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The role of excitatory amino acids in airway reflex responses in anesthetized dogs
Journal of the Autonomic Nervous System 67 Ž1997. 192–199
The role of excitatory amino acids in airway reflex responses in anesthetized dogs M.A. Haxhiu ) , B. Erokwu, I.A. Dreshaj Department of Medicine, Case Western ReserÕe UniÕersity School of Medicine, 10900 Euclid AÕenue, CleÕeland, OH 44106, USA Received 11 July 1997; revised 18 September 1997; accepted 24 September 1997
Abstract In these studies we examined the role of excitatory amino acids ŽEAAs. neurotransmission in communicating sensory inputs to the airway-related vagal preganglionic neurons, by examining the effects of either NMDA or AMPArkainate receptor blockade on reflex and chemical responses of tracheal smooth muscle. Experiments were performed in chloralose anesthetized, paralyzed and mechanically ventilated beagle dogs Ž n s 18., under hyperoxic, normocapnic, and normohydric conditions. Topical application or microinjection of NMDA receptor blockers, into the region of the ventrolateral medulla where airway-related vagal preganglionic neurons are located, insignificantly decreased the reflex changes in tracheal tone. However, topical application or microinjection of AMPArkainate subtype of glutamate receptor selective antagonists markedly reduced reflex increase in tracheal tone induced by Ž1. lung deflation, Ž2. stimulation of laryngeal cold receptors, and Ž3. activation of peripheral or central chemoreceptors. These effects were potentiated by prior NMDA receptor blockade. Findings indicate that an increase in central cholinergic outflow to the airways by a variety of excitatory afferent inputs is mediated via activation of EAA receptors, mainly AMPArkainate subtype of glutamate receptors. q 1997 Elsevier Science B.V. Keywords: Airway; Airway mechanoreceptors; Dog; Central chemoreceptors; Laryngeal cold receptors; Glutamate receptors: NMDA subtype receptor, AMPArkainate receptor; Nucleus ambiguus; Parasympathetic nervous system; Peripheral chemoreceptors; Rostral ventrolateral medulla; Tracheal tone
1. Introduction In recent years, considerable research effort has been made in elucidating the afferent and efferent innervation of the airways, and the physiology and pathophysiology of reflex responses w1,5,10,13,15,23,27,30,36 x. However, little is known about the primary neurochemicalŽs., and the receptor types involved in transmission of excitatory inputs that arise from the airway sensory fibers, peripheral and central chemoreceptors. This information is important in understanding the central nervous mechanisms that could initiate reflex airway responses, and in humans may influence the severity of airway diseases. Although a number of neurochemical signals may be implicated in mediating a reflexly induced increase in cholinergic outflow to the airways, the present study was
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0165-1838r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 7 . 0 0 1 1 0 - 0
focused on the role of excitatory amino acids ŽEAAs., since they are considered to be key neurotransmitters in almost all regions of the brain w17x. In addition, EAA receptors are widely distributed along the neuraxis w26,31,38x, and participate in most of the neural circuits, including those implicated in cardiorespiratory and airway control w2,4,12,18,20,22,29,34 x, and in mediating bronchodilator effects of hindlimb afferents stimulation w33x. Effects of released EAAs could be transmitted by activation of the inotropic or metabotropic receptors. The inotropic glutamate or ligand-gated ion channels, contain three known functional receptor subtypes: Ž1. N-methylD-aspartate ŽNMDA. receptors, which respond to glutamate and glutamate analogs such as NMDA, Ž2. the aam ino-3-hydroxy-5-m ethyl-4-isoxazolepropionate ŽAMPA., and Ž3. kainate receptor subtype. Although a fourth subtype of inotropic receptor exists, the delta receptor, it is not established as a functional ion channel w26x. The NMDA receptors are present in the brainstem w26,31x, and are activated by NMDA and glutamate, but do not respond to kainate and AMPA. Effects of activation of
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this functional receptor subtype in the ventrolateral medulla can be antagonized with competitive antagonists that act at the recognition site such as D-2-amino-5-phosphonovalerate ŽAP5., or D-2-amino-7-phosponohetanoate ŽAP7., and with a non-competitive channel blocking agent such as MK-801 w6,26,28,38x. The AMPA receptor, when expressed alone or in combination with other glutamate receptor subtypes, can be activated by quisqualate, AMPA, glutamate, and kainate. The quinoxalinediones such as 6,7-dinitroquinoxaline-2,3dione ŽDNQX ., 6-cyano-7-nitroquinoxaline-2,3-dione ŽCNQX., and 2,3-dihydroxy-6-nitro-7-sulphamoybenzoŽF. quinoxakine ŽNBQX. are potent antagonists of AMPA receptors w21,26,29x. This class of antagonists also blocks kainate receptors, but does not block NMDA receptors. Hence, it can be used to differentiate AMPArkainate receptor mediated effects from the NMDA signaling pathway. The kainate receptors respond to kainate, quisqualate, glutamate, and have lower responses to AMPA. Although in the CNS kainate receptors are about 50-fold less abundant than AMPA receptors w9,19x, it may play an important role in airway reflex responses. Recently it has been shown that kainate receptors can be activated synaptically, and might be involved in synaptic plasticity w37x. In the studies reported here we examined the role of EAA neurotransmission in communicating sensory inputs to airway-related vagal preganglionic neurons, by examining the effects of either NMDA or AMPArkainate receptor blockade on reflex responses of tracheal smooth muscle tone.
