Published ahead of print on November 25, 2003, doi:10.1164/rccm.200308-1139OC
© 2004 American Thoracic Society Passive Smoke Effects on Cough and Airways in Young Guinea PigsRole of Brainstem Substance PDepartments of Pediatrics, Pharmacology, and Internal Medicine, School of Medicine; and the Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California at Davis, Davis, California Correspondence and requests for reprints should be addressed to Jesse P. Joad, M.D., University of California, Davis, Department of Pediatrics, 2516 Stockton Boulevard, Sacramento, CA 95817. E-mail: jpjoad{at}ucdavis.edu
Children raised with extended exposure to environmental tobacco smoke (ETS) experience increased cough and wheeze. This study was designed to determine whether extended ETS exposure enhances citric acid–induced cough and bronchoconstriction in young guinea pigs via a neurokinin-1 (NK-1) receptor mechanism at the first central synapse of lung afferent neurons, the nucleus tractus solitarius. Guinea pigs were exposed to ETS from 1 to 6 weeks of age. At 5 weeks of age, guide cannulae were implanted bilaterally in the medial nucleus tractus solitarius at a site that produced apnea in response to the glutamate agonist D,L-homocysteic acid. At 6 weeks of age, either vehicle or a NK-1 receptor antagonist, SR 140333, was injected into the nucleus tractus solitarius of the conscious guinea pigs who were then exposed to citric acid aerosol. ETS exposure significantly enhanced citric acid–induced cough by 56% and maximal Penh (a measure of airway obstruction) by 43%, effects that were attenuated by the NK-1 receptor antagonist in the nucleus tractus solitarius. We conclude that in young guinea pigs extended exposure to ETS increases citric acid–induced cough and bronchoconstriction in part by an NK-1 receptor mechanism in the nucleus tractus solitarius.
Key Words: nucleus tractus solitarius • environmental tobacco smoke • substance P • guinea pig • development
Infants and young children raised in homes with smokers have an increased prevalence of cough, wheeze, and sputum production (1–4). They tend to have airway obstruction (5), airway hyperreactivity (6), and an increased prevalence and earlier onset of asthma (7). The mechanism by which this occurs is not known. Cough, wheeze, and mucus production involve both local and central nervous system (CNS) components. The neural afferent limb for the cough reflex and the central component of bronchoconstriction and mucus release are carried by sensory fibers from the larynx and airways in the vagus nerve to the nucleus of the solitary tract (NTS) in the brain stem (8). The NTS is the first site in the brain where incoming signals can be modulated before they are transmitted to more distal synapses for final coordinated motor and vagal output. The major sensory nerve fibers responsible for cough are the rapidly adapting receptors (RARs), myelinated fibers with conduction velocities in the A Substance P is found in nerve terminals in the NTS (12), some of which arise from vagal afferents (13). Furthermore, neurokinin-1 (NK-1) receptors, the receptors with greatest affinity for substance P, are also richly expressed in the NTS (12). In general, substance P effects have been reported to be excitatory in the NTS (14–16), enhancing the actions of glutamate, the major excitatory transmitter (17, 18), and reflex outputs (19, 20). We have shown that substance P injected into the NTS enhances C fiber–induced apnea, an effect attenuated by an NK-1 antagonist (20). Because substance P content in airways and nerve ganglia can be increased by exposure to allergens (21) and irritants (22), including mainstream smoke (23), environmental tobacco smoke (ETS) exposure may increase substance P or its effects in the NTS and thereby enhance neurotransmission and reflex responses. We therefore hypothesized that extended exposure to ETS increases cough and bronchoconstriction in young guinea pigs by its effects on substance P in the NTS. To evaluate this hypothesis, young guinea pigs were exposed to filtered air or to ETS. After the exposures, vehicle or an NK-1 receptor antagonist was injected into the NTS of the conscious unrestrained animals who were then exposed to an aerosol of citric acid to induce cough and bronchoconstriction.
