286

6I2.45:6I2.893

THE MECHANISM OF THE NERVOUS DISCHARGE

OF ADRENALINE.
BY W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
(From the National Institute for Medical Reseatch, London, N. W. 3.) 1
(Received March 12, 1934.)
RECENTLY two of us [W. F. and B. M., 1933] have shown that a substance
pharmacologically indistinguishable from acetylcholine can be detected
in the venous blood collected from the suprarenals during splanchnic
stimulation, when eserine is present, but that no such substance is present
in the absence of stimulation. It seemed to us that it should be possible
to demonstrate the effects of this liberated acetylcholine either on the
arterial blood-pressure or on a distant organ of the same animal, if the
blood from the suprarenals containing it were allowed to flow normally
into the general circulation. This possibility was tested in the experiments
described in the first part of this paper. To detect the acetylcholine by its
action on a distant organ we used the submaxillary gland.
Experiments thus to detect acetylcholine, by its action on other
systems of the animal into the blood of which it is liberated, are com-
plicated by the actions of adrenaline, the output of which from the
medullary cells is the main and more obvious effect of the splanchnic
stimulation. It is necessary in some way to suppress the actions of this
adrenaline, or, allowing it to produce its normal effects, to demonstrate
the action of acetylcholine only as a complication or phase of the total
effect. Both methods have been used.
The demonstration that acetylcholine is liberated from the suprarenal
bodies during splanchnic stimulation does not, however, provide a direct
proof that acetylcholine is the direct cause of the adrenaline discharge.
As evidence of this function of acetylcholine, we consider, in the second
part of this paper, the effect of eserine on the response of the suprarenals
to splanchnic stimulation by the discharge of adrenaline.
1 The experiments here recorded were begun in the Physiological
Institute of the
University of Berlin. When they were there interrupted, one of us (W.F.) had the privilege
of continuing and completing the work at the National Institute for Medical Research.
 DISCHARGE OF ADRENALINE. 287
Further experiments, which are described in the third part of the
paper, deal with the effects of nicotine and atropine on the actions of
acetylcholine and of splanchnic stimulation on the suprarenals.
It will be seen that, in addition to the predominant " nicotine " action
of acetylcholine on the medullary cells, there is evidence of a subsidiary
"muscarine" action, surviving nicotine paralysis and requiring atropine
for its suppression. In the same connection we have taken the oppor-
tunity of examining the action of certain other alkaloids on the medullary
cells.
METHODS.
The experiments were made on cats under chloralose ansesthesia or
on spinal cats. In the latter the preliminary operation under ether was
performed by removing the roof of the second cervical vertebra, cutting
the cord and destroying the brain through the foramen magnum. In
some experiments the whole spinal cord was destroyed as well.
For the stimulation of the splanchnic fibres to the suprarenal bodies,
the stomach, spleen, small and large intestine were removed, and the
vessels of the kidneys were tied in addition. The splanchnics were cut in
the chest, small incisions being made through the diaphragm. They were
tied and dissected for stimulation. Artificial respiration was used in all
these experiments. It has proved convenient to remove part of the liver,
especially of the left lobe, in order to obtain more room for the electrodes.
This was especially useful in the experiments in which the left splanchnic
was stimulated repeatedly with fluid electrodes, which had to be fixed in
position. The stimulation with fluid electrodes was only carried out when
the effect of repeated stimulations had to be compared quantitatively;
otherwise ordinary metal electrodes were used. In the earlier experiments
fluid electrodes of the type described by Keith Lucas, later those of
Brown and Garry [1932] as modified by Collison [1933] were used.
In the experiments in which the direct secretory action of drugs on
the medulla of the suprarenals was examined, we adopted a method in
which small amounts of the drug were injected into the abdo inal aorta
above the suprarenals. For this purpose the cat was eviscerated as
above, the renal vessels were tied near the kidneys, the abdominal aorta
and vena cava were tied below the suprarenals, and a cannula was tied
into the central end of the clamped coelic artery. Through this cannula
the injections were made. In the earlier experiments we used an ordinary
fine glass cannula, the free end of which was connected and closed with a
rubber tube. The injections were made through the rubber tube in the
same way as ordinary intravenous injections. Later we used the special
288 W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
cannula for arterial injections described by Chang and Gaddum [1933].
If a drug is injected in this way it reaches the suprarenals with relatively
little dilution by the blood. An action on the suprarenals can therefore
be obtained with amounts of the drug so small that, with intravenous
injection, they would cause little or no discharge of adrenaline. This kind
of injection has shown itself to be especially suitable if used for drugs with
strong depressor action [Feldberg and Minz, 1931; Szczygielski,
1932; Gutmann, 1932; Feldberg, 1932; Siehe, 1934]. These drugs
injected intravenously cause only a fall of pressure, by which their
actions on the suprarenals are completely masked; whereas the effect of
the same drug injected arterially, in the same or even in smaller doses,
may be mainly pressor, owing to the discharge of adrenaline. By com-
paring the effects of venous and arterial injections, the secretory action
of a drug on the suprarenals can thus be demonstrated. For the in-
travenous injections a cannula was tied into the jugular vein. For com-
plete evidence of action on the suprarenal medulla, it is necessary to
show, further, that the pressor action seen with arterial injection is
abolished when both suprarenals are removed.