2. Methods The studies were performed on 18 adult beagle dogs of either gender, weighing 8–12 kg, anesthetized by the administration of thiopental sodium Ž25 mgrkg iv. followed by a-chloralose Ž80 mgrkg iv.. Supplemental doses of a-chloralose Ž10 mgrkg iv. were given hourly to maintain anesthesia. A low tracheostomy was performed, and a tracheal tube was inserted. The dogs were artificially ventilated with a volume ventilator ŽHarvard Apparatus., delivering a constant volume of 10 mlrkg at a frequency of 20–25 breathsrmin, from a bag containing 50% O 2 in N2 Ž n s 16. or 100% O 2 Ž n s 2.. A femoral artery and a femoral vein were cannulated for measurement of blood pressure ŽDigi-Mede Blood Pressure Analyzer, MicroMed, Louisville, KY., blood gas sampling, or for administration of fluid and paralytic agent, gallamine triethiodide ŽFlaxedil, 4–6 mgrkg.. The body temperature was maintained between 37.5 and 38.58C, by means of a heating pad. Ventral aspect of the medulla oblongata was widely exposed, as described earlier w11x. An in vivo segment of extra thoracic trachea was prepared for isometric measurement of tracheal smooth muscle responses w3x. Changes in the force generated by
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tracheal smooth muscle contraction were measured by subtracting the initial preload tension Ž28–35 g. from the peak tension developed following excitation of airway sensory nerve fibers, peripheral or central chemoreceptors. In three of these dogs the fifth root of the phrenic nerve was isolated, cut, and desheathed. The central end of the desheathed phrenic nerve was placed on a bipolar hook electrode. Mass neural activity was amplified ŽP 511 amplifier, Grass instruments., filtered between 0.1 and 3 kHz, rectified and integrated Ž200 ms time constant. with Paynter filters ŽCharles Ward Enterprises.. Airway mechanoreceptors were stimulated by lung deflation, which was produced by stopping the ventilator during the deflation phase for a period of 30 to 60 s Ž n s 14.; laryngeal cold receptors were activated by changing laryngeal temperature from 37 to 10–88C for 10 to 15 s Ž n s 7.. This was achieved by delivering through a plastic tube into the larynx a flow of air, passed through a coiled copper tubing immersed in ground dry ice. Hypercapnic response was obtained by ventilating the animals with 7% CO 2 balance O 2 for 5 min Ž n s 7.; peripheral chemoreceptors were exited by ventilating animals with 10% O 2 balance N2 for 2 min Ž n s 5.. At the time when tracheal responses were recorded, the average arterial PO 2 was 206 " 26 mmHg, PCO 2 was 33 " 5 mmHg, and pH was 7.36 " 0.042 units Žmean " SEM.. The vehicle and the dissolved drugs were applied topically to the ventrolateral medulla or microinjected into the same region. Topical application was performed by a round-shaped pledget Ž2 mm diam.. made of Gel Foam and soaked with 10 m l of solution. Filter paper wicks were placed at the lateral and rostral margins of the exposed ventral medulla to prevent cerebrospinal fluid accumulation. Drugs were applied bilaterally on the area which corresponds to the region located 6–8 mm caudal to the foramen cecum, and 3.5–4.5 mm lateral to the midline. In a separate group of animals, drugs were microinjected in the same area, 1.5 mm deep to the surface. The volume of injectate was 100 nl, an amount that spreads about 1 mm in the medulla of the dog w10x. The NMDA receptors were blocked by topical application, or microinjections of MK-801, AP5 or AP7. The CNQX was used to antagonize the effects of AMPAr kainate receptor activation in all but one dog studied, in whom DNQX was employed. The first set of experiments Žseven dogs. addressed the potential role of NMDA receptors on airway reflex responses to lung deflation. Changes in the tracheal smooth muscle tension induced by lung deflation were recorded prior to and after topical administration or microinjections of AP5, AP-7 or MK-801. The second series of experiments Ž14 animals. examined the involvement of AMPAr kainate receptors in mediating airway reflex responses to lung deflation. In these animals CNQX was applied topically, or microinjected into the region in which airway-related vagal preganglionic cells are located. In
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seven of these dogs CNQX was administered after the application of NMDA receptor blockers. The effect of AMPArkainate receptor blockade on tracheal smooth muscle contraction induced by stimulation of laryngeal cold receptors was studied in eight dogs, effects on tracheal contraction caused by activation of peripheral chemoreceptors in five, and on changes induced by excitation of central chemoreceptors in seven animals. The airway responses were recorded before and after bilateral topical application or microinjection of AMPAr kainate receptor blockers. At the end of the experiment, the spread of topically applied drugs was tested by application of a pledget loaded with 10 m l of acridine orange dye solution. The dye encircled an area with a diameter of 2–3 mm. Solutions of drugs were freshly prepared from liquid aliquots ŽMK-801, AP5 or AP7: 100 mM; CNQX:4 and 50 mM; DNQX: 50 mM; NMDA: 50 mM.. 2.1. Data collection and analysis Records were analyzed to determine the airway responses to lung deflation, laryngeal cooling, stimulation of peripheral and central chemoreceptors, before and after interventions. Individual data and average values of all dogs are presented as mean change" standard error of mean Žmean " SEM. for each variable. Statistical comparisons were made with Student’s t-test. The criterion for statistical significance was p - 0.05.