To verify the specificity and dose of the NK-1 receptor antagonist (SR 140333; Sanofi Recherche, Montpellier, France), a preliminary study was performed on anesthetized male Dunkin-Hartley guinea pigs (Charles River Laboratories, Raleigh, NC). Injections of the glutamate receptor agonist D,L-homocysteic acid (DLH, 10 mM, 20–40 nl) were followed 5 minutes later by substance P (80 µM, 25 nl) injections in the same medial NTS site. The DLH and substance P injections were each repeated every hour for 3 hours (DLH1, DLH2, DLH3, SP1, SP2, and SP3); respiratory frequency was measured using a fitted nose cone and pressure transducer and the decrease in frequency expressed as the percentage change relative to an equivalent predrug control period. The first two injections of DLH or substance P were made to show repeatability of frequency slowing; the third injections were made to show specificity and adequate dose of the NK-1 receptor antagonist 1 hour after vehicle (10% dimethyl sulfoxide, 100 nl) or SR 140333 (1 mM, 100 nl) was injected into the NTS. Guinea pigs were exposed to filtered air (n = 16) or to aged and diluted sidestream cigarette smoke as a surrogate for ETS (n = 17) from 1 to 6 weeks of age. As in previous studies (24–26), ETS exposures were 1 mg/m3 of total suspended particulates for 6 hours per day 5 days per week. At 5 weeks of age, two guide cannulae used to direct injection needles to the NTS were surgically positioned and cemented in place using previously established methods (27). At 6 weeks of age, injection needles were inserted through the cannulae (cemented in place) to a depth that was 200 µm below the site established 1 week earlier to accommodate for growth, as determined in preliminary studies. SR 140333 (1 mM, 100 nl) or vehicle (10% dimethyl sulfoxide, 100 nl) was injected through the guide cannulae to assure delivery of neuroactive agents to the DLH-responsive site. Animals were then placed in a whole-body plethysmograph (Buxco Electronics, Sharon, CT) to measure cough and enhanced pause (Penh), a measure of airway obstruction (28, 29). To acclimate them, animals were first placed in the plethysmograph for 30 minutes and exposed to saline for 1.5 minutes; they were then exposed to saline aerosol for 3 minutes, followed by a 7-minute observation period. Because Penh did not change during this 10-minute period, the average Penh was used as the baseline. Animals were then exposed to citric acid aerosol (0.4 M) for 3 minutes, followed by a 12-minute observation period. Coughs were identified by the typical high-pitched sound amplified by a microphone in the chamber, by transient changes in airflow (rapid inspiration followed by rapid expiration), and by the characteristic body position (30). After the citric acid aerosol observation period, DLH was injected in the NTS. Only animals exhibiting a DLH-induced change in frequency in at least one NTS side were included. Data were analyzed by analysis of variance with post hoc analysis with Sheffe contrast tests among the treatment groups as appropriate (SAS; SAS Institute, Cary, NC). Significance was set at p < 0.05. Additional details are in the online supplement.
Effect of Substance P Acting via NK-1 Receptors in the NTS on Respiratory Rate As shown in the examples and grouped data in Figure 1 , an injection of substance P into the NTS decreased the respiratory rate, an effect that was attenuated by SR 140333. In contrast, DLH, which also decreased the respiratory rate, was not affected by SR 140333. Neither substance P nor DLH injected at hourly intervals caused tachyphylaxis (SP1 vs. SP2 and DLH1 vs. DLH2).
Exposures The ETS exposures were characterized by (mean ± SD) relative humidity 45.4 ± 12.1%, temperature 71.7 ± 2.0°C, carbon monoxide 5.95 ± 0.83 parts per million, nicotine 176 ± 43 µg/m3, and total suspended particulates 1.00 ± 0.05 mg/m3. During the exposure period, the weights of the animals increased from (mean ± SD) 129 ± 19 g at 1 week of age to 429 ± 39 g at 6 weeks of age, with no differences between the filtered-air and ETS-exposed animals.
