Demonstration of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig John
6. Richardson,
M.D.,
Ph.D.,
and
Th&&se
Bouchard
Montreal, Quebec, Ca?lada
A nonadrenergic inhibitory 7tervou.s system has been demonstrated in the gwinea pig trachea. E’lectrical field stimdation of this system, in the presence of ~~d?%nergiG and cholinergic bloclcade, resulted in relaxation of tracheal rings contracted by the mediators of immediate hypersensitivity or histamine. The relaxation was bloalced by tetrodotoxin, which indicated that nerve stimulation wa-s responsible for the relaxation. The gastrointestinal tract, which has a similar embryological origin to the respiratory tract, also has a nonadrenergic inhibitory system. In the gastrointestinal tract, this system is thought to be responsible for the relaxation phase of peristalsis, and absence of this system, in the colon and the rectum, is thought to be an explanation for the spastic bowel in Hirschsprmng’s disease. It is possible that an abnormality of the respiratory nonadrenergic inhibitory system may play a role in the pathogenesis of the hyperreactive airways in asthma. The airways, dzle to a lack of inhibition, may be either partially contracted or umablc to relax, and thus appear hyperreactive to stimdi.
The tracheobronchial tree arises from the ventral wall of the foregut and shares some of the anatomical and physiological features of the gastrointestinal tract, the most important being the responsesto the autonomic nervous system.l There has been recent interest in the nonadrenergic inhibitory system in the bowe12-4and the esophagus.5l6 This nonadrenergic inhibitory system has its neurones in the myenteric plexus of the intestine and it is thought to initiate the relaxation phase of peristalsis. 7 The spastic state of the colon and rectum in Hirschsprung’s diseaseis thought to be due to a lack of these neuroneqs and experimental anoxic destruction of these neurones results in the contraction of the portion of the bowel with necrotic neurones.” It has been suggested that a similar system exists in the lung,* and there is some evidence to show this.lO To test the hypothesis that this system exists in the lung and has strong, smooth muscle inhibitory actions, tracheal smooth muscle from both normal guinea pigs and guinea pigs immunized against ovalbumin was studied in a manner similar to the studies on the smooth muscle of the gastrointestinal tract. From the Departments of Pharmacology and Therapeutics and Pathology, McGill University. Supported by a grant from the Medical Research Council of Canada (MA 4536). The senior author is a Scholar of the Medical Research Council of Canada. Received for publication Sept. 3, 1974. Accepted for publication Oct. 31, 1974. Reprint requests to: Dr. John B. Richardson, 3655 Drummond St., Montreal, Quebec, Canada H3G lY6. Vol.
56, No.
6, pp. 473-480
474
Richardson
and
lg
J. ALLERGY
Bouchard
I +c31
-% propprop v
I
CLIN. IMMUNOL. DECEMBER 1975
atro
‘s.
I
5 min
ProV atro v phon
‘s’ 5min A
B
C
FIG. 1. The upper and lower tracings show the effects of a field stimulation (s) of 70 V, 20 Hz, and a pulse duration of 0.5 msec on tracheal chains. The upper tracings were made on a different time scale than the lower tracings. Stimulation of the tissue in the presence of no drugs (A) produced a contraction followed by a relaxation during the stimulation. Propranolol (prop) abolished the relaxation phase during the stimulation (B), which indicates that this relaxation was due to stimulation of adrenergic nerves. Atropine abolished the initial contraction due to stimulation of cholinergic nerves, and in the presence of atropine and propranolol stimulation of the tissue produced a relaxation (C). The lower right tissue (C) had phentolamine (phen) added to rule out an alpha adrenergic effect. All drug concentrations were 2 x lo-’ gm/ml, and at this concentration these drugs blocked the effects of the respective agonists when they were added to the bath.
Immunized animals were used to test if this system was capable of relaxing smooth muscle contracted by the mediators of immediate hypersensitivity. MATERIALS AND METHODS Guinea pigs of either sex and weighing 300 to 500 gm were used. To immunize the animals, 1 mg of ovalbumin (Sigma grade III) in 1 ml of saline was administered intraperitoneally, and 7 to 10 days later the guinea pigs were exsanguinated under ether anesthesia. The trachea, and the main bronchi were removed and made into tracheal chain preparation@ of 8 to 10 rings in length. The tracheal chains were mounted in lo-ml baths filled with Tyrode solution with millimole/liter concentrations (Na, 149.2; Cl, 145.3; K, 2.7; Ca, 3.6; Mg, 2.1; H,PO,, 0.4; HCO,, 11.9; dextrose, 5.0; and EDTA, 0.21) oxygenated with 95% 0, and 5% CO,. The temperature of the bath was maintained at 37” C, and the tissue was mounted with 0.5 gm of constant tension. Responses of the tissue were recorded through a strain gauge on a Grass polygraph (Model 79). The tracheal chains were mounted between two platinum wire electrodes, 1 mm in diameter and 6 mm apart, so that a field stimulation could be applied to the tissue. The field stimulation was produced by a Grass S 48 stimulator. The stimulation was with rectangular pulses of 40 to 70 V output from the stimulator, a frequency of 5 to 20 Hz and a pulse duration of 0.5 to 1.0 msec. This model of stimulator delivers rectangular pulses of current. The standard length of a period of stimulation was 60 sec. The following drugs were used: atropine sulfate, isoproterenol HCl, histamine dihydrochloride, phenoxybenzamine HCl, L-epinephrine bitartrate, phentolamine methanesulfonate, propranolol HCl, hexamethonium chloride, adenosine triphosphate disodium, adenosine monophosphate disodium,
VOLUME NUMBER
Nonadrenergic
56 6
TABLE 1. Degree of mediators of immediate
relaxation produced by field hypersensitivity or histamine
inhibitory
stimulation
tissue
contracted
Number
1 :
200 100 20
150 500 525
500 315 415
0.66 0.75 0.5
4 5
50 50
825 625
675 625
0.82 1.0
! ;
f8
550 300
300 415
0.86 1.o
400 500 875 300 750 1,150 1.000
225 175 675 250
0.45 0.44 0.77 0.83
100 10 10 10
Number
Histamine concentration h/ml)
Stimulation a pulse
Maximum
response (ma)
Maximum relaxation (ma)
system
Ovalbumin concentration (us/ml)
10 II
bath
of
nervous
by
Ratio of relaxation to contraction
(ma)
Ratio of relaxation to contraction
:
0.1
315 325
250 325
0.66 1.0
3 4 5
8:; 0.2 0.2
300 300 300
300 300 250
1.0 1.0 0.83 0.94 0.51 0.44
of the tracheal chain duration of 0.5 msec.
