INTRODUCTION

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Five decades after Knowlton and Larrabee (1) first described airway rapidly adapting receptors (RARs), questions remain concerning the contribution of these receptors to respiratory control despite progress in elucidating their functional roles (2). During the last two decades the opinion regarding whether RARs can cause bronchoconstriction (3, 4) was controversial. RARs have been suggested to cause bronchoconstriction since, under several conditions, their activation is accompanied by airway constriction (5). In addition, airway constriction is believed to stimulate RARs. Thus, a positive feedback system was postulated (6). For example, if activation of RARs causes bronchoconstriction, bronchoconstriction could cause further stimulation of RARs and further bronchoconstriction. However, no convincing evidence directly proving that activation of RARs causes airway constriction has been reported. Therefore, the question of RAR-mediated bronchoconstriction remains open.

Although our understanding of the functional role of RARs is limited, evidence suggests that RARs are the most important receptors found in the airway for the initiation of cough (2, 8). The theory that RARs cause bronchoconstriction has been periodically questioned (8, 11). It remains controversial that other modes of activating RARs such as decreasing lung compliance or increasing tidal volume do not evoke cough. One possible explanation is that the firing pattern of RARs evoked under various types of activation is different. When RARs are stimulated by mechanical probing, a large surge of discharges occurs. However, stimulation of RARs by decreasing lung compliance or increasing tidal volume causes a relatively regular discharge pattern with peak activity during each peak airway pressure. Research also indicates that the two reflex reactions, cough and bronchoconstriction, do not necessarily go hand in hand during chemical stimulation (9). Recently, however, the theory that RARs cause airway constriction has been challenged. Stimulation of RARs by decreasing lung compliance does not seem to produce bronchoconstriction in dogs (14). In the above study, RARs in the trachea and carina were not exposed to the stimuli because a Kortemere tube was inserted in the left and right bronchi. It is possible that RARs in different regions may evoke different reflexes, that is, the RARs in the carina and large bronchi may cause cough, whereas those in small airways mainly affect breathing pattern (15). Thus, the RARs in these unexposed regions may be responsible for bronchoconstriction.

In the present study, we used a different preparation, and stimulated a larger population of RARs to further test the hypothesis that stimulation of RARs causes bronchoconstriction. Experiments were conducted in anesthetized rabbits, a species frequently used to assess pulmonary reflexes. RARs in the lower trachea, carina, and peripheral airways were stimulated by decreasing lung compliance. We directly monitored airflow resistance to determine whether the stimulation produces bronchoconstriction. No changes in resistance were detected during the observation period, suggesting that bronchoconstriction (tracheal constriction is not examined in the present study) is not an important reflex component of RAR stimulation. We also activated RARs by mechanically probing the trachea, which is known to cause a surging discharge of RARs and causes cough. Our results show that cough was not accompanied by increases in lung resistance. This supports our previous conclusion that activation of RARs does not evoke bronchoconstriction (14).

	METHODS

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General

Experiments were conducted on male New Zealand white rabbits. The rabbits were initially injected intravenously with a mixture of ketamine (37.5 mg/kg) and xylazine (5 mg/kg). Surgical anesthesia was maintained by additional doses (10 mg) of sodium pentobarbital given intravenously. After completion of the surgery, anesthesia was maintained with -chloralose (1%) and urethan (10%) by intravenous infusion (1.4 ml/h), which were doses of about 5.6 and 56 mg/kg/h for chloralose and urethan, respectively. Measurements (see below) were obtained at least 90 min after the last dose of pentobarbital was given.

