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The reversal by atropine of ganglionic blockade produced by acetylcholine or methacholine
Life Sciences No, 12, pp" 677-6$2, 1962. Pergas~on Press, Inc. Printed in the United States .
T~ ESYFdiSAL BY ATBOPII~IE OF 6ANGLIONIC BLOCKADE P&ODUCF~ BY ACETYLCHOLINE ~ METHAC~HOLINE A. J. Pappsec and E. L. Volle Department of Pharmacology Schools of Medicine IIniversity of Pennsylvania Philadelphia, Pennsylvania (Received 15 No~ber 1962) IDI previous communications from this laboratory eridence xas presented xhich indicated the presence of txo pharmacologically distinctive receptors for acetylcholine .(ACh) in the sympathetic ganglia of cats . l ' 2,8
The primary
distinction betxeea the txo receptors xas the fact that one xas quite sensitive to blockade by small doses of atropine and the ether to the traditional ganglionic blocking drags. persisted far boars.
In addition, the blockade produced by atropine
Accordingly, the atropine-sensitive receptor possessed
features associated generally xith those located at the peripheral neuroeffector junctions of the parasympathetic division of the autonomic nervous system. The experiments described belox extend farther the pharmacological analysis of cholinoceptive sites in sy~athetic ganglia.
For this purpose,
studies xere made of the effects of atropine on the ganglionic responses produced by ACh and methacholine tacetyl-~ß-methacholine) .
The latter cholino"
mimetic agent is knoxn to possess prominent excitatory actions on parasympathetic neuro-effector junctions. Methods Two parameters of ganglionic function xere assessed in this study;
(a)
the asynchronous postganglionic discharge evoked by ACh and methacholine and
ATROPINE REVERSAL
67 8
No . 12
(b) the effects of these drugs on the postganglionic action potentials evoked by repetitive stimulation of the preganglionic nerve xith supramaximal volleys at a rate of 0.5 cps . Fourteen cats of both sexes xere used in thin study.
Folloxing the
indYCtion of anesthesia xith sodium diallylbarbiturate and urethane (Dial, 0.7 ml/kg, i .p .), the superior cervical ganglia xere exposed by surgical procedures The outlined in earlier reports fro® this laboratory . l' 2'3 cervical sympathetic nerve and the external carotid nerve (postganglionic) xere dissected free from underlying tissues and mounted on platinum electrodes .
Stimuli of 0,01 cosec duration xere applied to the preganglionic
nerve by means of a square rave generator and isolated from ground by means of an electronic isolation unit .
Action potentials evoked either by electric-
al stimulation or by chemical agents xere amplified by means of condenaorconpled prea®plifiers .
The amplified signals xere, in turn, coupled to a
dual-beam oscilloscope for visualization .
Permanent records xere made on
photographic paper with a kymographic camera . Drugs, dissolved in 0 .9~ NaCI solution, xere adniniatered by rapid igjection through a 27-gauge needle inserted into the common carotid artery . The volumes inj ected never exceeded 0.2 mi . The folloxing drugs xere used :
acetylcholine chloride (ACh), metha-
choline chloride, and atropine sulfate.
Doses are expressed as the salt .
The ranges of doses of acetylcholine and methacholine used in these experi ments xere 1 to 100 ~,g and 10 to 50 Wg, respectively . Results It is xell known that ACh and other cholinonimetic agents reduce markedly the amplitude of the postganglionic action potential evoked by preganglionic vo3leys .4
Furthermore, these agents evoke asynchronous post
ganglionic firing in resting, acutely decentralized ganglia .
In the present
series of experiments, the effects of small doses of atropine on these txo actions of cholinomimetic drugs xere studied.
No. 12
ATROPINE REVLR.SAL
679
u PTG. 1 Poatgaaglionic discharge of superior cervical ganglion evoked by acetylcholine (25Ng, i.e.) before (B) and after (A) atropine (1~g, i.a .) . oertioal and horisontal calibrations are 10 NV and 1 sec respectively. Dot at bottom of each record signals the injection of the aoetylcholine.
FIG. 2 Effects of soetylcholine (100~g, i.a .) on poetganglionic action potentials of superior cervical ganglion evoked by repetitive preganglionic stimulation (0.5 cps) before (B) and after (A) atropine (1~g, i .a.) . Same e=perfment as Fig. 1 . 4ertiael and horisontal calibrations are 500 rV sad 1 sea respectively. Dot at bottoa of each record signals the injection of acetylcholine .
