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Effect of changes in airway surface liquid on laryngeal receptors and muscles
ELSEVIER
Respiration Physiology 101 (1995) 31-39
Effect of changes in airway surface liquid on laryngeal receptors and muscles Franca B. Sant'Ambrogio a,*, James W. Anderson Giuseppe Sant'Ambrogio a
a,
Samuel T. Kuna b,
a Dept. of Physiology and Biophysics, University of Texas, Medical Branch, Galveston, TX 77555-0641, USA b Dept. of lnternal Medicine, University of Texas, Medical Branch, Galveston, TX 77555-0641, USA
Accepted 9 January 1995
Abstract
The effects of aerosolizing distilled water and isosmolal dextrose in the isolated larynx on the activity of pressure-responsive receptors and laryngeal muscles were studied in anesthetized dogs. Following water aerosolization, the mean discharge of pressure-responsive laryngeal mechanoreceptors during upper airway breathing and occlusion was 151% and 138% respectively of that present after saline aerosolization. During delivery of water aerosol, the peak activity of the posterior cricoarytenoid muscle increased to 229 _ 56% of control; no effects were present on the thyroarytenoid muscle activity. Saline or isosmolal dextrose aerosols did not have any effect on the activity of either muscle. The reflex increase in posterior cricoarytenoid muscle activity due to laryngeal negative pressure was enhanced (163%) when the negative pressure challenge was repeated following distilled water aerosol. These results suggest that alteration in laryngeal surface liquid composition modifies the response of pressure-responsive laryngeal receptors and thereby the reflex activation of airway patency maintaining muscles. Keywords: Airways; (pressure-responsive receptors); Larynx; (receptors); Mammals; (dog); Receptors; (airways; pressure); Upper airways;
(patency)
I. Introduction
Previous reports have examined the response of laryngeal receptors to changes in laryngeal mucosal surface liquid composition (Storey, 1968; Boushey et al., 1974; Harding et al., 1977; Boggs and Bartlett, 1982; Anderson et al., 1990). In anesthetized dogs spontaneously breathing through a tracheostomy, instillation of distilled water into the isolated in situ larynx stimulates both irritant and respiratory modu* Tel.: (409) 772-3398, Fax: (409) 772-3381.
lated pressure-responsive receptors, while instillation of isosmolal dextrose only stimulates irritant receptors (Anderson et al., 1990). These findings indicate that laryngeal pressure-responsive receptors are activated by hyposmolal solutions and irritant receptors are stimulated by solutions lacking small permeant anions, such as C1-. Stimulation of laryngeal receptors responding to intraluminal negative pressure is presumed to mediate a reflex activation of upper airway dilating muscles, such as the posterior cricoarytenoid (PCA) and the genioglossus (Mathew et al., 1982; Van
0034-5687/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0034-5687(95)00007-0
F. Sant'Ambrogio et aL / Respiration Physiology I01 (1995) 31-39
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Lunteren et al., 1984; Sant'Ambrogio et al., 1985). Stimulation of laryngeal irritant receptors elicits defense responses, such as cough and glottic closure (Karlsson et al., 1988), which involve activation of the thyroarytenoid (TA) muscle, a vocal cord adductor. The purpose of the current study is to ascertain if changes in laryngeal surface liquid composition alter the response of laryngeal receptors to negative pressure and modulate the reflex activation of laryngeal muscles.
2. Methods
General preparation Experiments were performed in 19 mongrel dogs of either sex in the 12-15 kg weight range. The animals were sedated with ketamine (10 mg/kg, i.m.), anesthetized intravenously with a mixture of a-chloralose (50 mg/kg) and urethane (500 mg/kg), and placed supine on an operating table. The trachea was exposed in the neck and cut longitudinally along its ventral aspect to insert a brass cannula with 3 sidearms (Fig. 1). A saline-filled catheter was placed in the mid-portion of the esophagus to record intrathoracic pressure. A large cannula (8.0 mm i.d.) was inserted into the oral cavity with its distal end dorsal to the epiglottis, facing the
aerosols ~52
k,,
~
~
PCA trodes
Fig. 1. Schematic representation of the setup. Aerosols were passed in the expiratory direction through S 1 while the larynx was functionally isolated by inflating the cuff of a Foley catheter between S 3 and $1; the dog was spontaneously breathing through S 2. Upper airway occlusions were performed by occluding the oral end of the oral cannula while the sidearms of the tracheal cannula were closed.
laryngeal opening; this cannula was then secured by constructing an air-tight face mask with quick-setting RTV Silicon Elastomer (Factor II, Lakeside, AZ, U.S.A.). Polyethylene catheters were introduced into the femoral vein and artery to administer supplementary doses of anesthetics, and monitor systemic arterial blood pressure, respectively.
A. Effect of distilled water aerosol on the response of pressure-responsive laryngeal receptors to negative pressure These experiments were performed in 7 dogs. Both branches of the superior laryngeal nerve (SLN) were cut 2 cm from their entrance into the larynx. The peripheral cut-end of the right SLN was placed on a dissecting platform and covered with mineral oil. With the aid of a dissecting microscope, thin filaments were separated until single unit action potentials were found. Receptors responding to negative pressure were identified by comparing their activity during tracheal and upper airway occlusion as described in a previous study (Sant'Ambrogio et al., 1983). Isosmolal saline or distilled water was aerosolized for 5-8 min with an ultrasonic nebulizer (DeVilbiss) used at a setting producing particles with a median size of 6 /zm. The aerosols were delivered through sidearm S 1 of the tracheal cannula in the expiratory direction while the larynx was functionally isolated (inflation of the balloon of a Foley catheter between S 1 and S 3) and the dog was breathing through sidearm S 2 (tracheal breathing; Fig. 1). The oral cannula and hypopharynx were then suctioned and the dog switched from tracheal to upper airway breathing (all tracheal cannula sidearms closed; Fig. 1). During upper airway breathing and occlusion, upper airway pressure was measured from sidearm S 3. Receptor activity was recorded during upper airway breathing and occlusion. One-two trials for each aerosol were performed at intervals of 10-15 min. Each trial consisted of 3 individual upper airway occlusions performed at intervals of 5 breaths. After a water aerosol trial, a brief bout of aerosolized saline was given to reestablish baseline conditions. Arterial blood pressure, action potentials, and upper airway and esophageal pressures were recorded on an electrostatic polygraph (Gould ES 1000).
