The Differential Effect of Gastroesophageal Reflux Disease on Mechanostimulation and Chemostimulation of the Laryngopharynx

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1 CHEST Original Research GASTROESOPHAGEAL REFLUX DISEASE The Differential Effect of Gastroesophageal Reflux Disease on Mechanostimulation and Chemostimulation of the Laryngopharynx Sin-Yong Phua, PhD ; Lorcan McGarvey, MD ; Meng Ngu, MBBS, PhD ; and Alvin Ing, MBBS, MD Objectives: Laryngo-hypopharyngeal sensitivity (LPS) as measured by thresholds to mechanostimulation and chemostimulation is important in the prevention of pulmonary aspiration. The presence of gastroesophageal reflux disease (GERD) increases thresholds to mechanostimulation. However, the effect of GERD on thresholds to chemostimulation remains unknown. The aim of this study was to compare laryngo-hypopharyngeal thresholds to chemostimulation in subjects with GERD with those of healthy subjects and to determine the relationship between thresholds to mechanostimulation and chemostimulation. Methods: Forty-eight patients with GERD and 18 control subjects without GERD underwent LPS testing using the Fiberoptic Endoscopic Evaluation of Swallowing with Sensory Testing technique. All 48 patients and 10 of the control subjects also underwent threshold testing for chemostimulation via hypopharyngeal infusions of normal saline and 0.1 N hydrochloric acid performed in a randomized, blinded manner. Thresholds to mechanical stimulation, as measured by the lowest air pressure level required to elicit the laryngeal adductor reflex (LAR), were determined before and after laryngo-hypopharyngeal infusions. Thresholds to chemical stimulation were measured by determining the infusion volume of acid or saline required to trigger an airway protection response. Results: The mean LAR threshold of the patient group was significantly higher compared with that of control subjects (9.5 mm Hg vs 3.9 mm Hg, P,.01). Compared with control subjects, significantly less acid (0.13 ml vs 0.21 ml, P,.01) was required to trigger airway protective responses in GERD subjects. There is a strong negative correlation between the volume of acid infused and the LAR thresholds in the control subjects ( r , P,.05). Conclusions: Compared with the control subjects, subjects with GERD have significantly increased thresholds to mechanical stimulation, suggesting reduced mechanosensitivity, but significantly reduced thresholds to chemical stimulation, suggesting heightened chemosensitivity. There is an inverse relationship between mechanosensitivity and chemosensitivity. This relationship may be integral in maintaining airway protection. Trial registry: Australian New Zealand Clinical Trials Registry (ANZCTR); Trial number: ACTRN ; URL: CHEST 2010; 138(5): Abbreviations: GERD 5 gastroesophageal reflux disease; HCl 5 hydrochloric acid; LAR 5 laryngeal adductor reflex; LPS 5 laryngo-hypopharyngeal sensitivity; NS 5 normal saline Laryngo-hypopharyngeal sensitivity (LPS) to both mechanical and chemical stimuli is important in the prevention of aspiration of foreign material into the airway. 1,2 LPS may be diminished by anesthesia, head and neck surgery, and neurologic impairment. It has been postulated that gastroesophageal reflux may also reduce LPS and so increase the risk of microaspiration of gastric refluxate. 3 Microaspiration has been implicated as one of the mechanisms in the pathogenesis of chronic cough, asthma, and idiopathic pulmonary fibrosis in subjects with gastroesophageal reflux disease (GERD). 4-6 We have previously reported that patients with GERD have significantly reduced laryngo-hypopharyngeal mechanosensitivity compared with healthy control subjects. 7 In the same study, we also demonstrated that in healthy subjects, 1180 Original Research

