of the experiments.

View larger version (18K): [in this window] [in a new window]   Figure 5.   Inhibitory effect of 8-OH-DPAT 30 µM (closed circles) and 8-OH-DPAT 10 µM (closed squares) on the cholinergic contraction, elicited by EFS (50 V at source, 0.5 ms, 0.5 to 32 Hz for 15 s every 4 min) in guinea pig tracheal strips in vitro. Pretreatment with SDZ 216-525 10 µM (open symbols) had no significant effect on the inhibition of the cholinergic contraction by 8-OH-DPAT 30 µM (open circles) and 8-OH-DPAT 10 µM (open squares). SDZ 216-525 alone (10 µM) (closed stars) did not modulate the cholinergic contraction in guinea pig airways in vitro. Points represent mean ± SEM of at least n = 5 observations.

Effects of capsaicin pretreatment on the 8-OH-DPAT-inhibition of the cholinergic contraction in guinea pig tracheal strips in vitro. Pretreatment of the tissues with capsaicin (10 µM) did not modulate the inhibitory effects of 8-OH-DPAT (10 to 30 µM) on the cholinergic contraction in guinea pig tracheal strips in vitro (n = 5, p = NS compared with 8-OH-DPAT alone) (Figure 6). Control tissues were treated with capsaicin only. A second addition of capsaicin (10 µM) did not produce any contraction, which confirms the depletion of neuropeptides by capsaicin pretreatment. The responses to EFS in vehicle-treated tissues remained stable throughout the period of the experiments.

View larger version (16K): [in this window] [in a new window]   Figure 6.   The effect of capsaicin pretreatment on the inhibitory effect of 8-OH-DPAT (10 to 30 µM) on the cholinergic contraction, elicited by EFS (50 V at source, 0.5 ms, 0.5 to 32 Hz for 15 s every 4 min) in guinea pig tracheal strips in vitro. Capsaicin pretreatment did not modulate the inhibitory effect of 8-OH-DPAT 30 µM (open circles) and 8-OH-DPAT 10 µM (open squares) when compared with the inhibition of the cholinergic contraction by 8-OH-DPAT 30 µM (closed circles) and 8-OH-DPAT 10 µM (closed squares). Points represent mean ± SEM of at least n = 5 observations.

Effects of 5-CT on the cholinergic contraction in guinea pig tracheal strips in vitro. Addition of high concentrations of 5-CT ( 30 µM) elicited a contraction that was completely antagonized by ketanserin (10 µM, a 5-HT₂ antagonist). Experiments with 5-CT were therefore performed in the presence of ketanserin (10 µM).

The effects of 5-CT on the cholinergic contraction elicited by EFS, after an incubation period of 15 min are shown in Figure 7. Preliminary experiments involving a time course of the inhibitory effects of 5-CT demonstrated no further inhibition of the cholinergic contraction with longer incubation time. 5-CT produced a maximal inhibition of the cholinergic contraction of 27.4% ± 6.6% at a concentration of 100 µM and at 0.5 Hz (n  5, p < 0.05 compared with control). These effects of 5-CT were especially prominent at lower stimulation frequencies (0.5 to 4 Hz) (Figure 7). The responses to EFS in vehicle-treated tissues remained stable throughout the period of the experiments.

View larger version (18K): [in this window] [in a new window]   Figure 7.   The effect of 5-CT (10 to 100 µM) on the cholinergic contraction, elicited by EFS (50 V at source, 0.5 ms, 0.5 to 32 Hz for 15 s every 4 min) in guinea pig tracheal strips in vitro, in the presence of ketanserin 10 µM. 5-CT produced a concentration-dependent inhibition of the cholinergic contraction. Curves are shown for 5-CT 100 µM (closed circles), 5-CT 30 µM (closed squares), 5-CT 10 µM (closed triangles). Points represent mean ± SEM of at least n = 5 observations. Significance of inhibition: *p < 0.05, **p < 0.01, compared with control.