EXPERIMENTAL.
(i) Direct evidence of liberation of acetyicholine.
(a) The effect on the arterial blood-pressure. In an eviscerated cat, the
pressor response of the arterial blood-pressure to stimulation of the
splanchnic nerves is caused only by the output of adrenaline from the
suprarenals. If the destruction of acetylcholine is inhibited by a previous
intravenous injection of eserine, this pressor response is modified in
different but characteristic ways. The rise of pressure may then be
delayed, or interrupted in its early stages, or preceded by a fall of pressure.
Further, if the blood-pressure of the cat has been made insensitive to
adrenaline itself, as during the persistent effect of an earlier supramaximal
injection of a few mg. of adrenaline, we obtain only the depressor action
of acetylcholine as the result of stimulating the splanchnic nerves. These
effects of the liberated acetylcholine are all abolished by atropine.
(1) On eviscerated cats with very low blood-pressure the rise of
pressure caused by splanchnic stimulation is often delayed after the
injection of eserine. This effect is shown in Fig. 1. In this and other
figures the beginning and end of stimulation are recorded not only by
the base-line signal but by vertical lines intersecting the blood-pressure
curve itself, produced by rapid compression and release of the rubber
 DISCHARGE OF ADRENALINE. 289
tubing leading to the manometer. The splanchnic nerves were stimulated
at I and II, for 30 sec. in each case, with the secondary coil at 10 cm.
Between I and II, 6 min. before II, 0.3 mg. of eserine was injected intra-
venously. At the first stimulation the blood-pressure rose during the
stimulation, the rise starting 21 sec. after the onset. At the second
stimulation the blood-pressure did not rise until 6 sec. after the stimu-

Fig. 1. Carotid blood-pressure of a cat (also in all other figures). Spinal eviscerated.
I and II stimulation ofleft splanchnic, 30 sec., coil at 10 cm. Between I and 110-3 mg.
eserine.

Fig. 2. Chloralose. Eviscerated. At I, II, III and IV stimulation of the left splanchnic,
10 sec., coil at 8 cm. Between I and II 0 4 mg. eserine, between III and IV 0 3 mg.
atropine intravenously.

lation had ceased, or 36 sec. after its onset. The pressor response was
therefore delayed by 15 sec. In other experiments the delay after eserine
was not quite so long, being between 7 and 12 sec. After an intravenous
injection of 0.1 mg. of atropine the delay produced by eserine was
abolished.
The delay is chiefly, but not wholly, due to the action of the acetyl-
choline liberated during the nerve stimulation and counteracting the
290 W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
pressor effect of adrenaline. A similar delay in the pressor response
occurs if a small amount of acetylcholine is mixed with adrenaline and
the two drugs are injected together [Feldberg and Minz, 1933]. A
part of the delay, however, is due to the slowing of the circulation which
occurs after eserine, which causes the liberated adrenaline to be carried
to the arteries more slowly than before the eserine injection. Thus the
pressor response to an intravenous injection of adrenaline showed also
a delay after eserine; but this amounted only to 24 sec., compared with
a delay of the splanchnic response, in the same experiments, lasting
12-15 sec.
(2) If the blood-pressure is not quite so low, or if the acetyl-
choline can act more vigorously, the rise caused by splanchnic stimulation
will be interrupted in its early stages. This is shown by the experiment
given in Fig. 2, which is also taken from an eviscerated spinal cat. At I,
II, III and IV the left splanchnic was stimulated for 10 sec., with the
secondary coil at 8 cm. The effect of the first stimulation (I) was an
uninterrupted rise of pressure, which began as soon as the stimulation
had stopped. After this the cat was given 0 4 mg. of eserine intravenously,
which caused a slight slowing of the heart beat. Five and 15 min. later
the second (II) and third (III) stimulations were made. The blood-
pressure began to rise, in both cases, as soon as the stimulation had
ceased, but after a few seconds the rise was interrupted by a slight
transitory fall, after which the main secondary rise set in. This began
about 12 sec. after the primary rise. In other experiments all types of
result between a simple delay and an actual interruption of the rise were
seen. Being due to the action of the acetylcholine, the fall is abolished
by atropine: 041 mg. of atropine was given intravenously 3 min. before
Fig. 2, IV. A similar interruption of a pressor response is sometimes
obtained if adrenaline is injected intravenously, mixed with acetyl-
choline.
(3) On eviscerated, spinal cats with high blood-pressure the
pressor response to splanchnic stimulation is sometimes preceded by
a pronounced fall of pressure, if eserine has been injected beforehand.
This fall is also due to the action of the acetylcholine liberated during the
stimulation of the nerve, and is abolished by atropine. These results are
more easily obtained on eviscerated cats under chloralose having a rather
high blood-pressure. Under these conditions, however, adrenaline itself
in small doses may have a depressor action, so that the adrenaline dis-
charged during splanchnic stimulation may by itself produce a fall of
pressure followed by a rise. This fall, however, is not abolished by
 DISCHARGE OF ADRENALINE. 291
atropine and is seen before the injection of eserine. It can, therefore,
easily be distinguished from the acetylcholine effect, which only occurs
after eserine and is abolished by atropine. If we obtain a fall of pressure
during splanchnic stimulation before the injection of eserine, due to the
depressor action of the commencing discharge of adrenaline itself, the
fall is greatly deepened after eserine; and this augmentation of the
depressor effect of the splanchnic stimulation is abolished by atropine,
and may accordingly be attributed to acetylcholine.

