THE POINT OF ATTACK OF CERTAIN DRUGS

ACTING ON THE PERIPHERY

II. ACTION ON THE RETRACTOR PENIS MUSCLE

OF THE DOG

CHARLES W. EDMUNDS
Ann Arbor, Michigan

Received for publication February 9, 1920

In an effort to shed more light upon the questions raised in

the first part of this paper a number of experiments were carried
out upon the action of these drugs upon the isolated retractor
penis muscle of the dog. This muscle has been recently examined
histologically by Fisher (7) who showed that the anterior three-
fifths is composed of non-striated fibers while in the posterior
two-fifths some cross striated fibers are to be found. The muscle
in a medium sized dog is about 50 mm. long and has a diameter
of about 3 mm. On account of its shape and the arrangement of
its fibers and its comparatively simple innervation, it is a very
suitable structure for such experimental studies.
Langley and Anderson (1) showed that the motor innervation
of the muscle was by way of the lumbar nerves while stimulation
of the sacral nerves (bc. cit, p. 104) produced inhibition of the
muscle. Fisher says he made systematic search for nerve ganglia
but could find them neither in the muscle nor in its sheath. The
innervation by way of the sacral nerves seems to have excited
most interest and the conclusions of Langley and Anderson as to
its inhibitory nature have been simply confirmed. A tracing
showing inhibition of the muscle upon sacral stimulation is
given by Starling (8) and also by Frohlich and Loewi (9).
Fletcher (10) demonstrated both motor and inhibitory nerves in
the hedgehog and rat retractor muscles and the subject is also
discussed by Br#{252}cke (11). The effect of stimulation of the
201
202 CHALRES W. EDMUNDS

sacrals upon the retractor muscle is of prime importance when

considering the point of action of some of the drugs to be con-
sidered, but a study of the references mentioned would not
seem to leave any doubt as to the correctness of Langley and
Anderson’s original findings.

FIG. 1. TRACING FROM BLADDER OF CAT

%Vell marked contraction with slight relaxation from the injection of adrenal
me. Lever moves up during contraction.

In my own experiments, the muscle was exposed immediately

after death of the dog and after being freed from its surrounding
covering, it was cut loose from its point of insertion and the
anterior two-thirds cut from the posterior portion-the lattet
 POINT OF ATTACK OF CERTAIN DRUGS 203

not being utilized at all in order not to complicate the results

upon the smooth muscle with any which might be given by the
striated fibers which mingle with the smooth in the posterior

Spontaneous rhythm. Inhibition and relaxation under influence of adrenaline.

Lever moves up as bladder contracts.

portion. The portion of muscle which was obtained was cut

into two or three pieces and those which were not to be used
at once were placed in cold Ringer’s solution and kept in an
ice-chest..
204 CHARLES w. EDMUNDS

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 POINT OF ATTACK OF CERTAIN DRUGS 205

The portion to be used was placed in warm oxygenated

Ringer’s solution and arranged to record its movements in the
usual manner. Along certain lines my experience with the
muscle agreed with those described by Bottazzi (12). These
points are especially those showing the necessity for abundant
oxygen as the muscle relaxed rapidly and became inactive if
this supply was not sufficient. Also, my experience with the
temperature necessary to produce best results was like that of
Bottazzi as the muscle maintained at a temperature of 3750

to 38#{176}C. was relatively inactive and very unsatisfactory but

when the solution was maintained at a temperature between
34.5#{176}and 35#{176}C.the tissue gave excellent. results. After being
placed in the bath under these conditions the muscle was inactive
for a variable time, half an hour or so, and then it would start
rhythmic contractions as have been described by Bottazzi and
by de Zilwa (14).

ADRENALINE

The first drug to be tested upon the muscle was adrenaline-

the commercial 1-1000 solution being used. The addition of 1
minim or often of small amounts of this solution to the Ringer’s
solution bathing the muscle produced either in the relatively
inactive muscle at 37.5#{176} or in the active muscle at 34.5#{176}
a vigor-
ous and somewhat lasting contraction. This result was in har-
mony with the effect of sympathetic stimulation as described
above. If now the muscle in fresh Ringer’s solution is subjected
to the action of ergotoxin for a short time and then in new
Ringer’s, is exposed to a second dose of adrenaline, it is found
that the adrenaline will no longer produce a contraction, the
motor sympathetic mechanism being paralyzed. Not only will
adrenaline after ergotoxin have no motor effect upon the re-
tractor (a result which was to be expected), but it will frequently
produce some relaxation. This latter effect was studied further
and is discussed in another paper upon the innervation of the
retractor muscle.