3. Results Only dogs in whom under hyperoxic, normocapnic and normohydric conditions, excitation of rapidly adapting receptors by lung deflation elicited significant tracheal smooth muscle contraction Ž) 15 g. were included in this report. 3.1. Effects of NMDA receptor blockade on airway reflex responses to lung deflation Lung deflation induced a reproducible increase in tracheal tone. In seven dogs the effects of NMDA receptor blockade on airway reflex responses to lung deflation were studied. Topical application of MK-801 Ž n s 2., AP5 Ž n s 2. or AP7 Ž n s 3., or microinjections of the AP7 10 nmolrsite Žin dogs following topical application of two other NMDA receptor blockers. caused a slight but insignificant reduction in tracheal smooth muscle response to lung deflation, expressed as a change from baseline tension Ž57.6 " 9 vs. 50.1 " 10.6 g; p ) 0.05.. Prior ipsilateral application of the same concentration of AP 5 or AP 7 blocked effects of unilateral topical administration of NMDA Ž4 nmol. to the ventrolateral medulla. However, NMDA topically applied on the contralateral site, follow-
ing vehicle administration, caused an increase in tracheal tension from the baseline by 67 " 17 g. CNQX had no effect on NMDA-induced increase in tracheal smooth muscle tension. 3.2. Effects of AMPA r kainate receptor blockade on airway reflex responses to lung deflation, and to laryngeal cooling Effects of AMPArkainate receptor blockade on airway reflex responses to lung deflation were studied in 14 dogs. In eight of these animals CNQX, and in one dog DNQX, were applied topically, and in the other five animals CNQX was microinjected into the rostral ventrolateral medulla. Blockade of AMPArkainate receptors elicited a concentration-dependent decrease in reflex tracheal smooth muscle response to lung deflation. An example of the effects of bilateral topical application of CNQX Ž1, 3 and 10 nmolrsite. on tracheal smooth muscle, and on arterial pressure responses to lung deflation is shown in Fig. 1ŽA.. Similarly, microinjections of CNQX in doses of 3–10 nmol diminished reflex airway constriction, and caused a decrease in inflation pressure. Concentrations of CNQX, which completely abolished airway response to lung deflation, had no effect on the amplitude of the phrenic nerve response, but increased the phrenic nerve discharge frequency. Topical application of lidocaine to the same sites abolished the phrenic nerve activity and also the response to lung deflation, as shown in Fig. 1ŽB.. Individual data for the tracheal smooth muscle response to lung deflation, expressed as a change in tracheal force from the baseline, are shown in Fig. 2 Župper left panel.. In the control period Žleft side of the figure., lung deflation increased the tracheal tension on average by 71 " 8.3 g. Following the AMPArkainate blockade, tracheal tension increased on average by 12.6 " 4.6 g, which is significantly less than in the control period Žright side of the figure; p - 0.05.. The degree of CNQX-induced inhibition of tracheal smooth muscle response to lung deflation tended to be higher when the drug was applied after the blockade of NMDA receptors than following vehicle administration, and differences when expressed as percent decrease in response to lung deflation were significant Ž92 " 4 vs. 73 " 9%; p 0.05.. Effects of the AMPArkainate receptor blockade on tracheal tone response to laryngeal cooling were studied in eight dogs ŽFig. 2, upper right panel.. In control conditions, laryngeal cooling increased tracheal tone. Repeated provocations, at five-minute intervals, caused reproducible responses. The blockade of AMPArkainate receptors by topical application of CNQX, in concentrations which diminished the response to lung deflation, also significantly attenuated the constrictive effect of laryngeal cooling on tracheal smooth muscle. In the control period, laryngeal cooling caused tracheal tension to increase on average by 52.2 " 7.2 g. Following the AMPArkainate
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Fig. 1. Tracings from paralyzed and artificially ventilated dogs, demonstrating the effect of bilateral topical application of CNQX to airway related ventrolateral medullary region on tracheal smooth muscle force ŽTf ., arterial pressure, and phrenic nerve activity responses to lung deflation. Dog A: a – control; b – after CNQX Ž1 nmol.; c – after CNQX Ž3 nmol.; and d – after CNQX Ž10 nmol.. Dog B: a – control; b – after CNQX 3 nmol; c – after CNQX Ž10 nmol., and d – after lidocaine Ž2%.. Pt – tracheal pressure; V – flow; Phr – moving average of the phrenic nerve activity. Horizontal bars: 30 s.
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3.4. Effects of NMDA and AMPAr kainate receptor blockade on arterial pressure and heart rate The NMDA receptor antagonists applied topically or microinjected within the ventrolateral region where the airway-related preganglionic cells are located, had no significant effect on mean arterial pressure. However, blockade of AMPArkainate receptors produced a decrease in baseline mean arterial pressure from 141 " 5 to 118 " 6 mmHg Ž p - 0.05., and had insignificant effects on the baseline heart rate Ž158 " 10 vs. 153 " 10 beats per min; p ) 0.05.. In addition, blockade of AMPArkainate receptors attenuated lung deflation-induced increase in arterial pressure.
4. Discussion
Fig. 2. Individual data of the effect of CNQX on tracheal smooth muscle response to lung deflation, laryngeal cooling, hypercapnic and hypoxic loading. Each pair of points connected by a line represents data from 1 animal; unfilled circles CNQX after AP7 Župper left panel.; horizontal bars, mean values for all dogs.
receptor blockade, this response was reduced to 3.5 " 1.3 g Ž p - 0.05.. 3.3. Effects of AMPAr kainate receptor blockade on tracheal tone responses to hypercapnic and hypoxic loading When animals were ventilated with 7% CO 2 in O 2 , tracheal tone increased, on average by 22.9 " 4.8 g Ž n s 7.. The blockade of AMPArkainate receptors reduced tracheal smooth muscle responses to hypercapnic stimulation. In these dogs, following CNQX administration, ventilation with 7% CO 2 in O 2 increased tracheal tone on average by 6.1 " 4.3 g, significantly less than in the control period Ž p - 0.05.. Individual data for the hypercapnia-induced changes in tracheal tension before and after AMPArkainate receptor blockade are presented in Fig. 2, lower left panel. The peripheral chemoreceptors were stimulated by ventilating animals for two min with 10% O 2 in N2 . As shown in Fig. 2 Žlower right panel., application of CNQX to the ventrolateral medulla attenuated the response to hypoxia, decreasing tracheal response from 35.9 " 7.8 to 8.4 " 6.7 g Ž p - 0.05..
