Journal of Applied Physiology AJP: Endocrinology and Metabolism
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J Appl Physiol 91: 1645-1654, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 4, 1645-1654, October 2001

Enhanced lung C-fiber responsiveness in sensitized adult guinea pigs exposed to chronic tobacco smoke

Dale R. Bergren

Department of Biomedical Sciences, School of Medicine, Creighton University, Omaha, Nebraska 68178


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Tobacco smoke (TS) exposure induces bronchoconstriction and increases airway secretions and plasma extravasation in certain sensitive individuals, particularly those with asthma. C-fiber activation also induces these effects. Although the mechanism by which chronic TS exposure induces airway dysfunction is not well understood, TS exposure may enhance C-fiber responsiveness. To investigate the effect of chronic TS exposure on C-fiber responsiveness to capsaicin and bradykinin, especially in atopic individuals, we exposed ovalbumin (OA)-sensitized guinea pigs to TS (5 mg/l air, 30 min/day for 7 days/wk) or to compressed air. Nonsensitized guinea pigs were also exposed to either compressed air or TS. Beginning after 120 days of exposure, C fibers and rapidly adapting receptors (RARs) were challenged with capsaicin and bradykinin. TS exposure enhanced sensory receptor and airway responsiveness to both intravenous capsaicin and bradykinin challenge. C-fiber, RAR, and airway responsiveness to capsaicin challenge was greatest in OA-sensitized guinea pigs exposed to TS. OA alone induced capsaicin hyperresponsiveness at 5 µg. Airway responsiveness to bradykinin was also greatest in OA-sensitized guinea pigs exposed to TS. OA alone enhanced C-fiber responsiveness to bradykinin at 5 and 10 µg. C-fiber activation by either agonist appeared direct, whereas RAR activation appeared indirect. Therefore, a mechanism of airway hyperirritability induced by the combination of OA sensitization and chronic TS exposure may include hyperirritability of lung C fibers.

pulmonary; tracheal pressure; C fibers; rapidly adapting receptors; capsaicin; bradykinin; ovalbumin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

CHRONIC TOBACCO SMOKE (TS) exposure increases the risk of developing dyspnea, bronchitis, and asthma in individuals who have never smoked (20). Acute TS exposure also induces pulmonary defense mechanisms, including bronchoconstriction and increased airway secretions and plasma extravasation, in susceptible individuals, especially those with asthma (13). Although the mechanism by which TS exposure induces airway dysfunction is poorly understood, the effects of TS exposure in sensitive individuals are similar to those induced by neurogenic inflammation. Therefore, airway hyperirritability induced by TS exposure may be partially mediated by enhanced neurogenic inflammation. Activation of sensory receptors in the airways called C fibers is reported to mediate neurogenic inflammation (2).

Defense reflexes attributed to lung C-fiber activation are bronchoconstriction, increased airway secretions, vasodilatation, and localized edema (12). Both centrally mediated and local axon reflexes are thought to induce these effects, with the latter involving release of neuropeptides such as substance P and neurokinin A (NKA) from C fibers (25). Few studies have attempted to determine whether C-fiber responsiveness is increased in airway disease or after chronic exposure to irritants such as TS. Although acute TS challenge activates C-fiber endings in the lungs (19), it is not known whether chronic TS exposure alters C-fiber responsiveness in airway disease. Chronic TS exposure increases airway responsiveness to capsaicin (7, 27). Capsaicin is a selective agent for activating C fibers (11, 17). Therefore, chronic TS exposure may enhance C-fiber responsiveness.

Rapidly adapting receptors (RARs) are a second category of sensory receptor in the lungs that has been thought to contribute to reflex bronchoconstriction (21). However, recent studies in guinea pigs suggest that activation of RARs to either histamine or capsaicin is secondary to changes in lung mechanics (4). However, acute TS challenge activates RARs (8), and chronic TS exposure enhances RAR activation by substance P (9) even without demonstrable changes in lung mechanics.

Therefore, I tested the hypothesis that daily exposure to TS increases airway C-fiber responsiveness to mediators known to induce neurogenic inflammation, i.e., those mediators thought to selectively activate airway C fibers, such as capsaicin and bradykinin. In addition, because neurogenic inflammation may become enhanced in atopic individuals, particularly those with asthma, I tested the hypothesis that airway sensitization and TS exposure act in a synergistic manner to either enhance C-fiber responsiveness or decrease the threshold of C-fiber activation. Because there remains a possibility that RARs affect airway tone and that RARs are stimulated by capsaicin (22), I also tested the hypothesis that chronic TS exposure alters RAR responsiveness to capsaicin.


    METHODS AND MATERIALS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Animals. The Creighton University Animal Use Committee approved the study. Male Hartley guinea pigs (Harlan, Minneapolis, MN) weighing ~300 g at the time of purchase were housed in the Creighton University School of Medicine Animal Resource Facility with food and water provided ad libitum.