2 brief exposure of the laryngo-hypopharyngeal mucosa to acid resulted in significant impairment of laryngeal mechanosensitivity. To our knowledge, there is to date no information on the effect of GERD on chemosensitivity in the laryngo-hypopharyngeal region. Sensory neural fibers with chemoreceptor characteristics have been identified throughout the laryngo-hypopharyngeal mucosa.8 They respond to both gaseous and aqueous chemical irritants. 9 In animals, chemical stimulation of the larynx has been known to initiate airway protective responses, including swallowing and coughing. 10,11 In newborn lambs, rapid swallowing has been observed in response to laryngeal chemostimulation. 11 It has also been documented that sour stimuli applied to the laryngeal mucosa facilitated the initiation of a pharyngeal swallow reflex. In addition, the number of swallows was found to be dependent on the concentration of the acid used, where the infusion of stronger concentrations resulted in an increased number of swallows. 10 Pharyngeal swallows in response to chemical stimuli may be a reflexive mechanism against pulmonary aspiration of noxious aqueous material. These studies suggest that chemoreceptors in the laryngo-hypopharyngeal region may have an active role in airway protection. It is unclear if chemoreceptors take on a greater role in airway protection in the presence of diminished mechanoreceptor sensitivity, such as that observed in subjects with GERD. However, given existing evidence that repeated acid exposure is associated with chemosensitivity, we hypothesize that chemosensitivity is heightened in patients GERD. The aims of this study were to: (1) determine the thresholds of laryngo-hypopharyngeal response to chemostimulation for healthy control subjects and subjects with GERD using 0.1 N hydrochloric acid (HCl) and normal saline (NS), and (2) study the relationship between the thresholds of response to laryngo-hypopharyngeal mechanostimulation and chemostimulation in both groups. To determine Manuscript received October 6, 2009; revision accepted April 16, Affiliations: From the Respiratory Investigation Unit, Department of Thoracic Medicine (Drs Phua and Ing) and the Department of Gastroenterology (Dr Ngu), Concord Repatriation General Hospital, Concord, NSW, Australia; and the Department of Medicine (Dr McGarvey), Institute of Clinical Science, The Queen s University Belfast, Northern Ireland. Funding/Support: This study was funded by the Respiratory Investigations Unit of Concord Hospital. Correspondence to: Alvin Ing, MBBS, MD, Respiratory Investigation Unit, Department of Thoracic Medicine, Concord Repatriation General Hospital, Hospital Rd, Concord, NSW, Australia, 2139; ajing@med.usyd.edu.au. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians ( site/misc/reprints.xhtml ). DOI: /chest laryngopharyngeal mechanosensitivity and chemosensitivity levels, we measured the threshold at which a response occurred in the laryngopharynx. We understand that the threshold at which a response occurs to either mechanical or chemical stimulation may not truly represent sensitivity because sensitivity is the slope of a stimulus-response relationship. What we are determining instead is the threshold at which a response occurs. However, because previously published papers, including our own work, 7 have termed a reduced threshold as suggesting increased sensitivity, we have used the term sensitivity in this article in this context. However, we acknowledge that the terms threshold and sensitivity are not always interchangeable. Study Subjects Materials and Methods Subjects with proven GERD and control subjects were recruited. The presence of GERD was determined using 24-h ambulatory esophageal ph studies (Digitrapper MK II; Synectics Medical; Stockholm, Sweden) or upper-gastrointestinal endoscopy. All subjects with GERD had either abnormal GERD parameters on esophageal ph study as defined by Johnson and DeMeester 12 or a minimum endoscopic finding of Grade 1 esophagitis. 