680
ATROPINE REVERSAL
No, 12
The records obtained in 1 out of 7 experiments xith ACh are illustrated in figures 1 and 2,
As expected, ACh (50 to 100 pg, i,a,) produced a marked
depression of the amplitude of the evoked action potentials and evoked directly asynchronous firing in the resting ganglion,
Ten minutes after the
administration of atropine (1 Wg, i,a,), ACh increased the amplitudes of the postganglionic spikes .
Under these conditions, the asynchronous firing
evoked by ACh xas not altered materially, Prior to the administration of atropine, the actions of methacholine on the ganglia were similar qualitatively to those of ACh,
Although methacholine
was somewhat less effective in evoking a postganglionic discharge than xas ACh it produced a more profound depression of the evoked spike.
Unlike ACh,
however, both the depression of the spike and the discharge produced by methacholine xere antagonized by small doses of atropine .
These actions of
methacholine were studied in 7 additional experiments, Discussion Oa the basis of existing knoxledge there are three possible explanations of the depression by ACh and methacholine of the postganglionic action potentials evoked by repetitive preganglionic volleys . Firstly, the possibility exists that these agents by depolarizing the ganglion cells produced a refractoriness of some of the ganglion cells and, accordingly, reduced the number of cells capable of responding to the preganglionic volleys,
The fact that agents of this type depolarize ganglion
cells forms the basis of this explanation, 4
Hoxever, the finding in ganglia
treated xith atropine that ACh increased the amplitude of the postganglionic spikes and evoked a postganglionic discharge cannot be explained reasonably in these terms, Secondly, it is possible that the depression of the action potentials xas dne to the liberation by ACh or methacholine of inhibitory substances in the ganglia,
Although pharmacological evidence has been presented which in
dicated the presence in sympathetic ganglia of inhibitory mechanisms, 5 'b it
ATROPINL REVERSAL
No . 12
681
is unlikely that the observations described in this report can be explained by these mechanisms .
It can be argued that if the depression of the action
potentials xas doe to the activation of inhibitory processes, then atropine, itself, xould be expected to enhance the ganglionic responses to the cholinomimetic agents, preganglionic stimulation, or both . Finally, and by exclusion, the findings can be explained by implicating a depolarization of both the pre- and postaynaptic components of the ganglia by the cholinomimetic agents .
In this regard, it is notexorthy that evidence
for a presynaptic locos of action of ACh, 7 ~ a aicotine,9 and atropine l0 has been described.
According to this proposal, the depolarization by ACh of the
nerve terminals resulted in a decrease in the amount of transmitter liberated by the nerve volley from the nerve terminals and, consequently, a decrease in the amplitude of the postganglionic action potential.
It folloxs also that
this action of ACh must be regarded as sensitive to blockade by small doses of atropine.
On the other hand, depolarization by ACh of the ganglion cells xas
not affected materially by atropine .
As a corollary to this xorking hypothesis,
the primary site of action of methacholine is the nerve terminals. Furthermore, the apecnlation can be made that both methacholine and ACh have a dnal action on the nerve terminals .
In addition to producing a
depolarization of the nerve terminals xhich interfered xith the nerve volley, it is conceivable that these agents are capable of causing the release of the transmitter anbatance from the nerve endings.
This latter possibility
is consistent xith the observed enhancement of the postganglionic action potentials produced by these agents in atropinized ganglia.
In this connect-
ion, it is of interest that carbachol has been reported to cause the release of ACh from resting perfused ganglia . ll Admittedly, the foregoing explanation is highly apecnlative .
Nonetheless,
it is consistent xith the present observations and those cited above.
Further-
more, it is a xorking hypothesis xhich can be subjected to further testing .
682
ATROPINE RL~7SRSAL
No . 12
References 1.
R, L. VOLLE, J, Pharmacol , 135, 45 (1962),
2.
A. KOI~ALAHIRANYA and R, L. VO[.L.E, J. Pharmacol, 138, 57 (1962),
3,
C. TAKESHIGE and R.L . VOLLE, J. Pharmacol . 138, 66 (1962),
4,
W,D,H. PATON and W.L,iI. PERKY, J. Physiol . 119. 43 (1953),
5,
E, COSTA, A. II. REYZIIV, S. YONTLlIAN, S. SPECTDB and B.B, BRODIE, Science _, 1822 (196.1) .
6.
R,il, ECCLES and B, LIBET, J, Physiol .
7,
R. L, YOLLE and G.B, KOE[.LE, J. Pharmacol, 133, 223 (1961) .
8,
R, L. YOLLE, J~ Pharmacol, 136, 68 (1962),
9.
E. W. PELIKAN, Ann, N,Y, Acad, Sciences . 90, 52 (1960),
~,
484 (1961),
10,
R, HQGHBS and B, C, WHALER, J, Physiol . 160, 221 (1962),
11 .