Airway sensitization. Two days after arrival, a randomly selected group of 22 guinea pigs were injected intraperitoneally with ovalbumin (OA; 10 µg) and aluminum hydroxide (100 mg) in distilled water (0.5 ml). A booster injection of 10 µg OA in 0.5 ml distilled water was administered 12 days later as per Andersson (1). Another 17 guinea pigs were injected with the OA vehicle and remained nonsensitized (NS) to OA. Airway responsiveness to OA was determined at 30, 60, and 90 days of the exposure period in the OA-sensitized guinea pigs. NS guinea pigs were challenged with the highest dose of OA only once. OA aerosol challenge induced airway responsiveness in OA-sensitized guinea pigs but not in vehicle-treated guinea pigs (Fig. 1). The data presented were obtained 30 days after administration of either OA or its vehicle.


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  		Fig. 1.   Ovalbumin aerosol challenge (30 s) of the airways in ovalbumin-sensitized guinea pigs exposed to tobacco smoke (OA-TS; n = 12), ovalbumin-sensitized guinea pigs exposed to compressed air (OA-A; n = 10), and nonsensitized guinea pigs exposed to compressed air (NS-A; n = 8). Values are means ± SE. Airway responsiveness is determined as the peak increase in enhanced pause (Penh) units (which are equal to pause × peak expiratory pressure/peak inspiratory pressure) within 5 min of OA aerosol administration (*P < 0.05 vs. base level). Although a statistical separation occurs between the peak Penh units of OA-A and OA-TS guinea pigs, the percent increase above base level is similar: 222.4% for OA-A and 234.8% for OA-TS guinea pigs.

TS exposure. After the initial injections, 12 OA-sensitized and 9 NS guinea pigs were exposed daily to mainstream TS from four standard 2R1 reference cigarettes (Tobacco and Health Research Institute, Lexington, KY) drawn into an exposure chamber (36 liters in volume). Exposures were 7 days/wk and 30 min/day. The concentration of TS inside the chamber was 5.3 ± 0.1 mg/l during the exposure (n = 10). The TS concentration was determined by weighing the cigarette before and after its burning and dividing by the total airflow, which was held constant through the exposure chamber during the time the cigarette burned. Temperature inside the chamber increased by only 1-2°C during the exposure. NS guinea pigs (n = 8) and OA-sensitized guinea pigs (n = 10) exposed to compressed air (A) in an identical manner served as controls. Bias flow in the chamber was 25 l/min, which was well above the estimated combined minute volume of 3-5 liters of 8 guinea pigs housed in the chamber at any one time. This prevented either a hypoxic or hypercapnic environment within the chamber. The latter was validated by monitoring CO2 content of the exhaust air from the chamber (LB-2 CO2 monitor, Beckman, Schiller Park, IL). CO2 content remained <1% of the exhaust air. To determine whether TS affected weight gain, guinea pigs were weighed on arrival and after 30, 60, 90, and 120 days. There was no difference in mean weights among treatment groups.

Whole body plethysmography. Airway responsiveness to OA, capsaicin, and bradykinin was determined by monitoring enhanced pause (Penh) units (which are equal to pause × peak expiratory pressure/peak inspiratory pressure) obtained from a plethysmograph that allows free movement of nonanesthetized animals (Buxco Electronics, Sharon, CT). Penh units and specific airway resistance are similar measurements of airway responsiveness (5).

Surgical preparations. After 120-156 days of exposure, guinea pigs were deeply anesthetized with pentobarbital sodium (50/kg ip; Astra, Arcadia, CA). Surgical anesthesia was maintained throughout the experiment with supplemental injections of one-quarter of the original dose at ~2-h intervals. Testing the withdrawal and corneal reflex assessed the level of anesthesia. Additional pentobarbital sodium was administered if either reflex was present. Skeletal muscle blockade was not utilized at any time during the study. After induction of a deep surgical stage of anesthesia, guinea pigs were placed in the supine position on an operating table. A heating pad (Gaymar, Orchard Park, NY) maintained normal body temperature. An anterior, midline incision was made in the neck. To monitor arterial blood pressure and heart rate, the right carotid artery was cannulated and connected to a pressure transducer attached to a polygraph (model 7D, Grass, Quincy, MA). The right jugular vein was cannulated for the purpose of drug injection. The trachea was cannulated and connected to a ventilator (rodent ventilator model 683, Harvard Apparatus, Holliston, MA). A pressure transducer connected by a "Y" tube in the expiratory line from the tracheal cannula monitored tracheal (insufflation) pressure (Ptr). The ventilator was set at 70 cycles/min with a stoke volume of 0.75 ml/100 mg body wt. The chest was opened by a midsternal incision to provide access to the lung parenchyma to establish that the receptive field of the fiber being studied was within the lungs. The expiratory line of the ventilator was placed under 3-5 cmH2O to maintain normal functional residual capacity. The lungs were frequently inflated to three times tidal volume to maintain a consistent compliance and volume history.

