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The changes in pulmonary C-fiber activity and lung mechanics induced by vagal stimulation in rabbits
Life Sciences, Vol. 61, No. 22, pp. 2189-2195, 1997 apyri@t 0 199-I ELcvier .s&tce Inc. PdeditttheUSA. AUri@mmerd am-32Mp $17.00 t .oo
PI1 s0024-3205(97)00921-1
ELSEVIER
THE CHANGES IN PULMONARY C-FIBER ACTIVITY AND LUNG MECHANICS INDUCED BY VAGAL STIMULATION IN RABBITS S. Matsumoto Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, l-9-20 Fujimi, Chiyoda-ku, Tokyo 102, Japan (Received in fmal form September 2, 1997) Summary The effect of unilateral stimulation of the vagus nerve on pulmonary C-fiber activity, total lung resistance (RI) and dynamic lung compliance (Cdyn) was studied in anesthetized, artificially ventilated rabbits. Vagal stimulation (4-30 Hz) increased both pulmonary C-fiber activity and RL but decreased Cdyn, and these changes were frequency-dependent. Atropine (2 mg/kg) blocked the responses of pulmonary Cfiber, RL and Cdyn to vagal stimulation at 4-30 Hz. Stimulation of pulmonary Cfiber activity by the higher volume-induced lung inflation was not significantly altered by atropine (2 mg/kg). A selective SP antagonist [D-Pro’, D-Try ,“I-SP (0.5 mg/kg) inhibited bronchoconstrictor responses to vagal stimulation at 30Hz but had no significant effect on those to the stimulation at 4-17 Hz. These results suggest that excitation of the rabbit pulmonary C-fiber activity by unilateral vagus nerve stimulation would occur as a result of the mechanical stimuli via a cholinergic neurotransmission and that during the stimulation at 30 Hz this excitation partly involves the local release of substance P (SP) to promote acetylcholine outflow. Key words:
atropine, pulmonary C-fiber, substance P antagonist, vagally mediated bronchoeonstriction
The predominant neuronal control of airway caliber is known to be exerted by the release of acetylcholine (ACh) from the postganglionic parasympathetic nerve fivers that innervate airway smooth muscle and that synapse in the paratracheal ganglia (1). In guinea-pigs, antidromic stimulation (preganglionically) of the vagus nerve causes the release of neuropeptides, for example, substance P (SP), neurokinin A and calcitonin gene-related peptide (CGRP), from capsaicin-sensitive nerves and ACh, indicating the presence of vagally mediated cholinergic and noncholinergic mechanisms (2, 3). The noncholinergic bronchoconstriction evoked by vagal stimulation disappears in capsaicin-treated animals (2), and administration of SP antagonists inhibits bronchoconstrictor responses to vagal stimulation and administration of capsaicin or SP (4). On the other hand, in several species the unilateral stimulation of the vagus nerve can elicit bronchoconstriction that is characterized by as an increase in pleural pressure, RL, or Cdyn (S-7). The right vagus nerve stimulation-induced bronchoconstriction in rabbits is abolished by administration of atropine (7), indicating that unilateral stimulation of the vagus nerve would not elicit the release of neurotransmitters originating from noncholinergic mechanisms. However, the contraction of airway smooth muscle is only an indirect measurement of the amount of ACh release and may be altered by other factors. Thus, it cannot completely rule out the possibility that noncholinergic-related substrates contribute partly to the bronchoconstriction evoked by unilateral vagus nerve stimulation. Correspondence: S. Matsumoto, Department of Physiology, Nippon Dental University, Dentistry at Tokyo, l-9-20 Fujimi, Chiyoda-ku, Tokyo 102, Japan
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Vagal Stimulation and Pulmonary C-fiber
Vol. 61, No. 22, 1997
Evidence that there is a close interaction between airway C-fiber activity and pulmonary edema via the local release of SP evoked by vagal stimulation has been reported by many investigators (g-10). Administration of SP is known to elicit bronchoconstriction, partly involving enhancement of ACh release form postganglionic parasympathetic nerve endings (1 l-13) but in in vivo experiments using rabbits, exogenous SP does not cause any significant increase in peak tracheal pressure (10). To determine whether excitation of vagal afferent C-fibers during unilateral vagus nerve stimulation is involved in the release of both cholinergic- and noncholinergic-related substrates, the present study was carried out to investigate the effects of antidromic stimulation of the vagus nerve with different frequencies on the responses of pulmonary C-fibers, total lung resistance (RL) and dynamic lung compliance (Cdyn) before and after administration of atropine or a SP antagonist as well as the effects of higher volume-induced lung inflations on the responses of pulmonary C-fibers before and after atropine. The experiments