bath
475
Maximum
response
(ms)
for
a period
of 60 set with
a stimulus
of 70 V, 20 Hz,
and
and tetrodotoxin. The tracheal chains were allowed to equilibrate in the bath for 1 hr before the addition of drugs or stimulation of the tissue. The final concentrations of the drugs in the bath are expressed as gm/ml of the salt. Twenty-seven chain preparations from 16 guinea pigs were tested. Four of these chains were from nonimmunized animals.
RESULTS Field stimulation of the tracheal chains in the absence of drugs produced an initial contraction followed by a relaxation, which increased markedly at the termination of the stimulation (Fig. 1, A). The relaxation during the stimulation did not occur when the beta adrenergic antagonist, propranolol, was present in the bath, but the marked relaxation at the end of the stimulation was still present (Fig. 1, B) . The addition of atropine to the bath, to block the effects of acetylcholine, prevented the initial contraction with stimulation, and instead there was a relaxation of the tracheal chain (Fig. 1, C) . The degree of relaxation in the presence of cholinergic and adrenergic blockade was slight when the chains were not in a contracted state but was clear when the tissue was in a contracted state. When the tracheal chains were contracted by histamine, in the presence of cholinergic and adrenergic blockade sufficient to block the effects of specific agonists added to the bath, stimulation caused the chains to relax (Fig. 2, A, B, and II). Upon cessation of the stimulation the relaxed chains contracted and then relaxed again upon later stimulation. The relaxation was
476
Richardson
and
J. ALLERGY
Bouchard
CLIN. IMMUNOL. DECEMBER 1975
J A
hi!+
iso ‘?
hist
‘?
B
1
alb
iso C
j,i;,
A
alb
ttx E
J--z2
alb
h&t F
4
G
FIG. 2. These tracings show the effects of field stimulation on tracheal chains contracted by either histamine or the mediators of immediate hypersensitivity in the presence of atropine, phentolamine, and propranolol at a concentration of 2 x 10e7 gm/mi. The top tracings show chains contracted by histamine at 10d7 gm/ml (A) and at 5 x 10m7 gm/ml (8). lsoproterenol (iso) was added at a bath concentration of 3 x 10F7 gm/ml and had no effect (A). Field stimulation for the periods indicated (s) produced relaxation of the chains. The addition of antigen (alb) to a tracheal chain from an immunized animal (C) caused the tissue to contract. lsoproterenol (iso) 2 x lo-’ gm/ml had no effect, but stimulation for the periods indicated (s) produced a relaxation of the chain (C). The effects of an increase in the pulse duration from 0.5 to 1.0 msec are shown in D. An increase in Tetrodotoxin at concentrations of 1 O-’ the pulse duration produced further relaxation. gm/ml (E) and 5 x 10m7 gm/ml (F) prevented the relaxation seen with stimulation (s). An increase in the pulse duration did not produce a relaxation in the presence of tetrodotoxin (F). Repeated stimulation (s) of a chain contracted after the addition of antigen (alb) showed that with time the amount of relaxation increased and the amount of contraction decreased (G).
complete with low concentrations of histamine, but with higher concentrations stimulation resulted in only 400/o to 50% relaxation of the chains (Table I). Further relaxation was, however, obtained when the pulse duration was increased from 0.5 msec to 1.0 msec (Fig. 2, D). Isoproterenol, when added to the bath to verify the beta blockade, did not relax the contracted tissue, which indicated an adequate block of the beta receptors. The addition of ovalbumin to
VOLUME NUMBER
Nonadrenergic
56 6
alb k4
I
A
inhibitory
nervous
amp
system
477
‘I’
FIG. 3. The upper tracing shows the effect of adenosine monophosphate (amp) on a tracheal chain contracted after the addition of the antigen (alb) in the presence of adrenergic and cholinergic blockade. Stimulation (s) produced a relaxation. The addition of amp, 10m5 gm/ml, produced a relaxation but stimulation increased the degree of relaxation (s). The lower tracing shows a similar experiment with adenosine triphosphate. In this experiment phenoxybenzamine, 10mb gm/ml, was used in addition to propranolol and atropine at concentrations of 2 x 10m7 gm/ml. The addition of antigen (alb) cause a slow rise in the tension due to the antihistaminic action of phenoxybenzamine. Isoproterenol, 2 x 10m7 gm/ml (iso), had no effect, but stimulation (s) caused the tissue to relax. Atp, 10m7 gm/ml, was added (second arrow) and the tissue relaxed. Further addition of atp to a final concentration of 5 x lo-’ gm/ml (third and fourth arrows) had no further effect, but stimulation (s) relaxed the tissue.