F. Sant'Ambrogio et aL / Respiration Physiology 101 (1995) 31-39
33
Hooked wire electrodes (38-gauge, Isomid Beldon) were implanted into the PCA and TA using a 25-gauge hypodermic needle as an introducer. The electrodes were inserted into the PCA by dissecting the left postero-lateral aspect of the caudal portion of the larynx free from the surrounding structures and gently rotating the larynx ventrally. The electrodes were inserted into the TA through the cricothyroid membrane. The needles were then withdrawn leaving the hooked wire electrodes embedded in the muscles. Correct electrode position was confirmed on autopsy. The electrical activity from each set of electrodes was amplified and filtered by an AC-coupled preamplifier, and integrated using a moving time averager (time constant 100 msec). Raw and integrated muscle activity, arterial blood pressure, esophageal pressure, and upper airway pressure were recorded on a thermal array recorder (Gould TA5000). The signals
The rate of receptor discharge was evaluated as the ratio between the number of action potentials during inspiration and inspiratory time, and standardized by dividing this ratio by peak upper airway pressure. When receptors were active during upper airway breathing, receptor activity was evaluated for the 2 breaths preceding each occlusion. Data were averaged for each receptor and are presented as mean _ SE. The values obtained following water and saline aerosols were compared using the Wilcoxon signed rank test. Differences were considered significant when P < 0.05. B. Effect of distilled water and isosmolal dextrose aerosols on PCA and TA activity Eight animals were used for this protocol; in 5, the internal branch of the SLN was isolated bilaterally for sectioning later in the experiment.
SALINE AEROSOL A.P.
(kPa)
Ill
-
Pe= (kPa)
II
i[ o[ ,\
-2
,
WATER AEROSOL
A.P.
i
I I
(kPa)
-
(kPa)
-2
I i
i
f ~
--
"
~
"/
"
'
~
" i
•
i
5s
Fig. 2. Effect of isosmolal saline and distilled water aerosols on a high threshold pressure-responsive receptor. In each panel: action potentials (A.P.), upper airway pressure (Pua) and esophageal pressure (Pes). Traces show upper airway breathing and 2 upper airway occlusions (indicated by the large swings in pressure). Pretreatment with water aerosol greatly enhances the response of this receptor to upper airway negative pressure; moreover, the receptor becomes active during upper airway breathing.
34
F. Sant'Ambrogio et al. // Respiration Physiology 101 (1995) 31-39
were also displayed and recorded for off-line analysis (AT-CODAS, Dataq Instrument, Inc.) on a personal computer (Dell 486P/50) at a sampling rate of 375 Hz. While the dog was breathing through sidearm S 2 (tracheostomy breathing), the functionally isolated larynx was challenged with 0.9% isosmolal saline, distilled water, or 5% isosmolal dextrose aerosolized for 5 min. After water or dextrose trials, a brief bout of saline aerosol was given to reestablish baseline conditions. One-two trials were performed with each aerosol before and after bilateral section of the internal branch of the SLN. Breathing frequency, esophageal pressure, and tonic (baseline activity above electrical zero) and phasic (difference between baseline and peak activity) PCA and TA activity were measured. PCA preactivation (time interval from the onset of phasic activity to the beginning of inspiration, as indicated by esophageal pressure) was also determined. The mean values of these parameters over 5 consecutive
I0 (o.u.)
o
Pus
o
E
breaths were evaluated prior to the aerosol challenge (control) and at the end of the 1st, 2nd, 3rd, 4th and 5th min of each trial. When more than 1 trial of the same aerosol was performed in the same dog, the data were averaged. Results from all dogs were evaluated for each aerosol challenge (saline, water and dextrose). Differences between control and test conditions were ascertained with Kruskal-Wallis test and StudentNewman-Keuls multiple comparisons method. The effect of saline and water aerosols on the response of the PCA to negative pressure was studied in 6 dogs (4 of these animals did not participate in the rest of protocol B). The protocol for these experiments was similar to that for protocol A with the exception that both SLN were left intact and electrodes were implanted into the PCA as described above. The peak PCA moving time average, expressed in arbitrary units (a.u.), was standardized by dividing it by the peak value of the upper airway pressure during the occlusion. Values were averaged
LLLLLLLW
. . . . . . .
(kPa) -1 E water
10E
PCA MTA
(ou)
o
Pus o (kPa) -1 E saline PCA
MTA
(o.~.) 0
Pus o (kPa) -~ E saline
water
I
I
20 sec Fig. 3. Effect of alternating distilled water and isosmolalsaline aerosolson PCA activity. Continuous recording of the movingtime average of the PCA (PCA~rrA) in arbitrary units (a.u.) and esophagealpressure (Pus) in kiloPascal(kPa). Note that changes in PCA activity are not accompanied by changes in breathing pattern.
F. Sant'Ambrogio et al. /Respiration Physiology 101 (1995) 31-39
for each dog and statistical differences between trials with saline and water aerosols were ascertained with the Wilcoxon signed rank test. Data from protocol B are presented as mean _ SE; differences are considered significant when P < 0.05.
35
lated by negative pressure and 2 of the 3 inhibited increased their activity after water aerosols. Fig. 2 depicts the effect of water aerosol on the response of one receptor to negative pressure. The mean discharge of the pressure-responsive laryngeal mechanoreceptors (n = 16) during upper airway breathing was 7.6 + 2.8 imp/sec after saline aerosol and increased to 11.5 + 3.4 imp/sec after water aerosol ( P = 0.01). During upper airway occlusion after saline and water aerosol the activity increased to 23.0 + 5.9 imp/sec and 31.7 + 7.2 imp/sec, respectively ( P = 0.002). Differences with upper airway occlusion following saline and water aerosols were also significant when the results were normalized by dividing inspiratory rate of discharge by
3. Results
A. Effect of distilled water aerosol on the response of pressure-responsive laryngeal receptors to negative pressure Sixteen receptors were studied; 13 were stimulated by negative pressure in the laryngeal airway and 3 were inhibited. Ten of the 13 receptors stimu-
1.0
30 Br.Fr.
P~s 0.8
20
(kPo)
0.6 0.4
10
0.2 0.0
saline
water
dextrose
saline
water
dextrose
water
dextrose
15
15
PCA phasic
PCA tonic
(a.u.) 10
10
salTne
water
saline
dextrose
1.0 PCA preoctivotion 0.8
(see)
0.6 0.4 0.2 0.0
saline
water
dextrose
Fig. 4. Summary of results with isosmolal saline, distilled water and isosmolal dextrose aerosols (n = 8 for the first 4 panels, n = 7 for PCA preactivation). In each group of columns, the hatched column represents the mean of 5 control breaths; the subsequent columns represent the mean of 5 breaths at the end of each minute of aerosolization. The increase in phasic PCA activity at each minute of aerosolization with water is statistically significant ( *, P = 0.006).