Effect of Extended Exposure to ETS and NTS Injection of an NK-1 Antagonist on Baseline Respiratory Pattern and Penh
Effect of Extended Exposure to ETS and NTS Injection of an NK-1 Antagonist on Citric Acid–induced Cough and Penh As shown in Figure 2 , young guinea pigs exposed to ETS (ETS-vehicle) coughed more frequently over the observation period (14.8 ± 2.2 coughs) than did guinea pigs exposed to filtered air (FA-vehicle, 9.5 ± 1.5 coughs, p = 0.03). In ETS-exposed guinea pigs, injection of the NK-1 receptor antagonist into the NTS (ETS-antagonist) decreased the number of citric acid–induced coughs from 14.8 ± 2.2 to 4.9 ± 0.9 coughs (p = 0.0003). In filtered air–exposed guinea pigs, an injection of the NK-1 receptor antagonist into the NTS did not statistically significantly reduce cough (9.5 ± 1.5 coughs in FA-vehicle vs. 7.3 ± 1.5 coughs in FA-antagonist, p = 0.37).
The time courses for the cough responses shown in Figure 3 demonstrate that ETS exposure prolonged the coughing response to citric acid, an effect attenuated by NK-1 receptor antagonist in the NTS. In general, guinea pigs from all groups did not cough during the 3-minute exposure to citric acid aerosol (CA on graph in Figure 3). After the citric acid aerosol was finished, the guinea pigs began to cough with the greatest frequency in the first 3 minutes after the aerosol (0- to 3-minute bin) and dropped off during the next 3 minutes (3- to 6-minute bin). In the filtered air–exposed guinea pigs (FA-vehicle and FA-antagonist) and in the ETS-exposed guinea pigs that received the NK-1 receptor antagonist into the NTS (ETS-antagonist), coughing mostly stopped thereafter (6- to 9-minute and 9- to 12-minute bins). In contrast, in the ETS-vehicle guinea pigs, cough continued during the 6- to 9-minute bin (p < 0.05, ETS-vehicle versus all other groups in 6- to 9-minute time bin).
As shown in Figure 4 , ETS exposure also enhanced Penh (ETS-vehicle vs. FA-vehicle, p = 0.02 during the 6- to 9-minute postaerosol peak response bin). This enhanced effect was attenuated by the NK-1 receptor antagonist in the NTS (ETS-vehicle vs. ETS-antagonist, p = 0.03). NK-1 receptor antagonist injected into the NTS caused a small, but not statically significant, decrease in Penh in filtered air–exposed animals (FA-vehicle versus FA-antagonist, p = 0.35).
The time course for the increase in Penh shown in Figure 4 was different from that of cough shown in Figure 3. Whereas cough peaked at 0 to 3 minutes after citric acid aerosol and then rapidly decreased, Penh increased steadily after citric acid aerosol to a plateau at 6 to 12 minutes. The Penh pattern was not affected by ETS exposure or NK-1 receptor antagonist in the NTS.