the baths with tracheal chains from immunized animals resulted in a contraction of the chains (Fig. 2, C, E, and F) . When a plateau state of contraction was obtained, stimulation for a period of 60 see, in the presence of cholinergic and adrenergic blockade, resulted in an abrupt relaxation that varied in amount with different chains. An increase in the pulse duration from 0.5 msec to 1.0 msec produced a greater degree of relaxation. Tetrodotoxin at concentrations of 10-? gm/ml and 5 x lo--? gm/ml blocked the relaxation produced by the field stimulation. This block was present even with a pulse duration of 2.0 msec (Fig. 2, E and P). Hexamethonium did not block the relaxation produced by the stimulation; amp and atp both produced some relaxation of the tracheal chains, but when the relaxation with these compounds was maximal field stimulation resulted in further relaxation (Fig. 3). No difference was found in the response of the tracheal tissue from normal animals and that from animals immunized against ovalbumin except in the response to this antigen. With the electrodes used, and with movement of the suspended tracheal chain in the bath due to small gas bubbles, the relaxation of the contracted tissue in the presence of the antagonists was greatest with a supramaximal voltage of 70 V, a frequency of 20 Hz, and a pulse duration of 1.0 msec. Relaxation of the contracted tissue was obtained with a frequency of 5 Hz, a 30 V output, and a pulse duration of 0.5 msec, but this was less than with the above stimulation. Since the tissue constantly moved in the bath, and thus the distance
478
Richardson
and
Bouchard
from the tissue to the electrodes was variable, no attempt the responses to variations in the field stimulation.
J. ALLERGY
was
made
CLIN. IMMUNOL. DECEMBER 1975
to quantify
DISCUSSION
These findings demonstrate the presence of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig. This system behaves in a manner similar to that in the gastrointestinal tract in that field stimulation of the tissue, in the presence of cholinergic and adrenergic blockade, results in relaxation of smooth muscle. The system in the trachea is capable of relaxing smooth muscle contracted by the mediators of immediate hypersensitivity or exogenous histamine. These findings confirm the suggestion of Burnstocl? and the findings of Coleman.1o The data in Table I show varied amounts of relaxation in relation to the amount of contraction. No definite conclusion can be made from these data, but generally the tissues which were contracted by a high concentration of histamine, or gave a very strong response to the mediators of immediate hypersensitivity, relaxed less in response to stimulation than those tissues contracted by a low concentration of histamine, or with a weak response to the mediators. A further increase in the amount of relaxation was always obtained with an increase in the pulse duration from 0.5 msec to 1.0 msec. In no case uas relaxation not present upon stimulation when the chains were contracted by either histamine or the mediators of the immediate hypersensitivity reaction. When the stimulation was terminated, the smooth muscle contracted again, due to either the presence of exogenous histamine or to the mediators. Subsequent stimulations produced relaxations which often increased in amount with an increase in time after the initial contraction and relaxation (Fig. 2, G). This effect was probably due to a combination of muscle fatigue and removal of the mediators and exogenous histamine. Inhibition of the stimulus-induced relaxation in the gastrointestinal tract by tetrodotoxin has been taken as evidence that nerves are stimulated.‘-’ Tctrodotoxin, a toxin isolated from puffer fish, blocks nerve conduction hy interfering with sodium conductance, and its ability to abolish a response indicates that the production of the response is nerve-mediated.‘z The inability of the ganglionic blocking agent, hexamethonium, to block the relaxation indicates that the stimulated nerves are not preganglionic. Coleman’” showed t,hat pretrcatmcnt of guinea pigs with drugs that deplete adrenergic stores or destroy the nerve endings also failed to inhihit the relaxation produced hey field stimulation. Catecholamines could be released by the stimulation of adrcnergic axons, lmt adrenergic-blocking agents would be expected to prevent the action of these endogcnous catecholamines. Blockade of endogenous catccholaminc action has been demonstrated in the gastrointestinal tract where the perivascular adrenergic nerves can be stimulated separately from the entire tissue and in the presence of adrenergic blockatle stimulation of these nerves produces no response. Blockade of endogenous catecholaminc action has not been demonstrated prcviously in the trachea, but the abolishment, by the addition of propranolol, of relaxation during field stimulation indicates that there is blockade of the
VOLUME NUMBER
56 6
Nonadrenergic
inhibitory
nervous
system
479
endogenous eatecholamines, and the persistence of the post-stimulation relaxation in the presence of this blockade indicates that this relaxation is not mediated by cndogenous ~atecholamines. Finally, pretreatment of the animal with &hytlrosytlopamine abolishes the catecholamine stores of the lung as judged by fluoresccncc mic,roscopy13 and the fact that relaxation of the trachea occurred after this treatment’” makes the release of endogenous catecholamines an unlikely mechanism for the inhibition seen in the preceding experiments. The chemical mediator of this inhibitory system is not known, but adcnosine triphosphate has been suggested as a mediator and there is evidence to support this suggestion” and evidence against it.14 In the present studies, atlenosinc monophosphate and adenosinc triphosphate both caused the smooth muscle to relax, but further relaxation was always obtained with field stimulation, and the action of these compounds might well be nonspecific. Although many other compounds have been tested in an effort to identify the mediator, none has, as yet, met the criteria. In asthma there is a hyperreactivity of the airways to a variety of stimu1i.l” The pathogenesis of this hyperreactivity is not known. The autonomic nervous system has been implicated in the asthmatic process either through the vagal activity”‘, I7 or through a defect of the beta adrenergic, receptors.‘” In the guinea pig, beta adrenergie blockade results in a hypersensitivity of the smooth muscle to histamine.1”-2i The exact mechanism, however, may not be just beta blockade since /3,-blocking agents also produce a hypersensitivity to histamin? and there is conflicting evidence in regard to the role of the central nervous system in this phcnomena.l”, 2o Propranolol has potent local anesthestic effects, and these may play a role in some of the results.22 There is no doubt that the adrcnergic system relaxes the respiratory smooth muscle, but it might not be the only system capable of this. The present findings demonstrate another inhibitory nervous system which shows similar characteristics to the nonadrenergic inhibitory system ill the gastrointestinal tract. The neurones of this system may be located in t,he peritracheal and pcrihronehial tissue where numerous ganglion cells are present.13* 2:~Absence of the ganglion cells in the colon results in contraction of the segment where they arc absent, and it has been postulated that a lack of the nonadrenergic system is the important cause of the contraction.8 Thus, in the gastrointestinal tract this system is thought to have a physiological function in the relaxation of the bowel wall both at rest and in peristalsis.7 If the nonadrcncrgic inhibitory system in the lung has a similar function to that in the gastrointestinal tract, then damage to the pulmonary system might result in either partially contracted airways or airways that are unable to dilate in response to a stimulus and thus appear to be hyperreactivc. Such damage might result from repeated infections such as often occur in asthma or chronic bronchitis. NOTE
ADDED
IN
PROOF
Since the submission tion
of
their
findings
of this (Rr. J.