D.N . 11C KINSTRY, E. KOENIG and G. B. KOH.i.E, Fed, Proc , =1 , 341 (1962),
T~ ESYFdiSAL BY ATBOPII~IE OF 6ANGLIONIC BLOCKADE P&ODUCF~ BY ACETYLCHOLINE ~ METHAC~HOLINE A. J. Pappsec and E. L. Volle Department of Pharmacology Schools of Medicine IIniversity of Pennsylvania Philadelphia, Pennsylvania (Received 15 No~ber 1962) IDI previous communications from this laboratory eridence xas presented xhich indicated the presence of txo pharmacologically distinctive receptors for acetylcholine .(ACh) in the sympathetic ganglia of cats . l ' 2,8
The primary
distinction betxeea the txo receptors xas the fact that one xas quite sensitive to blockade by small doses of atropine and the ether to the traditional ganglionic blocking drags. persisted far boars.
In addition, the blockade produced by atropine
Accordingly, the atropine-sensitive receptor possessed
features associated generally xith those located at the peripheral neuroeffector junctions of the parasympathetic division of the autonomic nervous system. The experiments described belox extend farther the pharmacological analysis of cholinoceptive sites in sy~athetic ganglia.
For this purpose,
studies xere made of the effects of atropine on the ganglionic responses produced by ACh and methacholine tacetyl-~ß-methacholine) .
The latter cholino"
mimetic agent is knoxn to possess prominent excitatory actions on parasympathetic neuro-effector junctions. Methods Two parameters of ganglionic function xere assessed in this study;
(a)
the asynchronous postganglionic discharge evoked by ACh and methacholine and
ATROPINE REVERSAL
67 8
No . 12
(b) the effects of these drugs on the postganglionic action potentials evoked by repetitive stimulation of the preganglionic nerve xith supramaximal volleys at a rate of 0.5 cps . Fourteen cats of both sexes xere used in thin study.
Folloxing the
indYCtion of anesthesia xith sodium diallylbarbiturate and urethane (Dial, 0.7 ml/kg, i .p .), the superior cervical ganglia xere exposed by surgical procedures The outlined in earlier reports fro® this laboratory . l' 2'3 cervical sympathetic nerve and the external carotid nerve (postganglionic) xere dissected free from underlying tissues and mounted on platinum electrodes .
Stimuli of 0,01 cosec duration xere applied to the preganglionic
nerve by means of a square rave generator and isolated from ground by means of an electronic isolation unit .
Action potentials evoked either by electric-
al stimulation or by chemical agents xere amplified by means of condenaorconpled prea®plifiers .
The amplified signals xere, in turn, coupled to a
dual-beam oscilloscope for visualization .
Permanent records xere made on
photographic paper with a kymographic camera . Drugs, dissolved in 0 .9~ NaCI solution, xere adniniatered by rapid igjection through a 27-gauge needle inserted into the common carotid artery . The volumes inj ected never exceeded 0.2 mi . The folloxing drugs xere used :
acetylcholine chloride (ACh), metha-
choline chloride, and atropine sulfate.
Doses are expressed as the salt .
The ranges of doses of acetylcholine and methacholine used in these experi ments xere 1 to 100 ~,g and 10 to 50 Wg, respectively . Results It is xell known that ACh and other cholinonimetic agents reduce markedly the amplitude of the postganglionic action potential evoked by preganglionic vo3leys .4
Furthermore, these agents evoke asynchronous post
ganglionic firing in resting, acutely decentralized ganglia .
In the present
series of experiments, the effects of small doses of atropine on these txo actions of cholinomimetic drugs xere studied.
No. 12
ATROPINE REVLR.SAL
679
u PTG. 1 Poatgaaglionic discharge of superior cervical ganglion evoked by acetylcholine (25Ng, i.e.) before (B) and after (A) atropine (1~g, i.a .) . oertioal and horisontal calibrations are 10 NV and 1 sec respectively. Dot at bottom of each record signals the injection of the aoetylcholine.
FIG. 2 Effects of soetylcholine (100~g, i.a .) on poetganglionic action potentials of superior cervical ganglion evoked by repetitive preganglionic stimulation (0.5 cps) before (B) and after (A) atropine (1~g, i .a.) . Same e=perfment as Fig. 1 . 4ertiael and horisontal calibrations are 500 rV sad 1 sea respectively. Dot at bottoa of each record signals the injection of acetylcholine .
680
ATROPINE REVERSAL
No, 12
The records obtained in 1 out of 7 experiments xith ACh are illustrated in figures 1 and 2,
As expected, ACh (50 to 100 pg, i,a,) produced a marked
depression of the amplitude of the evoked action potentials and evoked directly asynchronous firing in the resting ganglion,
Ten minutes after the
administration of atropine (1 Wg, i,a,), ACh increased the amplitudes of the postganglionic spikes .