Fig. 3. Chloralose. Eviscerated. At I, II and III stimulation of left splanchnic, 15 sec.,

coil at 6 cm. Between I and II 0.5 mg. of eserine intravenously. Level and position
of drum adjusted. Between II and III 0-2 mg. atropine.

In several cats under chloralose, however, adrenaline, as well as

splauchnic stimulation, caused only a rise of pressure. If eserine was
injected into these animals, splanchnic stimulation then caused a strong
fall followed by a rise. The depressor action was then abolished by atro-
pine. This is shown by the record given in Fig. 3. At I, and II and III
the left splanchnic is stimulated for 15 sec. with the secondary coil at
6 cm. The stimulations are made 8 min. from each other. Between I
and II O-5 mg. eserine, between II and III 041 mg. atropine was injected
intravenously. We see that the simple rise in I is preceded by a fall in II,
which is again absent after atropine at III.
In the experiments giving Figs. 1 and 3 we see, further, that the
292 W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
pressor response to splanchnic stimulation is greater after the injection
of eserine, whereas it is smaller in Fig. 2. We cannot conclude definitely
that these changes were due to the eserine, though it is possible. The
nerves were stimulated in these experiments with ordinary metal
electrodes, so that the effects of the different stimulations, in each
experiment, may not be strictly comparable. On the other hand we
might expect, on different occasions, either an increased or a decreased
pressor effect after eserine. Eserine causes an increased output of adrena-
line from splanchnic stimulation, as we shall see later, which will tend to
increase the pressor response. On the other hand, acetylcholine liberated
during the stimulation is not destroyed after eserine. According as the

-I

Fig. 4. Chloralose. Eviscerated. Arterial blood-pressure insensitive to adrenaline from

previous injections of 4-1 mg. in all. 20 min. before I 0.8 mg. eserine intravenously.
At I and III 1 mg. of adrenaline; at II and IV stimulation of both splanchnic nerves,
coil at 10 and 8 cm. Between II and III 0-1 mg. of atropine.