13 All control subjects had normal 24-h ambulatory esophageal ph monitoring studies. All study subjects were between 18 and 75 years old and had no history of previous head and neck surgery, cerebrovascular accidents, or other significant medical illness such as diabetes, ischemic heart disease, or renal impairment. Subjects with proven GERD had stopped taking all proton pump inhibitors and antireflux medications for a minimum of 4 days prior to beginning the study and did not take those medications for the study s duration. All subjects gave written informed consent. This study was approved by the Concord Hospital Ethics Review Committee. Determination of Laryngo-Hypopharyngeal Thresholds to Mechanical Stimulation All subjects underwent threshold testing for mechanical stimulation using the Fiberoptic Endoscopic Evaluation of Swallowing with Sensory Testing technique, as described in our previous study. 7 Laryngo-hypopharyngeal mechanosensitivity as determined by threshold testing was defined as the lowest air pressure level required to elicit an involuntary transient adduction of the vocal folds, known as the laryngeal adductor reflex (LAR). In brief, local anesthesia (Lignocaine, 4% topical) was applied to both nares only, and a nasoendoscope (Pentax FNL-10 AP Fiber-Naso-Pharyngo-Laryngoscope; Pentax Precision Instruments Corporation; New York City, NY), 3.2 mm in diameter, attached to the Pentax AP-4000 air-pulse sensory stimulator (Pentax Precision Instruments Corporation), was passed transnasally into the laryngopharynx to a position 3 mm above the arytenoid eminence. The Pentax AP-4000 was designed to deliver air pulses ranging between 2.0 and 10.0 mm Hg. An initial air pulse of 6.0 mm Hg was delivered. If an LAR was observed, the pressure was reduced in increments of 1.0 mm Hg until vocal cord adduction was no longer elicited. The air pressure delivered was then increased by increments of 0.5 mm Hg until an LAR was observed again. If an LAR was not observed at 6.0 mm Hg, the pressure was increased by 1.0 mm Hg increments until an LAR was observed. Once an LAR was CHEST / 138 / 5 / NOVEMBER,

3 observed, the pressure was reduced by increments of 1.0 mm Hg until an LAR was no longer elicited. The air pressure was then increased by 0.5 mm Hg increments until an LAR was observed again. If no LAR was observed at 10.0 mm Hg (the upper limit of air pressure delivery for the Pentax AP-4000), the procedure was ceased, and a threshold of mm Hg was recorded. The lowest air pressure required to trigger an LAR was documented as the LAR threshold pressure and used to define the laryngo-hypopharyngeal mechanosensitivity level for that study subject. The repeatability of the test was 0.96, the SD for measurement error was 0.34, and the coefficient of variation was 9.7%. 7 The Pentax AP-4000 was calibrated according to the manufacturer s instructions. Laryngo-hypopharyngeal threshold data from 10 of the 18 control subjects and 15 of the 48 GERD subjects has been previously reported. 7 Determination of Laryngo-Hypopharyngeal Thresholds to Chemostimulation All subjects underwent laryngo-hypopharyngeal threshold testing for chemical stimulation using acid (0.1 N HCl) and NS solutions. Each subject had both HCl and NS solutions infused into the pyriform sinus, with the order allocated randomly and the infusions performed on separate days approximately 1 week apart. The pyriform sinus is part of the hypopharynx, but it shares similar sensory innervation with the laryngopharynx. Both the subjects and the investigators were blinded to the fluid (NS or HCl solution) infused. A 5F nasogastric feeding tube (Indoplas; Maersk Indoplas Pty Limited; Sydney, NSW, Australia) was used to introduce fluids into the hypopharynx. The catheter was introduced transnasally through the other nostril. With the aid of the nasoendoscope, the catheter was guided and placed in the posterior hypopharynx, in the region of the pyriform sinus. Solutions used in the study were dyed blue for easy visualization. The mean ph of the HCl solution was 1.2 (ph range, ), and the mean ph of the saline solution was 7.2 (ph range ). Test solutions were delivered at a rate of 1 ml/min, regulated by an IMED 960 volumetric infusion pump (IMED Corporation; San Diego, CA). Test fluids were infused until a complete adduction of the vocal folds, an irrepressible swallow, or a cough was elicited. The volume of test solution infused (the threshold volume) was measured using a digital timer and defined as the laryngo-hypopharyngeal chemosensitivity for that subject. Statistical