were performed in anesthetized, bilaterally vagotomized, artificially ventilated rabbits. By the use of the same technique reported in a previous study (7) the activity of pulmonary C-fibers was recorded from the cut left vagus nerve, while the right vagus nerve was cut and electrically stimulated. Materials and Methods Animal oreoarations and lung mechanics Twenty rabbits, weighing 2.5-3.5 kg, were anesthetized with urethane (1 g/kg) given intraperitoneally (i.p.). Additional doses (0.1-0.2 glkgihour, i.v.) of this anesthetic agent were administered from a polyethylene catheter inserted into the femoral vein. The trachea was cannulated, and polyethylene catheters were inserted into the femoral artery for measurement of systemic arterial blood pressure (SAP), and into the right atrium through the external jugular vein for administration of drugs or a 0.9 % NaCl (saline) solution. Then the vagus nerves were identified and cut at the C2 level. Following intramuscular administration of suxamethonium (10 mglkg) the rabbits were artificially ventilated. Supplemental doses of this muscular relaxant were given with a constant infusion of 10 &kg/hour. The stroke volume of the respirator was set at 10 ml/kg and its frequency ranged between 30 and 35 cycles/min. Tracheal CO? partial pressufe was monitored and kept at 30-32 mmHg, by adjusting the ventilatory rate. Respiratory airflow (V) was measured by connecting the tracheal tube to a pneumotachograph and a differential pressure transducer, and the flow signal was integrated by means of an integrator to obtain tidal volume (VT). Tracheal pressure (Pr) was measured by connecting a polyethylene catheter inserted into the tracheal tube to a differential pressure transducer, in which one arm opened to the atmosphere. RI and Cdyn were calculated by using the manual graphic method (14). Electroohvsiotoaical exoeriments The peripheral end of the cut left vagus nerve was desheathed. A thin filament, containing afferent nerve fibers, was separated, placed on a unipolar silver electrode and submerged in warm liquid paraffin (37-38 “C). The small filament was split until the single unit activity of pulmonary Cfibers had been electrically isolated. The identification of those fibers was performed using the same technique, as described in previous studies (15, 16). First, the criteria for identification of the vagal C-fibers was confirmed based on their conduction velocities (0.8-2.3 m/set). Second, assessment of the pulmonary origin of C-fibers was made by determining injection response times (within l-3 set) after right atrial injection of phenylbiguanide (PBG, 30 @g/kg). Third, the identification of pulmonary C-fibers was further confirmed by an immediate increase of their activity during transient inhalation of hypercapnic gas mixtures (5 % CO2 in 95 % 02). The action potentials of pulmonary C-fibers were selected by a window discriminator, monitored on an oscilloscope and recorded on a polygraph. The peripheral end of the cut right vagus nerve was desheathed in a paraffin pool and placed on the stimulating electrodes. In order to prevent the electrical noise due to vagal stimulation, the stimulating electrodes were covered with a rubber sheet. The vagus nerve was stimulated at 15 V: 0.2 ms, 4-30 Hz for approximately 30 s. Exoerimentai design The experiments were performed to examine the responses of pulmonary C-fiber activity, RL and Cdyn to vagal stimulation with different frequencies (4, 10, 17 and 30 Hz) before and after administration of atropine (2 mg/kg) in six different pulmonary C-fibers in 6 rabbits or a selective SP antagonist [D-Pro-, D-Try’,‘]-SP (0.5 mgkg) in eight different pulmonary C-fibers in 8 rabbits
Vol. 61, No. 22, 1997
Vagal Stimulation and Pulmonary C-fiber
Ilpllses hlmnarv C-fiber activity fimp/sJ
+: 7o OK- 200 100 0
SAP hmtkl
Iwlses Pulmmw C-fiber activity (imp/s) SAP h&j
2191
I’!!+
‘1’
‘4
2s -
7. OK4 200 100 0
Fig. 1
in pulmonary C-fiber activity, tracheal pressure (PT), tidal volume (VT), respiratory airflow (V) to unilateral vagus nerve stimulation (15 V, 10 Hz, 0.2 ms) before (A) and after (B) administration of atropine (2 mg/kg). SAP, systemic arterial blood pressure. : Period of Response:
vagal stimulation.
Pulnanary
C-fiber
activity
hnp/s)
-50 -
-loo
lhtrol
4 Hz 10 Hz bad
c&l
17 Hz
30 Hz
ot imlation)
(xchsnge
htd
4 Hz IO Hz Ned
17 Hz
30 Hz
st inwlat ion)
Fig. 2 The effects of unilateral vagus nerve stimulation (15 V, 4-30 Hz, 0.2 ms) on the responses of pulmonary C-fiber activity, total lung resistance (RL) and dynamic lung compliance (Cdyn) before (open columns) and after (closed columns) administration of atropine (2 mgkg). Vertical bars are means t SEM (n=6). ‘Statistical significance from the control values (PcO.05). “Statistically different from that before atropine pretreatment (PcO.05).