increased output of adrenaline or the counteracting effect of the liberated

acetylcholine prevails, we may therefore obtain a stronger or weaker
pressor response.
(4) Fr6hlich and Pick [1913] have shown that the blood-pressure of
a cat will become insensitive to further injection of adrenaline after a
few mg. have been injected intravenously. These results were confirmed
by one of us [W. F.] in this laboratory six years ago. Under these con-
ditions, however, acetylcholine will still produce its depressor action,
though its effect is weakened. In this state of insensitiveness to adrena-
line, stimulation of the splanchnic ought to result only in a fall of blood-
pressure, if the destruction of the liberated acetylcholine is inhibited by
a previous injection of eserine. This is demonstrated by the experiment
illustrated by Fig. 4.
 DISCHARGE OF ADRENALINE. 293
The cat (3.2 kg.) was aneesthetized with chloralose and eviscerated.
It received first an intravenous injection of 01 mg. adrenaline. During
the rise which followed the injection, 1 mg. adrenaline was injected and
produced a further rise of pressure. A further injection of 1 mg. however,
given 1 min. later, was already ineffective. The injection was repeated
twice with the same result. Both splanchnic nerves were then stimulated
for 1 mi. with the secondary coil at 10 cm. The stimulation had no effect
on the arterial blood-pressure. The cat was then given 0-8 mg. eserine
intravenously, which caused a slight, gradual fall of pressure. A new
intravenous injection of 1 mg. adrenaline was still ineffective. This
injection is shown in Fig. 4, I. Two minutes later both splanchnics were
stimulated for about 80 sec. with the secondary coil at 10 cm. The
stimulation resulted in a prolonged fall of pressure, which started about
40 sec. after the beginning of the stimulation (II). The cat then received
an intravenous injection of 0.1 mg. atropine, after which the blood-
pressure rose again. A further injection of 1 mg. of adrenaline was still
without effect (III), and renewed stimulation of both splanchnics for
100 sec. with the coil at 8 cm. caused neither fall nor rise of pres-
sure (IV).
(b) The effect on the submaxillary gland. To demonstrate the sialogogue
effect of the acetylcholine liberated during splanchnic stimulation from
the suprarenals of a cat, one must exclude the effect of the discharged
adrenaline, which would itself produce a secretion of saliva. This can be
done by injecting intravenously a few mg. of ergotoxine. The salivary
glands poisoned by ergotoxine no longer react to adrenaline, but still
respond to chorda stimulation or acetylcholine. If eserine has also been
injected, the acetylcholine, which is now protected from esterase and
liberated into the blood stream during the stimulation, ought to cause a
secretion of saliva: and this secretory effect ought to be abolished again
by atropine.
These expectations were realized experimentally, and are demonstrated
by the experiment illustrated in Fig. 5, which is taken from a 3 kg. cat
in chloralose anaesthesia. The stomach, spleen, small and large intestine
of the cat were removed, and the circulation was further restricted by
tying the aorta and veha cava below the suprarenals. A cannula was
tied into the right Wharton's duct and connected with a long horizontal
glass tube with millimetre scale. The chorda-lingual nerve was cut on the
same side. An intravenous injection of 0.1 mg. adrenaline caused a
secretion of 0.8 c.c. of saliva, and stimulation of both splanchnics pro-
duced also a slight secretion of the salivary glands at that stage. These
294 W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
effects, however, were completely abolished after 3 mg. ergotoxine had
been injected intravenously, showing that the salivary glands had be-
come insensitive to adrenaline. The cat then received two intravenous
injections of 0 9 mg. eserine, which were given 25 mim. apart from each
other. Thereafter both splanchnics were stimulated anew, for 1 min.,
with the secondary coil at 7-5 cm. This stimulation resulted in a long-
lasting secretion of saliva as shown in Fig. 5. The secretion started about

Fig. 5. Arterial blood-pressure (upper tracing) and record of salivary secretion (middle
tracing). Chloralose. Evisceration and restriction of circulation. 1-8 mg. eserine
and 3 mg. ergotoxine given earlier. Stimulation of both splanchnic nerves, coil at
7-5 cm.

half a minute after the beginning of the stimulation and amounted to

0 5 c.c. of saliva, each mark in Fig. 5 corresponding to the secretion of
0-05 c.c. This secretion cannot be due to the effect of the discharged
adrenaline, because an intravenous injection of 0.1 mg. adrenaline, given
a few minutes after the stimulation, was ineffective, showing that the
salivary glands were still completely insensitive to adrenaline. The
secretion must be due to the acetylcholine liberated from the suprarenals
during the stimulation and protected from destruction by the eserine.
This effect, accordingly, was absent when the stimulation was repeated
after an injection of 0-1 mg. atropine. This is a further proof that it cannot
be due to the discharge of adrenaline, the secretory effect of which is not
affected by atropine. The fall of arterial pressure in Fig. 5 may also be
partly due to the acetylcholine liberated, but is due in part to the reversed
action, after ergotoxine, of adrenaline.
 DISCHARGE OF ADRENALINE. 295

(ii) Acetylcholine as the stimulus for adrenaline output.

