METHODS OF DEMONSTRATING ADRENAL SECRETION
AND ITS NERVOUS CONTROL
As stated in the first chapter, the inhibition of gastric secretion
produced by great excitement long outlasts the presence of the object
which evokes the excitement. The dog that was enraged by seeing a
cat for five minutes secreted only a few drops of gastric juice
during the next fifteen minutes. Why did the state of excitation
persist so long after the period of stimulation had ended? This
question, which presented itself to me while reading Bickel and
Sasaki’s paper, furnished the suggestion expressed at the close of
the last chapter, that the excitement might provoke a flow of adrenal
secretion, and that the changes originally induced in the digestive
organs by nervous impulses might be continued by circulating
adrenin. The prolongation of the effect might be thus explained.
Whether that idea is correct or not has not been tested. Its chief
service was in leading to an enquiry as to whether the adrenal
glands are in fact stimulated to action in emotional excitement.
The preganglionic fibres passing to the glands are contained in the
splanchnic nerves. What is the effect of splanchnic stimulation?
The Evidence that Splanchnic Stimulation
Induces Adrenal Secretion
It was in 1891 that Jacobi[29] described nerve fibres derived
from the splanchnic trunks which were distributed to the adrenal
glands. Six years later Biedl[30] found that these nerves conveyed
vasodilator impulses to the glands, and he suggested that they
probably conveyed also secretory impulses. Evidence in support of
this suggestion was presented the following year by Dreyer,[31] who
demonstrated that electrical excitation of the splanchnic nerves
produced in the blood taken from the adrenal veins an increased
amount of a substance having the power of raising arterial blood
pressure, and that this result was independent of accompanying
changes in the blood supply to the glands. The conclusion drawn
by Dreyer that this substance was adrenin has been confirmed in
various ways by later observers. Tscheboksaroff[32] repeated
Dreyer’s procedure and found in blood taken from the veins after
splanchnic stimulation evidences of the presence of adrenin that
were previously absent. Asher[33] observed a rise of blood pressure
when the glands were stimulated in such a manner as not to cause
constriction of the arteries--the rise was therefore assumed to be
due to secreted adrenin. Dilation of the pupil was used by Meltzer
and Joseph[34] to prove secretory action of the splanchnics on the
adrenal glands; they found that stimulation of the distal portion
of the cut splanchnic nerve caused the pupil to enlarge--an effect
characteristic of adrenin circulating in the blood. Elliott[35]
repeated this procedure, but made it a more rigorous proof of
internal secretion of the adrenals by noting that the effect failed
to appear if the gland on the stimulated side was removed. Additional
proof was brought by myself and Lyman[36] when we found that the
typical drop in arterial pressure produced in cats by injecting small
amounts of adrenin could be exactly reproduced by stimulating the
splanchnic nerves after the abdominal blood vessels, which contract
when these nerves are excited, were tied so that no changes in them
could occur to influence the rest of the circulation.
The problem of splanchnic influence on the adrenal glands Elliott
attacked by a still different method. Using, as a measure, the
graded effects of graded amounts of adrenin on blood pressure, he
was able to assay the quantity of adrenin in adrenal glands after
various conditions had been allowed to prevail. The tests were made
on cats. In these animals each adrenal gland is supplied only by
the splanchnic fibres of its own side, and the two glands normally
contain almost exactly the same amount of adrenin. Elliott[37]
found that when the gland on one side was isolated by cutting its
splanchnic supply, and then impulses were sent along the intact
nerves of the other side, either by disturbing the animal or by
artificial excitation of the nerves, the gland to which these
fibres reached invariably contained less adrenin, often very much
less, than the isolated gland. Results obtained by the method
employed by Elliott have been confirmed with remarkable exactness
in results obtained by Folin, Denis and myself,[38] using a highly
sensitive color test after adding the gland extract to a solution
of phosphotungstic acid.
All these observations, with a variety of methods, and by a
respectable number of reliable investigators, are harmonious in
bringing proof that artificial stimulation of the nerves leading to
the adrenal glands will induce secretory activity in the adrenal
medulla, and that in consequence adrenin will be increased in the
blood. The fact is therefore securely established that in the body
a mechanism exists by which these glands can be made to discharge
this peculiar substance promptly into the circulation.