Vagal Stimulation and Pulmonary C-fiber
,tdi I
IO Pulnwnar~ C-fiber activity (iiw/s)
5
t100
Vol. 61, No. 22, 1997
** ,eti mh *Y
60 Cdyn (X change of ccxltroll
1 0
-
I
-50 Control
w?LJ** P *& 4 Hz IO Hr
17 Hz 30 Hz
(Vasal stimulation)
Fig. 3 The effects of unilateral vagus nerve stimulation (15 V, 4-30 Hz, 0.2 ms) on the responses of pulmonary C-fiber activity, total lung resistance (RI.) and dynamic lung compliance (Cdyn) before (open columns) substance P (SP) antagonist pretreatment and after (closed columns) administration of a selective SP antagonist [D-Pro”, D-Try’,‘]-SP (0.5 mg/kg). Vertical bars are means ? SEM (n=8). ‘Statistical significance from the control values (P
LResults The increased activity ot.pulmonary C-fibers occurred after the onset of right vagus nerve (RVN) sttmulatton (15 V. IO Hz), with a latency being within 3 s. *The increase in pulmonary C-fiber activity coincided with an increase in PI and a decrease in V (Fig. 1A). As shown in Fig. lB,
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VagalStimulation andPulmonary C-fiber
12 10 Pulmonary C-fiber
activity
1
**
(in&s) 5
0
2193
2 VT
1 3 VT
Fig. 4 The effects of lung inflation at l-3 tidal volume (VT) on the responses of pulmonary C-fiber activity before (open columns) and after (closed columns) administration of atropine (2 m&g). Vertical bars are means + SEM (n=6). *Statistical significance from the control values (PcO.05).
atropine treatment (2 mg/kg) abolished excitation of the RVN stimulation-induced pulmonary Cfiber activity at the same stimulus. A summary of the results in 6 rabbits before and after administration of atropine (2 mg/kg) is shown in Fig. 2. The basal discharge of pulmonary Cfibers in the control was 0.9 f 0.2 imp/s. During RVN stimulation at 4, 10, 17 and 30 Hz the discharges of pulmonary C-fibers were increased to 1.8 2 0.4, 3.9 + 0.5,5.4 i: 0.5 and 7.2 + 0.7 imp/s, respectively. Baseline RL was 16.9 c 3.8 cmHzO/l/s and Cdyn was 2.9 2 0.5 ml/cmHzO. Electrical stimulation of RVN at 4, 10, 17 and 30 Hz increased RL by 18.3 + 5.7, 36.7 + 6.7, 53.3 + 8.3 and 78.9 ? 9.2 %, respectively, but decreased Cdyn by 8.6 + 2.1, 13.3 2 3.2, 21.7 + 3.7 and 30.6 + 3.9, respectively. Pretreatment with atropine (2 mg/kg) abolished changes in pulmonary C-fiber activity, RL and Cdyn induced by RVN stimulation (4-30 Hz). The summarized results of the effect of a selective SP antagonist [D-Pro’, D-Try’,“]-SP (0.5 mg/kg) in 8 rabbits are shown in Fig. 3. The discharge of pulmonary C-fibers during control was 1.0 + 0.4 imp/s and increased during RVN stimulation at 4, lo,17 and 30 Hz to 2.1 2 0.4,4.0 + 0.5, 5.8 -C0.5 and 7.4 + 0.7 imp/s, respectively. Basal values of RL and Cdyn were 16.6 Z!2.9 cmHzO/lls and 2.9 + 0.6 ml/cmHzO, respectively. The RVN stimulation at 4, 10, 17 and 30 Hz increased RL by 15.7 + 5.8,32.5 + 5.4,47.4 2 7.8 and 67.2 2 8.5 %, respectively, but decreased Cdyn by 8.7 2 2.9, 12.9 2 2.2, 23.4 2 2.8 and 30.5 2 3.5 %, respectively. A selective SP antagonist did not significantly alter the responses of pulmonary C-fiber activity and lung mechanics (RI_and Cdyn) to RVN stimulation at 4-17 Hz, but significantly inhibited those responses to the stimulation at 30 Hz. The effects of atropine (2 mg/kg) on the responses of pulmonary C-fibers to lung inflation at l-3 VT in 6 rabbits are summarized Fig. 4. The control discharge of pulmonary C-fibers was 0.8 + 0.2 imp/s and increased during lung inflation at 2 and 3 VT to 4.9 + 0.5 and 9.7 + 0.6 imp/s, receptively. The values of RL and Cdyn before lung inflation at higher volumes were 17.1 c 3.9 cmHzO/l/sec and 3.0 + 0.6 ml/cmH20. The discharges of pulmonary C-fibers during lung inflation at 2 and 3 VT in atropine-treated animals were 4.7 2 0.5 and 9.4 + 0.7 imp/s. Pretreatment with atropine had no significant effect on stimulation of pulmonary C-fiber activity by lung inflation at higher volumes.
