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Effect of chronic vagal denervation on drug-induced responses of isolated rabbit ileum
Life Sciences Vol . 7, Part I, pp . 1311-1315, 1968 . Printed in Great Britain.
Pergamon Press
EFFECT OF CHRONIC VAGAL DENEhVATION ON DRUG-INDUCED RESPONSES OF ISOLATED RABBIT ILEUM Marvin M. C*oldenberg Section of Pharmacology, Pharmacometrics Division, Research and Development Department The Norwich Pharmacal Company Norwich, New York (Received 5 August 1968 ; in final form 9 Septemk~er 1988) CLASSIFICATIJN of contractile responses of isolated intestines of various species as atropine sensitive or -resistant is wéll-documented . That is, excitation of the intrinsic motor nervous elements of these intestines by nicotine results in contractions which are either effectively antagonized, as in the
cat (1) and guinea pig (2) or relatively unaltered by atropine, as in the rabbit (3,4) . The non-parallel blocking activity of atropine among the intestines of various species suggests that cholinergic as well as non-cholinergic motor
neurons may be activated by nicotine . The present study is concerned with the atropine-resistant isolated rabbit ileum and the role played by the vague nerve (extrinsic) in determining the nature of the contractile response to nicotine .
Methods Ten male albino rabbits, 5 control and 5 vagally-denervated, ranging in weight from 1.5-4 Kg were killed by a blow to the back of the head; the abdomen was opened and the ileum removed. Each strip of ileum was sus-
pended in a 10 ml Anderson muscle bath containing Tyrode solution gassed by 95% 02 + 5% C02 at 36 °C. Isometric contractions were recorded by a Grass FT-03 force-displacement transducer monitored by a Grass poly-
graph. The initial tension on the muscle was adjusted to 6 g and contractions were expressed in terms of g of tension developed. In the preparation of the vagally-denervated ileum, the rabbit was anesthetized with ether and the vague nerves transected under aseptic conditions .
The vagi were excised (by removal of 3-5 mm pieces) in the
abdominal region at the point where they follow the esophagus just before entering the stomach. The nerves were allowed to degenerate for 23-34
days before the ileum was removed and suspended in the bath . Control contractions of approximately equal magnitude were obtained by adding alternatively acetylcholine (ACh) 0.1 ug/ml and nicotine 1 u q/ml . An interval of 8 min was allowed between the additions of the two agonists .
1311
1312
DRUG-INDUCED RESPONSES
Vol . 7, No . 23
A fter control responses were established, antagonists were added to the bath 8 min before introduction of an agonist . Tyrode solution of the following composition (g/1) was used in all experiments: NaCI, 8 ; KC1, 0.2; CaCl2, 0.2; ~NIgCl2 " 6H20, 0.1; NaH2P04 " H2O, 0.05; NaHC~, 1 .0; dextrose, 1 .0. Compounds used were: ACh chloride, nicotine salicylate, hexamethonium bromide, cocaine hydrochloride, atropine sulfate, morphine sulfate, and a .p~-bis - (dimethylammoniumacetaldehy>de diethylacetal)-p,p'-diacetylbiphenyl bromide (Di~+IAE) . :~11 concentrations of drugs refer to final concentrations of their salts and are expressed as ug/ml. hesults In the vagally-denervated ileal preparation the rate, frequency, or amplitude of spontaneous contractions did not appear to be different from that of normal ileum . However, the sensitivity of the vagally-denervated ileum to the contractile effect of ~Ch (0.1 ug/ml) or of nicotine (1 u g/ml) was significantly (P < 0.05) decreased as compared to that of normal preparations . fable 1 presents the results of the antagonists used on both types of ileal preparations . The significance of the difference in means was determined throughout using Student's t test (5), with a P value of 0.05 or less considered significant . The local anesthetic cocaine, 10 ug/ml, and the ganglionic blocking agent hexamethonium, 100 ug/ml, significantly inhibited nicotine-induced contraction of both the normal and the vagally-denervated ileum. However, the percentage reductions of the contractile response to nicotine in the two ileum preparations were not equal, in that a greater inhibition of the In both normal and vagallyresponse was noted in the normal ileum. denervated ilea atropine, 0.01 or 0.1 ug/ml, did not block the effect of nicotine to the same degree as was found for :1Ch (Table 1) . ~~ significant decrease in the contractile response to nicotine was evident, however, at either concentration of atropine in both strips . A'either the cholinergic neuronal blocking agent morphine, 1 ug/ml, or DYLaE, 10 u g/ml, which has been reported to possess specific antinicotine activity (6), specifically antagonized the effect of nicotine on either ileum. Significant inhibition of the response to nicotine was elicited by a combination of atropine (0.1 ug/ ml) and hexamethonium (100 u g/ml), which caused a greater degree of inhibition than either drug alone . :ACh-induced contraction of either ileum was not significantly affected by cocaine, hexamethonium, morphine, or DIVI_AE,