The results of this study indicate for the first time that excitatory amino acids, glutamate and possibly aspartate, are primary neurotransmitters in mediating reflex airway smooth muscle contraction. Furthermore, the present study demonstrated that AMPArkainate subtype of glutamate receptors is the major signaling pathway involved in transmission of excitatory reflex inputs from the nucleus tractus solitarius ŽnTS. neurons to airway-related vagal preganglionic cells, as illustrated in Fig. 3, and in mediating hypercapnia-induced airway constriction. Furthermore, these data support previous studies, showing that the ventral vagal nucleus is the main site for generation of cholinergic outflow to the airways w10–13,24x. Several lines of evidence support the idea that reflex tracheomotor responses triggered by sensory mechanisms in the lung and the airway can be amplified by many factors, including inputs from peripheral and central chemoreceptors. In experiments reported here, lung deflation-induced reflex tracheal constriction was abolished by blockade of AMPArkainate receptors signal transduction pathway. This reflex response, most likely initiated by withdrawal of inhibitory inputs to airway related vagal preganglionic neurons due to a decrease in activity of slowly adapting receptors, involves an interaction between lung receptors and changes in chemosensitive drive w23,36x. Hence, the present data indicate that AMPArkainate and partly NMDA receptors participate in this interaction that produce a constrictor response. Glutamate, and the EAA receptors are widely distributed along the neuraxis. They participate in most neuronal circuits, including those involved in control of respiratory activity, and cardiovascular and respiratory reflex responses w2,4,20,22x. Furthermore, it has been shown that NMDA receptors are involved in hypoxia induced Fos expression in the nTS w14x, and mediate respiratory responses to hypoxic stress w25,35x. However, present findings indicate that the NMDA receptor pathway is partly involved in transmission of excitatory inputs from nTS
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Fig. 3. Schematic presentation of the non-NMDA receptor signal transduction pathway involved in airway reflex responses.
neurons to airway-related vagal preganglionic cells, and in potentiation of the effects of AMPArkainate receptor activation. However, NMDA receptor pathway is not required for full expression of reflexly-induced airway constriction. This could be partly explained by the fact that the kinetics of NMDA receptors are slower than of the AMPArkainate receptors. Hence, the AMPArkainate receptors are more appropriate for transmission of fast signals, while NMDA receptors may mediate the slow and tonic influences. In addition, these experiments strongly indicate that the AMPArkainate subtype of glutamate receptors are involved in an hypercapnic-induced increase in the parasympathetic outflow to the airways. It is possible that central chemosensory neurons transmit chemical signals to airway-related preganglionic neurons via glutamate. Acetylcholine, as a neurotransmitter involved in central chemosensitivity, may facilitate glutamate release on nerve terminals that innervate airway-related vagal preganglionic cells. In the experiments reported here, a similarity exists between effects of the AMPArkainate receptor blockade by topical applications with that induced by microinjections of CNQX into the same region. This indicates that substances applied to the defined region of the ventral medullary surface reach the airway-related parasympathetic cells of the ventral vagal nucleus, some of which are found beneath the ventral medullary surface w13,15x. Unlike repeated microinjections, topical applications do not cause regional damage that may interfere with autonomic regulatory functions of the ventrolateral medullary network. In mammals, the rostral ventrolateral medulla is com-
posed of sets of neurons that integrate multiple visceral and motor functions, regulate parasympathetic tone w7,8,10–12x, and generate respiratory rhythm w32x. Stimulation of this region produces an increase in breathing activity and airway constriction w10,12x. Cooling of this area or topical application of g-amino butyric acid, or lidocaine, causes withdrawal of cholinergic outflow to the airways and inhibits tracheal and the phrenic nerve responses to stimulation of peripheral or central chemoreceptors w7,8,11x. Hence, changes in airway tone parallel those of the phrenic nerve output w11,24x. In addition, it has been shown that subpopulation of bulbospinal neurons project to both phrenic motoneurons and airway-related vagal preganglionic cells w16x. However, topical application or microinjections of AMPArkainate receptor antagonist to the same site abolished reflexly induced airway constriction, but had no discernible inhibitory effects on the phrenic nerve responses to lung deflation. This indicates that the circuit engaged in reflex control of airway smooth muscle tone might differ from that which regulates the response of the phrenic nerve output. In addition, these findings suggest that signal transduction pathways involved in neurotransmission of inputs from sensory system to the airway preganglionic cells and to the medullary neurons that control the phrenic nerve activity need not to be uniform. Modulation of airway reflex responses by blockade of AMPArkainate receptors at the rostral ventrolateral medulla could conceivably be due to changes in arterial pressure, which may result in reciprocal modulation of airway tone by a baroreceptor reflex w27x. In the present studies blockade of AMPArkainate receptors tended to cause a decrease in arterial blood pressure. Therefore, it
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seems unlikely that changes in vasomotor activity are responsible for the inhibition of airway reflex responses induced by AMPArkainate receptor blockade. In conclusion, the results of these studies provide new information demonstrating that the signal transduction pathwayŽs. linked to AMPArkainate glutamate receptor subtype plays a major role in mediating reflexly increased cholinergic outflow to the airways. Abnormalities in this central signaling pathway may lead to enhanced airway responsiveness, sustained obstruction of the airways and altered relaxation. Hence, these findings could influence the design of therapeutic interventions, and improve the treatment of airway obstructive disorders due to elevation of vagal tone.
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Acknowledgements This work was supported by the National Institute of Heart, Lung and Blood of the National Institutes of Health ŽHL 50527 and 56470.. We thank Ms. Cecily Lewis for secretarial support.