manuscript, Coleman Pharmacol.
52:
167,
and Levy have published a full descrip1974). In this paper they refer to :I
480
Richardson
prior demonstration There arc no basic
and
J. ALLERGY
Bouchard
of this differences
system by Coburn in the description
and Tomita (Am. of this inhibitory
J. Physiol. system.
CLIN. IMMUNOL. DECEMBER 1975
224:
1072,
1973).
REFERENCES of tracheobronchial smooth muscle, Physiol. Rev. 43: 1, 1 Widdicombe, J. G.: Regulation 1963. 2 Burnstock, G.: Purinergic nerves, Pharmacol. Rev. 24: 509, 1972. S.: Presence of a non-adrenergie 3 Crema, A., Del Tacea, M., Frigo, G. M., and Lecchini, inhibitory system in the human colon, Gut 9: 633, 1968. 4 Rikinaru, A., and Suzuki, T.: Neural mechanism of the relaxing responses of guinea pig taenin cob, Tohoku J. Exp. Med. 108: 303, 1971. 5 Christensen, J., Conklin, J., and Freeman, B.: Myoneural specialization at the esophagogastric junction in three species, J. Clin. Invest. 52: 19a, 1973. 6 Tuch, A., and Cohen, S.: Lower esophageal sphincter relaxation: Studies on the neurogenic inhibitory mechanism, J. Clin. Invest. 52: 14, 1973. analysis of the peristaltic 7 Crema, A., Frigo, G. M., and Lecchini, S.: A pharmacological reflex in the isolated colon of the guinea-pig or cat, Br. J. Pharmacol. 39: 334, 1970. 8 Frigo, G. M., Del Taeca, M., Lecchini, S., and Crema, A.: Some observations on the intrinsic nervous mechanism in Hirschsprung’s disease, Gut 14: 35, 1973. 9 Hukuhara, T., Kotani, S., and Sxto, G.: Effects of destruction of intramural ganglion cells on colon motility: Possible genesis of congenital megacolon, Jap. J. Physiol. 11: 635, 1961. 10 Coleman, R,. A. : Evidence for a non-adrenergic inhibitory nervous pathway in guinea-pig trachea, Br. J. Pharmacol. 48: 360, 1973. 11 Castillo, J. C., and de Beer, E. J.: The tracheal chain, J. Pharmacol. Exp. Ther. 90: 104, 1947. 12 Kao, C. Y.: Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomena, Pharmacol. Rev. 18: 997, 1966. 13 Rilva, D. G., and Ross, G.: Ultrastructural and fluorescence histochemical studies on the innervation of the tracheobroncheal muscle of normal cats and cats treated with 6hydroxydopamine, J. Ultrastruc. Res. 47: 310, 1974. 14 Wrisenthal, L. M., Hug, C. C., Weibrodt, N. W., and Bass, P.: Adrenergic mechanisms in the relaxation of the guinea-pig taenia. in vitro, J. Pharmacol. Exp. Ther. 178: 497, 1971. 15 Aas, K.: The biochemical and immunological basis of bronchial asthma, Springfield, Ill., 1972, Charles C Thomas, Publisher. 16 Mills, J. E., and Widdicombe, J. G.: Role of the vagus nerves in anaphylaxis and histamine-induced bronchoconstrictions in guinea pigs, Br. J. Pharmacol. 39: 724, 1970. 17 Gold, W. M., Kessler, G. F., and Yu, D. Y. C.: Role of vagus nerves in an experimental asthma in allergic dogs, J. Appl. Physiol. 33: 710, 1972. 18 Szentivanyi, A.: The beta adrenergic theory of the atopic abnormality in bronchial asthma,J. ALLERGY 42: 203,1968. 19 McCulloch, M. W., Proctor, C., and Rand, J.: Evidence for an adrenergic homeostatic bronchodilxtor reflex mechanism, Eur. J. Pharmacol. 2: 214, 1967. 20 Advenier, C., Boissier, J. R.., and Guidicelli, J. F.: Comparative study of six P-adrenergic antagonists on airway resistance and heart rate in the guinea-pig, Br. J. Pharmacol. 44: 642, 1972. 21 Diamond, L.: Potentiation of bronchomotor responses by beta adrenergic antagonists, J. Pharmacol. Exp. Tiler. 181: 434, 1972. 22 Morales-Aquilera, A., and Vaughan Williams, E. M.: The effects on cardiac muscle of beta receptor antagonists in relation to their activity as local anesthetics, Br. J. Pharmx(;ol. 24: 332, 1965. 23 Nagaishi, C.: Functional anatomy and histology of the lung, Baltimore, 1972, University Park Press.