Vagal Stimulation
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Discussion The analysis of the slowly adapting pulmonary stretch receptor @AR) activity recorded from the cut left vagus nerve (LVN) and the change in RL or Cdyn can provide the method to quantify the bronchoconstriction evoked by electrical stimulation of the cut right vagus nerve (RVN) in rabbits with artificial ventilation (7). During the bronchoconstriction evoked by RVN stimulation, reduced ventilation of the right lung would cause an increase in V.Sof the left lung. If such an effect occurs actually, it is impossible to explain evidence showing that atropine treatment completely blocked the changes in pulmonary C-fiber activity, RL and Cdyn in response to RVN stimulation. In addition, pretreatment with atropine did not significantly alter excitatory responses of pulmonary C-fibers to lung inflation at higher volumes (2 and 3 V.r). Furthermore, RVN stimulation at 30 Hz induced approximately 2-fold increase in RI,. The results in this study are consistent with those reported by other investigators (5, 6). Thus, it is most likely that the bronchoconstriction evoked by RVN stimulation results in an increase in pulmonary C-fiber activity. A noncholinergic innervation has been demonstrated in in vivo preparations during vagus nerve stimulation (2,4, 17), and noncholinergic contractions of airway smooth muscle are known to be inhibited by capsaicin or certain substance P antagonists (4, 18, 19). In an in vitro model to examine the vagal innervation of the guinea-pig bronchus, a&opine consistently inhibits the second-phase contraction which is abolished by capsaicin treatment (20). The results indicate that noncholinergic and cholinergic contractions are not entirely independent between intramural nerves in the smooth muscle. This may be consistent with the observations suggesting that the SPinduced bronchoconstriction is partly involved in the release of ACh in in vivo experiments of the rabbit tracheal smooth muscle (12, 13). In this study, RVN stimulation resulted in bronchoconstriction associated with an increase in pulmonary C-fiber activity, and those responses obtained were frequency-dependent. In addition, frequency-related responses of pulmonary Cfibers, RI, and Cdyn to vagal stimulation were abolished by prior treatment with atropine. In the rabbit bronchial ring preparation that keeps the vagus nerve connections intact, the maximal contractile responses to vagal stimulation are completely abolished by pretreatment with atropine (21). However, it is not easy to determine whether stimulation of pulmonary C-fibers by vagal stimulation is mediated by changes in lung mechanics and/or by the release of some mediators from the vasculature. In an in vivo study of guinea pig vagal afferents, Fox et al. (22) demonstrated that C- and A 6 -fibers were highly sensitive to mechanical stimuli but showed the marked difference on their chemosensitivity. They also stated that the A 6 fibers were insensitive to capsaicin, bradykinin, histamine and serotonin but that the C-fibers responded only to capsaicin and bradykinin (22). Thus, the results in this study suggest that excitation of the pulmonary Cfibers seen during RVN stimulation is due to the effect of mechanical stimuli coincident with the increased muscle contraction by ACh as shown in unilateral vagus nerve stimulation-induced bronchoconstriction. In the rabbit, there is evidence that a SP antagonist, [D-Pro’, D-Try7:‘]-SP, at the dosages of 0.3-0.5 mgikg does not cause any constrictor action but blocks stimulation of rapidly adapting pulmonary stretch receptor (RAR) activity by exogenous SP ranging from 0.3 to 3.0 .&kg as well as by endogenous release of SP due to aerosolized capsaicin (23). Stimulation of the airway C-fibers by vagus nerve stimulation is known to elicit the local release of sufficient SP to modify the plasma exudation in the trachea and bronchi (8,9,24). However, evidence for no inhibitory effect of [D-Pro’, D-Try’“]-SP was found on the responses of pulmonary C-fibers and lung mechanics to RVN stimulation at 4-17 Hz. This implies that when excitation of pulmonary C-fibers occurs during the stimulation at relatively lower frequencies, there is no release of endogenous SP from the rabbit airways and lungs. The changes in pulmonary C-fiber activity, RL and Cdyn in response to RVN at 30 Hz were significantly inhibited but not completely blocked by administration of a SP antagonist. Considering the atropine effect, taken together, the results lead to the suggestion that stimulation of the pulmonary C-fibers by the right vagus nerve stimulation at 30 Hz elicits the release of endogenous SP which may communicate with the postganglionic cholinergic fibers capable of releasing ACh, partly involving a possible action of SP on the mechanosensitivity of the C-fibers. However, the mechanism of a possible interaction between selective release of SP from the rabbit airways and vagal stimulation at a higher frequency (30 Hz) remains to be determined.