Nerve recording. The left vagus nerve was exposed in the cervical region and then cut near the nodose ganglion. The distal end of the nerve was freed from surrounding tissue and placed on a dissection platform. The skin surrounding the incision was elevated to form a trough that was filled with mineral oil to prevent drying of the nerve. Nerve fibers were dissected from the main nerve bundle and placed on bipolar, platinum recording electrodes held by a micromanipulator. The signal from the electrodes was relayed to a preamplifier (model AM502, Tektronix, Beaverton, OR) and then to an oscilloscope (model 5228, Tektronix). The output from the oscilloscope was separately relayed to an audiomonitor and to data acquisition hardware connected to a computer equipped with data acquisition software (Power Lab, Castle Hill, Australia). Both the polygraph and the computerized data acquisition system recorded nerve activity, heart rate, arterial blood pressure, and Ptr.

I utilized methods previously described to identify lung C fibers (4). Briefly, lung C fibers were identified by a conduction velocity indicative of a nonmyelinated fiber, a weaker response to lung hyperinflation compared with either slowly adapting pulmonary stretch receptors or RARs, and possessing a receptive field within the lung parenchyma. To determine the fiber's conduction velocity, stimulating electrodes were placed on the vagus nerve near its emergence from the thorax. The distance between the stimulating and recording electrodes was measured (usually ~20 mm). A signal generated by the activation of the stimulator was used to trigger a sweep of the oscilloscope. The conduction velocity was calculated by dividing the distance between the two electrodes by the time between the stimulus artifact and the onset of the action potential of the fiber. Fiber activity was recorded as the lungs were subjected to stepwise lung inflation to four times tidal volume, constant-pressure inflation of 20-30 cmH2O, lung deflation, and negative-pressure deflation of 5-10 cmH2O. The lungs were gently probed with a cotton pledget to determine the general location of the receptive field of the fiber being studied.

RARs were identified according to established criteria. (4, 6-8, 21, 26). Briefly, RARs were identified by their rapidly adapting response to constant-pressure lung inflations (i.e., adaptation of index >70%; Ref. 26), fiber conduction velocity indicative of Adelta fibers, and a receptive field within the lungs. Because of extensive anastomosis between the pulmonary and bronchial circulations in guinea pigs, I did not differentiate between fibers in the pulmonary and bronchial circulations. This is also true in other species (24). Both C fibers and RARs were then challenged with capsaicin, an agent selectively activating thin primary sensory neurons that are chemosensitive endings or nociceptors (11, 17). A short latency of activation (<3 s) suggested that the fibers were in the lungs. Fibers with latencies >3 s were not included in the data.

Mediator challenge. Solutions of capsaicin and bradykinin (0.1-0.2 ml) were loaded into the cannula in the jugular vein (0.2-ml dead space). The cannula was flushed with 0.2 ml of 0.9% NaCl. Dose-response curves for capsaicin were conducted first. If the preparation permitted, then dose-response curves were also conducted for bradykinin. Both agents were also aerosolized in a chamber (volume of 13 ml) connected in series with the inspiratory line using an ultrasonic nebulizer (model 65, DeVilbiss, Sommerset, PA). According to the manufacturer, the aerosol particulate size generated ranged from 1 to 5 µm in diameter. All agents were purchased from Sigma Chemical (St. Louis, MO) with the exception of pentobarbital sodium, which was purchased from Anpro Pharmaceuticals (Arcadia, CA). The experiments were terminated with an intravenous overdose of the anesthetic agent.

Total and differential cell counts of lung lavage fluid. After anesthetic overdose at the end of the experiment the lungs were lavaged through the tracheal cannula with Hanks' balanced salt solution (pH 7.4, 20 ml in 5-ml aliquots). The lavage fluid was then centrifuged at 2,000 rpm at 4°C. The supernatant was discarded, and the cell pellet was resuspended in 3 ml of Hanks' solution in preparation for total cell counts and differential cell counts. Total cell counts were determined by using a Neubauer chamber (American Optical, Southbridge, MA) after the addition of several drops of LeukoStat I and II stain (Leukostat, Fisher Diagnostics, Fair Lawn, NJ) to the cell suspension. Differential cell counts of 10-µl aliquots of the cell suspension were determined from slides placed in a cytocentrifuge set at 1,000 rpm for 6 min (Shandon Lipshaw Pittsburgh, PA). The cells on the slides were fixed and stained with the LeukoStat according to the manufacturer's instructions, and 300 cells/slide were counted under ×400 magnification. Cells were identified as mononuclear cells, eosinophils, and neutrophils. The absolute cell numbers and the percentage of each cell type were calculated.