6. Richardson,
M.D.,
Ph.D.,
and
Th&&se
Bouchard
Montreal, Quebec, Ca?lada
A nonadrenergic inhibitory 7tervou.s system has been demonstrated in the gwinea pig trachea. E’lectrical field stimdation of this system, in the presence of ~~d?%nergiG and cholinergic bloclcade, resulted in relaxation of tracheal rings contracted by the mediators of immediate hypersensitivity or histamine. The relaxation was bloalced by tetrodotoxin, which indicated that nerve stimulation wa-s responsible for the relaxation. The gastrointestinal tract, which has a similar embryological origin to the respiratory tract, also has a nonadrenergic inhibitory system. In the gastrointestinal tract, this system is thought to be responsible for the relaxation phase of peristalsis, and absence of this system, in the colon and the rectum, is thought to be an explanation for the spastic bowel in Hirschsprmng’s disease. It is possible that an abnormality of the respiratory nonadrenergic inhibitory system may play a role in the pathogenesis of the hyperreactive airways in asthma. The airways, dzle to a lack of inhibition, may be either partially contracted or umablc to relax, and thus appear hyperreactive to stimdi.
The tracheobronchial tree arises from the ventral wall of the foregut and shares some of the anatomical and physiological features of the gastrointestinal tract, the most important being the responsesto the autonomic nervous system.l There has been recent interest in the nonadrenergic inhibitory system in the bowe12-4and the esophagus.5l6 This nonadrenergic inhibitory system has its neurones in the myenteric plexus of the intestine and it is thought to initiate the relaxation phase of peristalsis. 7 The spastic state of the colon and rectum in Hirschsprung’s diseaseis thought to be due to a lack of these neuroneqs and experimental anoxic destruction of these neurones results in the contraction of the portion of the bowel with necrotic neurones.” It has been suggested that a similar system exists in the lung,* and there is some evidence to show this.lO To test the hypothesis that this system exists in the lung and has strong, smooth muscle inhibitory actions, tracheal smooth muscle from both normal guinea pigs and guinea pigs immunized against ovalbumin was studied in a manner similar to the studies on the smooth muscle of the gastrointestinal tract. From the Departments of Pharmacology and Therapeutics and Pathology, McGill University. Supported by a grant from the Medical Research Council of Canada (MA 4536). The senior author is a Scholar of the Medical Research Council of Canada. Received for publication Sept. 3, 1974. Accepted for publication Oct. 31, 1974. Reprint requests to: Dr. John B. Richardson, 3655 Drummond St., Montreal, Quebec, Canada H3G lY6. Vol.
56, No.
6, pp. 473-480
474
Richardson
and
lg
J. ALLERGY
Bouchard
I +c31
-% propprop v
I
CLIN. IMMUNOL. DECEMBER 1975
atro
‘s.
I
5 min
ProV atro v phon
‘s’ 5min A
B
C
FIG. 1. The upper and lower tracings show the effects of a field stimulation (s) of 70 V, 20 Hz, and a pulse duration of 0.5 msec on tracheal chains. The upper tracings were made on a different time scale than the lower tracings. Stimulation of the tissue in the presence of no drugs (A) produced a contraction followed by a relaxation during the stimulation. Propranolol (prop) abolished the relaxation phase during the stimulation (B), which indicates that this relaxation was due to stimulation of adrenergic nerves. Atropine abolished the initial contraction due to stimulation of cholinergic nerves, and in the presence of atropine and propranolol stimulation of the tissue produced a relaxation (C). The lower right tissue (C) had phentolamine (phen) added to rule out an alpha adrenergic effect. All drug concentrations were 2 x lo-’ gm/ml, and at this concentration these drugs blocked the effects of the respective agonists when they were added to the bath.
Immunized animals were used to test if this system was capable of relaxing smooth muscle contracted by the mediators of immediate hypersensitivity. MATERIALS AND METHODS Guinea pigs of either sex and weighing 300 to 500 gm were used. To immunize the animals, 1 mg of ovalbumin (Sigma grade III) in 1 ml of saline was administered intraperitoneally, and 7 to 10 days later the guinea pigs were exsanguinated under ether anesthesia. The trachea, and the main bronchi were removed and made into tracheal chain preparation@ of 8 to 10 rings in length. The tracheal chains were mounted in lo-ml baths filled with Tyrode solution with millimole/liter concentrations (Na, 149.2; Cl, 145.3; K, 2.7; Ca, 3.6; Mg, 2.1; H,PO,, 0.4; HCO,, 11.9; dextrose, 5.0; and EDTA, 0.21) oxygenated with 95% 0, and 5% CO,. The temperature of the bath was maintained at 37” C, and the tissue was mounted with 0.5 gm of constant tension. Responses of the tissue were recorded through a strain gauge on a Grass polygraph (Model 79). The tracheal chains were mounted between two platinum wire electrodes, 1 mm in diameter and 6 mm apart, so that a field stimulation could be applied to the tissue. The field stimulation was produced by a Grass S 48 stimulator. The stimulation was with rectangular pulses of 40 to 70 V output from the stimulator, a frequency of 5 to 20 Hz and a pulse duration of 0.5 to 1.0 msec. This model of stimulator delivers rectangular pulses of current. The standard length of a period of stimulation was 60 sec. The following drugs were used: atropine sulfate, isoproterenol HCl, histamine dihydrochloride, phenoxybenzamine HCl, L-epinephrine bitartrate, phentolamine methanesulfonate, propranolol HCl, hexamethonium chloride, adenosine triphosphate disodium, adenosine monophosphate disodium,
VOLUME NUMBER
Nonadrenergic
56 6
TABLE 1. Degree of mediators of immediate
relaxation produced by field hypersensitivity or histamine
inhibitory
stimulation
tissue
contracted
Number
1 :
200 100 20
150 500 525
500 315 415
0.66 0.75 0.5
4 5
50 50
825 625
675 625
0.82 1.0
! ;
f8
550 300
300 415
0.86 1.o
400 500 875 300 750 1,150 1.000
225 175 675 250
0.45 0.44 0.77 0.83
100 10 10 10
Number
Histamine concentration h/ml)
Stimulation a pulse
Maximum
response (ma)
Maximum relaxation (ma)
system
Ovalbumin concentration (us/ml)
10 II
bath
of
nervous
by
Ratio of relaxation to contraction
(ma)
Ratio of relaxation to contraction
:
0.1
315 325
250 325
0.66 1.0
3 4 5
8:; 0.2 0.2
300 300 300
300 300 250
1.0 1.0 0.83 0.94 0.51 0.44
of the tracheal chain duration of 0.5 msec.