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w35x A. Vardhan, A. Kachroo, H.N. Sapru, Excitatory amino acid receptors in commissural nucleus of the NTS mediate carotid chemoreceptor responses, Am. J. Physiol. 264 Ž1993. R41–R50. w36x E.H. Vidruk, Hypoxia potentiates, oxygen attenuates deflation-induced reflex tracheal constriction, J. Appl. Physiol. 59 Ž1985. 941– 946. w37x M. Vignes, G.L. Collingridge, The synaptic activation kainate receptors, Nature 388 Ž1997. 179–182. w38x E.H. Wong, J.A. Kemp, Sites for antagonism of the N-methyl-Daspartate recepto channel complex, Ann. Rev. Pharmacol. Toxicol. 31 Ž1991. 401–425.
The role of excitatory amino acids in airway reflex responses in anesthetized dogs M.A. Haxhiu ) , B. Erokwu, I.A. Dreshaj Department of Medicine, Case Western ReserÕe UniÕersity School of Medicine, 10900 Euclid AÕenue, CleÕeland, OH 44106, USA Received 11 July 1997; revised 18 September 1997; accepted 24 September 1997
Abstract In these studies we examined the role of excitatory amino acids ŽEAAs. neurotransmission in communicating sensory inputs to the airway-related vagal preganglionic neurons, by examining the effects of either NMDA or AMPArkainate receptor blockade on reflex and chemical responses of tracheal smooth muscle. Experiments were performed in chloralose anesthetized, paralyzed and mechanically ventilated beagle dogs Ž n s 18., under hyperoxic, normocapnic, and normohydric conditions. Topical application or microinjection of NMDA receptor blockers, into the region of the ventrolateral medulla where airway-related vagal preganglionic neurons are located, insignificantly decreased the reflex changes in tracheal tone. However, topical application or microinjection of AMPArkainate subtype of glutamate receptor selective antagonists markedly reduced reflex increase in tracheal tone induced by Ž1. lung deflation, Ž2. stimulation of laryngeal cold receptors, and Ž3. activation of peripheral or central chemoreceptors. These effects were potentiated by prior NMDA receptor blockade. Findings indicate that an increase in central cholinergic outflow to the airways by a variety of excitatory afferent inputs is mediated via activation of EAA receptors, mainly AMPArkainate subtype of glutamate receptors. q 1997 Elsevier Science B.V. Keywords: Airway; Airway mechanoreceptors; Dog; Central chemoreceptors; Laryngeal cold receptors; Glutamate receptors: NMDA subtype receptor, AMPArkainate receptor; Nucleus ambiguus; Parasympathetic nervous system; Peripheral chemoreceptors; Rostral ventrolateral medulla; Tracheal tone
1. Introduction In recent years, considerable research effort has been made in elucidating the afferent and efferent innervation of the airways, and the physiology and pathophysiology of reflex responses w1,5,10,13,15,23,27,30,36 x. However, little is known about the primary neurochemicalŽs., and the receptor types involved in transmission of excitatory inputs that arise from the airway sensory fibers, peripheral and central chemoreceptors. This information is important in understanding the central nervous mechanisms that could initiate reflex airway responses, and in humans may influence the severity of airway diseases. Although a number of neurochemical signals may be implicated in mediating a reflexly induced increase in cholinergic outflow to the airways, the present study was
) Corresponding author. Tel.: q1 216 3688630; fax: q1 216 3680034; e-mail: [email protected]
0165-1838r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 7 . 0 0 1 1 0 - 0
focused on the role of excitatory amino acids ŽEAAs., since they are considered to be key neurotransmitters in almost all regions of the brain w17x. In addition, EAA receptors are widely distributed along the neuraxis w26,31,38x, and participate in most of the neural circuits, including those implicated in cardiorespiratory and airway control w2,4,12,18,20,22,29,34 x, and in mediating bronchodilator effects of hindlimb afferents stimulation w33x. Effects of released EAAs could be transmitted by activation of the inotropic or metabotropic receptors. The inotropic glutamate or ligand-gated ion channels, contain three known functional receptor subtypes: Ž1. N-methylD-aspartate ŽNMDA. receptors, which respond to glutamate and glutamate analogs such as NMDA, Ž2. the aam ino-3-hydroxy-5-m ethyl-4-isoxazolepropionate ŽAMPA., and Ž3. kainate receptor subtype. Although a fourth subtype of inotropic receptor exists, the delta receptor, it is not established as a functional ion channel w26x. The NMDA receptors are present in the brainstem w26,31x, and are activated by NMDA and glutamate, but do not respond to kainate and AMPA. Effects of activation of
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this functional receptor subtype in the ventrolateral medulla can be antagonized with competitive antagonists that act at the recognition site such as D-2-amino-5-phosphonovalerate ŽAP5., or D-2-amino-7-phosponohetanoate ŽAP7., and with a non-competitive channel blocking agent such as MK-801 w6,26,28,38x. The AMPA receptor, when expressed alone or in combination with other glutamate receptor subtypes, can be activated by quisqualate, AMPA, glutamate, and kainate. The quinoxalinediones such as 6,7-dinitroquinoxaline-2,3dione ŽDNQX ., 6-cyano-7-nitroquinoxaline-2,3-dione ŽCNQX., and 2,3-dihydroxy-6-nitro-7-sulphamoybenzoŽF. quinoxakine ŽNBQX. are potent antagonists of AMPA receptors w21,26,29x. This class of antagonists also blocks kainate receptors, but does not block NMDA receptors. Hence, it can be used to differentiate AMPArkainate receptor mediated effects from the NMDA signaling pathway. The kainate receptors respond to kainate, quisqualate, glutamate, and have lower responses to AMPA. Although in the CNS kainate receptors are about 50-fold less abundant than AMPA receptors w9,19x, it may play an important role in airway reflex responses. Recently it has been shown that kainate receptors can be activated synaptically, and might be involved in synaptic plasticity w37x. In the studies reported here we examined the role of EAA neurotransmission in communicating sensory inputs to airway-related vagal preganglionic neurons, by examining the effects of either NMDA or AMPArkainate receptor blockade on reflex responses of tracheal smooth muscle tone.