Under these conditions, the asynchronous firing
evoked by ACh xas not altered materially, Prior to the administration of atropine, the actions of methacholine on the ganglia were similar qualitatively to those of ACh,
Although methacholine
was somewhat less effective in evoking a postganglionic discharge than xas ACh it produced a more profound depression of the evoked spike.
Unlike ACh,
however, both the depression of the spike and the discharge produced by methacholine xere antagonized by small doses of atropine .
These actions of
methacholine were studied in 7 additional experiments, Discussion Oa the basis of existing knoxledge there are three possible explanations of the depression by ACh and methacholine of the postganglionic action potentials evoked by repetitive preganglionic volleys . Firstly, the possibility exists that these agents by depolarizing the ganglion cells produced a refractoriness of some of the ganglion cells and, accordingly, reduced the number of cells capable of responding to the preganglionic volleys,
The fact that agents of this type depolarize ganglion
cells forms the basis of this explanation, 4
Hoxever, the finding in ganglia
treated xith atropine that ACh increased the amplitude of the postganglionic spikes and evoked a postganglionic discharge cannot be explained reasonably in these terms, Secondly, it is possible that the depression of the action potentials xas dne to the liberation by ACh or methacholine of inhibitory substances in the ganglia,
Although pharmacological evidence has been presented which in
dicated the presence in sympathetic ganglia of inhibitory mechanisms, 5 'b it
ATROPINL REVERSAL
No . 12
681
is unlikely that the observations described in this report can be explained by these mechanisms .
It can be argued that if the depression of the action
potentials xas doe to the activation of inhibitory processes, then atropine, itself, xould be expected to enhance the ganglionic responses to the cholinomimetic agents, preganglionic stimulation, or both . Finally, and by exclusion, the findings can be explained by implicating a depolarization of both the pre- and postaynaptic components of the ganglia by the cholinomimetic agents .
In this regard, it is notexorthy that evidence
for a presynaptic locos of action of ACh, 7 ~ a aicotine,9 and atropine l0 has been described.
According to this proposal, the depolarization by ACh of the
nerve terminals resulted in a decrease in the amount of transmitter liberated by the nerve volley from the nerve terminals and, consequently, a decrease in the amplitude of the postganglionic action potential.
It folloxs also that
this action of ACh must be regarded as sensitive to blockade by small doses of atropine.
On the other hand, depolarization by ACh of the ganglion cells xas
not affected materially by atropine .
As a corollary to this xorking hypothesis,
the primary site of action of methacholine is the nerve terminals. Furthermore, the apecnlation can be made that both methacholine and ACh have a dnal action on the nerve terminals .
In addition to producing a
depolarization of the nerve terminals xhich interfered xith the nerve volley, it is conceivable that these agents are capable of causing the release of the transmitter anbatance from the nerve endings.
This latter possibility
is consistent xith the observed enhancement of the postganglionic action potentials produced by these agents in atropinized ganglia.
In this connect-
ion, it is of interest that carbachol has been reported to cause the release of ACh from resting perfused ganglia . ll Admittedly, the foregoing explanation is highly apecnlative .
Nonetheless,
it is consistent xith the present observations and those cited above.
Further-
more, it is a xorking hypothesis xhich can be subjected to further testing .
682
ATROPINE RL~7SRSAL
No . 12
References 1.
R, L. VOLLE, J, Pharmacol , 135, 45 (1962),
2.
A. KOI~ALAHIRANYA and R, L. VO[.L.E, J. Pharmacol, 138, 57 (1962),
3,
C. TAKESHIGE and R.L . VOLLE, J. Pharmacol . 138, 66 (1962),
4,
W,D,H. PATON and W.L,iI. PERKY, J. Physiol . 119. 43 (1953),
5,
E, COSTA, A. II. REYZIIV, S. YONTLlIAN, S. SPECTDB and B.B, BRODIE, Science _, 1822 (196.1) .
6.
R,il, ECCLES and B, LIBET, J, Physiol .
7,
R. L, YOLLE and G.B, KOE[.LE, J. Pharmacol, 133, 223 (1961) .
8,
R, L. YOLLE, J~ Pharmacol, 136, 68 (1962),
9.
E. W. PELIKAN, Ann, N,Y, Acad, Sciences . 90, 52 (1960),
~,
484 (1961),
10,
R, HQGHBS and B, C, WHALER, J, Physiol . 160, 221 (1962),
11 .
D.N . 11C KINSTRY, E. KOENIG and G. B. KOH.i.E, Fed, Proc , =1 , 341 (1962),