Analysis Wilcoxon signed rank sum tests were used to analyze the difference in volumes of 0.1 N HCl and NS solutions required to be infused in control subjects and in subjects with GERD. The Mann-Whitney test was used to compare the difference in LAR thresholds between subjects with GERD and control subjects and the difference in volumes of 0.1 N HCl and NS solutions infused between subjects with GERD and control subjects. In addition, linear regressions were performed to analyze the relationship volumes of 0.1 N HCl and NS solutions required to be infused and LAR thresholds. Results Clinical Characteristics of Study Subjects Eighteen control subjects and 48 subjects with proven GERD were recruited. All control subjects underwent 24-h ambulatory esophageal ph monitoring, and their results were in the normal range. 12 Sub- jects with GERD underwent either 24-h ambulatory esophageal ph monitoring (n 5 12) or gastroscopy (n 5 36), and all met the criteria for GERD. The 36 subjects who underwent gastroscopy were found to have at least Grade 1 esophagitis. 13 The clinical characteristics of the study subjects are summarized in Table 1. Although all 18 control subjects underwent threshold testing for mechanostimulation, only 10 of the 18 control subjects agreed to have threshold testing for chemostimulation. There were no significant differences in baseline characteristics (age, sex, LAR thresholds) between those who agreed to threshold testing for chemostimulation testing and those who didn t. In view of protecting the airway from aspiration of the test fluids, any airway protection response was considered an outcome. In all instances, vocal adduction preceded swallowing. None of the subjects coughed as a result of exposure to acid or saline infusion. Comparison of Thresholds to Laryngo- Hypopharyngeal Mechanostimulation Between Study Groups The median LAR threshold of subjects with proven GERD was 10.0 mm Hg (range, mm Hg). This was significantly higher than the mean LAR threshold of 4.5 mmhg (range, mm Hg) as determined in control subjects ( P,.01). Comparisons of Thresholds to Laryngo- Hypopharyngeal Chemostimulation Between Groups Significantly smaller mean threshold volumes of HCl solution were recorded in subjects with proven Table 1 Clinical Characteristics of Subjects With GERD and Healthy Control Subjects Characteristics Subjects With GERD Healthy Control Subjects Number Male 31 7 Mean age, y Age range, y h esophageal ph monitoring, No. % of 24-h ph, 4 Mean (SD) 7.8 (2.2) a 0.95 (0.17) Range Mean number of reflux 56.3 a 16.4 events over 24 h Range Endoscopy grading n 536 Not performed Normal (grade 0) 0... Mild-moderate (grade 1-2) Severe (grade 3-4) 3... GERD 5 gastroesophageal reflux disease. adenotes significant difference, P, Original Research

4 GERD compared with control subjects (HCl solution [subjects with GERD] mean [SD] 0.13 [0.09] ml vs HCl solution [control subjects] 0.21 [0.05] ml, P,.01). In both subjects with GERD and control subjects, the threshold volumes for HCl solution were significantly smaller than the NS solution threshold volumes recorded (HCl solution [subjects with GERD] 0.13 [0.09] ml vs NS solution [subjects with GERD] 0.24 [0.15] ml, P,.001); HCl solution [control subjects] 0.21 [0.05] ml vs NS solution [control subjects] 0.80 [0.6] ml, P,.05). The volumes of 0.1 N HCl and NS solutions infused in both groups are shown in Figure 1. At the volumes of acid tested, the NS solution failed to evoke a response in both the subjects with GERD and the control subjects. In all subjects, vocal adduction preceded swallowing. None of the control subjects or subjects with GERD coughed as a result of exposure to acid or saline infusion. Correlation Between Thresholds of Response to Mechanostimulation and Chemostimulation in the Laryngopharynx In the control subjects, there was a strong negative correlation between the threshold volume of HCl solution infused and the LAR threshold (r , P,.05) ( Fig 2 ). There was no significant correlation between the threshold volume of the NS solution and the LAR threshold. Because of the ceiling effect in the determination of