THE JOUR. OF PHRM. AND EXPER. THERAP., VOL. XV, NO. 8

 206 CHARLES W. EDMUNDS

ATROPINE

Considerable interest attaches to the effect of atropine and

pilocarpine upon the muscle as they are supposed to act upon
the parasympathetic system, the stimulation of which in the case
of this muscle is to produce inhibition.
De Zilwa found that while his results were, at times, con-
flicting, in most cases 0.1 to 0.2 per cent atropine in defibrinated

FIG. 4. ISOLATED RETRACTOR MUSCLE

Active rhythm practically unaffected by atropine. Bath temperature 34.5#{176}C.

Lever moves down in contraction.

blood gave diminution of tone and in some cases abolition of

spontaneous rhythm. Also, that atropine injected into the cir-
culation did not effect the nerve endings but that the increase of
tone produced by such drugs as muscarine was removed by the
atropine. Fr#{246}hlich and Loewi found that the acraIs were un-
affected by atropine.
Bottazzi’s work with atropine deserves special comment. He
criticises de Zilwa’s results because he used atropine in defibri-
nated blood which is not an indifferent vehicle. He says that
 POINT OF ATTACK OF CERTAIN DRUGS 207

in his own experiments, in the greatest number of cases, atropine

raised the tone of the retractor and developed an active con-
tractile rhythm. In his more extensive paper he says that
acting alone, he has never observed inhibition from atropine;
that while it will remove the muscarine action yet alone it always
causes a contraction. A tracing of such a contraction is repro-
duced in his papers.
Before discussing these results, it may be well to give the
results of my own experiments. I have used atropine in varying
doses on the isolated muscle in numerous experiments, both in
connection with piocarpine and other drugs and also alone,
using it as the first drug in an experiment. I have used it on
inactive retractors and upon those exhibiting a spontaneous
rhythm and in no case has a contraction of the muscle been
obtained. My results agree in general with those of de Zilwa.
Used alone where the retractor was inactive it either had no
effect or it caused slight relaxation; in the case of muscles show-
ing a spontaneous rhythm there was frequently no effect while
in others the contraction would be lessened and the relaxation
possibly increased (Fig. 4). The contractions, or in other cases
increased tone, produced by pilocarpine and physostigmine were
promptly removed by it.

PILOCARPINE

The action of piocarpine is so closely connected with that of

atropine that they maybe best discussed together.
Bottazzi found that piocarpine ordinarily provoked strong
contractions if the muscle was not already in a state of con-
traction but that if it was contracted, it produced elongation of
the muscle which, he says, is not due to inhibition but to death
or paralysis of the muscle.
The first statement of Bottazzi, I have been able to abund-
antly confirm as even in relatively inactive muscles piocarpine
produced a contraction or an increase in tone and in spontane-
ously contracting muscles the contractions were strengthened.
In any dosage that I used I never saw the phenomenon described
208 CHARLES W. EDMUNDS

by him as elongation of the muscle due to death or paralysis from

the alkaloid.
In discussing the action of these drugs it would seem that
the findings of Bottazzi in regard to the action of atropine are
not only at variance with the results obtained by de Zilwa and
by myself but also they are not in harmony with pharmaco-
logical experience with this drug, especially in its relation to
piocarpine. The mere fact that atropine caused a primary
contraction even upon top of a piocarpine effect would not be
unusual and in fact, is an experience commonly met with. It
is of less frequent occurrence to have merely stimulation of
smooth muscle result from atropine but Kuroda (21) found
exactly this effect in the case of the ileo-colic sphincter in which
he always got contraction and increased movement. In most
experiments upon smooth muscle structures no effect has been
obtained from atropine unless pilocarpine had been given pre-
viously. According to Bottazzi piocarpine and atropine would
act in the same way which is unusual as elsewhere in the body
they are antagonistic and as a matter of fact, are perhaps the
best example of drugs presenting antagonistic actions to be
found anywhere. De Zilwa did not use piocarpine but he did
use muscarine which produced an increase in tone so that so far
as the effect of these two related alkaloids are concerned the
researches of de Zilwa, Bottazzi and myself are in agreement,
at least, in so far as they relate to the increased tone of the
muscle produced. As for the atropine effects my results agree
with those of de Zilwa and are opposed to those of Bottazzi.
The more important point is as to the location of the action
of these two drugs. According to an opinion still frequently
encountered, they must act upon the sacral nerves (parasympa-
thetic), pilocarpine stimulating and atropine removing its effect.
As the sacral nerves produce inhibition of the retractor, pilocar-
pine should have a similar action, but the results obtained by
its use are the reverse of this. This must eliminate then, all
the sacral nervous mechanism from consideration as possible
points of attack-both nerve endings and myoneural junctions
and leave only the muscle substance proper to be acted upon and
 POINT OF ATTACK OF CERTAIN DRUGS 209