F. Sant'Ambrogio et aL / Respiration Physiology 101 (1995) 31-39
36
upper airway pressure (11.9 + 2.5 vs 16.4 + 2.7 i m p / s e c / k P a , P -- 0.007). B. Effect of distilled water and isosmolal dextrose aerosols on PCA and TA activity The continuous recording in Fig. 3 shows the effect of brief applications of distilled water aerosol on PCA activity in one experiment. The initial application of water aerosol was associated with an increase in peak PCA activity which returned to baseline with administration of saline aerosol. Reapplication of water aerosol again resulted in an increase in PCA activity. The overall results for the 5 min aerosol trials are summarized in Fig. 4. Breathing frequency and
esophageal pressure were not affected by any of the aerosols. Water aerosol significantly increased phasic PCA activity ( P = 0.006; Fig. 4) for each min of the trial. This effect started 16.2 + 4.5 sec after the onset of the aerosol and peaked at 3.9 + 0.2 min; mean maximum peak PCA activity during water aerosol was 229.0 + 55.8% of control. Tonic PCA activity and PCA preactivation increased after water aerosol, but these changes did not reach statistical significance ( P = 0.216 and 0.063, respectively). Dextrose aerosol had only minimal and inconsistent effects on phasic PCA activity ( P = 0.82). With the exception of one experiment in which the TA acquired phasic expiratory activity with both water and dextrose aerosols, this muscle remained
H20 Aerosol B.P.
(kPo)
30 f 20 10
PCA MTA 15 ~(a.u.)
0
TA MTA
5
L
(o.u.)
o [
Pes (kPa)
o I -I
on
3'~s-
off
Isosmolot Dextrose Aerosol
B.P.
30
(kPo) 2o
10
PCA MTA15
(o.u.)
o
TA MTA 5 (a.u.) o Pes o (kPa) -2 on
Fig. 5. Effect of aerosolized distilled water (upper panels) and isosmolai dextrose (lower panel) on PCA and TA activity. In each panel from top to bottom: time in seconds, arterial blood pressure (B.P.), moving time average of PCA and TA muscles (PCAMT A and TAMTA), esophageal pressure (Pes). A break of 3 min and 38 sec between the left and the right upper panels is indicated. Water aerosol increases the inspiratory activity of the PCA. Neither water nor dextrose elicits coughing or TA activation. Note that mechanical stimulation of the laryngeal mucosa with the suction catheter (toward the end of the record in the upper panel) could elicit TA activity.
F. Sant'Ambrogio et al. / Respiration Physiology 101 (1995) 31-39 saline
aerosol
PCAMTA5 I ...... ~ . . . . . . (ou)
0
,,-.- ~[ ~.j
. . . . . . . . . .
Pua 0 - ~
~,L~L~l~
~m)L~
(kPo)
water aerosol PCAMTA (a.u.)
s
Pua C, ~ 1
~
~,x--
(kPa)
3 20 sec
Fig. 6. Interaction of distilled water aerosol and laryngeal collapsing pressure on PCA activation. Water aerosol enhances the negative pressure activation of the PCA.
silent in all trials. However, the TA could always be recruited by mechanical stimulation of the airway (Fig. 5). Saline aerosol did not have any effect on either PCA or TA activity. Bilateral section of the internal branch of the SLN eliminated all responses. Fig. 6 depicts the augmenting effect of water aerosol on the response of the PCA to negative pressure. Results from all dogs show that the peak activity of the PCA during upper airway occlusion was higher after water aerosol than after saline aerosol (31.2 + 3.9 vs 26.3 + 1.6 a.u.) but this difference did not reach statistical significance (P = 0.249). The change in upper airway pressure developed during upper airway occlusion was significantly lower after water aerosol than after saline aerosol (1.57 __+0.27 vs 1.96 ___0.24 kPa; P = 0.028). When PCA peak activity was normalized by dividing it by Pua, the augmenting effect of water aerosol became statistically significant (25.4 ___6.5 vs 15.6 ___ 2.7 a.u./kPa; P = 0.028). 4. Discussion
The purpose of this study was to determine the effect of reducing the osmolality of laryngeal surface
37
liquid on the response of laryngeal receptors to pressure and the reflex responses they mediate. The results clearly indicate that the response to pressure of laryngeal endings is enhanced following distilled water aerosol; even 2 of the 3 receptors inhibited by negative pressure increased their activity. Although a quantitative evaluation of the excitatory effects that water aerosol have on the response to pressure of laryngeal receptors goes beyond the scope of this study, the observation that such effects are absent or weaker during tracheal breathing (i.e. 0 transmural pressure), but become greater at higher pressures, suggests an increase in sensitivity of the pressure response relationship of these endings. However, an additional effect on the receptor threshold cannot be excluded. Stimulation of respiratory modulated pressure-responsive mechanoreceptors by negative pressure is thought to play a role in the activation of upper airway dilating muscles (Mathew et al., 1982; Van Lunteren et al., 1984; Sant'Ambrogio et al., 1985). In fact, laryngeal subatmospheric pressure stimulates pressure-responsive laryngeal receptors (Sant'Ambrogio et al., 1983) and reflexively enhances PCA activity thereby counteracting the collapsing effects on the larynx. The greater activation of laryngeal respiratory modulated mechanoreceptors by negative pressure following water aerosol corroborates the role attributed to these endings in preserving upper airway patency. It should be noted that during tracheostomy breathing and water aerosolization these effects occurred in the absence of changes in breathing pattern that per se could have modified upper airway muscle activity. However, when the dog performed inspiratory efforts against an occluded upper airway following distilled water aerosol, the peak subatmospheric pressures developed decreased (became less negative) compared to those after saline aerosol; this seems to be in line with the depressive effects on inspiratory activity occurring with upper airway negative pressure (Mathew et al., 1982; Van Lunteren et al., 1984; Sant'Ambrogio et al., 1985). Irritant receptors mediate defensive reflexes, such as laryngeal closure and cough; these responses are associated with activation of the TA, a vocal cord adductor. Most laryngeal irritant receptors are activated by solutions lacking chloride anions. Distilled water is both hypoosmolal and lacking chloride.