LAR thresholds, the correlation between the LAR thresholds and the 0.1 N HCl and NS solution volumes required to be infused in the subjects with GERD could not be determined. Figure 1. The distribution of normal saline and 0.1 N hydrochloric acid (HCl) infused in control subjects and in subjects with GERD. GERD 5 gastroesophageal reflux disease. Figure 2. The correlation between 0.1 N HCl (ml) and LAR thresholds (mm Hg) in healthy control subjects. LAR 5 laryngeal adductor reflex. See Figure 1 legend for expansion of other abbreviation. Safety No subject experienced any adverse reactions to the introduction of 0.1 N HCl solution. No episodes of laryngospasm were noted. On repeat nasoendoscopy, there were no signs of laryngeal irritation. Discussion The results of our study suggest that thresholds to laryngo-hypopharyngeal chemostimulation are significantly reduced in subjects with GERD, suggesting heightened chemosensitivity. Significantly smaller volumes of acid triggered an airway protective reflex such as vocal cord adduction or swallowing in patients with proven GERD compared with healthy control subjects. Furthermore, in all subjects, significantly smaller volumes of acid compared with saline were required to provoke these upper airway protective reflexes, suggesting a heightened laryngo-hypopharyngeal chemosensitivity to acid. We acknowledge that saline may provide a mechanical stimulus, but at the volumes of acid tested, NS failed to evoke a response in both subjects with GERD and control subjects. This implies that at volumes of acid infused, the stimulus for response was acid and not a mechanical event. We would again like to emphasize that this study investigated the differential effects of mechanostimulation and chemostimulation on the laryngopharynx and the elicited thresholds of response in both normal control subjects and patients with GERD. We determined the thresholds to response, and in previous studies, this has also been implied to represent sensitivity.7,14-17 We understand that sensitivity is the slope of a stimulus-response relationship, and we did not determine this relationship specifically. However, given the prior literature, we have used the term sensitivity to give our study context and recognize that thresholds to response may not always be a true measure of sensitivity. CHEST / 138 / 5 / NOVEMBER,

5 Our results are consistent with the evidence from experiments in animals that indicate a heightened response of the superior laryngeal nerve to acidic stimuli compared with saline stimuli. 14 Several lines of evidence suggest that chemoreceptors in the laryngeal region are adapted to detect nonsaline chemicals and have the primary function of preventing aspiration. 18,19 Detection of nonsaline chemicals such as acids may play a role in the prevention of aspiration of gastric contents. Although the mechanical stimulus occurred in the larynx (arytenoid eminence) and the chemical stimulus occurred in the hypopharynx (pyriform sinus), both sites are innervated by the same nerves (recurrent laryngeal nerve, internal laryngeal nerve, and branches of the superior laryngeal nerve). The term laryngo-hypopharyngeal sensitivity is thus applicable to this area. Mechanical deformation of the arytenoid eminence was observed as part of testing for LAR and thus as an assessment of mechanical sensitivity in both control subjects and patients with GERD. However, it is possible that other factors could be responsible for the LAR. The air pulse could have cooled the mucosal surface and stimulated temperature receptors. 20 In addition, nonhumidified air was used, and thus mucosal drying could have also played a role in the LAR response by stimulating sensory afferents. 