bath
475
Maximum
response
(ms)
for
a period
of 60 set with
a stimulus
of 70 V, 20 Hz,
and
and tetrodotoxin. The tracheal chains were allowed to equilibrate in the bath for 1 hr before the addition of drugs or stimulation of the tissue. The final concentrations of the drugs in the bath are expressed as gm/ml of the salt. Twenty-seven chain preparations from 16 guinea pigs were tested. Four of these chains were from nonimmunized animals.
RESULTS Field stimulation of the tracheal chains in the absence of drugs produced an initial contraction followed by a relaxation, which increased markedly at the termination of the stimulation (Fig. 1, A). The relaxation during the stimulation did not occur when the beta adrenergic antagonist, propranolol, was present in the bath, but the marked relaxation at the end of the stimulation was still present (Fig. 1, B) . The addition of atropine to the bath, to block the effects of acetylcholine, prevented the initial contraction with stimulation, and instead there was a relaxation of the tracheal chain (Fig. 1, C) . The degree of relaxation in the presence of cholinergic and adrenergic blockade was slight when the chains were not in a contracted state but was clear when the tissue was in a contracted state. When the tracheal chains were contracted by histamine, in the presence of cholinergic and adrenergic blockade sufficient to block the effects of specific agonists added to the bath, stimulation caused the chains to relax (Fig. 2, A, B, and II). Upon cessation of the stimulation the relaxed chains contracted and then relaxed again upon later stimulation. The relaxation was
476
Richardson
and
J. ALLERGY
Bouchard
CLIN. IMMUNOL. DECEMBER 1975
J A
hi!+
iso ‘?
hist
‘?
B
1
alb
iso C
j,i;,
A
alb
ttx E
J--z2
alb
h&t F
4
G
FIG. 2. These tracings show the effects of field stimulation on tracheal chains contracted by either histamine or the mediators of immediate hypersensitivity in the presence of atropine, phentolamine, and propranolol at a concentration of 2 x 10e7 gm/mi. The top tracings show chains contracted by histamine at 10d7 gm/ml (A) and at 5 x 10m7 gm/ml (8). lsoproterenol (iso) was added at a bath concentration of 3 x 10F7 gm/ml and had no effect (A). Field stimulation for the periods indicated (s) produced relaxation of the chains. The addition of antigen (alb) to a tracheal chain from an immunized animal (C) caused the tissue to contract. lsoproterenol (iso) 2 x lo-’ gm/ml had no effect, but stimulation for the periods indicated (s) produced a relaxation of the chain (C). The effects of an increase in the pulse duration from 0.5 to 1.0 msec are shown in D. An increase in Tetrodotoxin at concentrations of 1 O-’ the pulse duration produced further relaxation. gm/ml (E) and 5 x 10m7 gm/ml (F) prevented the relaxation seen with stimulation (s). An increase in the pulse duration did not produce a relaxation in the presence of tetrodotoxin (F). Repeated stimulation (s) of a chain contracted after the addition of antigen (alb) showed that with time the amount of relaxation increased and the amount of contraction decreased (G).
complete with low concentrations of histamine, but with higher concentrations stimulation resulted in only 400/o to 50% relaxation of the chains (Table I). Further relaxation was, however, obtained when the pulse duration was increased from 0.5 msec to 1.0 msec (Fig. 2, D). Isoproterenol, when added to the bath to verify the beta blockade, did not relax the contracted tissue, which indicated an adequate block of the beta receptors. The addition of ovalbumin to
VOLUME NUMBER
Nonadrenergic
56 6
alb k4
I
A
inhibitory
nervous
amp
system
477
‘I’
FIG. 3. The upper tracing shows the effect of adenosine monophosphate (amp) on a tracheal chain contracted after the addition of the antigen (alb) in the presence of adrenergic and cholinergic blockade. Stimulation (s) produced a relaxation. The addition of amp, 10m5 gm/ml, produced a relaxation but stimulation increased the degree of relaxation (s). The lower tracing shows a similar experiment with adenosine triphosphate. In this experiment phenoxybenzamine, 10mb gm/ml, was used in addition to propranolol and atropine at concentrations of 2 x 10m7 gm/ml. The addition of antigen (alb) cause a slow rise in the tension due to the antihistaminic action of phenoxybenzamine. Isoproterenol, 2 x 10m7 gm/ml (iso), had no effect, but stimulation (s) caused the tissue to relax. Atp, 10m7 gm/ml, was added (second arrow) and the tissue relaxed. Further addition of atp to a final concentration of 5 x lo-’ gm/ml (third and fourth arrows) had no further effect, but stimulation (s) relaxed the tissue.