Statistical analysis. Ptr was determined as the pressure swing during a single ventilatory cycle. Peak Ptr was the greatest pressure swing during with cycles of similar pressures on either side. Peak base-level Ptr was determined within 60 s immediately before mediator challenge. Peak Ptr after mediator challenge was determined within 60 s immediately after mediator challenge. Nerve activity (NA) was the average of peak activity averaged during a consecutive 6-s period. As with the determination of peak Ptr, NA activity was determined within 60 s immediately before (base-level NA) and 60 s immediately after (peak NA) mediator challenge. Data are presented as means ± SE. The Student's t-test was used for analysis of drug action within groups and between groups at drug doses when only two groups were challenged (28). A one-way ANOVA determined whether statistical separation occurred either within or between groups for both NA and Ptr. When the ANOVA indicated statistical separation either within or between treatment groups (P < 0.05), the Newman-Keuls test determined where statistical separation occurred (28).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

One hundred twenty-eight sensory nerve fibers were identified, characterized, and challenged with capsaicin and bradykinin in this study. Of these fibers, 95 were C fibers (25 in NS-A, 43 in OA-A, 16 in OA-TS, and 11 in NS-TS guinea pigs) and 33 were RARs (13 in NS-A, 14 in OA-A, and 6 in OA-TS guinea pigs).

C fibers. The averaged conduction velocity of the C fibers was 1.89 ± 0.36 m/s. Base-level activity of C-fibers during ventilation approximating eupneic conditions was 0.35 ± 0.05 impulses/s. There was no difference in base-level C-fiber activity among groups. The conduction velocities and base-level activities reported here are similar to that reported in dogs (10), rats (7), and guinea pigs (4, 8). Some C fibers responded to stepwise- and constant-pressure inflation of the lungs; however, the threshold of activation was usually at three to four times tidal volume or 30-40 cmH2O, and still the response was weak (usually only several action potentials), whereas other C fibers were unresponsive. The pattern of response to the high-pressure inflation of the lungs was not rapidly adapting (Fig. 2 at end of record, and see Figs. 4 and 7 at end of record and Fig. 7B).


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  		Fig. 2.   A: lung C-fiber response to capsaicin (0.5 µg) injected intravenously (indicated by the arrow at top) in a OA-TS guinea pig. From top to bottom are heart rate [HR; in beats/min (b/min)], arterial blood pressure (ABP), tracheal (insufflation) pressure (Ptr), and action potentials (AP). Note weak response of the C fiber to lung inflation at the end of the record. B: expanded section corresponds to the bar above. Note that C-fiber activity is independent of Ptr.

Response to intravenous capsaicin challenge. Intravenous capsaicin injection activated lung C fibers before detectable changes in lung mechanics (Fig. 2). Generally and qualitatively, C-fiber activity increased sharply and then decreased despite further increases in Ptr. In addition, capsaicin and bradykinin stimulated C fibers when no detectable change in Ptr occurred. Therefore, increased C-fiber activity was not dependent on changes in Ptr. These observations suggest that both capsaicin and bradykinin (results presented below) have direct action on C fibers rather than indirect action due to changes in lung mechanics.

To determine whether successive capsaicin challenges induced C-fiber tachyphylaxis, capsaicin injection of 1 µg was repeated 15 min after the first 1-µg injection in 10 guinea pigs. After the first injection, C-fiber activity increased from 0.2 ± 0.1 to 2.6 ± 0.7 impulses/s (P < 0.02) while Ptr increased from 6.2 ± 0.5 to 16.9 ± 5.4 cmH2O. After the second capsaicin injection of 1 µg, C-fiber activity increased from 0.5 ± 0.1 to 3.5 ± 0.9 impulses/s (P < 0.007) while Ptr increased from 6.9 ± 0.7 to 20.6 ± 7.3 cmH2O. There were no statistical differences between C-fiber responsiveness after the two challenges of 1 µg capsaicin.

C-fiber and airway responsiveness to intravenous capsaicin challenge were enhanced in OA-TS guinea pigs compared with OA-A, NS-TS, and NS-A guinea pigs (Fig. 3). C-fiber activity in OA-TS guinea pigs was enhanced at 0.25, 0.5, and 1.0 µg compared with OA-A guinea pigs and at 0.25 and 0.5 µg compared with NS-TS guinea pigs. Separation occurred between C-fiber activity of OA-A and NS-A guinea pigs at 5 µg. Chronic TS exposure enhanced Ptr responsiveness after capsaicin challenge in OA-TS guinea pigs compared with OA-A and NS-TS guinea pigs at 0.25, 0.5, and 1.0 µg. Ptr responsiveness was also enhanced at 0.5 and 1.0 µg capsaicin in NS-TS guinea pigs vs. NS-A guinea pigs. Statistical separation occurred with Ptr between OA-A and NS-A guinea pigs at 5 µg.


