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ANESTHETIC PHARMACOLOGY

The Effects of Fentanyl on the Contractile Response of Ovalbumin-Sensitized Rat Trachea

From the Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501, Japan.

Address correspondence to Osamu Shibata, MD, Department of Anesthesiology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. Address e-mail to opshiba{at}net.nagasaki-u.ac.jp.

	Abstract

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BACKGROUND: It is not clear whether fentanyl affects a hyperresponsive airway. We examined the effects of fentanyl on the contractile response of ovalbumin (OA)-sensitized rat tracheas.

METHODS: Rats were sensitized with a single intraperitoneal injection of 10 µg of OA mixed with adjuvant. Fourteen days later, the trachea was cut into 3-mm-wide rings. The OA-induced tension was measured, and the effects of fentanyl were studied in the presence of naloxone. Second, the role of cholinergic nerves and serotonin in the contraction and the effects of fentanyl were examined using tetrodotoxin and ketanserin. Third, lungs of sensitized rats were ventilated, and respiratory system resistance was calculated before and after the administration of OA in the presence of fentanyl.

RESULTS: Fentanyl dose-dependently attenuated the OA-induced contraction, and naloxone partly reversed it. Both tetrodotoxin and ketanserin attenuated the contraction. Fentanyl had no further effect on the contraction in the presence of tetrodotoxin, whereas the contraction was nearly abolished by fentanyl in the presence of ketanserin. OA increased respiratory system resistance in sensitized rats, and this effect was attenuated by fentanyl.

CONCLUSIONS: Fentanyl attenuates the airway hyperresponsiveness of sensitized rat trachea through the inhibition of cholinergic nerves on the smooth muscle.

	Introduction

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Although commonly used in general anesthesia for asthmatic patients, fentanyl is reported to increase airway resistance (1–3), and enhance the bronchoconstrictictor effects of serotonin (5-HT) (4). However, fentanyl and opioids are also reported to attenuate contractile responses to electrical field stimulation (EFS) (5,6), acetylcholine (ACh) (5), and carbachol (7) in isolated airways.

Antigen induces an immunoglobulin E (IgE)-dependent activation of mast cells to release mediators that induce bronchoconstriction (8). Antigen-induced contractile responses in isolated airways are reported in mice (9) and rats (10,11), and these reports suggest that the antigen-induced tracheal contraction is mediated by 5-HT and ACh (9,11). Immunization increases antigen-specific IgE levels in the ovalbumin (OA)-sensitized asthma model (8).

Although fentanyl affects the airway, the mechanisms by which it does so are not fully understood. This study was performed to determine the effects of fentanyl on antigen-induced contractile responses using OA-sensitized rat trachea in vitro and in vivo.

	METHODS

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Experiments were conducted under guidelines approved by the Institutional Animal Care Committee. Fifty-five male Wistar rats weighing 200–250 g were used for experiments in vitro, and 56 male Wistar rats weighing 300–350 g were used for experiment in vivo.

Sensitization Procedure
The rats were sensitized with a single intraperitoneal injection of 10 µg of OA mixed with 10 mg of aluminum hydroxide as adjuvant. Fourteen days later, anaphylactic responses were tested. This period was chosen on the basis of the work of Coleman et al. (12), who demonstrated that, in rats, the levels of circulating IgE antibodies increased rapidly 7–14 days after the intraperitoneal injection of OA. Nonsensitized rats were injected with the same volume of normal saline intraperitonealy, and were also used 14 days later.

Anaphylactic Contractile Response In Vitro
The rats were exsanguinated under anesthesia with intraperitoneal pentobarbital, and the trachea was rapidly isolated. The trachea was cut into 3-mm-wide rings with a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, UK). In each experiment, eight rings from three rats were used in eight organ chambers. We used only the distal three rings of the trachea, i.e., within 9 mm from the carina, since the contractile responses to OA differ between the proximal and distal segments (10). The tracheal rings were placed between two stainless steel hooks in a 5-mL water-jacketed organ chamber (Kishimotoika, Kyoto, Japan) containing Krebs–Henseleit solution (composition: NaCl 118 mM, KCl 4.7 mM, CaCl₂ 1.3 mM, KH₂PO₄ 1.2 mM, MgSO₄ 1.2 mM, NaHCO₃ 25 mM, glucose 11 mM, Na₂-EDTA 0.05 mM). The solution was continuously aerated with 95% O₂ and 5% CO₂, and the temperature was maintained at 37°C. Isometric tensions were measured using an isometric transducer (Kisimotoika, Kyoto, Japan) and recorded using a MacLab system (Milford, MA). The resting tension was adjusted periodically to 0.5 g during the equilibration period. The rings were washed every 15 min and reequilibrated to resting tension for 60 min.