38
F. Sant'Ambrogio et al. / Respiration Physiology 101 (1995) 31-39
Therefore, we expected to find an increase in TA activity during water aerosol. Although a clear enhancement of PCA activation was present, we failed to detect a recruitment of the TA, which nevertheless could always be activated by mechanical stimulation of the carina. The different time course of the response to water of the two types of receptors (short duration for the irritant, long lasting for the respiratory modulated endings) is such that the response of the latter could be present at a time when that of the former has already subsided (Anderson et al., 1990). The activation of the PCA rather than the TA with distilled water suggests that respiratory modulated receptors, not the irritant receptors, elicit this reflex response. The possibility exists that the reflex effect of irritant receptors is masked by that of the respiratory modulated receptors. However, this is not supported by the finding that isosmolal dextrose, which only stimulates irritant receptors, was also ineffective in recruiting the TA, even at the start of aerosolization. An alternate explanation for the lack of a reflex activation of the TA may be sought in a greater depressive effect of anesthesia on laryngeal adductor as compared to laryngeal abductor muscle activity (Nakamura et al., 1958; Eyzaguirre and Taylor, 1963; Wyke, 1974; Sherrey and Megirian, 1974). The enhanced response of pressure-responsive receptors and the PCA to negative pressure with water aerosol is an interesting example of interaction between stimuli. A similar interaction between negative pressure and CO 2 on laryngeal receptors has been anticipated by several authors (Anderson et al., 1990; Bradford et al., 1990; Bartlett and Knuth, 1992) and demonstrated by Ghosh and Mathew (1994). Furthermore, Nolan et al. (1990) have shown that CO 2 in the isolated upper airway increases the activity of the genioglossus and concomitantly depresses ventilation. This response is similar to the effect of negative pressure and the authors suggest that, in the case of obstructive apnea, the interaction of the two stimuli could act in combination to restore upper airway patency (Nolan et al., 1990). The augmented effect of negative pressure on PCA activity after aerosolization of the larynx with water points to the importance of the physico-chemical characteristics of the airway surface liquid on reflex responses. Considering that the composition of
the airway surface liquid can be modified by the relative humidity of the inspired air, mouth breathing vs nose breathing (Man et al., 1979), airway secretion and various pharmacological agents (Marin, 1986) we envisage the possibility for various interventions to enhance or suppress reflex responses mediated by laryngeal receptors. For example, it was recently shown in this laboratory that furosemide, an agent that inhibits the activity of the Na+-2C1--K ÷ co-transport system, decreases the stimulation of laryngeal irritant receptors by isosmolal dextrose (Sant'Ambrogio et al., 1993). In addition, Ventresca et al. (1990) showed that furosemide diminishes the tussigenic effect of low chloride aerosols. These results along with those in the current study, suggest that pharmacological interventions might be used to modify upper airway surface liquid composition in order to activate upper airway dilating muscles and insure upper airway patency.
Acknowledgements Supported by NIH Grants HL-20122, HL-27520, Bridging Grant no. 2503-94 from the John Sealy Memorial Endowment Fund, and Moody Foundation Grant 94-255. Part of this work was presented at the 1991 FASEB meeting, 1991 Oxford Conference (Fuji Institute of Training, Japan), and International Union of Physiological Sciences in Glasgow (U.K.) August, 1993.
References Anderson J.W., F.B. Sant'Ambrogio, O.P. Mathew and G. Sant'Ambrogio (1990). Water-responsive laryngeal receptors in the dog are not specialized endings. Respir. Physiol. 79: 33-44. Bartlett, D., Jr. and S.L. Knuth (1992). Responses of laryngeal receptors to intralaryngeal CO 2 in the cat. J. Physiol. (London) 457: 187-193. Boggs, D.F. and D. Bartlett, Jr. (1982). Chemical specificity of a laryngeal apneic reflex in puppies. J. Appl. Physiol. 53: 455462. Boushey, H.A., P.S. Richardson, J.G. Widdicombe and J.C.M. Wise (1974). The response of laryngeal afferent fibres to mechanical and chemical stimuli. J. Physiol. (London) 240: 153-175. Bradford, A., P. Nolan, D. McKeogh, C. Bannon and R.G.
F. Sant'Ambrogio et al. /'Respiration Physiology 101 (1995) 31-39 O'Regan (1990). The responses of superior laryngeal nerve afferent fibres to laryngeal airway CO 2 concentration in the anesthetized cat. Exptl. Physiol. 75: 267-270. Eyzaguirre, C. and J.R. Taylor (1963). Respiratory discharge of some vagal motoneurons. J. Neurophysiol. 26: 61-78. Ghosh, T.K. and O.P. Mathew (1994). Influence of intralaryngeal CO 2 on the response of laryngeal afferents to upper airway negative pressure. J. Appl. Physiol. 76: 2720-2725. Harding, R., P. Johnson and M.E. McClelland (1977). Liquid-sensitive laryngeal receptors in the developing sheep, cat and monkey. J. Physiol. (London) 277: 409-422. Karlsson, J.-A., G. Sant'Ambrogio and J.G. Widdicombe (1988). Afferent neural pathways in cough and reflex bronchoconstriction. J. Appl. Physiol. 65: 1007-1023. Man, S.F.P., G.K. Adams III and D.F. Proctor (1979). Effects of temperature, relative humidity, and mode of breathing on canine airway secretions. J. Appl. Physiol. 46: 205-210. Marin, M.G. (1986). Pharmacology of airway secretion. Pharmacol. Rev. 38: 273-289. Mathew, O.P., Y.K. Abu-Osba and B.T. Thach (1982). Genioglossus muscle responses to upper airway pressure changes: afferent pathways. J. Appl. Physiol. 52: 445-450. Nakamura, F., Y. Uyeda and Y. Sonoda (1958). Electromyographic study on respiratory movements of the intrinsic laryngeal muscles. Laryngoscope. 68: 109-119. Nolan, P., A. Bradford, R.G. O'Regan and D. McKeogh (1990). The effects of changes in laryngeal airway CO 2 concentration on genioglossus muscle activity in the anesthetized cat. Exptl. Physiol. 75: 271-274.
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Sant'Ambrogio, F.B., O.P. Mathew, W.D. Clark and G. Sant'Ambrogio (1985). Laryngeal influences on breathing pattern and posterior cricoarytenoid muscle activity. J. Appl. Physiol. 58: 1298-1304. Sant'Ambrogio, F.B., G. Sant'Ambrogio and J.W. Anderson (1993). Effect of furosemide on the response of laryngeal receptors to low-chloride solutions. Eur. Respir. J. 6: 11511155. Sant'Ambrogio, G., O.P. Mathew, J.T. Fisher and F.B. Sant'Ambrogio (1983). Laryngeal receptors responding to transmural pressure, airflow and local muscle activity. Respir. Physiol. 54: 317-330. Sherrey, J.H. and D. Megirian (1974). Spontaneous and reflexly evoked laryngeal abductor and adductor muscle activity of cat. Exp. Neurol. 43: 487-498. Storey, A.T. (1968). A functional analysis of sensory units innervating epiglottis and larynx. Exptl. Neurol. 20: 366-383. Van Lunteren, E., W.B. Van de Graaff, D.M. Parker, J. Mitra, M.A. Haxhiu, K.P. Strohl and N.S. Cherniack (1984). Nasal and laryngeal reflex responses to negative upper airway pressure. J. Appl. Physiol. 56: 746-752. Ventresca, P.G., G.M. Nichol, P.J. Barnes and K.F. Chung (1990). Inhaled furosemide inhibits cough induced by low chloride content solutions but not by capsaicin. Am. Rev. Respir. Dis. 142: 143-146. Wyke, B. (1974). Respiratory activity of intrinsic laryngeal muscles: an experimental study. In: Ventilatory and Phonatory Control Systems, edited by B. Wyke. London, New York, Toronto: Oxford University Press, pp. 408-429.