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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
J.B. RICHARDSON, Am Rev Respir Dis 119 785-802 (1979). J.M. LUNDBERG and A. SARIA, Acta Physiol Stand 116 473-476 (1982). J.M. LUNDBERG and A. SARIA, Ann Rev Physiol @ 557-572 (1987). J.M. LUNDBERG, A. SARIA, E. BRODIN, S. ROSELL and K. FOLKERS, Proc Nat1 Acta Sci USA&J 1120-1124 (1983). CR. OLSEN, H.J.H. COLEBATCH, P.E. MEBEL, J.A. NADEL and N.C. STAUB, J Appl Physiol 2n 202-208 (1965). R.E.J. TEN BERGE, M. KRIKKE, A.C.H. TEISMAN, A.F. ROFFEL and J. ZAAGSMA, Eur J Pharmacol318 131-139 (1996). S. MATSUMOTO, Lung 124 333-344 (1996). C.T. KAPPAGODA, G.C.W. MAN, K.K. TEO, J Physiol (Lond) 394 249-265 (1987). C.A. MAGGI, A. GIACHETI’I, R.D. DEY and S.I. SAID, Physiol Rev 76 277-322 (1995). A.C. BONHAM, K.S. KOTT, K. RAVI, C.T. KAPPAGODA and J.P. JOAD, J Physiol (Lond) &j 229-238 (1996). P. ANDERSSON and H. PERSSON, Acta Pharmacol Toxic01 41444-448 (1977). D.T. TANAKA and M.M. GRUNSTEIN, J Appl Physiol 511551-1557 (1984). D.T. TANAKA and M.M. GRUNSTEIN, J Appl Physiol 60 458-463 (1986). 0. NORLANDER, P. HERZOG, I. NORDEN, G. HOSSLI, H. SCHAER and R. GA’ITIKER, Acta Anaesth Stand 12 735-752 (1968). S. DELPIERRE, C.H. GRIMAUD, Y. JAMMES and N. MEI, J Physiol (Land) 316 61-74 (1981). S. MATSUMOTO, T. KANNO, M. YAMASAKI, T. NAGAYAMA and TSHIMIZU, Respir Physiol fl165-181(1992). J.M. LUNDBERG, E. BRODIN and A. SARIA, Acta Physiol Stand 119 243-252 (1983). J. SZOLCSANYI and L. BARTHO, Neurosci Lett 3 247-251 (1982). J.-A. KARLSSON, M.J.B. FINNEY, G.A. PERSSON and C. POST, Life Sci 35 2681-2691 (1984). B.J. UNDEN, A.C. MYERS, H. BARTHLOW and D. WEINREICH, J Appl Physiol @ 13361346 (1990). J.W. BLOOM, C. BAUMGARTENER-FOLKERTS, J.D. PALMER, H.I. YAMAMURA and M. HALONEN, J Appl Physiol 65 2144-2150 (1988). A.J. FOX, P.J. BARNES, L. URBAN and A. DRAY, J Physiol (Lond) 469 21-35 (1933). S. MATSUMOTO, M. YAMASAKI, T. KANNO, T. NAGAYAMA, T. TANNO and S. SHIMIZU, Lung 122 31-45 (1994). L.S. NGUYEN, A.C. VILLABLANCA and J.C. RUTLEDGE, Am J Physiol268 RlO60R1068 (1995).
PI1 s0024-3205(97)00921-1
ELSEVIER
THE CHANGES IN PULMONARY C-FIBER ACTIVITY AND LUNG MECHANICS INDUCED BY VAGAL STIMULATION IN RABBITS S. Matsumoto Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, l-9-20 Fujimi, Chiyoda-ku, Tokyo 102, Japan (Received in fmal form September 2, 1997) Summary The effect of unilateral stimulation of the vagus nerve on pulmonary C-fiber activity, total lung resistance (RI) and dynamic lung compliance (Cdyn) was studied in anesthetized, artificially ventilated rabbits. Vagal stimulation (4-30 Hz) increased both pulmonary C-fiber activity and RL but decreased Cdyn, and these changes were frequency-dependent. Atropine (2 mg/kg) blocked the responses of pulmonary Cfiber, RL and Cdyn to vagal stimulation at 4-30 Hz. Stimulation of pulmonary Cfiber activity by the higher volume-induced lung inflation was not significantly altered by atropine (2 mg/kg). A selective SP antagonist [D-Pro’, D-Try ,“I-SP (0.5 mg/kg) inhibited bronchoconstrictor responses to vagal stimulation at 30Hz but had no significant effect on those to the stimulation at 4-17 Hz. These results suggest that excitation of the rabbit pulmonary C-fiber activity by unilateral vagus nerve stimulation would occur as a result of the mechanical stimuli via a cholinergic neurotransmission and that during the stimulation at 30 Hz this excitation partly involves the local release of substance P (SP) to promote acetylcholine outflow. Key words:
atropine, pulmonary C-fiber, substance P antagonist, vagally mediated bronchoeonstriction
The predominant neuronal control of airway caliber is known to be exerted by the release of acetylcholine (ACh) from the postganglionic parasympathetic nerve fivers that innervate airway smooth muscle and that synapse in the paratracheal ganglia (1). In guinea-pigs, antidromic stimulation (preganglionically) of the vagus nerve causes the release of neuropeptides, for example, substance P (SP), neurokinin A and calcitonin gene-related peptide (CGRP), from capsaicin-sensitive nerves and ACh, indicating the presence of vagally mediated cholinergic and noncholinergic mechanisms (2, 3). The noncholinergic bronchoconstriction evoked by vagal stimulation disappears in capsaicin-treated animals (2), and administration of SP antagonists inhibits bronchoconstrictor responses to vagal stimulation and administration of capsaicin or SP (4). On the other hand, in several species the unilateral stimulation of the vagus nerve can elicit bronchoconstriction that is characterized by as an increase in pleural pressure, RL, or Cdyn (S-7). The right vagus nerve stimulation-induced bronchoconstriction in rabbits is abolished by administration of atropine (7), indicating that unilateral stimulation of the vagus nerve would not elicit the release of neurotransmitters originating from noncholinergic mechanisms. However, the contraction of airway smooth muscle is only an indirect measurement of the amount of ACh release and may be altered by other factors. Thus, it cannot completely rule out the possibility that noncholinergic-related substrates contribute partly to the bronchoconstriction evoked by unilateral vagus nerve stimulation. Correspondence: S. Matsumoto, Department of Physiology, Nippon Dental University, Dentistry at Tokyo, l-9-20 Fujimi, Chiyoda-ku, Tokyo 102, Japan
School of
2190