First, after the equilibration period, the nonsensitized rat tracheal rings were challenged with OA (final concentration, 50 µg/mL). To the sensitized tracheal rings, fentanyl was added in final concentrations of 0, 0.001, 0.01, 0.1, and 1 µM. Fifteen minutes later, OA was added in a final concentration of 50 µg/mL and ring tension was measured for 15 min. Thereafter, tissues were washed with fresh Krebs–Henseleit solution three times in 15 min, and then OA was added again at the same concentration as mentioned above and ring tension was measured. In addition, to examine whether the effect of fentanyl is mediated by the µ-opioid receptor, tracheal rings of sensitized rats were pretreated with naloxone (10 or 100 µM) before fentanyl and OA were added.

Second, the role of cholinergic nerves and 5-HT in the contraction, and the effects of fentanyl were examined with tetrodotoxin and ketanserin. Tetrodotoxin, a sodium channel blocker, is used as the denervating drug in the isolated tracheal experiments. Because parasympathetic postganglionic neurons are considered to be close to the targeted end-organ, tracheal rings used in the present study would contain parasympathetic postganglionic neurons. In the previous study, tetrodotoxin, 1 µM, abolished the contraction induced by EFS (5). Thus, we used tetrodotoxin, 1 µM, to completely block the parasympathetic nervous system in isolated trachea. On the other hand, we used ketanserin, a 5-HT receptor antagonist, to evaluate the effect of fentanyl on the contraction mediated by 5-HT. Eum et al. (9) reported that the mechanism of OA-induced airway smooth muscle contraction would involve the 5-HT receptor. After the equilibration period, drugs were administered at a 15 min interval in five separate experiments as follows: (1) tetrodotoxin, 0.1 µM, followed by fentanyl, 1 µM, (2) tetrodotoxin, 0.1 µM, followed by the solution, (3) ketanserin, 1 µM, followed by the solution, (4) ketanserin, 1 µM, followed by fentanyl, 1 µM, (5) ketanserin, 1 µM, followed by tetrodotoxin, 0.1 µM. Another 15 min later, OA, 50 µg/mL, was added and the ring tension was measured.

Effects of Fentanyl on the OA-Induced Airway Constriction In Vivo
The rats were anesthetized with intraperitoneal pentobarbital, 50 mg/kg, and underwent a tracheotomy. The trachea was cannulated and connected to a ventilator (EVM-50A type1, Aika Medical, Tokyo, Japan). Polyethylene catheters were inserted into the left femoral artery for recording arterial blood pressure and into the left femoral vein for drug administration. No experiment lasted longer than 90 min, during which time surgical anesthesia was maintained without the need for supplementary anesthesia. Body temperature was maintained at 37°C and mean arterial blood pressure was maintained at 110 ± 30 mm Hg with the infusion of warmed lactated Ringer's solution. Airway pressure, airflow, and end-tidal CO₂ tension were measured at a sampling rate of 100 Hz (CO₂SMO⁺, Novametrix Medical Systems, Wallingford, CT). The rats were paralyzed with a continuous infusion of vecuronium, and their lungs were inflated to 30 cm H₂O for the first minute to prevent atelectasis. Thereafter, the lungs were ventilated at a rate of 40 per minute with a tidal volume of 10 mL/kg. Inspiratory time was 0.6 s and the end-inspiratory plateau ratio was adjusted to 30%. Thirty-five sensitized rats were divided into five groups. One of the groups received 1.5 MAC of sevoflurane (3.6% end-tidal concentration) (13). Each of the other groups received IV normal saline, fentanyl, 3 or 10 µg/kg, or atropine, 0.01 mg/kg, respectively. Nonsensitized rats received 1.5 MAC of sevoflurane, normal saline or fentanyl, 10 µg/kg. Ten minutes later, OA, 1 mg/kg, was administered IV and the measurement was continued for another 15 min. Respiratory system resistance (Rrs) was calculated from the obtained Airflow (V) and the difference between end-inspiratory peak airway pressure (P_peak) and plateau pressure (P_plat): Rrs = (P_peak – P_plat)/V. The Rrs was calculated at 1 min before drug administration (T₁), 1 min before OA administration (T₂), the time of maximal airway pressure obtained after OA administration (T_max), and 10 min after T_max (T₃). Five respiratory cycles were averaged to provide one data point.