Confirmation of Injection Sites in the NTS
This study demonstrated that ETS exposure in young guinea pigs increases citric acid–induced cough and bronchoconstriction, in part by NK-1 receptor actions in the NTS. These findings add to previous work by linking ETS exposure and substance P. We previously characterized the effects of ETS on other aspects of the neural control of airways and breathing pattern using this same exposure protocol. Our electrophysiologic studies of nerve activity showed that ETS exposure increased the excitability of the primary afferent nerve fibers, the RARs in response to substance P (24) and C fibers in response to hyperinflation and to injection of capsaicin into the left atrium (25). We also found that ETS exposure augmented the peak and duration of the number of action potentials in the second and higher order neurons in the NTS (26). This body of evidence suggests that ETS increases the responsiveness of the sensory nerves to stimuli and that the increase is at least relayed at the level of the NTS. These studies do not allow us to determine, however, whether neurotransmission in the NTS is altered and if it is by what mechanism. These findings suggest that ETS does increase neurotransmission in the NTS and that it is substance P acting on NK-1 receptors in the NTS that is at least partially responsible. For methodologic reasons, our previous studies have been performed in anesthetized guinea pigs. In one of the studies, we showed that ETS exposure enhanced the apnea produced by stimulation of C fibers with capsaicin in the left atrium. Although this finding has possible implications for the etiology of the relationship between ETS exposure and sudden infant death syndrome, the findings could not be appropriately generalized to the respiratory symptoms of cough and wheeze experienced by children who live with exposure to ETS. This study was designed specifically to evaluate cough and airway obstruction in conscious, unrestrained animals, to mimic the human condition. The exposure was done to replicate that of a child exposed to ETS concentrations approximating a person who smokes with a personal particulate cloud of 1–2 mg/m3 of total suspended particulates. We adapted a method we used in rats for conscious microinjections into a precise area of the NTS (27). We functionally confirmed the appropriate placement of needles for making injections in the NTS by injecting DLH bilaterally to evoke a respiratory response rather than through histologic analysis. Although postmortem histologic analysis provides confirmation of appropriate placement of the cannulae, functional verification, particularly obtained by injections made through the same cannulae cemented in the same place, provides equally strong and often more reliable evidence that the injections were made in the NTS. We also took advantage of the Penh measurement, which allows the guinea pig to be unrestrained. Penh is a considered "an empiric parameter that reflects changes in waveform of the measured box pressure signal that are a consequence of bronchoconstriction" (28). A direct correlation has been demonstrated between pulmonary resistance and Penh, and a doubling of Penh has been shown to relate to a doubling of RL (28). Penh has come under criticism as a measure of airway obstruction (36–38), especially in mice. Pressure changes within the box can be due to either expansion of alveolar gas or heating and humidification of the inspired VT (36). Because guinea pigs are larger than mice, the relative contribution of expansion of alveolar gas should be greater in guinea pigs than mice (38). In addition, our guinea pigs were acclimated to the box for 30 minutes and exposed to an aerosol before any measurements to minimize the effects of humidification and heating of inspired VT. We studied substance P because it is a known neuromodulator in the NTS and is produced by C fibers and under certain conditions by RARs (39, 40). NTS microinjections of substance P or capsaicin (which releases neurokinins including substance P) (41) have been shown to augment lung C fiber–evoked lengthening of expiratory time (20) or to slow respiratory rate or lengthen expiratory time (17, 42, 43). Local excitation of NTS neurons by substance P (14–16, 44) or selective NK-1 receptor agonists (45) has further confirmed an excitatory effect on neuronal activity. On the other hand, activation of NK-1 receptors on vagal afferent neurons in the nodose ganglia has been shown to activate a calcium-dependent potassium current (46) and suppress a noninactivating hyperpolarization-activated inward current (47), suggesting that substance P effects on afferent fibers could be inhibitory. Finally, when an NK-1 receptor antagonist was nonspecifically injected into the brain via the vertebral artery, the number of coughs produced by mechanical stimulation of the intrathoracic trachea was decreased (48). We chose SR 140333, a potent, selective, and long-lasting NK-1 receptor antagonist (49) and a dose that was able to block completely the change in respiratory pattern produced by substance P injected into the NTS while sparing a similar change induced by DLH activation of ionotropic glutamate receptors. The block of the