Vagal Stimulation and Pulmonary C-fiber
Vol. 61, No. 22, 1997
Evidence that there is a close interaction between airway C-fiber activity and pulmonary edema via the local release of SP evoked by vagal stimulation has been reported by many investigators (g-10). Administration of SP is known to elicit bronchoconstriction, partly involving enhancement of ACh release form postganglionic parasympathetic nerve endings (1 l-13) but in in vivo experiments using rabbits, exogenous SP does not cause any significant increase in peak tracheal pressure (10). To determine whether excitation of vagal afferent C-fibers during unilateral vagus nerve stimulation is involved in the release of both cholinergic- and noncholinergic-related substrates, the present study was carried out to investigate the effects of antidromic stimulation of the vagus nerve with different frequencies on the responses of pulmonary C-fibers, total lung resistance (RL) and dynamic lung compliance (Cdyn) before and after administration of atropine or a SP antagonist as well as the effects of higher volume-induced lung inflations on the responses of pulmonary C-fibers before and after atropine. The experiments were performed in anesthetized, bilaterally vagotomized, artificially ventilated rabbits. By the use of the same technique reported in a previous study (7) the activity of pulmonary C-fibers was recorded from the cut left vagus nerve, while the right vagus nerve was cut and electrically stimulated. Materials and Methods Animal oreoarations and lung mechanics Twenty rabbits, weighing 2.5-3.5 kg, were anesthetized with urethane (1 g/kg) given intraperitoneally (i.p.). Additional doses (0.1-0.2 glkgihour, i.v.) of this anesthetic agent were administered from a polyethylene catheter inserted into the femoral vein. The trachea was cannulated, and polyethylene catheters were inserted into the femoral artery for measurement of systemic arterial blood pressure (SAP), and into the right atrium through the external jugular vein for administration of drugs or a 0.9 % NaCl (saline) solution. Then the vagus nerves were identified and cut at the C2 level. Following intramuscular administration of suxamethonium (10 mglkg) the rabbits were artificially ventilated. Supplemental doses of this muscular relaxant were given with a constant infusion of 10 &kg/hour. The stroke volume of the respirator was set at 10 ml/kg and its frequency ranged between 30 and 35 cycles/min. Tracheal CO? partial pressufe was monitored and kept at 30-32 mmHg, by adjusting the ventilatory rate. Respiratory airflow (V) was measured by connecting the tracheal tube to a pneumotachograph and a differential pressure transducer, and the flow signal was integrated by means of an integrator to obtain tidal volume (VT). Tracheal pressure (Pr) was measured by connecting a polyethylene catheter inserted into the tracheal tube to a differential pressure transducer, in which one arm opened to the atmosphere. RI and Cdyn were calculated by using the manual graphic method (14). Electroohvsiotoaical exoeriments The peripheral end of the cut left vagus nerve was desheathed. A thin filament, containing afferent nerve fibers, was separated, placed on a unipolar silver electrode and submerged in warm liquid paraffin (37-38 “C). The small filament was split until the single unit activity of pulmonary Cfibers had been electrically isolated. The identification of those fibers was performed using the same technique, as described in previous studies (15, 16). First, the criteria for identification of the vagal C-fibers was confirmed based on their conduction velocities (0.8-2.3 m/set). Second, assessment of the pulmonary origin of C-fibers was made by determining injection response times (within l-3 set) after right atrial injection of phenylbiguanide (PBG, 30 @g/kg). Third, the identification of pulmonary C-fibers was further confirmed by an immediate increase of their activity during transient inhalation of hypercapnic gas mixtures (5 % CO2 in 95 % 02). The action potentials of pulmonary C-fibers were selected by a window discriminator, monitored on an oscilloscope and recorded on a polygraph. The peripheral end of the cut right vagus nerve was desheathed in a paraffin pool and placed on the stimulating electrodes. In order to prevent the electrical noise due to vagal stimulation, the stimulating electrodes were covered with a rubber sheet. The vagus nerve was stimulated at 15 V: 0.2 ms, 4-30 Hz for approximately 30 s. Exoerimentai design The experiments were performed to examine the responses of pulmonary C-fiber activity, RL and Cdyn to vagal stimulation with different frequencies (4, 10, 17 and 30 Hz) before and after administration of atropine (2 mg/kg) in six different pulmonary C-fibers in 6 rabbits or a selective SP antagonist [D-Pro-, D-Try’,‘]-SP (0.5 mgkg) in eight different pulmonary C-fibers in 8 rabbits
Vol. 61, No. 22, 1997
Vagal Stimulation and Pulmonary C-fiber
Ilpllses hlmnarv C-fiber activity fimp/sJ
+: 7o OK- 200 100 0
SAP hmtkl
Iwlses Pulmmw C-fiber activity (imp/s) SAP h&j
2191
I’!!+
‘1’
‘4
2s -
7. OK4 200 100 0
Fig. 1
in pulmonary C-fiber activity, tracheal pressure (PT), tidal volume (VT), respiratory airflow (V) to unilateral vagus nerve stimulation (15 V, 10 Hz, 0.2 ms) before (A) and after (B) administration of atropine (2 mg/kg). SAP, systemic arterial blood pressure. : Period of Response:
vagal stimulation.