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  		Fig. 3.   Dose-response relationship of intravenous capsaicin challenge vs. C-fiber nerve activity [NA; in impulses/s (imp/s); top] and Ptr (bottom) in OA-TS, OA-A, nonsensitized exposed to TS (NS-TS), and NS-A guinea pigs. Values are means ± SE. The number of OA-TS fibers is 10, 10, 7, 16, and 11; the number of OA-A fibers is 33, 33, 34, 33, 43, and 42; the number of NS-TS fibers is 10, 10, 9, 11, and 8; and the number of NS-A fibers is 16, 25, 22, and 9, respectively, for each dose of capsaicin administered. · P < 0.05 vs. base level. + P < 0.05 vs. NS-A guinea pigs. *P < 0.05 vs. NS-A and OA-A guinea pigs. ++ P < 0.05 vs. NS-A, OA-A, and NS-TS guinea pigs or all other groups at that dose.

Response to intravenous bradykinin challenge. Bradykinin challenge also increased both C-fiber activity and Ptr (Fig. 4). Qualitatively, C-fiber activation was comparatively slower in onset after bradykinin injection, and its activation tended to last longer than that of capsaicin in all groups of guinea pigs. As was the case with capsaicin, the effect of bradykinin on C-fiber activation appears direct without respiratory modulation. Changes in Ptr also tended to be slower in onset and longer in duration compared qualitatively with the effect of capsaicin. Such statistical comparisons were performed previously that support these observations (4).


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  		Fig. 4.   A: lung C-fiber response to bradykinin injection (0.5 µg) injected intravenously (indicated by the arrow at top) in an OA-TS guinea pig. From top to bottom are HR, ABP, Ptr, and action potentials (AP). Note weak response of fiber to lung inflation at the end of the record and more prolonged activity compared with capsaicin injection shown in Fig. 2B. Expanded section (B) corresponds to the bar above. Note C-fiber activity is independent of Ptr.

C-fiber and airway responsiveness to bradykinin challenge were enhanced by both chronic TS exposure and OA sensitization (Fig. 5). C-fiber responsiveness reached statistical separation from base level at 0.1 and 0.2 µg only for OA-TS guinea pigs but not for other groups. C-fiber activity in NS-TS guinea pigs was enhanced at 1 µg vs. C-fiber activity in NS-A guinea pigs. C-fiber activity in OA-A guinea pigs was enhanced at 5 and 10 µg vs. C-fiber activity in NS-A guinea pigs. Chronic TS exposure enhanced airway responsiveness to bradykinin challenge at 0.25-1.0 µg in OA-TS guinea pigs vs. OA-A guinea pigs. Ptr increased above base level after bradykinin challenge at 0.1 and 0.25 µg for NS-TS and at 0.1-1 µg in OA-TS guinea pigs. However, Ptr did not increase above base level until the 5-µg injection in OA-A guinea pigs and not at all in NS-A guinea pigs.


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  		Fig. 5.   Dose-response relationship of intravenous bradykinin challenge vs. NA (top) and Ptr (bottom) in OA-TS, NS-TS, and NS-A guinea pigs. Values are means ± SE. The number of OA-TS fibers is 6, 6, 6, 4, and 3; the number of OA-A fibers is 15, 15, 31, 32, 31, 27, and 5; the number of NS-TS fibers is 6, 6, 6, 3, and 3; and the number of NS-A fibers is 8, 6, 8, and 4, respectively, for each dose of bradykinin administered. · P < 0.05 vs. base level. + P < 0.05 vs. NS-A guinea pigs. *P < 0.05 vs. OA-A guinea pigs. ++ P < 0.05 vs. NS-A, OA-A, and NS-TS guinea pigs or all other groups at that dose.

Aerosol challenge of C-fibers. Capsaicin aerosol challenge increased C-fiber activity and Ptr in both OA-TS and NS-A guinea pigs. In OA-TS guinea pigs, capsaicin aerosol (0.001%, 30 s) increased C-fiber activity from 0.39 ± 0.12 to 1.17 ± 0.44 impulses/s and Ptr from 8.8 ± 0.7 to 20.1 ± 4.3 cmH2O (n = 11; P < 0.05 in each case). Capsaicin aerosol of 0.01% was administered in both of these groups. These data are presented in Fig. 6. Peak Ptr was greater during capsaicin aerosol challenge at this concentration in OA-TS vs. NS-A guinea pigs (Fig. 6, bottom). Unlike the response to intravenous administration of capsaicin, C-fiber responsiveness was similar in both groups (Fig. 6, top). Only one dose of bradykinin aerosol was administered during the study and only to the OA-TS group. Bradykinin aerosol challenge (0.01%, 30 s) increased C-fiber activity from 0.20 ± 0.04 to 4.98 ± 0.94 impulses/s and Ptr from 7.9 ± 0.5 to 32.4 ± 6.0 cmH2O (n = 11; P < 0.05 in each case).


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  		Fig. 6.   NA (top) and Ptr (bottom) in response to capsaicin aerosol challenge (0.01%, 30 s) to the airways in OA-TS (n = 12) and NS-A (n = 11) guinea pigs. Values are means ± SE. · P < 0.05 vs. base level. *P < 0.05 OA-TS guinea pigs vs. NS-A guinea pigs.