The trachea was cannulated low in the neck. The lungs were ventilated by a small animal ventilator (Model 683; Harvard Apparatus Co., South Natick, MA) in which the expiratory outlet was connected to a positive end-expiratory pressure (PEEP) of 3 to 4 cm H₂O. Tidal volume was set at 10 ml/kg body weight. Ventilatory frequency on average was 15 to 19 cycles/min. Blood PCO₂ and PO₂ were maintained at 50 ± 2 and 96 ± 7 mm Hg, respectively. The chest was opened by a midline incision. The opening was covered with a cloth soaked with saline to prevent drying of the tissue. A femoral artery was cannulated to monitor blood pressure and to take arterial blood samples to monitor blood gas. Blood pressure, airway pressure, airflow, and tidal volume were recorded by a Grass recorder (Grass Instruments, Quincy, MA).

Pulmonary mechanics, i.e., dynamic lung compliance and total lung resistance, were estimated by a modification of the method of Amdur and Mead (16). Air flow was measured by a pneumotachograph connected to a Statham differential pressure transducer (PM15; Statham Instruments, Oxnard, CA). Airway pressure was monitored by a pressure transducer attached to a side arm of the tracheal tube. Airflow and airway pressure signals were connected to an analogue Pulmonary Function Analyzer (LS-20; Buxco Electronics, Sharon, CT), which provided a continuous breath-by-breath analysis of airway pressure, flow, tidal volume, resistance, and dynamic lung compliance. In the report, the resistance from the tracheal tube was subtracted from the measured resistance. The calibration scale for the analyzer was obtained by previous manual calculations according to the method of Amdur and Mead (16).

Stimulation of RARs

To determine whether an increase in RAR activity can reflexly constrict the low airways, we mechanically stimulated RARs and monitored total lung resistance. Each experiment was started by hyperinflating the lungs with three tidal volumes above functional residual capacity. RARs were then stimulated by the following method: The lungs were deflated to a lower volume by disconnecting the outlet tube of the respirator with the PEEP for 10 to 15 ventilatory cycles. The PEEP was added back at the end of 10 to 15 ventilatory cycles. For simplicity, this procedure is referred to as the PEEP maneuver (Figure 1) in the following text. This PEEP maneuver increases airway pressure swings significantly. Whereas the PEEP maneuver is not absolutely specific for RARs, it has been shown to favor their stimulation (17). After the PEEP maneuver, RAR activity increases substantially, whereas C-fiber activity and the averaged PSR activity do not change (see Table 1 in Reference 17). At the end of an experiment, the lungs were hyperinflated with three tidal volumes, which is known to restore RAR activity to the control level (17). In addition, RARs were stimulated by strokes of a cotton tip applicator stick in the upper portion of the trachea through the opening above the point of tracheal cannulation. The cotton tip was soaked with normal saline before applying for mechanical stimulation. Care was taken to ensure that the force and duration of stimulation remained as constant as possible throughout the experiments.

View larger version (118K): [in this window] [in a new window]   Figure 1.   An illustration of PEEP maneuver. From top down the traces are: VT tidal volume; Paw, tracheal pressure, and , airflow. Period A is control; period B is an experimental period. Note that after removal and then replacement of PEEP, airway pressure swing is increased because of a decrease in dynamic lung compliance. The compliance is restored (airway pressure swing returned back to the control level) after hyperinflation of the lung with three tidal volumes.

Right Atrial Injection of Histamine

To confirm that the vagal reflex for airway constriction was intact in our experimental preparation, we monitored lung resistance in response to histamine (10 µg in 0.2 ml, flushed by 0.5 ml saline) injected into the right atrium. Histamine is known to stimulate bronchial and pulmonary C-fibers as well as RARs (18). The recorded respiratory variables were averaged over 30 s as controls. The responses were measured at 10-s intervals after injection. We examined the histamine effects in two separate groups of rabbits with a different level of anesthesia. One group received anesthetic as stated in METHODS; the other group received fives times more anesthetic (1% -chloralose and 10% urethane). Both groups received histamine injection before and after vagotomy. At least 20 min were allowed between injections of histamine.