Pulnanary
C-fiber
activity
hnp/s)
-50 -
-loo
lhtrol
4 Hz 10 Hz bad
c&l
17 Hz
30 Hz
ot imlation)
(xchsnge
htd
4 Hz IO Hz Ned
17 Hz
30 Hz
st inwlat ion)
Fig. 2 The effects of unilateral vagus nerve stimulation (15 V, 4-30 Hz, 0.2 ms) on the responses of pulmonary C-fiber activity, total lung resistance (RL) and dynamic lung compliance (Cdyn) before (open columns) and after (closed columns) administration of atropine (2 mgkg). Vertical bars are means t SEM (n=6). ‘Statistical significance from the control values (PcO.05). “Statistically different from that before atropine pretreatment (PcO.05).
Vagal Stimulation and Pulmonary C-fiber
,tdi I
IO Pulnwnar~ C-fiber activity (iiw/s)
5
t100
Vol. 61, No. 22, 1997
** ,eti mh *Y
60 Cdyn (X change of ccxltroll
1 0
-
I
-50 Control
w?LJ** P *& 4 Hz IO Hr
17 Hz 30 Hz
(Vasal stimulation)
Fig. 3 The effects of unilateral vagus nerve stimulation (15 V, 4-30 Hz, 0.2 ms) on the responses of pulmonary C-fiber activity, total lung resistance (RI.) and dynamic lung compliance (Cdyn) before (open columns) substance P (SP) antagonist pretreatment and after (closed columns) administration of a selective SP antagonist [D-Pro”, D-Try’,‘]-SP (0.5 mg/kg). Vertical bars are means ? SEM (n=8). ‘Statistical significance from the control values (P
LResults The increased activity ot.pulmonary C-fibers occurred after the onset of right vagus nerve (RVN) sttmulatton (15 V. IO Hz), with a latency being within 3 s. *The increase in pulmonary C-fiber activity coincided with an increase in PI and a decrease in V (Fig. 1A). As shown in Fig. lB,
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**
(in&s) 5
0
2193
2 VT
1 3 VT
Fig. 4 The effects of lung inflation at l-3 tidal volume (VT) on the responses of pulmonary C-fiber activity before (open columns) and after (closed columns) administration of atropine (2 m&g). Vertical bars are means + SEM (n=6). *Statistical significance from the control values (PcO.05).
atropine treatment (2 mg/kg) abolished excitation of the RVN stimulation-induced pulmonary Cfiber activity at the same stimulus. A summary of the results in 6 rabbits before and after administration of atropine (2 mg/kg) is shown in Fig. 2. The basal discharge of pulmonary Cfibers in the control was 0.9 f 0.2 imp/s. During RVN stimulation at 4, 10, 17 and 30 Hz the discharges of pulmonary C-fibers were increased to 1.8 2 0.4, 3.9 + 0.5,5.4 i: 0.5 and 7.2 + 0.7 imp/s, respectively. Baseline RL was 16.9 c 3.8 cmHzO/l/s and Cdyn was 2.9 2 0.5 ml/cmHzO. Electrical stimulation of RVN at 4, 10, 17 and 30 Hz increased RL by 18.3 + 5.7, 36.7 + 6.7, 53.3 + 8.3 and 78.9 ? 9.2 %, respectively, but decreased Cdyn by 8.6 + 2.1, 13.3 2 3.2, 21.7 + 3.7 and 30.6 + 3.9, respectively. Pretreatment with atropine (2 mg/kg) abolished changes in pulmonary C-fiber activity, RL and Cdyn induced by RVN stimulation (4-30 Hz). The summarized results of the effect of a selective SP antagonist [D-Pro’, D-Try’,“]-SP (0.5 mg/kg) in 8 rabbits are shown in Fig. 3. The discharge of pulmonary C-fibers during control was 1.0 + 0.4 imp/s and increased during RVN stimulation at 4, lo,17 and 30 Hz to 2.1 2 0.4,4.0 + 0.5, 5.8 -C0.5 and 7.4 + 0.7 imp/s, respectively. Basal values of RL and Cdyn were 16.6 Z!2.9 cmHzO/lls and 2.9 + 0.6 ml/cmHzO, respectively. The RVN stimulation at 4, 10, 17 and 30 Hz increased RL by 15.7 + 5.8,32.5 + 5.4,47.4 2 7.8 and 67.2 2 8.5 %, respectively, but decreased Cdyn by 8.7 2 2.9, 12.9 2 2.2, 23.4 2 2.8 and 30.5 2 3.5 %, respectively. A selective SP antagonist did not significantly alter the responses of pulmonary C-fiber activity and lung mechanics (RI_and Cdyn) to RVN stimulation at 4-17 Hz, but significantly inhibited those responses to the stimulation at 30 Hz. The effects of atropine (2 mg/kg) on the responses of pulmonary C-fibers to lung inflation at l-3 VT in 6 rabbits are summarized Fig. 4. The control discharge of pulmonary C-fibers was 0.8 + 0.2 imp/s and increased during lung inflation at 2 and 3 VT to 4.9 + 0.5 and 9.7 + 0.6 imp/s, receptively. The values of RL and Cdyn before lung inflation at higher volumes were 17.1 c 3.9 cmHzO/l/sec and 3.0 + 0.6 ml/cmH20. The discharges of pulmonary C-fibers during lung inflation at 2 and 3 VT in atropine-treated animals were 4.7 2 0.5 and 9.4 + 0.7 imp/s. Pretreatment with atropine had no significant effect on stimulation of pulmonary C-fiber activity by lung inflation at higher volumes.