RARs. RARs in the lungs were identified by their rapidly adapting responses to stepwise hyperinflation, constant-pressure inflation, and negative-pressure challenges of the lungs as described in earlier studies (4, 7, 8). The average conduction velocity of the RARs was 16.25 ± 1.66 m/s. Base-level activity of the RARs during the prescribed eupneic ventilatory pattern of the respirator was 0.24 ± 0.06 impulses/s.

RAR activation by intravenous capsaicin challenge coincided with increased Ptr (Fig. 7A). The pattern of activation of the RARs typically reflected changes in airway pressures during the ventilatory cycle (Fig. 7C). Both airway and RAR responsiveness after capsaicin challenge were shifted to the left in OA-TS guinea pigs compared with either OA-A or NS-A guinea pigs (Fig. 8). After the capsaicin challenge (0.5 µg), RAR activity increased by 2,187% in OA-TS guinea pigs but by only 248% in OA-A guinea pigs. Ptr increased by 203% in OA-TS guinea pigs but by only 22% in OA-A guinea pigs. The ratio of the peak percent RAR activity to peak Ptr was 10.8 in OA-TS guinea pigs and was 11.3 in OA-A guinea pigs. This ratio further indicates that RAR activation correlates with changes in lung mechanics and that TS did not enhance RAR responsiveness.


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  		Fig. 7.   A: response of a lung rapidly adapting receptor to capsaicin challenge (0.5 µg) injected intravenously (indicated by the arrow at top) in an OA-TS guinea pig. From top to bottom are HR, ABP, Ptr, and AP. Note response of fiber to lung inflation at the end of the record. Smaller action potential is that of a C fiber. Note C-fiber activity is both earlier in response and unrelated to ventilatory modulation compared with that of the rapidly adapting receptor activity. B: response of RAR and C fiber to a 5-s constant-pressure lung inflation of 30 cmH2O. C: expanded section corresponds to the bar above. Note that rapidly adapting receptor activity displays a pattern of activity related to lung expansion.



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  		Fig. 8.   Dose-response relationship of intravenous capsaicin challenge and rapidly adapting receptor nerve activity (NA; top) and Ptr (bottom) in OA-TS, OA-A, and NS-A guinea pigs. Values are means ± SE. The number of OA-TS fibers is 6, 6, and 5; the number of OA-A fibers is 12, 12, 14, 14, and 14; and the number NS-A fibers is 13, 6, 13, 11, and 7, respectively, for each dose of capsaicin. · P < 0.05 vs. base level. *P < 0.05 vs. NS-A guinea pigs.

Cardiovascular responses. Injection of either capsaicin or bradykinin generally induced bradycardia and hypotension in all guinea pigs. For example, in OA-TS guinea pigs, capsaicin injection of 1 µg decreased heart rate from 229 ± 3.1 to 212.7 ± 5.4 beats/min and decreased arterial blood pressure from 50/31 ± 2/1 to 42/25 ± 2/2 mmHg. In NS-A animals, capsaicin injection of 1 µg decreased heart rate from 225 ± 10 to 217 ± 10 beats/min and decreased arterial blood pressure from 57/34 ± 1/1 to 53/28 ± 2/1 mmHg. Although the changes in heart rate and arterial blood pressure were numerically greater in OA-TS guinea pigs than in the other groups, statistical separation was not demonstrable for either agent at any dose.

Total and differential cell count. The total cell count of 80.1 ± 9.5 × 105 cells in OA-TS (n = 10) and 78.2 ± 18.4 × 105 cells in OA-A guinea pigs (n = 10) was higher than the total cell count of 43.9 ± 5.5 × 105 cells in NS-TS (n = 9) and 24.8 ± 3.3 × 105 cells in NS-A guinea pigs (n = 10; P < 0.05). However, the total cell counts of NS-TS guinea pigs was higher than that of NS-A guinea pigs (P < 0.05). The percentage of mononuclear cell was 73.7 ± 4.8% in the NS-A guinea pigs and 71.6 ± 2.5% in the NS-TS guinea pigs compared with 45.5 ± 3.5% in OA-A and 50.4 ± 6.2% in OA-TS guinea pigs (P < 0.05). The decrease in the percentage of mononuclear cells in OA-TS guinea pigs was because of a dramatic increase in the number of eosinophils. Eosinophils were 20.1 ± 4.0% of the total number of cells in NS-A guinea pigs and 24.2 ± 1.1% in NS-TS whereas eosinophils were 51.2 ± 6.0% in OA-A and 48.3 ± 6.0% of the total number of cells in OA-TS guinea pigs (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