Data Analysis
Values were expressed as means ± sd. The results were analyzed by one-way analysis of variance. A value of P < 0.05 was considered statistically significant.

	RESULTS

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The contraction induced by OA was 1.6 ± 0.29 g in the sensitized tracheal rings, while OA did not change the tension of nonsensitized tracheal rings. Fentanyl dose-dependently attenuated the OA-induced contraction of sensitized rat trachea (Fig. 1). Figures 2a and b show typical recordings for the experiments with 0 and 1 µM fentanyl followed by OA, respectively. The second application of OA performed after washing out the first did not change the tension. Figure 3 shows the effect of naloxone on the attenuation by fentanyl. Naloxone, 10 µM, partly reversed this effect of fentanyl, but could not fully reverse it even at a higher dose. Figure 4 shows the effects of tetrodotoxin (a) and ketanserin (b) on the contractile response. Tetrodotoxin, 0.1 µM, attenuated the contraction induced by OA (0.90 ± 0.12 g), and fentanyl had no further effect on the contraction in the presence of tetrodotoxin (0.95 ± 0.12 g). Ketanserin, 1 µM, also attenuated the contraction induced by OA (0.54 ± 0.12 g), and the contractile response to OA was nearly abolished by 1 µM ketanserin in the presence of 0.1 µM tetrodotoxin or 1 µM fentanyl. Fentanyl had no effect on Rrs in either the sensitized or nonsensitized rats, but sevoflurane decreased Rrs at T_max and T₃ compared with those of fentanyl-treated group in nonsensitized rats. OA did not change Rrs in the nonsensitized rats, but increased Rrs in the sensitized rats at T_max. The increase in Rrs was inhibited by fentanyl, 10 µg/kg, atropine and sevoflurane (Table 1).

View larger version (24K): [in this window] [in a new window]   Figure 1. The effects of fentanyl on the contraction induced by ovalbumin (OA), 50 µg/mL. **P < 0.001, versus control. Thirty tracheal rings from 10 rats were used.

View larger version (13K): [in this window] [in a new window]   Figure 2. Typical recordings of contractile response induced by ovalbumin (OA) in the absence (a) or presence (b) of fentanyl 1 µM.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of naloxone on the attenuation of ovalbumin (OA)-induced contraction by fentanyl (1 µM). *P < 0.05, **P < 0.001, vs. control. The data for the control and fentanyl were traced from Figure 1. Twelve tracheal rings from four rats were additionally used.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effects of tetrodotoxin (a) and ketanserin (b) on the contractile response induced by ovalbumin (OA). Tetrodotoxin: 0.1 µM, fentanyl: 1 µM, ketanserin: 1 µM. **P < 0.001, versus control. P < 0.05, P < 0.001, versus ketanserin. The data for the control was traced from Figure 1. Thirty tracheal rings from 10 rats were additionally used.

	DISCUSSION

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The present results show that fentanyl attenuates the OA-induced contraction of sensitized rat trachea. It was reported that the OA sensitization increased the circulating and tissue IgE levels in rats, and that the OA challenge produced marked mast cell degranulation (12,14,15). Thus, the OA-induced contraction of rat trachea in the present study would be due to an immediate type of allergic reaction mediated by IgE, which triggers release of mediators from mast cells.

The possible mechanisms involved in the inhibitory action of fentanyl are as follows (Fig. 5). First, fentanyl may inhibit the release of ACh from cholinergic nerves in the smooth muscle. Toda and Hatano (5) reported that cholinergic nerves of smooth muscle mediated the contractile response to EFS in isolated trachea, and this response was abolished by tetrodotoxin, 0.1 µM. In the present study, when cholinergic nerves were blocked beforehand by tetrodotoxin, fentanyl had no effect on the OA-induced contraction. It suggests that fentanyl would inhibit the release of ACh from cholinergic nerves in the smooth muscle of OA-sensitized rats. Nagase et al. (13) reported that methysergide, a 5-HT receptor antagonist, and ketanserin inhibited the OA-induced contraction of bronchial rings of sensitized rat similar to our model. Eum et al. (9) also investigated the effect of methysergide and atropine on a similar model with mice. They concluded that OA provoked bronchoconstriction in sensitized animals by stimulating the release of 5-HT which, in turn, acted via the cholinergic mediator, ACh. In the present study, similar results were observed, i.e., ketanserin attenuated the OA-induced contraction, and the contractile response to OA was nearly abolished by 1 µM ketanserin in the presence of 0.1 µM tetrodotoxin or 1 µM fentanyl. Thus, these results support the notion that fentanyl attenuates OA-induced contraction through the inhibition of ACh release from the cholinergic nerves.