Effects of 8-OH-DPAT on the inhibitory NANC contraction in guinea pig tracheal strips in vitro. In the presence of atropine (10 µM), EFS (50 V, 0.5 ms, 1, 2, 4, or 8 Hz for 30 s every 4 min) caused a relaxation in guinea pig upper tracheal strips. Hexamethonium (10 µM), a ganglion blocker, had no effect on the NANC relaxation elicited by EFS but the responses to EFS were completely blocked by tetrodotoxin (1 µM) confirming their neuronal origin.

8-OH-DPAT (10 to 30 µM) had no effect on the EFS- induced nonadrenergic relaxation at 1, 2, 8, and 16 Hz in guinea pig trachea (data not shown). The responses to EFS in vehicle-treated tissues remained stable throughout the period of the experiments.

Effects of 8-OH-DPAT on the cholinergic contraction in human bronchial rings in vitro. EFS in human bronchial rings (50 V, 0.5 ms, 1 to 32 Hz for 15 s every 4 min) resulted in a cholinergic contraction which increased in amplitude with increasing frequencies of stimulation and ranged from 0.2 ± 0.1 g tension at 1 Hz to 1.8 ± 0.5 g tension at 32 Hz. This contraction was abolished by pretreatment of the tissues with atropine (1 µM) while hexamethonium (10 µM) had no effect on the cholinergic contraction elicited by EFS, which confirms that the contractile responses were mediated by the release of ACh from postganglionic cholinergic nerves. The responses to EFS were completely blocked by tetrodotoxin (1 µM) confirming their neuronal origin.

8-OH-DPAT (10 to 30 µM) inhibited the cholinergic contraction with a maximal inhibition of 46.2% ± 7.4% at 1 Hz (n  5, p < 0.01 compared with control) (Figure 8). This inhibition was more pronounced at lower frequencies of stimulation. Pretreatment with SDZ 216-525 (10 µM) had no effect either on the cholinergic contraction or on the inhibition of the cholinergic contraction by 8-OH-DPAT (30 µM). Pretreatment with methysergide (30 µM) had no effect on the cholinergic contraction itself but significantly attenuated the inhibitory effects of 8-OH-DPAT (30 µM) on the cholinergic contraction in human bronchial rings at lower stimulation frequencies (1 to 4 Hz) (n  5, p < 0.05 compared with control) (Figure 8). The responses to EFS in vehicle-treated tissues remained stable throughout the period of the experiments.

View larger version (17K): [in this window] [in a new window]   Figure 8.   Inhibitory effect of 8-OH-DPAT 30 µM (closed circles) and 8-OH-DPAT 10 µM (closed squares) on the cholinergic contraction, elicited by EFS (50 V at source, 0.5 ms, 1 to 32 Hz for 15 s every 4 min) in human bronchial rings in vitro, and attenuation of the 8-OH-DPAT (30 µM)-induced inhibition by methysergide 30 µM (open circles). Methysergide 30 µM alone (closed stars) had no effect on the cholinergic contraction in human airways in vitro. Pretreatment with SDZ 216-525 10 µM (inverted triangles) had no effect on the inhibition of the cholinergic contraction by 8-OH-DPAT 30 µM. Points represent mean ± SEM of at least n = 5 observations. Significance of inhibition: *p < 0.05, **p < 0.01, compared with 8-OH-DPAT 30 µM.

Concentration-Response Curve to Acetylcholine

Effects of 8-OH-DPAT on the concentration-response curve to acetylcholine in guinea pig tracheal strips in vitro. Acetylcholine produced a concentration-dependent contraction in guinea pig tracheal strips with a maximal contraction of 1.7 ± 0.2 g tension at a concentration of ACh  1 mM. Pretreatment with 8-OH-DPAT (30 µM) did not significantly alter the contractile responses to incremental concentrations of acetylcholine (10 nM to 30 mM) in guinea pig tracheal strips in vitro (n = 5, p = NS compared with control) (Figure 9).

View larger version (17K): [in this window] [in a new window]   Figure 9.   Effect of 8-OH-DPAT 30 µM on the cumulative concentration-response curve to exogenously applied ACh (1 nM to 1 mM) in guinea pig tracheal strips in vitro. Effects are displayed as a percentage of the maximum to ACh 10 mM. Curves are shown for ACh in the absence (open circles) and in the presence (closed circles) of 8-OH-DPAT 30 µM. Points represent mean ± SEM of at least n = 5 observations.