2. Methods The studies were performed on 18 adult beagle dogs of either gender, weighing 8–12 kg, anesthetized by the administration of thiopental sodium Ž25 mgrkg iv. followed by a-chloralose Ž80 mgrkg iv.. Supplemental doses of a-chloralose Ž10 mgrkg iv. were given hourly to maintain anesthesia. A low tracheostomy was performed, and a tracheal tube was inserted. The dogs were artificially ventilated with a volume ventilator ŽHarvard Apparatus., delivering a constant volume of 10 mlrkg at a frequency of 20–25 breathsrmin, from a bag containing 50% O 2 in N2 Ž n s 16. or 100% O 2 Ž n s 2.. A femoral artery and a femoral vein were cannulated for measurement of blood pressure ŽDigi-Mede Blood Pressure Analyzer, MicroMed, Louisville, KY., blood gas sampling, or for administration of fluid and paralytic agent, gallamine triethiodide ŽFlaxedil, 4–6 mgrkg.. The body temperature was maintained between 37.5 and 38.58C, by means of a heating pad. Ventral aspect of the medulla oblongata was widely exposed, as described earlier w11x. An in vivo segment of extra thoracic trachea was prepared for isometric measurement of tracheal smooth muscle responses w3x. Changes in the force generated by
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tracheal smooth muscle contraction were measured by subtracting the initial preload tension Ž28–35 g. from the peak tension developed following excitation of airway sensory nerve fibers, peripheral or central chemoreceptors. In three of these dogs the fifth root of the phrenic nerve was isolated, cut, and desheathed. The central end of the desheathed phrenic nerve was placed on a bipolar hook electrode. Mass neural activity was amplified ŽP 511 amplifier, Grass instruments., filtered between 0.1 and 3 kHz, rectified and integrated Ž200 ms time constant. with Paynter filters ŽCharles Ward Enterprises.. Airway mechanoreceptors were stimulated by lung deflation, which was produced by stopping the ventilator during the deflation phase for a period of 30 to 60 s Ž n s 14.; laryngeal cold receptors were activated by changing laryngeal temperature from 37 to 10–88C for 10 to 15 s Ž n s 7.. This was achieved by delivering through a plastic tube into the larynx a flow of air, passed through a coiled copper tubing immersed in ground dry ice. Hypercapnic response was obtained by ventilating the animals with 7% CO 2 balance O 2 for 5 min Ž n s 7.; peripheral chemoreceptors were exited by ventilating animals with 10% O 2 balance N2 for 2 min Ž n s 5.. At the time when tracheal responses were recorded, the average arterial PO 2 was 206 " 26 mmHg, PCO 2 was 33 " 5 mmHg, and pH was 7.36 " 0.042 units Žmean " SEM.. The vehicle and the dissolved drugs were applied topically to the ventrolateral medulla or microinjected into the same region. Topical application was performed by a round-shaped pledget Ž2 mm diam.. made of Gel Foam and soaked with 10 m l of solution. Filter paper wicks were placed at the lateral and rostral margins of the exposed ventral medulla to prevent cerebrospinal fluid accumulation. Drugs were applied bilaterally on the area which corresponds to the region located 6–8 mm caudal to the foramen cecum, and 3.5–4.5 mm lateral to the midline. In a separate group of animals, drugs were microinjected in the same area, 1.5 mm deep to the surface. The volume of injectate was 100 nl, an amount that spreads about 1 mm in the medulla of the dog w10x. The NMDA receptors were blocked by topical application, or microinjections of MK-801, AP5 or AP7. The CNQX was used to antagonize the effects of AMPAr kainate receptor activation in all but one dog studied, in whom DNQX was employed. The first set of experiments Žseven dogs. addressed the potential role of NMDA receptors on airway reflex responses to lung deflation. Changes in the tracheal smooth muscle tension induced by lung deflation were recorded prior to and after topical administration or microinjections of AP5, AP-7 or MK-801. The second series of experiments Ž14 animals. examined the involvement of AMPAr kainate receptors in mediating airway reflex responses to lung deflation. In these animals CNQX was applied topically, or microinjected into the region in which airway-related vagal preganglionic cells are located. In
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seven of these dogs CNQX was administered after the application of NMDA receptor blockers. The effect of AMPArkainate receptor blockade on tracheal smooth muscle contraction induced by stimulation of laryngeal cold receptors was studied in eight dogs, effects on tracheal contraction caused by activation of peripheral chemoreceptors in five, and on changes induced by excitation of central chemoreceptors in seven animals. The airway responses were recorded before and after bilateral topical application or microinjection of AMPAr kainate receptor blockers. At the end of the experiment, the spread of topically applied drugs was tested by application of a pledget loaded with 10 m l of acridine orange dye solution. The dye encircled an area with a diameter of 2–3 mm. Solutions of drugs were freshly prepared from liquid aliquots ŽMK-801, AP5 or AP7: 100 mM; CNQX:4 and 50 mM; DNQX: 50 mM; NMDA: 50 mM.. 2.1. Data collection and analysis Records were analyzed to determine the airway responses to lung deflation, laryngeal cooling, stimulation of peripheral and central chemoreceptors, before and after interventions. Individual data and average values of all dogs are presented as mean change" standard error of mean Žmean " SEM. for each variable. Statistical comparisons were made with Student’s t-test. The criterion for statistical significance was p - 0.05.