21 Our study did not differentiate between these potential mechanisms, but it is likely that mechanical stimulation played a role, given the observed deformation in all subjects. We also acknowledge that the precise chemical stimulus in GERD is unclear. Although we chose HCl to test, other components of the gastric refluxate, such as bile and pepsin, could well be important, as could the osmolality of the refluxate. Sensitization of chemoreceptors may play a crucial role in the detection of proximal gastroesophageal reflux. Little is known about laryngo-hypopharyngeal chemosensitivity in humans, while relatively more is known regarding esophageal chemosensitivity. Sensitization of esophageal chemoreceptors to acid in subjects with GERD 15,22, and heightened sensitivity as a result of repeated acid exposure have been previously reported. 15,16,23 Significantly smaller volumes of acid perfused into the esophagus were required to induce dyspeptic symptoms in patients with GERD compared with healthy control subjects. 22 Progressive sensitization of esophageal chemoreceptors, as defined by pain perception, has been previously demonstrated. 15 It has been postulated that increased sensitization of chemoreceptors could be linked to direct exposure to acid or could be a result of the release of inflammatory mediators. 23 It is likely that sensitization could be a result of increased permeability of the squamous epithelium, allowing better access of protons to the sensory endings. 16 The relationship between mechanosensitivity and chemosensitivity in the laryngopharynx in humans has not been previously described. In this study, we established that compared with healthy control subjects, subjects with GERD had heightened thresholds of response to the presence of air-pulse stimuli, but exhibited reduced thresholds of response to the presence of acid in the laryngopharynx. In addition, in healthy subjects there was a statistically significant inverse correlation between thresholds to mechanostimulation and chemostimulation to acid. We suggest that heightened chemoreceptor activity may be a compensatory mechanism in the presence of diminished mechanosensitivity. It is possible that mechanoreceptors become desensitized in the presence of some noxious chemical stimuli, such as acid. In a study of laryngeal mechanoreceptor activity in cats, it was found that mechanoreceptor activity was unchanged in the presence of histamine solution or ammonia vapor, but that mechanoreceptors become desensitized when exposed to tobacco smoke. 17 The authors postulate that while mechanoreceptors become desensitized or have inhibitive responses to chemical stimuli, silent receptors in the region are sensitized and respond to the presence of chemical irritants. 17 Chemosensitivity in the laryngopharynx could be due to the recruitment of polymodal receptors in the region to detect noxious chemical stimuli. Irritant receptors, or rapid-adapting receptors, in the upper airway are mainly polymodal and respond to mechanical and chemical irritants. 9 It is likely that in the presence of noxious chemical stimuli, these polymodal receptors become sensitized to the chemical stimuli and relinquish their ability to detect mechanical stimuli. The exact mechanism remains unknown. Central mechanisms may also play a role. Brainstem pathways exist and have been characterized using c-fos staining following mechanical and acid stimulation of both the esophagus and the upper airways.24 It is thus plausible that afferent feedback from the inflamed esophagus could also sensitize brainstem pathways, which could result in increased laryngeal and hypopharynx responsiveness. Conclusions Subjects with GERD had significantly reduced thresholds to the presence of acid infused into the laryngopharynx compared with healthy control subjects. Overall, subjects with GERD had increased thresholds to mechanostimulation, but reduced thresholds to chemostimulation. This could imply that there is a strong negative relationship between chemosensitivity and mechanosensitivity, as demonstrated in 1184 Original Research