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Discussion The analysis of the slowly adapting pulmonary stretch receptor @AR) activity recorded from the cut left vagus nerve (LVN) and the change in RL or Cdyn can provide the method to quantify the bronchoconstriction evoked by electrical stimulation of the cut right vagus nerve (RVN) in rabbits with artificial ventilation (7). During the bronchoconstriction evoked by RVN stimulation, reduced ventilation of the right lung would cause an increase in V.Sof the left lung. If such an effect occurs actually, it is impossible to explain evidence showing that atropine treatment completely blocked the changes in pulmonary C-fiber activity, RL and Cdyn in response to RVN stimulation. In addition, pretreatment with atropine did not significantly alter excitatory responses of pulmonary C-fibers to lung inflation at higher volumes (2 and 3 V.r). Furthermore, RVN stimulation at 30 Hz induced approximately 2-fold increase in RI,. The results in this study are consistent with those reported by other investigators (5, 6). Thus, it is most likely that the bronchoconstriction evoked by RVN stimulation results in an increase in pulmonary C-fiber activity. A noncholinergic innervation has been demonstrated in in vivo preparations during vagus nerve stimulation (2,4, 17), and noncholinergic contractions of airway smooth muscle are known to be inhibited by capsaicin or certain substance P antagonists (4, 18, 19). In an in vitro model to examine the vagal innervation of the guinea-pig bronchus, a&opine consistently inhibits the second-phase contraction which is abolished by capsaicin treatment (20). The results indicate that noncholinergic and cholinergic contractions are not entirely independent between intramural nerves in the smooth muscle. This may be consistent with the observations suggesting that the SPinduced bronchoconstriction is partly involved in the release of ACh in in vivo experiments of the rabbit tracheal smooth muscle (12, 13). In this study, RVN stimulation resulted in bronchoconstriction associated with an increase in pulmonary C-fiber activity, and those responses obtained were frequency-dependent. In addition, frequency-related responses of pulmonary Cfibers, RI, and Cdyn to vagal stimulation were abolished by prior treatment with atropine. In the rabbit bronchial ring preparation that keeps the vagus nerve connections intact, the maximal contractile responses to vagal stimulation are completely abolished by pretreatment with atropine (21). However, it is not easy to determine whether stimulation of pulmonary C-fibers by vagal stimulation is mediated by changes in lung mechanics and/or by the release of some mediators from the vasculature. In an in vivo study of guinea pig vagal afferents, Fox et al. (22) demonstrated that C- and A 6 -fibers were highly sensitive to mechanical stimuli but showed the marked difference on their chemosensitivity. They also stated that the A 6 fibers were insensitive to capsaicin, bradykinin, histamine and serotonin but that the C-fibers responded only to capsaicin and bradykinin (22). Thus, the results in this study suggest that excitation of the pulmonary Cfibers seen during RVN stimulation is due to the effect of mechanical stimuli coincident with the increased muscle contraction by ACh as shown in unilateral vagus nerve stimulation-induced bronchoconstriction. In the rabbit, there is evidence that a SP antagonist, [D-Pro’, D-Try7:‘]-SP, at the dosages of 0.3-0.5 mgikg does not cause any constrictor action but blocks stimulation of rapidly adapting pulmonary stretch receptor (RAR) activity by exogenous SP ranging from 0.3 to 3.0 .&kg as well as by endogenous release of SP due to aerosolized capsaicin (23). Stimulation of the airway C-fibers by vagus nerve stimulation is known to elicit the local release of sufficient SP to modify the plasma exudation in the trachea and bronchi (8,9,24). However, evidence for no inhibitory effect of [D-Pro’, D-Try’“]-SP was found on the responses of pulmonary C-fibers and lung mechanics to RVN stimulation at 4-17 Hz. This implies that when excitation of pulmonary C-fibers occurs during the stimulation at relatively lower frequencies, there is no release of endogenous SP from the rabbit airways and lungs. The changes in pulmonary C-fiber activity, RL and Cdyn in response to RVN at 30 Hz were significantly inhibited but not completely blocked by administration of a SP antagonist. Considering the atropine effect, taken together, the results lead to the suggestion that stimulation of the pulmonary C-fibers by the right vagus nerve stimulation at 30 Hz elicits the release of endogenous SP which may communicate with the postganglionic cholinergic fibers capable of releasing ACh, partly involving a possible action of SP on the mechanosensitivity of the C-fibers. However, the mechanism of a possible interaction between selective release of SP from the rabbit airways and vagal stimulation at a higher frequency (30 Hz) remains to be determined.
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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
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