If the output of adrenaline from the suprarenals, caused by splanchnic
stimulation, is actually due to the liberation of acetylcholine to act as the
direct stimulant of the medullary cells, eserine would be expected to
increase the effect. Similarly eserine ought to increase the effect of acetyl-
choline artificially injected, in causing an output of adrenaline from the
glands. Even this latter effect of eserine, however, has not hitherto been
proved. There is only the statement of White and Stedman [1931] that
the pressor action of an intravenous injection of acetylcholine, in atro-
pinized cats, is increased by a previous injection of miotine, a synthetic
urethane compound, which has an eserine-like action; but the pressor
response of acetylcholine is only in part attributable to the effect of
acetylcholine on the suprarenals. It seemed, therefore, necessary to
determine whether the secretory effect of acetylcholine on the suprarenal
bodies is actually increased by eserine. As controls for these experiments
with acetylcholine, we have tested the effect of other drugs, such as
choline and histamine, which also cause secretion from the medulla of
the suprarenals.
It was necessary first to be certain that the effect of adrenaline itself
is not sensitized by eserine. Intravenous injections of the same dose of
adrenaline, given before and after an intravenous injection of eserine,
showed no differences in the pressor responses. Any change that might
occur after eserine in the pressor effects of histamine, choline, acetyl-
choline or splanchnic stimulation on the suprarenals, must therefore be
due to alterations in the output of adrenaline, and not to changes in the
effects of adrenaline itself.
(a) The effect of eserine on the adrenaline discharge caused by acetyl-
choline. If acetylcholine is injected through the cannula tied into the
central end of the cceliac artery of an eviscerated cat, the circulation of
which has been restricted (see "Methods"), its pressor effect is wholly due
to its secretory action on the suprarenals. This pressor response is greatly
enhanced by eserine, as is shown by the experiment given in Fig. 6 from
a cat under atropine. Injections of 0-02 mg. acetylcholine were repeated
several times before eserine with constant effect. The last two are shown
in Fig. 6, A and B. Between B and C, 1 mg. eserine was given intra-
venously. Five and 25 mi. later 002 mg. acetylcholine was injected
again. It can be seen that the pressor responses to these injections are
greater than those before the eserine injection. The pressure rises higher
and the effect lasts longer. At E the injection of acetylcholine was
296 W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
repeated after removal of the suprarenals. The lack of any pressor
response shows that those caused by the previous injections of acetyl-
choline were wholly due to excretory effects on the suprarenals.
Under the same conditions we have studied the effects of choline and
histamine separately on the suprarenals, and have found that eserine
produced no increase of the pressor responses to these substances.
(b) The effect of eserine on the adrenaline discharge caused by splanchnic
stimulation. These experiments have also been carried out on cats under
chloralose or with the brain destroyed. The cats were eviscerated, the
vessels of the kidneys were tied, atropine was injected intravenously
before stimulation, and the left splanchnic was stimulated with fluid
electrodes. Under these conditions repeated stimulations, for equal

Fig. 6. Chloralose. Eviscerated. Renal vessels, abdominal aorta and vena cava tied below
the suprarenals. 0-2 mg. atropine intravenously. At A, B, C, D, and B arterial
(celiac) injections of 0-02 mg. of acetylcholine. Between C and D 1 mg. eserine
intravenously. Between D and E both suprarenals removed.

lengths of time and with the same strength of current, gave practically
identical pressor responses, wholly due to the output of adrenaline from
the suprarenals. These pressor responses were increasled after an injection
of eserine. In some experiments the increased effect was chiefly revealed
by the higher maximum in others by the greater persistence of the
pressor effect. These results are demonstrated in Fig. 7, from a cat under
chloralose. Before the stimulation 0-2 mg. atropine was injected intra-
venously. At I, II, III and IV the left splanchnic was stimulated, for
20 sec. in each case, with the secondary coil at 12 cm. Between II and III
1 mg. eserine, and between III and IV a further 0-2 mg. atropine was
injected. After eserine the blood-pressure rises higher and stays much
longer at the high level than at I and II. Further, instead of the small
interruption which takes place at I and II in the early stages of the rises,
 ~ ~ ~ ~.
DISCHARGE OF ADRENALINE. 297
we obtain in III and IV a strong fall preceding the rise of pressure. This
must be interpreted as also due to an increased output of adrenaline,
since the atropine excludes its production by acetylcholine. A similar
depressor effect, followed by a rise of pressure, was also obtained before
the injection of eserine, when the splanchnic nerve was stimulated with
the coil at 9 cm. instead of 12 cm. The injection of atropine was repeated
after III, but the depressor action persisted at IV. The depressor effect is
therefore, in this case, due to the commencing output of adrenaline.

Fig. 7. Chloralose. Eviscerated, renal vessels tied. 0-2 mg. atropine intravenously. At
I, II, III and IV stimulation of left splanchnic (fluid electrodes) for 20 sec., coil at
12 cm. Between II and III 1 mg. eserine intravenously. Between III and IV 0-2 mg.
atropine intravenously.