if this is true of pilocarpine, the same must be true of atropine

and its point of attack would also be upon the muscle substance
proper.
There is, however, one other possibility and that is that these
drugs may act in the case of the retractor muscle upon the
sympathetic mechanism in the same way as Cushny (15) has
shown that they act upon the uterus in the cat where he found
that pilocarpine causes contraction or inhibition according to the
condition of the organ in this resembling the effects of adrenaline
and hypogastric stimulation. The contractile effects of pilo-
carpine are antagonized by ergotoxin while both motor and in-
hibitory effects are removed by atropine. From these results,
Cushny draws the conclusion that in the case of the pregnant
uterus of the cat pilocarpine acts upon the sympathetic mechan-
ism. These results are very important as they constitute the
only instance (with the exception of the sweat glands) in which
pilocarpine acts upon the sympathetic (dorso-lumbar) mechan-
ism. To see whether the retractor would form another example
of this class, pieces of the muscle suspended in Ringer’s solution
were treated with ergotoxin until the motor effect of adrenalin
was abolished and then to the muscle in fresh Ringer’s pilocar-
pine was added and the alkaloid still producing its customary
contraction eliminated the sympathetic mechanism as a possible
point of attack from consideration and localized the effect as
being one upon the muscle substance proper.

PHYSOSTIGMINE

The effects of physostigmine upon the retractor were much the

same as those produced by pilocarpine with the usual differences
between the two drugs which are shown elsewhere. It produced
an increase in tone or an increased strength of contraction in the
same way as piocarpine had done and was more active in an-
tagonizing atropine than was the latter drug. These effects also
could not have been produced by stimulation of the nerve
ending or myoneual junction of the sacral nerves, and must,
therefore, have been upon the muscle substance proper.
210 CHARLES W. EDMUNDS

In the same way as was done with philocarpine the sympa-

thetic nerves were eliminated from consideration as possible point
of attack by the use of ergotoxin and physostigmine still pro-
ducing a contraction, could not have acted upon that mechan-
ism. In contra-distinction to his findings with pilocarpine,
Cushny found that the motor effects of physostigmine upon the
pregnant cat’s uterus were not affected by ergotoxin,-his results
therefore with this alkaloid being in agreement with mine upon
the retractor.
MORPHINE

The effect of this alkaloid was in general to produce a stimu-

lation although many times it would fail to have any effect,
but in most cases, its effects were unmistakable (fig. 5). The
results as far as certainty of reaction were concerned compared
very closely with those obtained upon the bladder where too,
they were nil at times. The morphine contractions were not
effected by the previous use of ergotoxin so that the alkaloid
did not act upon the sympathetics; they could not have been
produced by stimulation of the sacral mechanism, and must
therefore be due to a direct action upon the muscle.
Macht (16) in his work upon the action of morphine upon the
ureter eliminated the sympathetics as a possible point of attack
by the use of ergotoxin but apparently did not decide whether
it acted upon the sacral innervation or upon the muscle of the
ureter. Neither did Waddell (17) attempt to localize the action
of morphine in his experiments upon the pharmacology of the
vagina.
NICOTINE

The effects of nicotine upon the retractor were studied upon

the isolated muscle in which the addition of small amounts
(0.5 mgm.) to the perfusion bath was followed by a strong con-
traction (fig. 6). Later additions than the primary one were not
so active. Fresh preparations of the retractor subjected to
ergotoxin until all motor effects of adrenaline.were removed and
then subjected to nicotine, also gave strong contractions. This
 POINT OF ATTACK OF CERTAIN DRUGS 211

FIG. 5. MORPHINE UPON THE ISOLATED RETRACTOR MUSCLE

Bath temperature 34.5#{176}C. Lever moves down in contraction

FIG. 6. NICOTINE UPON A RELATIVELY INACTIVE RETRACTOR MUSCLg

Lever moves down in contraction

212 CHARLES W. EDMUNDS

result demonstrated that the action of nicotine must be one

direct upon the muscle since the motor sympathetics were
paralyzed and stimulation of the parasympathetics would have
caused relaxation.
This result is in agreement with the findings of Kuroda (21)
upon the ileo-colic sphincter where he concludes that the action
of the alkaloid in the case of that structure is one direct upon
the muscle. And such a conclusion which does not demand the
presence of ganglion cells would be in harmony with the histo-
logical researches of Fisher (7) in which he made serial sections
of the muscle but was unable to demonstrate any ganglion
cells. Fletcher (10) also stated that in his study of the nervous
apparatus of the retractor of the rat, no nerve cells were seen.