Respiration Physiology 101 (1995) 31-39
Effect of changes in airway surface liquid on laryngeal receptors and muscles Franca B. Sant'Ambrogio a,*, James W. Anderson Giuseppe Sant'Ambrogio a
a,
Samuel T. Kuna b,
a Dept. of Physiology and Biophysics, University of Texas, Medical Branch, Galveston, TX 77555-0641, USA b Dept. of lnternal Medicine, University of Texas, Medical Branch, Galveston, TX 77555-0641, USA
Accepted 9 January 1995
Abstract
The effects of aerosolizing distilled water and isosmolal dextrose in the isolated larynx on the activity of pressure-responsive receptors and laryngeal muscles were studied in anesthetized dogs. Following water aerosolization, the mean discharge of pressure-responsive laryngeal mechanoreceptors during upper airway breathing and occlusion was 151% and 138% respectively of that present after saline aerosolization. During delivery of water aerosol, the peak activity of the posterior cricoarytenoid muscle increased to 229 _ 56% of control; no effects were present on the thyroarytenoid muscle activity. Saline or isosmolal dextrose aerosols did not have any effect on the activity of either muscle. The reflex increase in posterior cricoarytenoid muscle activity due to laryngeal negative pressure was enhanced (163%) when the negative pressure challenge was repeated following distilled water aerosol. These results suggest that alteration in laryngeal surface liquid composition modifies the response of pressure-responsive laryngeal receptors and thereby the reflex activation of airway patency maintaining muscles. Keywords: Airways; (pressure-responsive receptors); Larynx; (receptors); Mammals; (dog); Receptors; (airways; pressure); Upper airways;
(patency)
I. Introduction
Previous reports have examined the response of laryngeal receptors to changes in laryngeal mucosal surface liquid composition (Storey, 1968; Boushey et al., 1974; Harding et al., 1977; Boggs and Bartlett, 1982; Anderson et al., 1990). In anesthetized dogs spontaneously breathing through a tracheostomy, instillation of distilled water into the isolated in situ larynx stimulates both irritant and respiratory modu* Tel.: (409) 772-3398, Fax: (409) 772-3381.
lated pressure-responsive receptors, while instillation of isosmolal dextrose only stimulates irritant receptors (Anderson et al., 1990). These findings indicate that laryngeal pressure-responsive receptors are activated by hyposmolal solutions and irritant receptors are stimulated by solutions lacking small permeant anions, such as C1-. Stimulation of laryngeal receptors responding to intraluminal negative pressure is presumed to mediate a reflex activation of upper airway dilating muscles, such as the posterior cricoarytenoid (PCA) and the genioglossus (Mathew et al., 1982; Van
0034-5687/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0034-5687(95)00007-0
F. Sant'Ambrogio et aL / Respiration Physiology I01 (1995) 31-39
32
Lunteren et al., 1984; Sant'Ambrogio et al., 1985). Stimulation of laryngeal irritant receptors elicits defense responses, such as cough and glottic closure (Karlsson et al., 1988), which involve activation of the thyroarytenoid (TA) muscle, a vocal cord adductor. The purpose of the current study is to ascertain if changes in laryngeal surface liquid composition alter the response of laryngeal receptors to negative pressure and modulate the reflex activation of laryngeal muscles.
2. Methods
General preparation Experiments were performed in 19 mongrel dogs of either sex in the 12-15 kg weight range. The animals were sedated with ketamine (10 mg/kg, i.m.), anesthetized intravenously with a mixture of a-chloralose (50 mg/kg) and urethane (500 mg/kg), and placed supine on an operating table. The trachea was exposed in the neck and cut longitudinally along its ventral aspect to insert a brass cannula with 3 sidearms (Fig. 1). A saline-filled catheter was placed in the mid-portion of the esophagus to record intrathoracic pressure. A large cannula (8.0 mm i.d.) was inserted into the oral cavity with its distal end dorsal to the epiglottis, facing the
aerosols ~52
k,,
~
~
PCA trodes
Fig. 1. Schematic representation of the setup. Aerosols were passed in the expiratory direction through S 1 while the larynx was functionally isolated by inflating the cuff of a Foley catheter between S 3 and $1; the dog was spontaneously breathing through S 2. Upper airway occlusions were performed by occluding the oral end of the oral cannula while the sidearms of the tracheal cannula were closed.
laryngeal opening; this cannula was then secured by constructing an air-tight face mask with quick-setting RTV Silicon Elastomer (Factor II, Lakeside, AZ, U.S.A.). Polyethylene catheters were introduced into the femoral vein and artery to administer supplementary doses of anesthetics, and monitor systemic arterial blood pressure, respectively.
A. Effect of distilled water aerosol on the response of pressure-responsive laryngeal receptors to negative pressure These experiments were performed in 7 dogs. Both branches of the superior laryngeal nerve (SLN) were cut 2 cm from their entrance into the larynx. The peripheral cut-end of the right SLN was placed on a dissecting platform and covered with mineral oil. With the aid of a dissecting microscope, thin filaments were separated until single unit action potentials were found. Receptors responding to negative pressure were identified by comparing their activity during tracheal and upper airway occlusion as described in a previous study (Sant'Ambrogio et al., 1983). Isosmolal saline or distilled water was aerosolized for 5-8 min with an ultrasonic nebulizer (DeVilbiss) used at a setting producing particles with a median size of 6 /zm. The aerosols were delivered through sidearm S 1 of the tracheal cannula in the expiratory direction while the larynx was functionally isolated (inflation of the balloon of a Foley catheter between S 1 and S 3) and the dog was breathing through sidearm S 2 (tracheal breathing; Fig. 1). The oral cannula and hypopharynx were then suctioned and the dog switched from tracheal to upper airway breathing (all tracheal cannula sidearms closed; Fig. 1). During upper airway breathing and occlusion, upper airway pressure was measured from sidearm S 3. Receptor activity was recorded during upper airway breathing and occlusion. One-two trials for each aerosol were performed at intervals of 10-15 min. Each trial consisted of 3 individual upper airway occlusions performed at intervals of 5 breaths. After a water aerosol trial, a brief bout of aerosolized saline was given to reestablish baseline conditions. Arterial blood pressure, action potentials, and upper airway and esophageal pressures were recorded on an electrostatic polygraph (Gould ES 1000).