Vol. 7, No. 23
DRUG-IIdDUCED RESPONSES
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1314
DRUG-INDUCED RESPONSES
Vol. 7, No . 23
Discussion and Summary Degeneration of the abdominal vagi of the rabbit (3~ to 5 weeks after sectioning) resulted in a decreased responsiveness of the ileum to nicotine . The lowered sensitivity to nicotine cannot be attributed solely to a lose of intrinsic neuronal function of the ileum by means of degenerative changes after preganglionic parasympathetic section, since the action of ACh, mediated via muscarinic smooth muscle receptors, was similarly diminished. Interpretation of these data can be only tentative, but it may the be that disproportionately larger number of nerve cells in the plexus of
the
rabbit
intestine as compared to preganglionic vagal fibers (7) is
capable of activation by nicotine, although the function of the va~i has been interrupted. On this basis, a reduction of the effect of nicotine may reflect an impairment of the intestinal smooth muscle which is unable to respond to indirect neuronal stimulation any more than to direct stimulation by ACh. It
was of interest to note that the per cent change .in contractile . response to nicotine for the different ilea was different for control and for the antagonists, cocaine or hexamethonium . More specifically, the amount by which cocaine or hexamethonium inhibited the response to nicotine in the vagally-denervated ileum was not greater than for the normal one. No such finding was apparent after the employment of the other antagonists. Inhibition of the contractile response to nicotine to the same absolute
level in that the
both ilea by a local anesthetic or a ganglion blocker suggests intrinsic motor nerve supply may be unaltered after vagal de-
nervation. The present findings also indicate that a functional extrinsic parasympathetic preganglionic nerve (the vague) is not necessary in revealing the relatively atropine-resistant response of the rabbit intestine. In addi tion, neither morphine, which reduces the output of ACh from postganglionic
cholinergic nerve endings (8), or DMAE, an antinicotine agent (8), significantly antagonized the contractile response to nicotine of the vagallyThat portion of the effect of nicotine which denervated or normal ileum. is atropine resistant and which is not altered by vagal denervation may be due to activation of a cholinergic-like intrinsic nerve supply which can function independently of the vague. It is concluded that chronic denervation of the preganglionic abdominal vague does not interfere with the demonstration of atropine-resistant nicotine-induced contraction of the isolated rabbit ileum.
Vol. 7, No . 23
DRUG-IIdDUCED RESPONSES
1315
Another interesting observation was that nicotine-induced contraction of both ilea was more susceptible to inhibition by a combination of atropine
and hexamethonium than to inhibition by either drug alone. This observa
tion suggests that nicotine has more than one site of action on the rabbit ileum (i .e., ganglionic receptors and nerve terminal receptors) and thus the two antagonists
can more effectively antagonize the overall contractile Aside from the classical ganglionic action of nicotine, been reported to excite postganglionic nerve terminals in
effect of nicotine .
the drug has other isolated tissue (9, 10).
Acknowledgment I thank Dr. J. P. Long of the College of Medicine, University of Iowa for providing the DMAE . References 1. 2. 3. 4. 5. 6.
7.
N. AMBACHE and J. EDWARDS, Brit . J. Pharmacol. 6, 311 (1951) . S . ELLLS, Science 114, 325 (1951) . U. S. VON EDLER, Acta Pharmacol. et Toxicol. 1, 29 (1945) .
S . ELLLS and H . RASMUSSEN, J. Pharmacol. ~0 j 259 (1951) . G. VG . SNEDECOR and W . G . COCHRAN, Statistical Methods p. 339, The Iowa State University Press (1967) . S . WONG and J. P. LONG, J. Pharmacol. ~, 469 (1967) . D.H .L . EVANS and J. G. MURRAY, J. Anat . Lond. 88, 320 (1954) .
W. SCHAUMANN, Nature, Lond. 178, 1121 (1956) . U. SCHAEPPI, B. L. DENNISON, and M. DODD, J. Pharmacol . ~, 216 (1966) . 10 . J.H . BURN, E.H . LEACH, M.J . RAND, and J.W . THOMPSON, J. Physiol. 14g, 332 (1959) . 8. 9.