3. Results Only dogs in whom under hyperoxic, normocapnic and normohydric conditions, excitation of rapidly adapting receptors by lung deflation elicited significant tracheal smooth muscle contraction Ž) 15 g. were included in this report. 3.1. Effects of NMDA receptor blockade on airway reflex responses to lung deflation Lung deflation induced a reproducible increase in tracheal tone. In seven dogs the effects of NMDA receptor blockade on airway reflex responses to lung deflation were studied. Topical application of MK-801 Ž n s 2., AP5 Ž n s 2. or AP7 Ž n s 3., or microinjections of the AP7 10 nmolrsite Žin dogs following topical application of two other NMDA receptor blockers. caused a slight but insignificant reduction in tracheal smooth muscle response to lung deflation, expressed as a change from baseline tension Ž57.6 " 9 vs. 50.1 " 10.6 g; p ) 0.05.. Prior ipsilateral application of the same concentration of AP 5 or AP 7 blocked effects of unilateral topical administration of NMDA Ž4 nmol. to the ventrolateral medulla. However, NMDA topically applied on the contralateral site, follow-
ing vehicle administration, caused an increase in tracheal tension from the baseline by 67 " 17 g. CNQX had no effect on NMDA-induced increase in tracheal smooth muscle tension. 3.2. Effects of AMPA r kainate receptor blockade on airway reflex responses to lung deflation, and to laryngeal cooling Effects of AMPArkainate receptor blockade on airway reflex responses to lung deflation were studied in 14 dogs. In eight of these animals CNQX, and in one dog DNQX, were applied topically, and in the other five animals CNQX was microinjected into the rostral ventrolateral medulla. Blockade of AMPArkainate receptors elicited a concentration-dependent decrease in reflex tracheal smooth muscle response to lung deflation. An example of the effects of bilateral topical application of CNQX Ž1, 3 and 10 nmolrsite. on tracheal smooth muscle, and on arterial pressure responses to lung deflation is shown in Fig. 1ŽA.. Similarly, microinjections of CNQX in doses of 3–10 nmol diminished reflex airway constriction, and caused a decrease in inflation pressure. Concentrations of CNQX, which completely abolished airway response to lung deflation, had no effect on the amplitude of the phrenic nerve response, but increased the phrenic nerve discharge frequency. Topical application of lidocaine to the same sites abolished the phrenic nerve activity and also the response to lung deflation, as shown in Fig. 1ŽB.. Individual data for the tracheal smooth muscle response to lung deflation, expressed as a change in tracheal force from the baseline, are shown in Fig. 2 Župper left panel.. In the control period Žleft side of the figure., lung deflation increased the tracheal tension on average by 71 " 8.3 g. Following the AMPArkainate blockade, tracheal tension increased on average by 12.6 " 4.6 g, which is significantly less than in the control period Žright side of the figure; p - 0.05.. The degree of CNQX-induced inhibition of tracheal smooth muscle response to lung deflation tended to be higher when the drug was applied after the blockade of NMDA receptors than following vehicle administration, and differences when expressed as percent decrease in response to lung deflation were significant Ž92 " 4 vs. 73 " 9%; p 0.05.. Effects of the AMPArkainate receptor blockade on tracheal tone response to laryngeal cooling were studied in eight dogs ŽFig. 2, upper right panel.. In control conditions, laryngeal cooling increased tracheal tone. Repeated provocations, at five-minute intervals, caused reproducible responses. The blockade of AMPArkainate receptors by topical application of CNQX, in concentrations which diminished the response to lung deflation, also significantly attenuated the constrictive effect of laryngeal cooling on tracheal smooth muscle. In the control period, laryngeal cooling caused tracheal tension to increase on average by 52.2 " 7.2 g. Following the AMPArkainate
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Fig. 1. Tracings from paralyzed and artificially ventilated dogs, demonstrating the effect of bilateral topical application of CNQX to airway related ventrolateral medullary region on tracheal smooth muscle force ŽTf ., arterial pressure, and phrenic nerve activity responses to lung deflation. Dog A: a – control; b – after CNQX Ž1 nmol.; c – after CNQX Ž3 nmol.; and d – after CNQX Ž10 nmol.. Dog B: a – control; b – after CNQX 3 nmol; c – after CNQX Ž10 nmol., and d – after lidocaine Ž2%.. Pt – tracheal pressure; V – flow; Phr – moving average of the phrenic nerve activity. Horizontal bars: 30 s.
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3.4. Effects of NMDA and AMPAr kainate receptor blockade on arterial pressure and heart rate The NMDA receptor antagonists applied topically or microinjected within the ventrolateral region where the airway-related preganglionic cells are located, had no significant effect on mean arterial pressure. However, blockade of AMPArkainate receptors produced a decrease in baseline mean arterial pressure from 141 " 5 to 118 " 6 mmHg Ž p - 0.05., and had insignificant effects on the baseline heart rate Ž158 " 10 vs. 153 " 10 beats per min; p ) 0.05.. In addition, blockade of AMPArkainate receptors attenuated lung deflation-induced increase in arterial pressure.
4. Discussion
Fig. 2. Individual data of the effect of CNQX on tracheal smooth muscle response to lung deflation, laryngeal cooling, hypercapnic and hypoxic loading. Each pair of points connected by a line represents data from 1 animal; unfilled circles CNQX after AP7 Župper left panel.; horizontal bars, mean values for all dogs.
receptor blockade, this response was reduced to 3.5 " 1.3 g Ž p - 0.05.. 3.3. Effects of AMPAr kainate receptor blockade on tracheal tone responses to hypercapnic and hypoxic loading When animals were ventilated with 7% CO 2 in O 2 , tracheal tone increased, on average by 22.9 " 4.8 g Ž n s 7.. The blockade of AMPArkainate receptors reduced tracheal smooth muscle responses to hypercapnic stimulation. In these dogs, following CNQX administration, ventilation with 7% CO 2 in O 2 increased tracheal tone on average by 6.1 " 4.3 g, significantly less than in the control period Ž p - 0.05.. Individual data for the hypercapnia-induced changes in tracheal tension before and after AMPArkainate receptor blockade are presented in Fig. 2, lower left panel. The peripheral chemoreceptors were stimulated by ventilating animals for two min with 10% O 2 in N2 . As shown in Fig. 2 Žlower right panel., application of CNQX to the ventrolateral medulla attenuated the response to hypoxia, decreasing tracheal response from 35.9 " 7.8 to 8.4 " 6.7 g Ž p - 0.05..