DISCUSSION

The results of the work upon the retractor would point to the
muscle substance proper as being the point of attack of pio-
carpine, physostigmine, morphine, nicotine, and atropine as no
nervous structure can be concerned in the production of their
effects. These conclusions can probably be safely transferred
to the bladder as the effects in general are in harmony with the
experimental findings upon that organ, although in the case of
nicotine, the action upon the ganglia doubtless also plays a part.
But when we come to transfer the results to other organs and
tissues, we find that difficulties arise, especially as regards the
localization of the action of piocarpine and atropine.
In most of the instances in which these drugs have been em-
ployed, especially in working with the abdominal and pelvic
viscera, no effort seems to have been made to localize exactly
the point of their attack. Reference, however, has been made
to Cushny’s work, placing the piocarpine action (and pre-
sumably its antagonist, atropine) in the case of the uterus upon
the sympathetic myoneural junctions. However, with the ex-
ception of the strutures mentioned (sweat glands and uterus),
the sympathetics (dorso-lumbar) fibers may perhaps be safely
eliminated from consideration as possible points of attack for
 POINT OF ATTACK OF CERTAIN DRUGS 213

the pilocarpine group, but the question of parasympathetic nerve

endings, myoneural junctions or muscle still remain.
Satani (18) in his recent paper upon the ureter, argues the
presence of parasympathetic nerve endings from a positive
action of physostigmine and atropine upon that structure. That
piocarpine may act upon nerve endings is supported, by Dixon
and Brodie’s (19) work upon the action of this alkaloid upon the
bronchial muscles. These workers found that if the vagi were
cut the pilocarpine stimulant effect was lost uniformly with loss
of vagus response to stimulation, and they therefore concluded
that it acts upon the nerve terminations of the contractor fibers.
Opposed to this view are the experimental results of Anderson
(20) upon the iris. He found that piocarpine produced a
greater and a longer contraction upon the denervated pupil than
it did upon the control. This result was obtained even after the
diary nerves had been cut for three or four months and which,
therefore, had completely degenerated. This action of piocar-
pine was antagonized by atropine and therefore both alkaloids
act upon some substance peripheral to the nerve ending which
has degenerated. (Physostigmine, however, lost its action a
few days after the ciliary ganglion had been removed and all
ciliary nerves cut and, therefore, its action must be upon the his-
tological nerve endings.) The action of pilocarpine and atropine
could not be upon the contractile substance proper as the iris is
contracted by dyspnoea even after it has been completely
atropinized. So that we have on the iris, according to this
view, physostigmine acting on the nerve endings while pilo-
carpine and atropine act upon some non-degenerating inter-
mediate substance (myoneural junction?).
For an action by these alkaloids upon the muscle cell direct,
there is the effect upon the retractor muscle to which Bottazi
has also called attention, although he mentions the possibility of
the action being upon the sympathetic.
In addition, Kuroda (21) working on the effect of drugs on
the ileo-colic sphincter comes to the conclusion that the seat of
the action of atropine, pilocarpine, nicotine, and cocaine is not
on the nervous mechanism, but upon the muscular. He is led
214 CHARLES W. EDMUNDS

to take this view by the fact that the effects of these alkaloids
upon the sphincter are very similar to those upon the intestine
and yet the innervation of the sphincter is entirely different
from that of the intestine. “

We have, therefore, evidence for the localization of the action

of atropine and of pilocarpine upon the sympathetic (dorso-
lumbar) myoneural junctions; upon the parasympathetic nerve
endings; upon the parasympathetic myoneural junction, and
upon the muscle substance proper. Nothing could illustrate
more clearly the danger of using these and allied alkaloids for
“revealing the presence of parasympathetic nerve endings” or
of referring to them as the “parasympathetic stimulants,” or
again of saying that they are “generally recognized as exercising
a selective action on the myoneural elements of the parasym-
pathetic nervous system.” Such statements may be correct
when applied to certain structures in the body and incorrect in
other locations.
Physostigmine
When it comes to an exact localization of the physostigmine
point of attack on the periphery, there is much the same
difficulty encountered that there is with piocarpine, excepting
that there is no evidence of the sympathetic mechanism being
involved. But we have an action upon the nerve endings in the
case of the iris (Anderson) and upon the muscle substance proper
in the case of the retractor. While the alkaloid has been used in
experiments upon other smooth muscle organs, such as the
ureter, vagina, uterus, etc., an exact localization seems not to
have been made in these cases. In addition to the work upon
the iris, retractor, and other smooth muscle, there is the series
of experiments (Edmunds and Roth (22) ) upon the action of the
alkaloid upon certain muscles of the fowl. These experiments
showed an action upon the receptive substance (myoneural junc-
tion), but it would be rather dangerous to transfer this effect
upon voluntary muscles to the smooth muscle structures.
Finally, much the same references are made to physostigmine
in recent papers as being a “stimulant of the parasympathetic
nerve endings” as are made to pilocarpine and with the same
dangers attended thereto.
 POINT OF ATTACK OF CERTAIN DRUGS 215