Effects of Vagotomy and Atropine

It is known that the vagal efferent nerves supply airway smooth muscle with a tonic tone. To confirm that our preparation has basal vagal tone, we compared lung mechanics before and after bilateral vagotomy in nine rabbits. We also compared lung mechanics before and after intravenously administered atropine (1 mg) in four rabbits.

Electrical Stimulation of Vagal Efferents

To confirm that signal transmission between vagal efferents and airway smooth muscle was intact, in seven rabbits that underwent bilateral vagotomy, we electrically stimulated the peripheral end of the severed vagus nerve while continuously monitoring airway pressure, airway resistance, and dynamic compliance. The nerve was stimulated at an intensity of a constant current of 10 mA with a frequency of 10 Hz and a duration of 1 ms (14) until a peak response was achieved (usually within 1 min).

Effects of Acetylcholine

Acetylcholine (ACh) is known to cause airway constriction and thus to increase airway resistance. To confirm that we could detect changes in airway resistance, we examined lung mechanics before and after intravenous administration of ACh (0.1 ml of 10⁴ M to 10³ M) in 10 additional rabbits with intact vagi.

Protocols

Our experiments usually started about 1 h after completion of surgery. This was more than 90 min after the last dose of pentobarbital and when anesthesia was maintained with -chloralose and urethan. The rabbits were lightly anesthetized. Respiratory reflexes such as the cough and bronchoconstriction are intact under these conditions. We started the experiments with a hyperinflation of the lung to three tidal volumes above functional residual capacity. This procedure standardizes lung mechanics (14). Then, we examined the response to PEEP maneuver, mechanical stimulation of the tracheal mucosa, or injection of histamine.

Data are presented as mean ± SEM. Student's paired t test was used to compare two groups of data. Wilcoxon's paired test was used to compare the percentage change of a variable from its control value. A p value less than 0.05 was considered as statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

During control, arterial blood pressure was 65 ± 2.6 mm Hg. The baselines for the airway pressure swing, dynamic lung compliance, and lung resistance were 8.7 ± 0.7 cm H₂O, 3.4 ± 0.2 ml/cm H₂O, and 0.045 ± 0.004 cm H₂O/ml/s (n = 15), respectively. Alteration of lung mechanics by removing and replacing PEEP (PEEP maneuver; see Figure 1) increased the airway pressure swing (16.4 ± 4% above control; n = 15, p = 0.0016), which was accompanied by a decrease in lung compliance (15.2 ± 2.8% below control; p = 0.0002) without a significant influence on the resistance (+8.0 ± 4.4%; p = 0.85). In seven of the 15 rabbits, we examined the effects of the PEEP maneuver on these variables before and after bilateral vagotomy. Vagotomy did not significantly alter the pattern (Figure 2), suggesting that vagal efferent control on broncholar smooth muscle is not a part of the reflex evoked by the PEEP maneuver.

View larger version (11K): [in this window] [in a new window]   Figure 2.   Effects of PEEP maneuver on airway pressure swing, dynamic lung compliance, and lung resistance before (left) and after (right) bilateral vagotomy (n = 7). The percentage changes in the above variables were calculated by comparing them with the control period before the maneuver in each case (comparing the Period B with Period A in Figure 1). *p < 0.05. Note that the patterns of response to PEEP maneuver are similar before and after vagotomy.

Effects of Tickling the Airway

We examined the mechanically induced cough in the open-chest rabbit preparation. As expected, tickling the airway evoked cough. Typically, the rabbits showed one or two expiratory movements, manifested as an increase in the positive pressure swing during the inflation phase of the ventilator (Figures 3 and 4). However, respiratory movements also occurred during deflation phase. The mechanically induced cough was not accompanied by an increase in total lung resistance. There were no detectable changes in airflow, airway pressure swing, and total lung resistance during tickling the airway after the rabbit was paralyzed by succinylcholine (Table 1).