change in respiratory pattern was not due to the well-described tachyphylaxis to substance P, as spacing substance P injections 1 hour apart did not diminish the effect (Figure 1). Extended exposure to smoke clearly affects RARs and C fibers peripherally. Using electrophysiologic techniques, we have shown that ETS exposure increases the sensitivity of C fibers (25) and RARs (24). Kwong and colleagues (23) exposed adult guinea pigs to mainstream smoke or air for 2 weeks. In the mainstream smoke-exposed animals, intravenous capsaicin-induced increase in tracheal pressure was enhanced, an effect that was attenuated by the combined intravenous administration of NK-1 and NK-2 receptor antagonists. Interestingly, the NK-1 antagonist used, CP 99,994, crosses the blood brain barrier (50) and thus could have been acting centrally. They further found that mainstream smoke exposure increased substance P in the airway tissue and ß-preprotachykinin gene expression in the jugular ganglia. If ETS caused the same effects in our young guinea pigs, the increased production of substance P may have been transported also to the NTS to enhance neurotransmission. Bergren (51) exposed adult guinea pigs to mainstream smoke for at least 17 weeks. He found that Penh responses to capsaicin, bradykinin, and a neurokinin A fragment aerosols were enhanced after the mainstream smoke exposure. Interestingly, he found that neither atropine nor lidocaine attenuated the augmented response to capsaicin, suggesting that in contrast to this study, there was no CNS reflex component. This difference between the Bergren study and our own suggests that young animals, whose CNS development continues postnatally, may have a greater neuroplastic response to environmental factors than adult animals. In addition, the effects of mainstream smoke on the CNS may differ from those of ETS because of the marked differences in the proportion of their constituents (52). The patterns for cough and airway obstruction in response to citric acid were very different in our study. The guinea pigs had very little cough or change in Penh during the citric acid exposure, suggesting that citric acid either took a long time to diffuse to its receptors or produced a mediator or physiologic change such as airway edema (53), which then stimulated the afferent fibers. After the citric acid aerosol, the cough response peaked in the first 3 minutes then rapidly waned while the bronchoconstrictor response slowly increased to a maximum 6 to 12 minutes after citric acid. A possible explanation is that in the early phase, the cough and bronchoconstriction were evoked via the CNS reflex, whereas in the later phase, the local neurokinin-induced bronchoconstriction predominated resulting in the prolonged Penh response. However, the shape of the curve in Figure 3 suggests that the NK-1 receptor antagonist in the NTS reduced bronchoconstriction throughout the 12 minutes after citric acid, suggesting that the CNS reflex persisted throughout the observation period. Another explanation is that citric acid affected different types of receptors (RARs, nociceptors) in different locations (larynx, trachea, bronchi) at different times. Exposure to ETS did not alter baseline rate or depth of breathing. This is in agreement with our other work that showed no difference between ETS- and FA-exposed guinea pigs on baseline activity of RARs (24), C fibers (25), or NTS neurons (26). The baseline Penh was decreased by 15% in the ETS-exposed animals. Because Penh is a newly developed measure reflecting effort of breathing that is proportional to bronchoconstrictor-induced changes in airway resistance and RL (28, 29), it would be inappropriate to try to interpret the meaning of a change in baseline Penh. Interestingly, in our previous work using the same exposure protocol, we found that isolated lungs from guinea pigs exposed to ETS had a 17% increase in baseline dynamic compliance but no difference in baseline RL (54). In conclusion, the findings of this study provide new information obtained in conscious guinea pigs showing that exposing young guinea pigs to ETS increased cough and bronchoconstriction in response to citric acid. The mechanism includes neuroplastic changes in the NTS involving enhanced substance P and/or NK-1 receptors or their function.
J.P.J. has no declared conflict of interest; P.A.M. has no declared conflict of interest; J.M.B. has no declared conflict of interest; S.J.E. has no declared conflict of interest; K.E.P. has no declared conflict of interest; C-Y.C. has no declared conflict of interest; A.C.B. has no declared conflict of interest. The authors thank Mike Goldsmith for the ETS exposures and Dr. Pamela Eisele for her assistance with developing the anesthesia method.
Supported by the University of California Tobacco-Related Research Disease Grant 9RT-0010, a Health Services Research Award from the University of California Davis Medical Center, and a gift of SR 140333 from Sanofi Recherche, Montpellier France. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form August 15, 2003; accepted in final form November 24, 2003
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ABSTRACT
METHODS
range, and cell bodies in the nodose ganglia (