6 control subjects. This relationship could play a role in the prevention of aspiration in subjects with reduced mechano-laryngo-hypopharyngeal sensitivity, such as subjects with GERD. Acknowledgments Author contributions: Dr Phua had full access to all of the data in the study and takes full responsibility for the integrity of all of the data and the accuracy of the data analysis, including and especially any adverse effects. Dr Phua: was involved in the design, patient recruitment, data collection, statistical analysis, and writing of this article. Dr McGarvey: was involved in the design, statistical analysis of the data, and the writing of this article. Dr Ngu: was involved in the design, patient recruitment, and writing of this article. Dr Ing: was involved in the design, patient recruitment, data collection, and writing of this article. Financial /nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. References 1. Aviv JE, Martin JH, Sacco RL, et al. Supraglottic and pharyngeal sensory abnormalities in stroke patients with dysphagia. Ann Otol Rhinol Laryngol ;105 (2 ): Shaker R, Milbrath M, Ren J, et al. Esophagopharyngeal distribution of refluxed gastric acid in patients with reflux laryngitis. Gastroenterology ;109 (5 ): Aviv JE, Liu H, Parides M, Kaplan ST, Close LG. Laryngopharyngeal sensory deficits in patients with laryngopharyngeal reflux and dysphagia. Ann Otol Rhinol Laryngol ; 109 (11 ): Tobin RW, Pope CE II, Pellegrini CA, Emond MJ, Sillery J, Raghu G. Increased prevalence of gastroesophageal reflux in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med ;158 (6 ): Harding SM, Richter JE. The role of gastroesophageal reflux in chronic cough and asthma. Chest ;111 (5 ): Ing AJ. Interstitial lung disease and gastroesophageal reflux. Am J Med ;111 (Suppl 8A ):41S-44S. 7. Phua SY, McGarvey LP, Ngu MC, Ing AJ. Patients with gastrooesophageal reflux disease and cough have impaired laryngopharyngeal mechanosensitivity. Thorax ;60 (6 ): Widdicombe J. Airway receptors. Respir Physiol ; 125 (1-2 ): Sant Ambrogio G, Widdicombe J. Reflexes from airway rapidly adapting receptors. Respir Physiol ;125 (1-2 ): Kajii Y, Shingai T, Kitagawa J, et al. Sour taste stimulation facilitates reflex swallowing from the pharynx and larynx in the rat. Physiol Behav ;77 (2-3 ): Kovar I, Selstam U, Catterton WZ, Stahlman MT, Sundell HW. Laryngeal chemoreflex in newborn lambs: respiratory and swallowing response to salts, acids, and sugars. Pediatr Res ;13 (10 ): Johnson LF, DeMeester TR. Development of the 24-hour intraesophageal ph monitoring composite scoring system. J Clin Gastroenterol ;8 (Suppl 1 ): Savary M, Miller G. The Oesophagus. Handbook and Atlas of Endoscopy. Solothrun, Switzerland : Verlag Gassman ; Hanamori T, Kunitake T, Kato K, Kannan H. Neurons in the posterior insular cortex are responsive to gustatory stimulation of the pharyngolarynx, baroreceptor and chemoreceptor stimulation, and tail pinch in rats. Brain Res ;785 (1 ): Yoshida Y, Tanaka Y, Hirano M, Nakashima T. Sensory innervation of the pharynx and larynx. Am J Med ;108 (Suppl 4a ): 51S-61S. 16. Bradley RM. Sensory receptors of the larynx. Am J Med ;108 (Suppl 4a ):47S-50S. 17. Sant Ambrogio G, Brambilla-Sant Ambrogio F, Mathew OP. Effect of cold air on laryngeal mechanoreceptors in the dog. Respir Physiol ;64 (1 ): Shephard KL, Rahmoune H. Evaporation-induced changes in airway surface liquid on an isolated guinea pig trachea. J Appl Physiol ;76 (3 ): Helm JF, Dodds WJ, Hogan WJ. Salivary response to esophageal acid in normal subjects and patients with reflux esophagitis. Gastroenterology ;93 (6 ): Siddiqui MA, Johnston BT, Leite LP, Katzka DA, Castell DO. Sensitization of esophageal mucosa by prior acid infusion: effect of decreasing intervals between infusions. Am J Gastroenterol ;91 (9 ): Partosoedarso ER, Blackshaw LA. Vagal efferent fibre responses to gastric and oesophageal mechanical and chemical stimuli in the ferret. J Auton Nerv Syst ;66 (3 ): Fass R, Naliboff B, Higa L, et al. Differential effect of longterm esophageal acid exposure on mechanosensitivity and chemosensitivity in humans. Gastroenterology ;115 (6 ): Davis PJ, Nail BS. The sensitivity of laryngeal epithelial receptors to static and dynamic forms of mechanical stimulation. In: Fujimura O, ed. Vocal Physiology: Vocal Production, Mechanisms and Functions. New York, NY : Raven Press ; 1988 : Suwanprathes P, Ngu M, Ing A, Hunt G, Seow F. c-fos immunoreactivity in the brain after esophageal acid stimulation. Am J Med ;115 (Suppl 3A ):31S-38S. CHEST / 138 / 5 / NOVEMBER,