View larger version (93K): [in this window] [in a new window]   Figure 3.   Effect of intravenous injection of succinylcholine (1 mg) on changes in breathing during tickling of tracheal mucosa in an anesthetized, open-chest, and artificially ventilated rabbit. From top to bottom, the traces are airway pressure, Paw; airflow, ; arterial blood pressure, BP. The horizontal bar at the bottom denotes the duration of tickling. A, control; B, 2 min after succinylcholine. Note a gradual increase in response; C, 3 min after the end of panel B. Note a complete recovery of cough reflex after neuromuscular blockade wore off.

View larger version (18K): [in this window] [in a new window]   Figure 4.   Effect of mechanical stimulation of trachea (tickling) on lung mechanics in open-chest rabbits (n = 8). Fpi, peak inspiratory airflow; Fpe, peak expiratory airflow. All differences are significant (p < 0.001).

Effects of Histamine

In six lightly anesthetized rabbits and eight deeply anesthetized rabbits, we examined the effects of intravenous injection of histamine on airway resistance. Histamine increased total lung resistance, which was more pronounced with vagal nerves intact (Figure 5). This suggests involvement of a neural reflex component; however, this increase in resistance was only present if the animal was lightly anesthetized. The neural reflex component could not be detected during deep anesthesia (Figure 5).

View larger version (14K): [in this window] [in a new window]   Figure 5.   Right atrial injection of histamine (10 µg) evoked reflex increase in airway resistance under light anesthesia. Bilateral vagotomy greatly suppressed the increase in airway resistance after histamine. The difference in responses before and after vagotomy is significant (p = 0.02). Note that during deep anesthesia, the response to histamine were the same before and after vagotomy.

Effects of Vagotomy and Atropine on Lung Mechanics

In nine rabbits, we examined the effects of vagal efferent activity on lung mechanics. We found that a basal airway tone was exerted by vagal efferent activity since bilateral vagotomy decreased both airway pressure swing and the resistance. Percent changes in these variables after vagotomy are illustrated in Figure 6A. The same trend was also observed in four additional rabbits after intravenous administration of atropine (Figure 6B). These changes approached, but did not reach, statistical significance between the controls and after atropine (p = 0.06 for the three variables), probably because of the small sample size.

View larger version (15K): [in this window] [in a new window]   Figure 6.   Effects of bilateral vagotomy (n = 9) and intravenous injection of atropine (1 mg) (n = 4) on lung mechanics. Note that both vagotomy (A) and atropine (B) decreased airway pressure swing and lung resistance, and increased lung compliance. *p < 0.016.

Effects of Electrical Stimulation of Vagal Efferents

In seven rabbits, we electrically stimulated the efferent ends of the severed vagus nerves. In five rabbits we stimulated both right and left vagus nerves, respectively, and in the remaining two rabbits we stimulated only the left vagus nerve. The stimulation increased mainly the resistance, although it also increased airway pressure swing and decreased dynamic compliance (Figure 7A).

View larger version (17K): [in this window] [in a new window]   Figure 7.   Effects of electrical stimulation (10 Hz, 1 ms, 10 mA) of the vagus nerves (n = 12) and intravenous injection of acetylcholine (n = 9) on the lung mechanics. Data from electrical stimulation (A) are pooled from seven rabbits. Five rabbits have 2 data points, with each from the stimulation of each vagus; whereas in the other two rabbits only one nerve was stimulated. Note that the electrical stimulation mainly affects lung resistance. *p = 0.0022. Note that acetylcholine (B) also has a predominant effect to increase resistance. *p = 0.0051.

Effects of ACh on Lung Mechanics

In 10 additional rabbits, we examined the effects of ACh on lung mechanics. Like electrical stimulation of vagal efferents, intravenous injection of ACh mainly increased the resistance. ACh also increased airway pressure swing and decreased lung compliance, but to a much lesser extent than the increased resistance (Figure 7B).