In general, the combination of OA sensitization and TS exposure induced the greatest C-fiber and Ptr responsiveness to intravenous capsaicin challenge. In OA-TS guinea pigs, the threshold responsiveness of both variables decreased. C-fiber activation by capsaicin has been shown to be direct on C fibers with little or no direct activation of airway smooth muscle (4, 5, 15, 17). Aerosol challenge with capsaicin, on the other hand, produced similar C-fiber responsiveness in OA-TS guinea pigs and NS-A guinea pigs, although airway responsiveness increased in OA-TS guinea pigs. Airway responsiveness to intravenous bradykinin challenge was also enhanced in OA-TS guinea pigs. C-fiber hyperresponsiveness to bradykinin challenge and airway hyperresponsiveness to both capsaicin and bradykinin challenge at 1 µg occurred in NS-TS guinea pigs compared with NS-A guinea pigs. RAR activation was enhanced in OA-TS guinea pigs compared with either OA-A or NS-A guinea pigs. However, enhanced responsiveness of RARs to capsaicin challenge appeared to result from enhanced airway responsiveness rather than by direct action. In other studies, RAR activation by capsaicin has been shown to be indirect (3, 15).

Airway hyperresponsiveness and increased secretions and plasma extravasation occur in asthmatic individuals exposed to TS as well as other inhaled irritants (13, 20). Activation of lung C fibers induces similar reactions in the airways (2). In the present study, enhanced C-fiber and airway responsiveness to capsaicin occurred in OA-TS guinea pigs compared with OA-A, NS-A, or NS-TS groups of guinea pigs. In previous studies, airway responsiveness to capsaicin and bradykinin aerosol challenges was also greatest in conscious OA-TS guinea pigs compared with OA-A, NS-A, or NS-TS groups of guinea pigs (5). Because capsaicin is a selective agent that activates C fibers (4, 15, 17), our hypothesis that chronic TS exposure enhances C-fiber hyperresponsiveness was confirmed, particularly in guinea pigs sensitized to OA.

Mechanisms of enhanced responsiveness of airway sensory receptors. Chronic TS exposure may induce changes in the transduction properties of the nerve endings. Possibilities include the number of ligand receptors in the cell membrane, changes in the resting membrane potential of the nerve cell, or changes in the threshold of activation of the ion channels that lead to cell depolarization. Such possibilities have been discussed thoroughly by Mutoh et al. (23).

Mechanisms of enhanced C-fiber responsiveness resulting from chronic TS exposure conceivably could include enhanced accessibility of inhaled irritants to C-fiber endings. The airway epithelia of OA-TS guinea pig display secretory cell hyperplasia and hypertrophy and enhanced airway secretions (unpublished data). An enhanced airway and epithelial barrier may contribute to the failure of the capsaicin aerosol challenge to induce enhanced C-fiber responsiveness in OA-sensitized guinea pigs chronically exposed to TS, despite the observation of enhanced C-fiber responsiveness after its intravenous administration. Similar results have been reported in juvenile guinea pigs by Mutoh et al. (23) for both intravenous and aerosol challenge of capsaicin. Although it is difficult to know the precise doses of intravenous capsaicin challenge used by Mutoh et al., it appears to be 0.5 µg in most cases. If so the response reported as impulses per second of the C fibers for both A- and TS-exposed juvenile guinea pigs is remarkably similar to the adult guinea pigs observed in the present study.

Unlike the apparent direct activation of C fibers by capsaicin, activation of RARs by capsaicin appeared to result from changes in lung mechanics. The response of Ptr to capsaicin was greater in OA-TS guinea pigs than in OA-A and NS-A guinea pigs. Because RARs are sensitive mechanoreceptors then enhanced RAR activation would be expected in OA-TS guinea pigs. However, it has been suggested that capsaicin activates RARs directly (22). On the other hand, I reported that RAR activation in guinea pigs by capsaicin was dependent on changes in lung mechanics because prior administration of beta -adrenergic agonists blocked both increased Ptr and RAR activation by capsaicin challenges (4). In an in vitro, open tracheal preparation in which bronchoconstriction could not increase transmural tension in the airways, myelinated mechanoreceptors believed to be that of RARs in the trachea and bronchi of guinea pigs were insensitive to capsaicin challenge (15). Therefore, in agreement with previous studies (4, 15), RARs are apparently activated indirectly by capsaicin through changes in lung mechanics in guinea pigs exposed to either A or TS.

Enhanced airway responsiveness in guinea pigs chronically exposed to TS. Chronic TS exposure may enhance airway responsiveness to capsaicin and bradykinin challenge after C-fiber activation through central and/or local "axon" reflexes. The present study suggests that the central reflex is enhanced by TS exposure. Recently. Mutoh et al. (23) reported that TS exposure for 5 wk enhances C-fiber responsiveness but not airway responsiveness to either capsaicin or bradykinin challenge in juvenile, NS guinea pigs exposed to sidestream smoke. The differences in airway responsiveness between these two studies may be due to differences in the guinea pig ages, airway sensitization, and duration of TS exposure (17-22 wk for the present study) or to dosages of irritant agents used. However, both studies support that TS exposure enhances the central reflex arc of C-fiber activation.