F. Sant'Ambrogio et aL / Respiration Physiology 101 (1995) 31-39
33
Hooked wire electrodes (38-gauge, Isomid Beldon) were implanted into the PCA and TA using a 25-gauge hypodermic needle as an introducer. The electrodes were inserted into the PCA by dissecting the left postero-lateral aspect of the caudal portion of the larynx free from the surrounding structures and gently rotating the larynx ventrally. The electrodes were inserted into the TA through the cricothyroid membrane. The needles were then withdrawn leaving the hooked wire electrodes embedded in the muscles. Correct electrode position was confirmed on autopsy. The electrical activity from each set of electrodes was amplified and filtered by an AC-coupled preamplifier, and integrated using a moving time averager (time constant 100 msec). Raw and integrated muscle activity, arterial blood pressure, esophageal pressure, and upper airway pressure were recorded on a thermal array recorder (Gould TA5000). The signals
The rate of receptor discharge was evaluated as the ratio between the number of action potentials during inspiration and inspiratory time, and standardized by dividing this ratio by peak upper airway pressure. When receptors were active during upper airway breathing, receptor activity was evaluated for the 2 breaths preceding each occlusion. Data were averaged for each receptor and are presented as mean _ SE. The values obtained following water and saline aerosols were compared using the Wilcoxon signed rank test. Differences were considered significant when P < 0.05. B. Effect of distilled water and isosmolal dextrose aerosols on PCA and TA activity Eight animals were used for this protocol; in 5, the internal branch of the SLN was isolated bilaterally for sectioning later in the experiment.
SALINE AEROSOL A.P.
(kPa)
Ill
-
Pe= (kPa)
II
i[ o[ ,\
-2
,
WATER AEROSOL
A.P.
i
I I
(kPa)
-
(kPa)
-2
I i
i
f ~
--
"
~
"/
"
'
~
" i
•
i
5s
Fig. 2. Effect of isosmolal saline and distilled water aerosols on a high threshold pressure-responsive receptor. In each panel: action potentials (A.P.), upper airway pressure (Pua) and esophageal pressure (Pes). Traces show upper airway breathing and 2 upper airway occlusions (indicated by the large swings in pressure). Pretreatment with water aerosol greatly enhances the response of this receptor to upper airway negative pressure; moreover, the receptor becomes active during upper airway breathing.
34
F. Sant'Ambrogio et al. // Respiration Physiology 101 (1995) 31-39
were also displayed and recorded for off-line analysis (AT-CODAS, Dataq Instrument, Inc.) on a personal computer (Dell 486P/50) at a sampling rate of 375 Hz. While the dog was breathing through sidearm S 2 (tracheostomy breathing), the functionally isolated larynx was challenged with 0.9% isosmolal saline, distilled water, or 5% isosmolal dextrose aerosolized for 5 min. After water or dextrose trials, a brief bout of saline aerosol was given to reestablish baseline conditions. One-two trials were performed with each aerosol before and after bilateral section of the internal branch of the SLN. Breathing frequency, esophageal pressure, and tonic (baseline activity above electrical zero) and phasic (difference between baseline and peak activity) PCA and TA activity were measured. PCA preactivation (time interval from the onset of phasic activity to the beginning of inspiration, as indicated by esophageal pressure) was also determined. The mean values of these parameters over 5 consecutive
I0 (o.u.)
o
Pus
o
E
breaths were evaluated prior to the aerosol challenge (control) and at the end of the 1st, 2nd, 3rd, 4th and 5th min of each trial. When more than 1 trial of the same aerosol was performed in the same dog, the data were averaged. Results from all dogs were evaluated for each aerosol challenge (saline, water and dextrose). Differences between control and test conditions were ascertained with Kruskal-Wallis test and StudentNewman-Keuls multiple comparisons method. The effect of saline and water aerosols on the response of the PCA to negative pressure was studied in 6 dogs (4 of these animals did not participate in the rest of protocol B). The protocol for these experiments was similar to that for protocol A with the exception that both SLN were left intact and electrodes were implanted into the PCA as described above. The peak PCA moving time average, expressed in arbitrary units (a.u.), was standardized by dividing it by the peak value of the upper airway pressure during the occlusion. Values were averaged
LLLLLLLW
. . . . . . .
(kPa) -1 E water
10E
PCA MTA
(ou)
o
Pus o (kPa) -1 E saline PCA
MTA
(o.~.) 0
Pus o (kPa) -~ E saline
water
I
I
20 sec Fig. 3. Effect of alternating distilled water and isosmolalsaline aerosolson PCA activity. Continuous recording of the movingtime average of the PCA (PCA~rrA) in arbitrary units (a.u.) and esophagealpressure (Pus) in kiloPascal(kPa). Note that changes in PCA activity are not accompanied by changes in breathing pattern.
F. Sant'Ambrogio et al. /Respiration Physiology 101 (1995) 31-39
for each dog and statistical differences between trials with saline and water aerosols were ascertained with the Wilcoxon signed rank test. Data from protocol B are presented as mean _ SE; differences are considered significant when P < 0.05.
35
lated by negative pressure and 2 of the 3 inhibited increased their activity after water aerosols. Fig. 2 depicts the effect of water aerosol on the response of one receptor to negative pressure. The mean discharge of the pressure-responsive laryngeal mechanoreceptors (n = 16) during upper airway breathing was 7.6 + 2.8 imp/sec after saline aerosol and increased to 11.5 + 3.4 imp/sec after water aerosol ( P = 0.01). During upper airway occlusion after saline and water aerosol the activity increased to 23.0 + 5.9 imp/sec and 31.7 + 7.2 imp/sec, respectively ( P = 0.002). Differences with upper airway occlusion following saline and water aerosols were also significant when the results were normalized by dividing inspiratory rate of discharge by
3. Results
A. Effect of distilled water aerosol on the response of pressure-responsive laryngeal receptors to negative pressure Sixteen receptors were studied; 13 were stimulated by negative pressure in the laryngeal airway and 3 were inhibited. Ten of the 13 receptors stimu-
1.0
30 Br.Fr.
P~s 0.8
20
(kPo)
0.6 0.4
10
0.2 0.0
saline
water
dextrose
saline
water
dextrose
water
dextrose
15
15
PCA phasic
PCA tonic
(a.u.) 10
10
salTne
water
saline
dextrose
1.0 PCA preoctivotion 0.8
(see)
0.6 0.4 0.2 0.0
saline
water
dextrose
Fig. 4. Summary of results with isosmolal saline, distilled water and isosmolal dextrose aerosols (n = 8 for the first 4 panels, n = 7 for PCA preactivation). In each group of columns, the hatched column represents the mean of 5 control breaths; the subsequent columns represent the mean of 5 breaths at the end of each minute of aerosolization. The increase in phasic PCA activity at each minute of aerosolization with water is statistically significant ( *, P = 0.006).