Pergamon Press
EFFECT OF CHRONIC VAGAL DENEhVATION ON DRUG-INDUCED RESPONSES OF ISOLATED RABBIT ILEUM Marvin M. C*oldenberg Section of Pharmacology, Pharmacometrics Division, Research and Development Department The Norwich Pharmacal Company Norwich, New York (Received 5 August 1968 ; in final form 9 Septemk~er 1988) CLASSIFICATIJN of contractile responses of isolated intestines of various species as atropine sensitive or -resistant is wéll-documented . That is, excitation of the intrinsic motor nervous elements of these intestines by nicotine results in contractions which are either effectively antagonized, as in the
cat (1) and guinea pig (2) or relatively unaltered by atropine, as in the rabbit (3,4) . The non-parallel blocking activity of atropine among the intestines of various species suggests that cholinergic as well as non-cholinergic motor
neurons may be activated by nicotine . The present study is concerned with the atropine-resistant isolated rabbit ileum and the role played by the vague nerve (extrinsic) in determining the nature of the contractile response to nicotine .
Methods Ten male albino rabbits, 5 control and 5 vagally-denervated, ranging in weight from 1.5-4 Kg were killed by a blow to the back of the head; the abdomen was opened and the ileum removed. Each strip of ileum was sus-
pended in a 10 ml Anderson muscle bath containing Tyrode solution gassed by 95% 02 + 5% C02 at 36 °C. Isometric contractions were recorded by a Grass FT-03 force-displacement transducer monitored by a Grass poly-
graph. The initial tension on the muscle was adjusted to 6 g and contractions were expressed in terms of g of tension developed. In the preparation of the vagally-denervated ileum, the rabbit was anesthetized with ether and the vague nerves transected under aseptic conditions .
The vagi were excised (by removal of 3-5 mm pieces) in the
abdominal region at the point where they follow the esophagus just before entering the stomach. The nerves were allowed to degenerate for 23-34
days before the ileum was removed and suspended in the bath . Control contractions of approximately equal magnitude were obtained by adding alternatively acetylcholine (ACh) 0.1 ug/ml and nicotine 1 u q/ml . An interval of 8 min was allowed between the additions of the two agonists .
1311
1312
DRUG-INDUCED RESPONSES
Vol . 7, No . 23
A fter control responses were established, antagonists were added to the bath 8 min before introduction of an agonist . Tyrode solution of the following composition (g/1) was used in all experiments: NaCI, 8 ; KC1, 0.2; CaCl2, 0.2; ~NIgCl2 " 6H20, 0.1; NaH2P04 " H2O, 0.05; NaHC~, 1 .0; dextrose, 1 .0. Compounds used were: ACh chloride, nicotine salicylate, hexamethonium bromide, cocaine hydrochloride, atropine sulfate, morphine sulfate, and a .p~-bis - (dimethylammoniumacetaldehy>de diethylacetal)-p,p'-diacetylbiphenyl bromide (Di~+IAE) . :~11 concentrations of drugs refer to final concentrations of their salts and are expressed as ug/ml. hesults In the vagally-denervated ileal preparation the rate, frequency, or amplitude of spontaneous contractions did not appear to be different from that of normal ileum . However, the sensitivity of the vagally-denervated ileum to the contractile effect of ~Ch (0.1 ug/ml) or of nicotine (1 u g/ml) was significantly (P < 0.05) decreased as compared to that of normal preparations . fable 1 presents the results of the antagonists used on both types of ileal preparations . The significance of the difference in means was determined throughout using Student's t test (5), with a P value of 0.05 or less considered significant . The local anesthetic cocaine, 10 ug/ml, and the ganglionic blocking agent hexamethonium, 100 ug/ml, significantly inhibited nicotine-induced contraction of both the normal and the vagally-denervated ileum. However, the percentage reductions of the contractile response to nicotine in the two ileum preparations were not equal, in that a greater inhibition of the In both normal and vagallyresponse was noted in the normal ileum. denervated ilea atropine, 0.01 or 0.1 ug/ml, did not block the effect of nicotine to the same degree as was found for :1Ch (Table 1) . ~~ significant decrease in the contractile response to nicotine was evident, however, at either concentration of atropine in both strips . A'either the cholinergic neuronal blocking agent morphine, 1 ug/ml, or DYLaE, 10 u g/ml, which has been reported to possess specific antinicotine activity (6), specifically antagonized the effect of nicotine on either ileum. Significant inhibition of the response to nicotine was elicited by a combination of atropine (0.1 ug/ ml) and hexamethonium (100 u g/ml), which caused a greater degree of inhibition than either drug alone . :ACh-induced contraction of either ileum was not significantly affected by cocaine, hexamethonium, morphine, or DIVI_AE,