the baths with tracheal chains from immunized animals resulted in a contraction of the chains (Fig. 2, C, E, and F) . When a plateau state of contraction was obtained, stimulation for a period of 60 see, in the presence of cholinergic and adrenergic blockade, resulted in an abrupt relaxation that varied in amount with different chains. An increase in the pulse duration from 0.5 msec to 1.0 msec produced a greater degree of relaxation. Tetrodotoxin at concentrations of 10-? gm/ml and 5 x lo--? gm/ml blocked the relaxation produced by the field stimulation. This block was present even with a pulse duration of 2.0 msec (Fig. 2, E and P). Hexamethonium did not block the relaxation produced by the stimulation; amp and atp both produced some relaxation of the tracheal chains, but when the relaxation with these compounds was maximal field stimulation resulted in further relaxation (Fig. 3). No difference was found in the response of the tracheal tissue from normal animals and that from animals immunized against ovalbumin except in the response to this antigen. With the electrodes used, and with movement of the suspended tracheal chain in the bath due to small gas bubbles, the relaxation of the contracted tissue in the presence of the antagonists was greatest with a supramaximal voltage of 70 V, a frequency of 20 Hz, and a pulse duration of 1.0 msec. Relaxation of the contracted tissue was obtained with a frequency of 5 Hz, a 30 V output, and a pulse duration of 0.5 msec, but this was less than with the above stimulation. Since the tissue constantly moved in the bath, and thus the distance
478
Richardson
and
Bouchard
from the tissue to the electrodes was variable, no attempt the responses to variations in the field stimulation.
J. ALLERGY
was
made
CLIN. IMMUNOL. DECEMBER 1975
to quantify
DISCUSSION
These findings demonstrate the presence of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig. This system behaves in a manner similar to that in the gastrointestinal tract in that field stimulation of the tissue, in the presence of cholinergic and adrenergic blockade, results in relaxation of smooth muscle. The system in the trachea is capable of relaxing smooth muscle contracted by the mediators of immediate hypersensitivity or exogenous histamine. These findings confirm the suggestion of Burnstocl? and the findings of Coleman.1o The data in Table I show varied amounts of relaxation in relation to the amount of contraction. No definite conclusion can be made from these data, but generally the tissues which were contracted by a high concentration of histamine, or gave a very strong response to the mediators of immediate hypersensitivity, relaxed less in response to stimulation than those tissues contracted by a low concentration of histamine, or with a weak response to the mediators. A further increase in the amount of relaxation was always obtained with an increase in the pulse duration from 0.5 msec to 1.0 msec. In no case uas relaxation not present upon stimulation when the chains were contracted by either histamine or the mediators of the immediate hypersensitivity reaction. When the stimulation was terminated, the smooth muscle contracted again, due to either the presence of exogenous histamine or to the mediators. Subsequent stimulations produced relaxations which often increased in amount with an increase in time after the initial contraction and relaxation (Fig. 2, G). This effect was probably due to a combination of muscle fatigue and removal of the mediators and exogenous histamine. Inhibition of the stimulus-induced relaxation in the gastrointestinal tract by tetrodotoxin has been taken as evidence that nerves are stimulated.‘-’ Tctrodotoxin, a toxin isolated from puffer fish, blocks nerve conduction hy interfering with sodium conductance, and its ability to abolish a response indicates that the production of the response is nerve-mediated.‘z The inability of the ganglionic blocking agent, hexamethonium, to block the relaxation indicates that the stimulated nerves are not preganglionic. Coleman’” showed t,hat pretrcatmcnt of guinea pigs with drugs that deplete adrenergic stores or destroy the nerve endings also failed to inhihit the relaxation produced hey field stimulation. Catecholamines could be released by the stimulation of adrcnergic axons, lmt adrenergic-blocking agents would be expected to prevent the action of these endogcnous catecholamines. Blockade of endogenous catccholaminc action has been demonstrated in the gastrointestinal tract where the perivascular adrenergic nerves can be stimulated separately from the entire tissue and in the presence of adrenergic blockatle stimulation of these nerves produces no response. Blockade of endogenous catecholaminc action has not been demonstrated prcviously in the trachea, but the abolishment, by the addition of propranolol, of relaxation during field stimulation indicates that there is blockade of the
VOLUME NUMBER
56 6
Nonadrenergic
inhibitory
nervous
system
479
endogenous eatecholamines, and the persistence of the post-stimulation relaxation in the presence of this blockade indicates that this relaxation is not mediated by cndogenous ~atecholamines. Finally, pretreatment of the animal with &hytlrosytlopamine abolishes the catecholamine stores of the lung as judged by fluoresccncc mic,roscopy13 and the fact that relaxation of the trachea occurred after this treatment’” makes the release of endogenous catecholamines an unlikely mechanism for the inhibition seen in the preceding experiments. The chemical mediator of this inhibitory system is not known, but adcnosine triphosphate has been suggested as a mediator and there is evidence to support this suggestion” and evidence against it.14 In the present studies, atlenosinc monophosphate and adenosinc triphosphate both caused the smooth muscle to relax, but further relaxation was always obtained with field stimulation, and the action of these compounds might well be nonspecific. Although many other compounds have been tested in an effort to identify the mediator, none has, as yet, met the criteria. In asthma there is a hyperreactivity of the airways to a variety of stimu1i.l” The pathogenesis of this hyperreactivity is not known. The autonomic nervous system has been implicated in the asthmatic process either through the vagal activity”‘, I7 or through a defect of the beta adrenergic, receptors.‘” In the guinea pig, beta adrenergie blockade results in a hypersensitivity of the smooth muscle to histamine.1”-2i The exact mechanism, however, may not be just beta blockade since /3,-blocking agents also produce a hypersensitivity to histamin? and there is conflicting evidence in regard to the role of the central nervous system in this phcnomena.l”, 2o Propranolol has potent local anesthestic effects, and these may play a role in some of the results.22 There is no doubt that the adrcnergic system relaxes the respiratory smooth muscle, but it might not be the only system capable of this. The present findings demonstrate another inhibitory nervous system which shows similar characteristics to the nonadrenergic inhibitory system ill the gastrointestinal tract. The neurones of this system may be located in t,he peritracheal and pcrihronehial tissue where numerous ganglion cells are present.13* 2:~Absence of the ganglion cells in the colon results in contraction of the segment where they arc absent, and it has been postulated that a lack of the nonadrenergic system is the important cause of the contraction.8 Thus, in the gastrointestinal tract this system is thought to have a physiological function in the relaxation of the bowel wall both at rest and in peristalsis.7 If the nonadrcncrgic inhibitory system in the lung has a similar function to that in the gastrointestinal tract, then damage to the pulmonary system might result in either partially contracted airways or airways that are unable to dilate in response to a stimulus and thus appear to be hyperreactivc. Such damage might result from repeated infections such as often occur in asthma or chronic bronchitis. NOTE
ADDED
IN
PROOF
Since the submission tion
of
their
findings
of this (Rr. J.