	DISCUSSION

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In open-chest, artificially ventilated rabbits that were lightly anesthetized, we tested the hypothesis that activation of RARs can reflexly constrict airways. We measured lung resistance and compliance during maneuvers known to stimulate RARs. The values of lung resistance and compliance obtained in this study are similar to those reported for rabbits (5). We stimulated RARs by stroking the trachea and by decreasing lung compliance, which favors activation of RARs without changing mean activities of PSRs and C-fibers (17). Because we did not find an increase in resistance during mechanical stimulation of RARs, we conclude that bronchoconstriction is not a major reflex component of RAR stimulation in rabbits.

There is a report (19) in which the response of tracheal smooth muscle tension is shown to be more sensitive than that of pulmonary resistance to methacholine or norepinephrine challenges. In our study, the trachea was cannulated low in the neck, and the upper trachea was excluded from the resistance measurement. Therefore, our results do not exclude the possibility that stimulation of RARs causes tracheal constriction. In addition, in our experiments it still could be argued that the anesthesia, even though light, could have affected the sensitivity of the airway resistance measurement differently from that in a conscious or decorticated animal. However, our results demonstrate that our preparation was capable of reflexly increasing airway tone in response to histamine and that the cough reflex was evoked by activating pulmonary afferents. Under these conditions, we could not observe an increase in lung resistance during activation of RARs.

In open-chest, artificially ventilated rabbits (20), removal followed by replacement of PEEP (PEEP maneuver) increased airway pressure swing. This increased pressure swing could be interpreted as a decrease in dynamic lung compliance or, alternatively, as an increase in airway resistance. Because this maneuver strongly stimulates RARs, delineation of these two possible components will help us to understand the mechanisms of mechanical activation of RARs. In the present study, we directly measured both dynamic compliance and lung resistance before and after the PEEP maneuver. We found that the PEEP maneuver decreased lung compliance without increasing the resistance. These results confirm that the PEEP maneuver stimulates RARs by decreasing dynamic lung compliance (20), and not by increasing resistance.

In the present experiments, mechanical stimulation of RARs in the rabbit did not increase lung resistance and, thus, does not appear to cause airway constriction. Some existing data are consistent with our conclusion. Pisarri and colleagues (21) found in dogs that application of nonisotonic solution into airways stimulated RARs and C-fibers. Whereas water mainly activates RARs, hypertonic solution mainly activates C-fibers. If RARs have a greater effect than C-fibers on bronchoconstriction, we anticipate a larger increase in tracheal tension after applying water. However, their data have shown that the increased tension was similar when water and 1,200 mosmol/L saline (which both caused activation of C-fibers to a similar extent) were applied. The increased tension was greatest when 2,400 mosmol/L saline (when the C-fibers were greatly stimulated) was applied. These results suggest that C-fibers are responsible for airway constriction. If any airway constriction could be ascribed to RARs, it would be small. On the other hand, Kappagoda and colleagues (22) reported that stimulation of RARs by pulmonary congestion can reflexly induce a vagally mediated tracheal contraction. The contraction was abolished by cooling the vagus nerve to 8 to 9° C, which blocks most RARs. It is reasonable to interpret that contraction was due to activation of RARs. However, pulmonary congestion also stimulates pulmonary C-fibers (23). The temperature that blocks RARs also reduces activity of C-fibers (24). Therefore, the contraction could also be interpreted as a result of activation of C-fibers.

The most important receptors responsible for cough seem to be RARs, although other airway receptors also contribute to the cough reflex (2, 8, 10, 25). It is widely accepted that stimulation of RARs by mechanically tickling the trachea can cause cough and bronchoconstriction. Experimentally, directly touching the receptor field evokes a surging discharge of the RARs. If it is the surging discharge of RARs responsible for both cough and bronchoconstriction, failure to observe both cough and increases in lung resistance during activation of RARs by decreasing lung compliance could be due to the absence of this surge. In the present study, RARs in the upper portion of trachea were stimulated by direct mechanical strokes with a cotton-tipped applicator, we did not detect an increase in resistance while cough was elicited. The absence of an increase in resistance was confirmed by the abolition of any increases in airway pressure after paralysis. It suggests that bronchoconstriction is not an important reflex component of the RARs. It can always be argued that our method of measuring lung resistance is not sensitive enough to detect the increase. However, if there is an increase in resistance, it must be small because our method detected neurally mediated changes in resistance to histamine and vagotomy.