The effect of stimulation with the coil at 12 cm. being much increased
by eserine, we moved the secondary coil outwards till an effect was
obtained, at a distance of 18 cm., approximately equal to that obtained
before eserine with the coil at 12 cm.
In order to show that eserine has generally no sensitizing action on
sympathetic nerve effects, we examined its effect on the vaso-constriction
caused by stimulating sympathetic nerves. We stimulated either the
abdominal sympathetic chains on eviscerated cats, or the left splanchnic
nerve after removal of the left suprarenal body, with the viscera intact.
The vaso-constriction produced by the abdominal sympathetic chains
298 W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
resulted only in a slight rise of arterial blood-pressure, due to vaso-
constriction chiefly in the hindlegs, whereas stimulation of the left
splanchnic, after removal of the left suprarenal body, caused a large rise
of pressure, due to vaso-constriction in the abdominal viscera. These
stimulations were all carried out with fluid electrodes, and practically
identical pressor responses could be obtained if the stimulations were
repeated with the same strength of current and the same duration. If
an intravenous injection of 0*5-1 mg. of eserine was given between two
such stimulations, the pressor responses were usually unaffected. In a
few experiments only, in which the left splanchnic was stimulated, the
fall of the pressure to the original level, after the stimulation, seemed to
be slower when eserine was present. This effect, however, was not constant
and was not very marked, compared with the strong sensitization of the
adrenaline discharge. On the other hand, it must be remembered that, in
stimulating the splanchnic nerves, we are dealing with preganglionic vaso-
constrictor fibres, and from the experiments of Feldb erg and Gaddum
[1933] we can assume that the impulses passing from preganglionic to
ganglion cells are transmitted by acetylcholine. The slight sensitization
observed occasionally may, therefore, be due to an effect of eserine on the
ganglion.
We may here draw attention to an observation which, though it has
no relation to our present enquiry, may have some significance not yet
clear. Much weaker currents (secondary coil at 20-24 cm.) were sufficient
for stimulating the directly vaso-constrictor fibres in the splanchnic than
were required for its secretory effect on the suprarenals (secondary coil
at 8-12 cm.).
(iii) "Muscarine" and "nicotine " actions on the suprarenal medulla.
The expression "muscarine-" and "nicotine-like actions " was first
used by Dale [1914] in his study of the pharmacological action of choline
and its derivatives. He found that the depressor action of these drugs
on the arterial blood-pressure was completely abolished by small doses
of atropine, whereas the pressor action which then remained, and which
was assumed to be chiefly due to an action on the sympathetic ganglia,
and the suprarenal medulla, was completely abolished by nicotine. This
distinction, which is based on the sensitiveness of the effect to atropine
and nicotine, has been generally accepted. One must take into account,
however, the fact that even the nicotine-like action can be abolished if
very large doses of atropine are used [Feldberg and Minz, 1931]. On
the other hand, it seems that very large doses of nicotine have some
 DISCHARGE OF ADRENALINE. 299
weakening effect on the muscarine-like action. The distinction between
the muscarine-like and nicotine-like actions is, therefore, not absolutely
sharp, but if the overlapping effects of very large doses of atropine and
nicotine are excluded, it holds true. As the nicotine-like action of
splanchnic stimulation and acetylcholine are well known, the following
experiments are especially concerned with the muscarine-like action of
these two, i.e. with that part of the effect, which is sensitive to small
doses of atropine.
(a) Splanchnic stimulation. The " muscarine-like " effect of splanchnic
stimulation on the suprarenal medulla cannot so easily be demonstrated.
If we compare the rise of pressure caused in an eviscerated spinal cat,
by stimulating the left splanchnic with fluid electrodes before and after
an intravenous injection of a small dose of atropine, no very convincing
reduction of the pressor effect will be seen.
Even after large doses of nicotine, which almost completely abolish
the effect of splanchnic stimulation on the suprarenal medulla, the
remaining effect will only be slightly but definitely reduced further by an
intravenous injection of atropine. It seems as if the large doses of
nicotine which are necessary to abolish the nicotine-like action also affect
the slight muscarine-like effect. The best way of demonstrating the
existence of a muscarine component is as follows. In an eviscerated cat
large doses of nicotine are first injected, till the pressor response to
splanchnic stimulation is almost completely abolished. If eserine is then
injected in addition the surviving remnant of the pressor response is
greatly enhanced. This effect, however, is then completely abolished by
a small dose of atropine. Fig. 8 is a record from sueb an experiment. The
tracing is taken from an eviscerated spinal cat. The strong pressor
response, which was obtained at the beginning of the experiment by
stimulating the left splanchnic nerve with fluid electrodes for 20 sec.,
with the secondary coil at 8-5 cm., was nearly completely abolished by
five previous intravenous injections each of 5 mg. nicotine. Only the
slight effect seen at Fig. 8 remained: 0-8 mg. of eserine was then injected,
and a few minutes later the left splanchnic was stimulated again, at II,
with the same strength of current and for the same length of time, and
now produced a strong pressor response. This was not reduced, if 5 mg.
of nicotine were again injected intravenously. If, however, a small dose
of atropine (0-1 mg.) was injected, the stimulation became ineffective
(Fig. 8, III).
(b) Acetylcholine. The experiment illustrated in Fig. 9 demonstrates
the "muscarine" action of acetylcholine on the suprarenal medulla.
PH. LXXXT. 20
300 W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
Small doses of nicotine and acetylcholine were given to the glands by
arterial injection, using the method already described. These doses were