The results of this study indicate for the first time that excitatory amino acids, glutamate and possibly aspartate, are primary neurotransmitters in mediating reflex airway smooth muscle contraction. Furthermore, the present study demonstrated that AMPArkainate subtype of glutamate receptors is the major signaling pathway involved in transmission of excitatory reflex inputs from the nucleus tractus solitarius ŽnTS. neurons to airway-related vagal preganglionic cells, as illustrated in Fig. 3, and in mediating hypercapnia-induced airway constriction. Furthermore, these data support previous studies, showing that the ventral vagal nucleus is the main site for generation of cholinergic outflow to the airways w10–13,24x. Several lines of evidence support the idea that reflex tracheomotor responses triggered by sensory mechanisms in the lung and the airway can be amplified by many factors, including inputs from peripheral and central chemoreceptors. In experiments reported here, lung deflation-induced reflex tracheal constriction was abolished by blockade of AMPArkainate receptors signal transduction pathway. This reflex response, most likely initiated by withdrawal of inhibitory inputs to airway related vagal preganglionic neurons due to a decrease in activity of slowly adapting receptors, involves an interaction between lung receptors and changes in chemosensitive drive w23,36x. Hence, the present data indicate that AMPArkainate and partly NMDA receptors participate in this interaction that produce a constrictor response. Glutamate, and the EAA receptors are widely distributed along the neuraxis. They participate in most neuronal circuits, including those involved in control of respiratory activity, and cardiovascular and respiratory reflex responses w2,4,20,22x. Furthermore, it has been shown that NMDA receptors are involved in hypoxia induced Fos expression in the nTS w14x, and mediate respiratory responses to hypoxic stress w25,35x. However, present findings indicate that the NMDA receptor pathway is partly involved in transmission of excitatory inputs from nTS
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Fig. 3. Schematic presentation of the non-NMDA receptor signal transduction pathway involved in airway reflex responses.
neurons to airway-related vagal preganglionic cells, and in potentiation of the effects of AMPArkainate receptor activation. However, NMDA receptor pathway is not required for full expression of reflexly-induced airway constriction. This could be partly explained by the fact that the kinetics of NMDA receptors are slower than of the AMPArkainate receptors. Hence, the AMPArkainate receptors are more appropriate for transmission of fast signals, while NMDA receptors may mediate the slow and tonic influences. In addition, these experiments strongly indicate that the AMPArkainate subtype of glutamate receptors are involved in an hypercapnic-induced increase in the parasympathetic outflow to the airways. It is possible that central chemosensory neurons transmit chemical signals to airway-related preganglionic neurons via glutamate. Acetylcholine, as a neurotransmitter involved in central chemosensitivity, may facilitate glutamate release on nerve terminals that innervate airway-related vagal preganglionic cells. In the experiments reported here, a similarity exists between effects of the AMPArkainate receptor blockade by topical applications with that induced by microinjections of CNQX into the same region. This indicates that substances applied to the defined region of the ventral medullary surface reach the airway-related parasympathetic cells of the ventral vagal nucleus, some of which are found beneath the ventral medullary surface w13,15x. Unlike repeated microinjections, topical applications do not cause regional damage that may interfere with autonomic regulatory functions of the ventrolateral medullary network. In mammals, the rostral ventrolateral medulla is com-
posed of sets of neurons that integrate multiple visceral and motor functions, regulate parasympathetic tone w7,8,10–12x, and generate respiratory rhythm w32x. Stimulation of this region produces an increase in breathing activity and airway constriction w10,12x. Cooling of this area or topical application of g-amino butyric acid, or lidocaine, causes withdrawal of cholinergic outflow to the airways and inhibits tracheal and the phrenic nerve responses to stimulation of peripheral or central chemoreceptors w7,8,11x. Hence, changes in airway tone parallel those of the phrenic nerve output w11,24x. In addition, it has been shown that subpopulation of bulbospinal neurons project to both phrenic motoneurons and airway-related vagal preganglionic cells w16x. However, topical application or microinjections of AMPArkainate receptor antagonist to the same site abolished reflexly induced airway constriction, but had no discernible inhibitory effects on the phrenic nerve responses to lung deflation. This indicates that the circuit engaged in reflex control of airway smooth muscle tone might differ from that which regulates the response of the phrenic nerve output. In addition, these findings suggest that signal transduction pathways involved in neurotransmission of inputs from sensory system to the airway preganglionic cells and to the medullary neurons that control the phrenic nerve activity need not to be uniform. Modulation of airway reflex responses by blockade of AMPArkainate receptors at the rostral ventrolateral medulla could conceivably be due to changes in arterial pressure, which may result in reciprocal modulation of airway tone by a baroreceptor reflex w27x. In the present studies blockade of AMPArkainate receptors tended to cause a decrease in arterial blood pressure. Therefore, it
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seems unlikely that changes in vasomotor activity are responsible for the inhibition of airway reflex responses induced by AMPArkainate receptor blockade. In conclusion, the results of these studies provide new information demonstrating that the signal transduction pathwayŽs. linked to AMPArkainate glutamate receptor subtype plays a major role in mediating reflexly increased cholinergic outflow to the airways. Abnormalities in this central signaling pathway may lead to enhanced airway responsiveness, sustained obstruction of the airways and altered relaxation. Hence, these findings could influence the design of therapeutic interventions, and improve the treatment of airway obstructive disorders due to elevation of vagal tone.
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Acknowledgements This work was supported by the National Institute of Heart, Lung and Blood of the National Institutes of Health ŽHL 50527 and 56470.. We thank Ms. Cecily Lewis for secretarial support.
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