F. Sant'Ambrogio et aL / Respiration Physiology 101 (1995) 31-39
36
upper airway pressure (11.9 + 2.5 vs 16.4 + 2.7 i m p / s e c / k P a , P -- 0.007). B. Effect of distilled water and isosmolal dextrose aerosols on PCA and TA activity The continuous recording in Fig. 3 shows the effect of brief applications of distilled water aerosol on PCA activity in one experiment. The initial application of water aerosol was associated with an increase in peak PCA activity which returned to baseline with administration of saline aerosol. Reapplication of water aerosol again resulted in an increase in PCA activity. The overall results for the 5 min aerosol trials are summarized in Fig. 4. Breathing frequency and
esophageal pressure were not affected by any of the aerosols. Water aerosol significantly increased phasic PCA activity ( P = 0.006; Fig. 4) for each min of the trial. This effect started 16.2 + 4.5 sec after the onset of the aerosol and peaked at 3.9 + 0.2 min; mean maximum peak PCA activity during water aerosol was 229.0 + 55.8% of control. Tonic PCA activity and PCA preactivation increased after water aerosol, but these changes did not reach statistical significance ( P = 0.216 and 0.063, respectively). Dextrose aerosol had only minimal and inconsistent effects on phasic PCA activity ( P = 0.82). With the exception of one experiment in which the TA acquired phasic expiratory activity with both water and dextrose aerosols, this muscle remained
H20 Aerosol B.P.
(kPo)
30 f 20 10
PCA MTA 15 ~(a.u.)
0
TA MTA
5
L
(o.u.)
o [
Pes (kPa)
o I -I
on
3'~s-
off
Isosmolot Dextrose Aerosol
B.P.
30
(kPo) 2o
10
PCA MTA15
(o.u.)
o
TA MTA 5 (a.u.) o Pes o (kPa) -2 on
Fig. 5. Effect of aerosolized distilled water (upper panels) and isosmolai dextrose (lower panel) on PCA and TA activity. In each panel from top to bottom: time in seconds, arterial blood pressure (B.P.), moving time average of PCA and TA muscles (PCAMT A and TAMTA), esophageal pressure (Pes). A break of 3 min and 38 sec between the left and the right upper panels is indicated. Water aerosol increases the inspiratory activity of the PCA. Neither water nor dextrose elicits coughing or TA activation. Note that mechanical stimulation of the laryngeal mucosa with the suction catheter (toward the end of the record in the upper panel) could elicit TA activity.
F. Sant'Ambrogio et al. / Respiration Physiology 101 (1995) 31-39 saline
aerosol
PCAMTA5 I ...... ~ . . . . . . (ou)
0
,,-.- ~[ ~.j
. . . . . . . . . .
Pua 0 - ~
~,L~L~l~
~m)L~
(kPo)
water aerosol PCAMTA (a.u.)
s
Pua C, ~ 1
~
~,x--
(kPa)
3 20 sec
Fig. 6. Interaction of distilled water aerosol and laryngeal collapsing pressure on PCA activation. Water aerosol enhances the negative pressure activation of the PCA.
silent in all trials. However, the TA could always be recruited by mechanical stimulation of the airway (Fig. 5). Saline aerosol did not have any effect on either PCA or TA activity. Bilateral section of the internal branch of the SLN eliminated all responses. Fig. 6 depicts the augmenting effect of water aerosol on the response of the PCA to negative pressure. Results from all dogs show that the peak activity of the PCA during upper airway occlusion was higher after water aerosol than after saline aerosol (31.2 + 3.9 vs 26.3 + 1.6 a.u.) but this difference did not reach statistical significance (P = 0.249). The change in upper airway pressure developed during upper airway occlusion was significantly lower after water aerosol than after saline aerosol (1.57 __+0.27 vs 1.96 ___0.24 kPa; P = 0.028). When PCA peak activity was normalized by dividing it by Pua, the augmenting effect of water aerosol became statistically significant (25.4 ___6.5 vs 15.6 ___ 2.7 a.u./kPa; P = 0.028). 4. Discussion
The purpose of this study was to determine the effect of reducing the osmolality of laryngeal surface
37
liquid on the response of laryngeal receptors to pressure and the reflex responses they mediate. The results clearly indicate that the response to pressure of laryngeal endings is enhanced following distilled water aerosol; even 2 of the 3 receptors inhibited by negative pressure increased their activity. Although a quantitative evaluation of the excitatory effects that water aerosol have on the response to pressure of laryngeal receptors goes beyond the scope of this study, the observation that such effects are absent or weaker during tracheal breathing (i.e. 0 transmural pressure), but become greater at higher pressures, suggests an increase in sensitivity of the pressure response relationship of these endings. However, an additional effect on the receptor threshold cannot be excluded. Stimulation of respiratory modulated pressure-responsive mechanoreceptors by negative pressure is thought to play a role in the activation of upper airway dilating muscles (Mathew et al., 1982; Van Lunteren et al., 1984; Sant'Ambrogio et al., 1985). In fact, laryngeal subatmospheric pressure stimulates pressure-responsive laryngeal receptors (Sant'Ambrogio et al., 1983) and reflexively enhances PCA activity thereby counteracting the collapsing effects on the larynx. The greater activation of laryngeal respiratory modulated mechanoreceptors by negative pressure following water aerosol corroborates the role attributed to these endings in preserving upper airway patency. It should be noted that during tracheostomy breathing and water aerosolization these effects occurred in the absence of changes in breathing pattern that per se could have modified upper airway muscle activity. However, when the dog performed inspiratory efforts against an occluded upper airway following distilled water aerosol, the peak subatmospheric pressures developed decreased (became less negative) compared to those after saline aerosol; this seems to be in line with the depressive effects on inspiratory activity occurring with upper airway negative pressure (Mathew et al., 1982; Van Lunteren et al., 1984; Sant'Ambrogio et al., 1985). Irritant receptors mediate defensive reflexes, such as laryngeal closure and cough; these responses are associated with activation of the TA, a vocal cord adductor. Most laryngeal irritant receptors are activated by solutions lacking chloride anions. Distilled water is both hypoosmolal and lacking chloride.