Effects of 8-OH-DPAT on the concentration-response curve to acetylcholine in human bronchial rings in vitro. Acetylcholine produced a concentration-dependent contraction in human bronchial rings with a maximal contraction of 2.4 ± 0.4 g tension at a concentration of ACh  1 mM. Pretreatment with 8-OH-DPAT (30 µM) had no significant effect on the contractile responses to incremental concentrations of acetylcholine (10 nM to 30 mM) in human bronchial rings in vitro (n = 5, p = NS compared with control) (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study isometric contraction of guinea pig tracheal strips and human bronchial rings was induced by EFS. This contraction was antagonized by atropine but not by hexamethonium, which indicates that these contractile responses were modulated by the release of ACh from postganglionic parasympathetic nerves.

Previous studies have demonstrated that 5-HT in vitro facilitated cholinergic neurotransmission in airway tissue of a number of species (10). The 5-HT receptor subtype responsible for the potentiation of cholinergic responses appeared to differ according to the species that was investigated. In the present study, we have investigated the effects of the selective 5-HT agonists 8-OH-DPAT and 5-CT on the cholinergic contraction in both guinea pig and human airways in vitro. By using several 5-HT antagonists, we have also tried to determine the receptor subtype involved.

In guinea pig tracheal strips 8-OH-DPAT produced a concentration-dependent inhibition of the cholinergic contraction which was only significant at relatively high concentrations. Similar effects of 8-OH-DPAT were also present in human bronchial rings in vitro although this inhibition was less pronounced than in guinea pig tracheal strips. The effects of 8-OH-DPAT on the cholinergic neurotransmission were frequency-dependent with a more pronounced inhibition at lower frequencies of stimulation. This suggests a prejunctional effect, which was further confirmed by the lack of effect of 8-OH-DPAT on the concentration-response curve to ACh. A maximal concentration of 8-OH-DPAT, which produced a potent inhibition of the EFS-induced cholinergic contraction, did not affect the cumulative concentration-response curve to exogenously applied ACh in both guinea pig and human airways.

8-OH-DPAT is a 5-HT receptor agonist, which shows some selectivity for the 5-HT_1A subtype (25). Recently it has also been demonstrated that 8-OH-DPAT can act in some systems as a partial agonist at 5-HT₇ receptors (7). In our study, we were unable to modulate the effects of 8-OH-DPAT on the cholinergic contraction by pretreatment with SDZ 216-525 and pindobind, two selective 5-HT_1A antagonists (22, 23). Methysergide, a nonselective 5-HT antagonist at 5-HT₁, 5-HT₂, and 5-HT₇ receptors, on the other hand, significantly antagonized the inhibitory effects of 8-OH-DPAT in a concentration-dependent manner whereas antagonists at 5-HT₂, 5-HT₃, and 5-HT₄ receptors had no effect. These data suggest that, although the 8-OH-DPAT-induced inhibition of the cholinergic neurotransmission is probably mediated through stimulation of prejunctional 5-HT receptors, these effects could not be explained by an action on 5-HT_1A subtype receptors. On the basis of the pharmacological profile of 5-HT₇ receptors (7), our findings could, however, be compatible with an action on a 5-HT₇ receptor subtype. This hypothesis is consistent with our previous data on the effects of epinastine on cholinergic neurotransmission in both guinea pig and human airways (18, 19).

Epinastine is an antiallergic drug with histamine H₁-receptor blocking effects and with binding affinities for 5-HT₂ and 5-HT₇ receptors, but not for 5-HT₁ receptors (26). Epinastine inhibited cholinergic contraction in both guinea pig and human airways in vitro, which was antagonized by pretreatment of the tissues with methysergide (18, 19).