Curara

With curara the difficulties of localizing its point of attack

are also great and probably at the present time, impossible of
a completely satisfactory solution. Dixon in his paper (23)
upon the subject also comes to the same conclusion. Upon
the fowl’s muscle, an action is evident upon the myoneural
junctions, as it antagonized physostigmine completely, but as
pointed out by the writer elsewhere and again later by Dixon,
there is also much evidence in favor of an action upon nerve
endings proper.
In the present work, there is the incomplete anatgonism to
pilocarpine and physostigmine when the drugs are given in the
body but little antagonism so far as my experiments go when
used against those alkaloids on the isolated organs and it is not
impossible that its action is upon more than one constituent or
structure.
Morphine

The effects of morphine when acting upon the periphery have

not been examined so extensively as the other alkaloids referred
to, but there is no evidence of any action other than one direct
upon the muscular substance proper-neither nerve endings nor
receptive substances being involved.

CONCLUSION

In the case of several drugs possessing a peripheral action, it is

not possible in the present state of our knowledge to make a rule
for a specific drug which will localize its action as being upon one
special structure no matter in what part of the body it may be
found.
However, where the action of adrenaline is concerned or the
action of morphine upon smooth muscle is concerned, a defi-
nite statement may perhaps be made which wifi fit all cases.
But, with pilocarpine, physostigmine, atropine, and nicotine,
the case is otherwise. Upon the retractor penis muscle of the
dog the action is a direct one upon the muscle proper and there
216 CHARLES W. EDMUNDS

is nothing to indicate that the same may not hold good for their
action upon the bladder with the possible exception of nicotine
which may also act upon the ganglia along the course of the
nerves going to that organ.
In the case of other organs and structures such as the iris,
bronchial muscles, uterus, etc., the same general rule will
not hold as the action may be one upon nerve endings or upon
myoneural junctions, or upon the muscle substance proper. It
is therefore dangerous to speak of these alkaloids as being
stimulants of the “parasympathetic nerve endings” or of the
“myoneural junctions.”

REFERENCES

(1) LANGLEY AND ANDERSON: J. Physiol., 1895, xix, 73.

(2) ELLIOTT: J. Physiol., 1907, xxxv, 367.
(3) ELLI0Tr: J. Physiol., 1905, xxxii, 404.
(4) STREULI: Zeitsch. f. Biol., 1915, lxvi, 167.
(5) ADLER, LEO: Arch. f. exp. Path. u. Pharm., 1918, lxxxiii, 252.
(6) ABELIN: Zeitsch. f. Biol., 1919, lxix, 391.
(7) FISHER: Anat. Record, 1917, xiii, 69.
(8) STARLING: Textbook of Physiology, 1907, 156.
(9) FROHLICR AND LoEwl: Arch. f. exp. Path. u. Pharm., 1908, lix, 38.
(10) FLETCHER: Proc. Physiol. Soc., 1898, xxii, xxxvii.
(11) BRtCKE: Pfluger Arch., 1910, cxxxvi, 502.
(12) BorrAzzi: Memorie della R. Accad. dei Lincei (5), 1915, xi, 43.
(13) BOTTAZZI: Arch. Ital. de Biol., 1916, lxv, 265.
(14) DE ZILWA: J. Physiol., 1901, xxvii, 200.
(15) CUSHNY: J. Physiol., 1910, xli, 233.
(16) MACHT: J. Pharm. & Exp. Therap., 1916, ix, 198.
(17) WADDELL: J. Pharm. & Exp. Therap., 1917, ix, 423.
(18) SATANI: Am. J. Physiol., 1919, lxix, 474.
(19) DIXON AND BRODIE: J. Physiol., 1903, xxix, 141.
(20) ANDERSON: J. Physiol., 1905, xxxiii, 414.
(21) KURODA: J. Pharm. & Exp. Therap., 1916, ix, 187.
(22) EDMUNDS AND Roni: Am. J. Physiol., 1908, xxiii, 28.
(23) DIXON: Proc. Royal Soc. of Med., 1912, vi, 1.