View larger version (17K): [in this window] [in a new window]   Figure 5. Schematic diagram of the ovalbumin (OA)-induced contraction of airway smooth muscle. The possible effects are shown in arrows, and broken arrows mean inhibition of the effects. IgE: immunoglobulin E, ACh: acetylcholine, 5-HT: serotonin receptor, M2: muscarinic M2 receptor, M3: muscarinic M3 receptor.

Second, fentanyl may inhibit the release of ACh from cholinergic nerves in smooth muscle through opioid receptors. The inhibition of cholinergic neurotransmission by opioids was previously reported in humans (16), and animals (6,17). In the present study, naloxone partly reversed the effects of fentanyl on the contraction induced by OA. This observation is similar to the effect of naloxone on the attenuation by fentanyl of contraction induced by EFS, reported by Toda and Hatano (5). Since there are reports of naloxone-insensitive inhibition of the contraction of airway smooth muscle by endogenous opioids (18,19), further examination is required to clarify the role of the µ-opioid receptor, or other opioid receptor subtypes, in this inhibition of the contractile response.

Third, fentanyl may inhibit the release of mediators from mast cells, resulting in an attenuation of the OA-induced contraction of sensitized rat trachea. In the present study, the first challenge of OA induced only a brief contraction in sensitized rat trachea and the second challenge, performed after washing out the first application of OA, did not elicit the contraction. These results indicate that the number of mast cells in the isolated trachea is limited, and that the mediators are exhausted when the second challenge of OA is performed. If the release of mediators were inhibited by fentanyl, the mediators would be saved and the second challenge of OA could elicit the contraction. Thus, it is unlikely that fentanyl inhibits the release of mediators from mast cells.

Fourth, fentanyl may affect the novel mechanism derived from the sensitization-induced change of the smooth muscle structure or the function of ACh receptors. Some previous reports suggest that sensitization changes the smooth muscle structure (20) or the function of the ACh receptor (21). However, in our previous study, the concentration of ACh or 5-HT to cause a 50% effect for contraction in the sensitized rat trachea was not significantly different from that in the nonsensitized one (22). These findings indicate that the sensitization by OA would not change the smooth muscle structure or the function of ACh receptors in our model.

Contrary to the findings in vitro, some previous in vivo studies reported that fentanyl induced airway constriction in humans (1–3) and animals (4). In the present study, fentanyl did not increase the Rrs either in sensitized or nonsensitized rats. This contradiction might be due to differences in species, model, bronchoconstrictor stimulus or measurement techniques. However, OA increased the Rrs at Tmax in sensitized rats, an increase attenuated by fentanyl and atropine. This result supports our in vitro finding that although the cholinergic nervous system is activated in the antigen-induced airway, fentanyl would inhibit this action. Consequently, in the hyperresponsive condition, fentanyl would attenuate an antigen-induced asthmatic attack. Considering the clinical dose, atropine appears to be more effective than fentanyl, but fentanyl would be a relatively useful IV analgesic for patients with antigen-induced asthma. In the anesthetic management of asthmatic patients, volatile anesthetics such as sevoflurane are recommended for the effect of bronchodilatation. In the present study, sevoflurane decreased Rrs at T_max and T₃ compared with fentanyl in nonsensitized rats. Moreover, in sensitized rats, sevoflurane produced a nearly 60% reduction in the increase of Rrs at T_max from the normal saline-treated group. Although the effect of fentanyl seems to be less than that of sevoflurane, fentanyl would have a significant effect in inhibiting OA-induced airway constriction.

In conclusion, fentanyl attenuates the OA-induced contractile response of the trachea and OA-induced increase in Rrs in the sensitized rat. The mechanism involved in fentanyl's action would be the inhibition of the OA-induced activation of cholinergic nerves in airway smooth muscle.

	Footnotes

 
Accepted January 18, 2007.

Supported by grant 15591638 and 18791084 from the Ministry of Education, Science and Culture, Japan.

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