38
F. Sant'Ambrogio et al. / Respiration Physiology 101 (1995) 31-39
Therefore, we expected to find an increase in TA activity during water aerosol. Although a clear enhancement of PCA activation was present, we failed to detect a recruitment of the TA, which nevertheless could always be activated by mechanical stimulation of the carina. The different time course of the response to water of the two types of receptors (short duration for the irritant, long lasting for the respiratory modulated endings) is such that the response of the latter could be present at a time when that of the former has already subsided (Anderson et al., 1990). The activation of the PCA rather than the TA with distilled water suggests that respiratory modulated receptors, not the irritant receptors, elicit this reflex response. The possibility exists that the reflex effect of irritant receptors is masked by that of the respiratory modulated receptors. However, this is not supported by the finding that isosmolal dextrose, which only stimulates irritant receptors, was also ineffective in recruiting the TA, even at the start of aerosolization. An alternate explanation for the lack of a reflex activation of the TA may be sought in a greater depressive effect of anesthesia on laryngeal adductor as compared to laryngeal abductor muscle activity (Nakamura et al., 1958; Eyzaguirre and Taylor, 1963; Wyke, 1974; Sherrey and Megirian, 1974). The enhanced response of pressure-responsive receptors and the PCA to negative pressure with water aerosol is an interesting example of interaction between stimuli. A similar interaction between negative pressure and CO 2 on laryngeal receptors has been anticipated by several authors (Anderson et al., 1990; Bradford et al., 1990; Bartlett and Knuth, 1992) and demonstrated by Ghosh and Mathew (1994). Furthermore, Nolan et al. (1990) have shown that CO 2 in the isolated upper airway increases the activity of the genioglossus and concomitantly depresses ventilation. This response is similar to the effect of negative pressure and the authors suggest that, in the case of obstructive apnea, the interaction of the two stimuli could act in combination to restore upper airway patency (Nolan et al., 1990). The augmented effect of negative pressure on PCA activity after aerosolization of the larynx with water points to the importance of the physico-chemical characteristics of the airway surface liquid on reflex responses. Considering that the composition of
the airway surface liquid can be modified by the relative humidity of the inspired air, mouth breathing vs nose breathing (Man et al., 1979), airway secretion and various pharmacological agents (Marin, 1986) we envisage the possibility for various interventions to enhance or suppress reflex responses mediated by laryngeal receptors. For example, it was recently shown in this laboratory that furosemide, an agent that inhibits the activity of the Na+-2C1--K ÷ co-transport system, decreases the stimulation of laryngeal irritant receptors by isosmolal dextrose (Sant'Ambrogio et al., 1993). In addition, Ventresca et al. (1990) showed that furosemide diminishes the tussigenic effect of low chloride aerosols. These results along with those in the current study, suggest that pharmacological interventions might be used to modify upper airway surface liquid composition in order to activate upper airway dilating muscles and insure upper airway patency.
Acknowledgements Supported by NIH Grants HL-20122, HL-27520, Bridging Grant no. 2503-94 from the John Sealy Memorial Endowment Fund, and Moody Foundation Grant 94-255. Part of this work was presented at the 1991 FASEB meeting, 1991 Oxford Conference (Fuji Institute of Training, Japan), and International Union of Physiological Sciences in Glasgow (U.K.) August, 1993.
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F. Sant'Ambrogio et al. /'Respiration Physiology 101 (1995) 31-39 O'Regan (1990). The responses of superior laryngeal nerve afferent fibres to laryngeal airway CO 2 concentration in the anesthetized cat. Exptl. Physiol. 75: 267-270. Eyzaguirre, C. and J.R. Taylor (1963). Respiratory discharge of some vagal motoneurons. J. Neurophysiol. 26: 61-78. Ghosh, T.K. and O.P. Mathew (1994). Influence of intralaryngeal CO 2 on the response of laryngeal afferents to upper airway negative pressure. J. Appl. Physiol. 76: 2720-2725. Harding, R., P. Johnson and M.E. McClelland (1977). Liquid-sensitive laryngeal receptors in the developing sheep, cat and monkey. J. Physiol. (London) 277: 409-422. Karlsson, J.-A., G. Sant'Ambrogio and J.G. Widdicombe (1988). Afferent neural pathways in cough and reflex bronchoconstriction. J. Appl. Physiol. 65: 1007-1023. Man, S.F.P., G.K. Adams III and D.F. Proctor (1979). Effects of temperature, relative humidity, and mode of breathing on canine airway secretions. J. Appl. Physiol. 46: 205-210. Marin, M.G. (1986). Pharmacology of airway secretion. Pharmacol. Rev. 38: 273-289. Mathew, O.P., Y.K. Abu-Osba and B.T. Thach (1982). Genioglossus muscle responses to upper airway pressure changes: afferent pathways. J. Appl. Physiol. 52: 445-450. Nakamura, F., Y. Uyeda and Y. Sonoda (1958). Electromyographic study on respiratory movements of the intrinsic laryngeal muscles. Laryngoscope. 68: 109-119. Nolan, P., A. Bradford, R.G. O'Regan and D. McKeogh (1990). The effects of changes in laryngeal airway CO 2 concentration on genioglossus muscle activity in the anesthetized cat. Exptl. Physiol. 75: 271-274.
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Sant'Ambrogio, F.B., O.P. Mathew, W.D. Clark and G. Sant'Ambrogio (1985). Laryngeal influences on breathing pattern and posterior cricoarytenoid muscle activity. J. Appl. Physiol. 58: 1298-1304. Sant'Ambrogio, F.B., G. Sant'Ambrogio and J.W. Anderson (1993). Effect of furosemide on the response of laryngeal receptors to low-chloride solutions. Eur. Respir. J. 6: 11511155. Sant'Ambrogio, G., O.P. Mathew, J.T. Fisher and F.B. Sant'Ambrogio (1983). Laryngeal receptors responding to transmural pressure, airflow and local muscle activity. Respir. Physiol. 54: 317-330. Sherrey, J.H. and D. Megirian (1974). Spontaneous and reflexly evoked laryngeal abductor and adductor muscle activity of cat. Exp. Neurol. 43: 487-498. Storey, A.T. (1968). A functional analysis of sensory units innervating epiglottis and larynx. Exptl. Neurol. 20: 366-383. Van Lunteren, E., W.B. Van de Graaff, D.M. Parker, J. Mitra, M.A. Haxhiu, K.P. Strohl and N.S. Cherniack (1984). Nasal and laryngeal reflex responses to negative upper airway pressure. J. Appl. Physiol. 56: 746-752. Ventresca, P.G., G.M. Nichol, P.J. Barnes and K.F. Chung (1990). Inhaled furosemide inhibits cough induced by low chloride content solutions but not by capsaicin. Am. Rev. Respir. Dis. 142: 143-146. Wyke, B. (1974). Respiratory activity of intrinsic laryngeal muscles: an experimental study. In: Ventilatory and Phonatory Control Systems, edited by B. Wyke. London, New York, Toronto: Oxford University Press, pp. 408-429.