Fig. 8. Chloralose. Eviscerated. At I, II and III stimulation of left splanchnic (fluid

electrodes), 20 sec., coil at 8*5 cm. Cat had previously received 25 mg. nicotine in all
intravenously. Between I and II 0*8 mg. eserine intravenously, between II and III
0.1 mg. atropine intravenously.

Fig. 9. Chloralose. Artificial respiration, eviscerated. Renal vessels, abdominal aorta and
vena cava tied below suprarenals. At A and C arterial (cceliac) injection of 0.1 mg.
nicotine; at B, D and F arterial injection of 0X02 mg. acetylcholine; between B and C
12 mg. nicotine in all intravenously. At E 0 05 mg. atropine intravenously.

chosen so as to give approximately equal outputs of adrenaline, as shown

by the pressor responses. The comparison could not be made with great
accuracy, because the pressor effect of adrenaline liberated in response
to acetylcholine was complicated by preliminary depressor effects, due to
 DISCHARGE OF ADRENALINE. 301
traces of acetylcholine escaping into the general circulation, whereas
nicotine similarly escaping would have an additional pressor action.
Subject to such corrections, it may be said that the output of adrenaline
produced by 0.1 mg. nicotine, at Fig. 9, A, is of the same order as that
produced by 0-02 mg. of acetylcholine at B. Between B and C 12 mg. of
nicotine were given intravenously. This sufficed to annul the effect of a
further 01 mg. nicotine injected arterially at C. It may be noted that
even much larger doses of nicotine would not suppress the effect of larger
(3-5 mg.) doses of that alkaloid, given arterially, on the suprarenal
medulla. The point of interest for our present purpose, however, is that

3I P ~~~~~~~L
Fig. 10. As in Fig. 9. At A intravenous, at B, C and D arterial (cceliac) injection of 0 1 mg.
arecoline. Between B and C 0*08 mg. atropine. Between C and D 15 mg. nicotine
inltravenously.

the effect of 0.1 mg. was annulled (C) by treatment which left a sub-
stantial part of the action on the suprarenal medulla of 0-02 mg. of
acetylcholine (at D) a dose which had earlier been roughly equivalent
in action to 0.1 mg. nicotine. An injection of Olmg. of atropine, at E,
now suppresses almost the whole of the action of 0-02 mg. of acetyl-
choline, given again at F, which had survived the nicotine paralysis.
Nearly the whole of the action at D, therefore, must have been of the
"muscarine" type.
(c) Muscarine. A few experiments were tried with arterial injections
of true muscarine. This alkaloid, however, is so stable, and its depressant
effects on the heart and general circulation were so prolonged, that it
was difficult to be certain of its action on the suprarenal medulla. In
several cases, however, we have recorded an undoubted, though weak,
20-2
302 W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
pressor effect, begnning about 30 sec. after the arterial injection, and,
like the direct effects of muscarine, abolished by atropine. The obser-
vation only has interest in showing that a pure muscarine action, without
any suspicion of a "nicotine" component, does stimulate to some extent
the output of adrenaline from the medullary cells.
(d) Pilocarpine. Dale and Laidlaw [1912] have shown that pilo-
carpine causes a secretion of adrenaline from the suprarenals. They,
however, did not study the effect more in detail. We found that the
effect is a peripheral one and that it is completely abolished by a small
dose of atropine. This effect appears, therefore, to be of the muscarine
type, and is not as strong as that of acetylcholine.
(e) Arecoline. Arecoline, of which hitherto only parasympatho-
mimetic effects have been described, has a strong secretory action on the
suprarenal medulla. This effect is stronger than that produced by pilo-
carpine, but weaker than that produced by acetylcholine. It differs from
the secretory action of muscarine and pilocarpine on the suprarenals in
that it is resistant to small doses of atropine (Fig. 10). The action of
arecoline on the suprarenal medulla is, therefore, chiefly, if not wholly, of
the nicotine type. This observation corresponds with more recent ones,
to be published later, on the action of arecoline on ganglion cells.