Chronic TS exposure may also enhance the local or axon reflex. One likely mechanism for TS enhancement of the axon reflex is inhibition of neutral endopeptidase (NEP). Dusser et al. (14) determined that TS decreases neuropeptide metabolism by inhibiting NEP. Neuropeptides are released from C fibers (2, 12). Indeed, airway responsiveness is enhanced in guinea pigs chronically exposed to TS when challenged with NKA fragment 4-10 (5), suggesting a reduced rate of NKA metabolism. A-exposed guinea pigs treated with the NEP inhibitor then challenged with the NKA fragment duplicated qualitatively the effect of chronic TS exposure.

Inhibition of NEP appears to be only one of the mechanisms for TS-induced airway hyperirritability. Chronic TS exposure of OA-sensitized guinea pigs increased neuropeptide "overflow" into the lung perfusate during acute TS challenge in groups pretreated with NEP inhibitors (5). These results imply enhanced C-fiber responsiveness and possibly enhanced tachykinins content in guinea pig lungs chronically exposed to TS. The latter may be true because airway responsiveness to capsaicin was only modestly enhanced in NS guinea pigs exposed to A after NEP inhibition. Therefore, airway hyperresponsiveness observed in OA-TS guinea pigs may result from enhanced tachykinin release from C fibers as well as from impaired tachykinin metabolism.

OA sensitization and sensory receptor responsiveness. This is the first study to report the effects of OA sensitization on either C-fiber or RAR responsiveness to mediator challenges. The data support the hypothesis that airway sensitization enhances C-fiber responsiveness to capsaicin in OA-A guinea pigs compared with NS-A guinea pigs. Enhanced C-fiber responsiveness to 5 µg capsaicin and to 5 and 10 µg bradykinin challenge occurred in OA-A guinea pigs compared with NS-A guinea pigs. Enhanced Ptr responsiveness occurred only with capsaicin injection at 5 and 10 µg. The mechanisms of C-fiber and airway hyperresponsiveness induced by either TS exposure or OA sensitization are unknown.

In addition, the synergistic effect of TS exposure and airway sensitization on either C-fiber or airway responsiveness is poorly understood. Certain asthmatic individuals are hypersensitive to TS (13). With airway sensitization comes an influx of polymorphonuclear cells, especially eosinophils. Activated eosinophils or their secretory products in cell culture can stimulate sensory neurons to release neuropeptides (16). It is possible that chronic TS exposure affects eosinophil activation or enhances mediator release. Such events may subsequently activate C fibers as well as enhance airway responsiveness to inhaled irritants. However, the effect of capsaicin or TS exposure on eosinophil activation is unknown.

Chronic TS exposure and cardiovascular function. Guinea pigs have low blood pressure compared with humans or laboratory animals such as rats. In awake, nonsedated guinea pigs mean blood pressure is 60 mmHg (18). Anesthesia decreases arterial pressure. The guinea pigs were deeply anesthetized to eliminate the use of an antagonist of diaphragm and skeletal muscle contraction. This prevented muscle movement that would endanger the nerve recording preparation and allowed uninterrupted assessment of the level of anesthesia during artificial ventilation. However, arterial pressures of the present study are similar to those reported previously (6). Neither chronic TS exposure nor OA sensitization altered cardiovascular responses to either capsaicin or bradykinin challenges. These results are in agreement with those reported by Mutoh et al. (23). It is likely that arterial pressure had little influence on C-fiber or RAR responsiveness to either capsaicin or bradykinin challenges because similar sensory nerve responsiveness is reported in an isolated, in vitro tracheal nerve preparation (15).

Conclusion. The combination of chronic TS exposure and OA sensitization enhanced C-fiber and airway hyperresponsiveness to both capsaicin and bradykinin when intravenously injected. OA sensitization alone enhanced both C-fiber and Ptr responsiveness to capsaicin but at higher dosages than that required in OA-TS guinea pigs. Enhanced Ptr responsiveness did not occur in OA-A guinea pigs after bradykinin injection. Capsaicin aerosol challenge induced Ptr but not C-fiber hyperresponsiveness in OA-TS guinea pigs. The combination of chronic TS exposure and OA sensitization may enhance both the central reflex arc and axon reflex responsiveness through enhanced C-fiber responsiveness; however, data from previous studies suggest the local reflex mechanism appears to dominate in guinea pigs (3).


    ACKNOWLEDGEMENTS

Quenton Tanko expertly provided technical assistance.


    FOOTNOTES

This study was funded by a grant from the State of Nebraska's Department of Health and Human Service Cancer and Smoking Disease Program.

Address for reprint requests and other correspondence: D. R. Bergren, Dept. of Biomedical Sciences, School of Medicine, Creighton Univ., Omaha, NE 68178 (E-mail: dbergren{at}creighton.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 June 2000; accepted in final form 30 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
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J APPL PHYSIOL 91(4):1645-1654
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society


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