In the present study, we have also demonstrated that 5-CT, another 5-HT agonist with a similar profile on 5-HT_1A and 5-HT₇ receptors as 8-OH-DPAT (21) and additional affinities at 5-HT_1B and 5-HT_1D and probably other 5-HT receptors, inhibited the cholinergic contraction in guinea pig tracheal strips. The inhibition by 5-CT, which is generally considered as a more potent agonist at 5-HT_1A and 5-HT₇ receptors than 8-OH-DPAT, was, however, less pronounced than the inhibition by 8-OH-DPAT. Although this could argue against the involvement of 5-HT₇ receptors, the inhibitory effects of 5-CT on the cholinergic contraction in our study could be partially antagonized by a simultaneous action of 5-CT on 5-HT₄ receptors (21), which have been shown to enhance the cholinergic neurotransmission (13).

We have also considered alternative mechanisms of action for the inhibitory effects of 8-OH-DPAT on the cholinergic contraction. Numerous prejunctional receptors on cholinergic nerves have been described to influence neurotransmitter release (27). An effect through one of these receptors seems unlikely as 8-OH-DPAT has not been shown to act on any of these receptors and as it could not account for the protective effects of methysergide on the 8-OH-DPAT-induced inhibition of the cholinergic contraction. 5-CT and 8-OH-DPAT have also been found to inhibit the release of neuropeptides from sensory nerve endings in guinea pig tracheal strips in vitro through stimulation of 5-HT₁-like (5-HT₇?) receptors (6). Sensory neuropeptides released from C-fibers may facilitate cholinergic neurotransmission (28). To exclude an indirect effect of 8-OH-DPAT and 5-CT on the cholinergic contraction by interfering with the release of neuropeptides, we have also investigated the effects of capsaicin pretreatment on the 8-OH-DPAT-induced inhibition of the cholinergic contraction. Capsaicin pretreatment, which depletes sensory nerves of neuropeptides, did not modulate the inhibitory effects of 8-OH-DPAT on the cholinergic contraction.

On the other hand, the possibility also existed that the cholinergic contraction was counteracted by an enhancement of the nonadrenergic relaxation. Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) have been implicated as neurotransmitters for the nonadrenergic relaxation in guinea pig airways (29), whereas in human airways only NO seems to be involved (30). Both VIP and NO have also been demonstrated to be able to modulate the cholinergic neurotransmission in guinea pig tracheal strips as well as in human tracheal strips (31, 32). Therefore, we hypothesized that 5-HT might inhibit the cholinergic contraction through stimulation of VIP and NO release. We could, however, exclude this possibility, because we have also shown, in the present experiments, that 8-OH-DPAT did not modulate the EFS-induced nonadrenergic relaxation in guinea pig tracheal strips.

The presence of 5-HT receptors inhibiting cholinergic neurotransmission has also been demonstrated in isolated colon ascendens and ileum preparations of guinea pig. As demonstrated by Fozard and Kilbinger, 8-OH-DPAT caused an inhibition of the electrically evoked neurotransmitter release from enteric cholinergic neurons, which was inhibited by metergoline and mehiothepin (17). In guinea pig tracheal strips pretreated with ketanserin and ondansetron, respectively a 5-HT₂ and a 5-HT₃ antagonist, 5-HT and 5-CT produced a relaxation of ACh-precontracted tissues (14). Administration of urapidil, a 5-HT_1A agonist, induced a moderate bronchodilatation in patients with obstructive airway disease (15). Our results, however, suggest that 8-OH-DPAT inhibits the cholinergic contraction in guinea pig and human airways in vitro through stimulation of prejunctional 5-HT receptors, possibly 5-HT₇ subtype, located on postganglionic cholinergic nerves. Indeed the pharmacological profile of the 5-HT receptors that were involved in our study was not compatible with that of 5-HT_1A receptors. Recently genes encoding the 5-ht_1E and the 5-ht_1F receptors have been cloned (21) and it is still possible that the receptor responsible for the inhibition of the cholinergic contraction could correspond to one of these novel 5-HT receptors. However, definitive characterization awaits further functional correlates on these recently described receptors and development of selective receptor ligands. This might also explain why the identification of 5-HT receptor subtypes based on pharmacological data is sometimes not evident and should also be taken into consideration when interpreting our results.