Vol. 7, No. 23
DRUG-IIdDUCED RESPONSES
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1314
DRUG-INDUCED RESPONSES
Vol. 7, No . 23
Discussion and Summary Degeneration of the abdominal vagi of the rabbit (3~ to 5 weeks after sectioning) resulted in a decreased responsiveness of the ileum to nicotine . The lowered sensitivity to nicotine cannot be attributed solely to a lose of intrinsic neuronal function of the ileum by means of degenerative changes after preganglionic parasympathetic section, since the action of ACh, mediated via muscarinic smooth muscle receptors, was similarly diminished. Interpretation of these data can be only tentative, but it may the be that disproportionately larger number of nerve cells in the plexus of
the
rabbit
intestine as compared to preganglionic vagal fibers (7) is
capable of activation by nicotine, although the function of the va~i has been interrupted. On this basis, a reduction of the effect of nicotine may reflect an impairment of the intestinal smooth muscle which is unable to respond to indirect neuronal stimulation any more than to direct stimulation by ACh. It
was of interest to note that the per cent change .in contractile . response to nicotine for the different ilea was different for control and for the antagonists, cocaine or hexamethonium . More specifically, the amount by which cocaine or hexamethonium inhibited the response to nicotine in the vagally-denervated ileum was not greater than for the normal one. No such finding was apparent after the employment of the other antagonists. Inhibition of the contractile response to nicotine to the same absolute
level in that the
both ilea by a local anesthetic or a ganglion blocker suggests intrinsic motor nerve supply may be unaltered after vagal de-
nervation. The present findings also indicate that a functional extrinsic parasympathetic preganglionic nerve (the vague) is not necessary in revealing the relatively atropine-resistant response of the rabbit intestine. In addi tion, neither morphine, which reduces the output of ACh from postganglionic
cholinergic nerve endings (8), or DMAE, an antinicotine agent (8), significantly antagonized the contractile response to nicotine of the vagallyThat portion of the effect of nicotine which denervated or normal ileum. is atropine resistant and which is not altered by vagal denervation may be due to activation of a cholinergic-like intrinsic nerve supply which can function independently of the vague. It is concluded that chronic denervation of the preganglionic abdominal vague does not interfere with the demonstration of atropine-resistant nicotine-induced contraction of the isolated rabbit ileum.
Vol. 7, No . 23
DRUG-IIdDUCED RESPONSES
1315
Another interesting observation was that nicotine-induced contraction of both ilea was more susceptible to inhibition by a combination of atropine
and hexamethonium than to inhibition by either drug alone. This observa
tion suggests that nicotine has more than one site of action on the rabbit ileum (i .e., ganglionic receptors and nerve terminal receptors) and thus the two antagonists
can more effectively antagonize the overall contractile Aside from the classical ganglionic action of nicotine, been reported to excite postganglionic nerve terminals in
effect of nicotine .
the drug has other isolated tissue (9, 10).
Acknowledgment I thank Dr. J. P. Long of the College of Medicine, University of Iowa for providing the DMAE . References 1. 2. 3. 4. 5. 6.
7.
N. AMBACHE and J. EDWARDS, Brit . J. Pharmacol. 6, 311 (1951) . S . ELLLS, Science 114, 325 (1951) . U. S. VON EDLER, Acta Pharmacol. et Toxicol. 1, 29 (1945) .
S . ELLLS and H . RASMUSSEN, J. Pharmacol. ~0 j 259 (1951) . G. VG . SNEDECOR and W . G . COCHRAN, Statistical Methods p. 339, The Iowa State University Press (1967) . S . WONG and J. P. LONG, J. Pharmacol. ~, 469 (1967) . D.H .L . EVANS and J. G. MURRAY, J. Anat . Lond. 88, 320 (1954) .
W. SCHAUMANN, Nature, Lond. 178, 1121 (1956) . U. SCHAEPPI, B. L. DENNISON, and M. DODD, J. Pharmacol . ~, 216 (1966) . 10 . J.H . BURN, E.H . LEACH, M.J . RAND, and J.W . THOMPSON, J. Physiol. 14g, 332 (1959) . 8. 9.