Airway tone is under neural and humeral regulation (3, 26). Activation of pulmonary afferents can influence airway tone (3, 9). Activation of airway C-fibers increases airway tone in dogs (2), whereas activation of slowly adapting stretch receptors relaxes airway smooth muscle (27). Activation of RARs is believed to cause airway constriction, but there is no convincing evidence showing a direct cause-effect relationship between stimulation of RARs and an increase in airway tone. The perception that RARs cause bronchoconstriction is mainly based on the observation that under several conditions, activation of RARs is accompanied by airway constriction (5, 6). As pointed out by Coleridge and Coleridge (13), such a conclusion cannot be drawn by indirect association. In the present study, our data clearly showed that the airway was under the influence of vagal nerve efferent activity. Vagotomy or atropine decreased lung resistance (Figure 6), and electrical stimulation of vagal efferents increased resistance (Figure 7). These responses together with the changes in lung mechanics recorded after intravenous injections of ACh indicate our recording system is able to detect changes in lung resistance. In addition, our preparation had an intact vagal afferent pathway as demonstrated by the existence of cough upon mechanical stimulation of RARs in an upper portion of the trachea.

More importantly, our preparation demonstrated that bronchoconstriction in response to right atrial injection of histamine has a vagal reflex component. In our study, the decreased responses to histamine after vagotomy cannot be explained by tachyphylaxis because under the same circumstances the responses to histamine before and after vagotomy were the same under deep anesthesia. Thus, the difference in bronchoconstriction elicited from histamine injections before and after vagotomy is a vagally mediated response. Although histamine activates both C-fibers and RARs (18), in the present study, activation of C-fibers probably contributed to the bronchoconstriction induced by histamine. This notion is consistent with the observation of Karczewski and Widdicombe (8), in which histamine-induced increase in airway resistance became smaller after cooling the vagus nerves to 8° C, and even small after further vagotomy, suggesting that C-fibers played a role in this vagally mediated airway constriction. Our studies indicate that RARs do not play a significant role in bronchoconstriction, unless the bronchoconstrictive response evoked from RARs is much more susceptible to anesthesia than the bronchoconstrictive response from C-fiber stimulation, and than the cough response from stimulation of RARs. Once again, our results cannot exclude the possibility that activation of RARs can cause tracheal constriction, which is not tested in the present study.

Although bronchoconstriction may often be associated with cough, they are not necessarily triggered and mediated by the same afferents (2, 8). These two responses can be dissociated by aerosol of hypotonic saline (28) and distilled water (29). Thus, a question is raised whether the two responses, cough and bronchoconstriction, are mediated through two different sets of receptors. In the present study, we have further shown that these two responses dissociate during the stimulation of tracheal mucosa by mechanical means.

Recently, Yu and Mink (14) could not detect any increase in airway tone in one lung while RARs in the other lung were stimulated by altering lung mechanics in dogs whose lungs were separately ventilated. In the present study, we used a different preparation with a different anesthetic and a different species to test the same hypothesis. We used the rabbit, which has strong vagal airway tone (30) and possesses a cough reflex. With this preparation we attained similar results and reached the same conclusion that mechanical activation of RARs does not cause significant bronchoconstriction.

Our study did not demonstrate that bronchoconstriction occurs in response to mechanical stimulation of RARs. Because it has also been reported that contraction of the trachealis muscle does not activate RARs (31), it is unlikely that RARs form a positive-feedback loop facilitating airway constriction.