manuscript, Coleman Pharmacol.
52:
167,
and Levy have published a full descrip1974). In this paper they refer to :I
480
Richardson
prior demonstration There arc no basic
and
J. ALLERGY
Bouchard
of this differences
system by Coburn in the description
and Tomita (Am. of this inhibitory
J. Physiol. system.
CLIN. IMMUNOL. DECEMBER 1975
224:
1072,
1973).
REFERENCES of tracheobronchial smooth muscle, Physiol. Rev. 43: 1, 1 Widdicombe, J. G.: Regulation 1963. 2 Burnstock, G.: Purinergic nerves, Pharmacol. Rev. 24: 509, 1972. S.: Presence of a non-adrenergie 3 Crema, A., Del Tacea, M., Frigo, G. M., and Lecchini, inhibitory system in the human colon, Gut 9: 633, 1968. 4 Rikinaru, A., and Suzuki, T.: Neural mechanism of the relaxing responses of guinea pig taenin cob, Tohoku J. Exp. Med. 108: 303, 1971. 5 Christensen, J., Conklin, J., and Freeman, B.: Myoneural specialization at the esophagogastric junction in three species, J. Clin. Invest. 52: 19a, 1973. 6 Tuch, A., and Cohen, S.: Lower esophageal sphincter relaxation: Studies on the neurogenic inhibitory mechanism, J. Clin. Invest. 52: 14, 1973. analysis of the peristaltic 7 Crema, A., Frigo, G. M., and Lecchini, S.: A pharmacological reflex in the isolated colon of the guinea-pig or cat, Br. J. Pharmacol. 39: 334, 1970. 8 Frigo, G. M., Del Taeca, M., Lecchini, S., and Crema, A.: Some observations on the intrinsic nervous mechanism in Hirschsprung’s disease, Gut 14: 35, 1973. 9 Hukuhara, T., Kotani, S., and Sxto, G.: Effects of destruction of intramural ganglion cells on colon motility: Possible genesis of congenital megacolon, Jap. J. Physiol. 11: 635, 1961. 10 Coleman, R,. A. : Evidence for a non-adrenergic inhibitory nervous pathway in guinea-pig trachea, Br. J. Pharmacol. 48: 360, 1973. 11 Castillo, J. C., and de Beer, E. J.: The tracheal chain, J. Pharmacol. Exp. Ther. 90: 104, 1947. 12 Kao, C. Y.: Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomena, Pharmacol. Rev. 18: 997, 1966. 13 Rilva, D. G., and Ross, G.: Ultrastructural and fluorescence histochemical studies on the innervation of the tracheobroncheal muscle of normal cats and cats treated with 6hydroxydopamine, J. Ultrastruc. Res. 47: 310, 1974. 14 Wrisenthal, L. M., Hug, C. C., Weibrodt, N. W., and Bass, P.: Adrenergic mechanisms in the relaxation of the guinea-pig taenia. in vitro, J. Pharmacol. Exp. Ther. 178: 497, 1971. 15 Aas, K.: The biochemical and immunological basis of bronchial asthma, Springfield, Ill., 1972, Charles C Thomas, Publisher. 16 Mills, J. E., and Widdicombe, J. G.: Role of the vagus nerves in anaphylaxis and histamine-induced bronchoconstrictions in guinea pigs, Br. J. Pharmacol. 39: 724, 1970. 17 Gold, W. M., Kessler, G. F., and Yu, D. Y. C.: Role of vagus nerves in an experimental asthma in allergic dogs, J. Appl. Physiol. 33: 710, 1972. 18 Szentivanyi, A.: The beta adrenergic theory of the atopic abnormality in bronchial asthma,J. ALLERGY 42: 203,1968. 19 McCulloch, M. W., Proctor, C., and Rand, J.: Evidence for an adrenergic homeostatic bronchodilxtor reflex mechanism, Eur. J. Pharmacol. 2: 214, 1967. 20 Advenier, C., Boissier, J. R.., and Guidicelli, J. F.: Comparative study of six P-adrenergic antagonists on airway resistance and heart rate in the guinea-pig, Br. J. Pharmacol. 44: 642, 1972. 21 Diamond, L.: Potentiation of bronchomotor responses by beta adrenergic antagonists, J. Pharmacol. Exp. Tiler. 181: 434, 1972. 22 Morales-Aquilera, A., and Vaughan Williams, E. M.: The effects on cardiac muscle of beta receptor antagonists in relation to their activity as local anesthetics, Br. J. Pharmx(;ol. 24: 332, 1965. 23 Nagaishi, C.: Functional anatomy and histology of the lung, Baltimore, 1972, University Park Press.