DISCUSSION.
The demonstration that acetylcholine is liberated from the suprarenal
glands during splanchnic stimulation does not, by itself, prove that it is
actually the direct stimulant of the medullary cells to secrete adrenaline.
The liberation of acetylcholine might be an incidental effect of the nerve
stimulation; it might not even be liberated in relation to the medullary
cells at all, but in the cortex of the gland. This possibility reminds us of
the old conception of Lohmann [1907], now obsolete, that choline is
the hormone of the cortex, which thus would liberate a hormone acting
like parasympathetic nerve stimulation, and antagonistic to adrenaline,
the "sympathetic" hormone of the medulla. This theory was based on
the observation that the cortex contained a large amount of choline,
whereas the medulla contained very little. Recent observations, as yet
unpublished, by H. Schild and one of us [W. F.], however, have shown
that the concentration of acetylcholine, or of a substance pharma-
cologically indistinguishable from it, is twice to four times as high in the
medulla as in the cortex. This points strongly in the direction of the
medulla as the seat of the acetylcholine liberation. The experiments of
 DISCHARGE OF ADRENALINE. 303
the present paper, in which the influence of eserine on the adrenaline
discharge caused by splanchnic stimulation is shown, provide additional
evidence for the view that acetylcholine is the direct stimulant of the
medullary cells transmitting the effect of the nerve stimulation. After
eserine the acetylcholine liberated would not be destroyed so easily, and
should therefore be able to act longer and more vigorously. We have
shown that the adrenaline discharge caused by splanchnic stimulation is,
in fact, increased by eserine.
A more detailed analysis of the action of acetylcholine and splanchnic
stimulation on the suprarenal medulla showed that the action in both
is due mainly to the "nicotine" aspect of its action, which is abolished by
large doses of nicotine, but partly also to a "muscarine" action, which is
abolished by small doses of atropine. This parallelism of a double action,
in the case of acetylcholine and of splanchnic stimulation, is a further
indication that the adrenaline discharge is due to the same direct
stimulus in both cases.
All our experiments, therefore, point in the same direction. They
show that the impulses travelling down the splanchnic nerve fibres to the
suprarenal medulla are chemically transmitted to the effector cells, by
liberation of something indistinguishable from acetylcholine.
Hitherto acetylcholine has been regarded as the humoral transmitter
of parasympathetic effects only. The splanchnic fibres to the suprarenals
are sympathetic. The fact that impulses travelling down these fibres are
also chemically transmitted by acetylcholine provides a first example of
the transmission of preganglionic impulses by liberation of that substance.
A further example, of more general significance, is presented in the
adjoining paper by Feldberg and Gaddum.

SUMMARY.
1. The liberation of acetylcholine from the suprarenals during
splanclnic stimulation is shown by its effect on the arterial blood-
pressure and the salivary glands of the same animal.
2. The adrenaline discharge caused by splanchnic stimulation is
greatly enhanced by eserine.
3. The action of acetylcholine as well as of splanchnic stimulation on
the suprarenal medulla is mainly a "inicotine" action, which can be
abolished by larger doses of nicotine; it has also, however, a "muscarine"
component, which is abolished by small doses of atropine.
4. It is concluded that acetylcholine is the humoral transmitter of
304 W. FELDBERG, B. MINZ AND H. TSUDZIMURA.
splanchnic impulses to the suprarenal medulla, so that the preganglionic
sympathetic fibres are cholinergic.
5. Muscarine and pilocarpine cause an output of adrenaline from the
suprarenals. These effects are completely abolished by atropine. Arecoline
also causes a discharge of adrenaline, which is wholly or mainly resistant
to atropine, but abolished by nicotine.
We desire to make grateful acknowledgment of our debt to the Medical Research
Council for hospitality and to Sir Henry Dale for help and advice.

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Brown, G. L. and Garry, R. C. (1932). J. Phy8iol. 75, 213.
Chang, H. C. and Gaddum, J. H. (1933). Ibid. 79, 255.
Collison, L. W. (1933). Ibid. 80, 14P.
Dale, H. H. (1914). J. Pharmacol., Baltimore, 6, 147.
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Feldberg, W. (1932). Arch. exp. Path. Pharmak. 168, 287.
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