The Question of Adrenal Secretion in Emotional Excitement
As we have already seen, the phenomena of a great emotional
disturbance in an animal indicate that sympathetic impulses dominate
the viscera. When, for example, a cat becomes frightened, the pupils
dilate, the activities of the stomach and intestines are inhibited,
the heart beats rapidly, the hairs of the back and tail stand
erect--from one end of the animal to the other there are abundant
signs of nervous discharges along sympathetic courses. Do not the
adrenal glands share in this widespread subjugation of the viscera
to sympathetic control?
This question, whether the common excitements of an animal’s life
might be capable of evoking a discharge of adrenin, was taken up by
D. de la Paz and myself in 1910. We made use of the natural enmity
between two laboratory animals, the dog and the cat, to pursue our
experiments. In these experiments the cat, fastened in a comfortable
holder (the holder already mentioned as being used in X-ray studies
of the movements of the alimentary canal), was placed near a barking
dog. Some cats when thus treated showed almost no signs of fear;
others, with scarcely a movement of defense, presented the typical
picture. In favorable cases the excitement was allowed to prevail
for five or ten minutes, and in a few cases longer. Samples of
blood were taken within a few minutes before and after the period.
The Method of Securing Blood from Near the Adrenal Veins
The blood was obtained from the inferior vena cava anterior to
the opening of the adrenal veins, i. e., at a point inside the
body near the level of the notch at the lower end of the sternum.
To get the blood so far from the surface without disturbing the
animal was at first a difficult problem. We found, however, that by
making anesthetic with ethyl chloride the skin directly over the
femoral vein high in the groin, the vein could be quickly bared,
cleared of connective tissue, tied, and opened without causing any
general disturbance whatever. A long, fine, flexible catheter (2.4
millimeters in diameter) which had previously been coated with
vaseline inside and out, to lubricate it and to delay the clotting
of blood within it, was now introduced into the opening in the
femoral vein, thence through the iliac and on into the inferior
cava to a point near the level of the sternal notch. A thread
tied around this tube where, after being inserted to the proper
distance, it disappeared into the femoral vein, marked the extent of
insertion, and permitted a later introduction to the same extent.
This slight operation--a venesection, commonly practised on our
ancestors--consumed only a few minutes, and as the only possibility
of causing pain was guarded against by local anesthesia, the animal
remained tranquil throughout. Occasionally it was necessary to
stroke the cat’s head gently to keep her quiet on the holder, and
under such circumstances I have known her to purr during all the
preparations for obtaining the blood, and while the blood was being
taken.
The blood (3 or 4 cubic centimeters) was slowly drawn through the
catheter into a clean glass syringe. Care was taken to avoid any
marked suction such as might cause collapse of the vein near the
inner opening of the tube. As soon as the blood was secured, the
catheter was removed and the vein tied loosely, to prevent bleeding.
The blood was at once emptied into a beaker, and the fibrin whipped
from it by means of fringed rubber tubing fitted over a glass rod.
Since this defibrinated blood was obtained while the animal was
undisturbed, it was labelled “quiet blood.”
The animal was then exposed to the barking dog, as already described,
and immediately thereafter blood was again removed, from precisely
the same region as before. This sample, after being defibrinated,
was labelled “excited blood.” The two samples, the “quiet” and
the “excited,” both obtained in the same manner and subsequently
treated in the same manner, were now tested for their content of
adrenin.
The Method of Testing the Blood for Adrenin
It was desirable to use as a test tissues to which the blood was
naturally related. As will be recalled, adrenin affects viscera
even after they have been removed from the body, just as if they
were receiving impulses via sympathetic fibres, and further, that
sympathetic fibres normally deliver impulses which cause contraction
of the internal genitals and relaxation of the stomach and
intestines. The uterus has long been employed as a test for adrenin,
the presence of which it indicates by increased contraction. That
isolated strips of the longitudinal muscle of the intestine, which
are contracting rhythmically, are characteristically inhibited by
adrenin in dilutions of 1 part in 20 millions, had been shown by
Magnus in 1905. Although, previous to our investigation in 1910,
this extremely delicate reaction had not been used as a biological
signal for adrenin, it possesses noteworthy advantages over other
methods. The intestine is found in all animals and not in only
half of them, as is the uterus; it is ready for the test within a
few minutes, instead of the several hours said to be required for
the best use of the uterus preparation;[39] and it responds by
relaxing. This last characteristic is especially important, for
in defibrinated blood there are, besides adrenin, other substances
capable of causing contraction of smooth muscle,[40] and liable
therefore to lead to erroneous conclusions when a structure which
responds by contracting, such as uterus or artery, is used to prove
whether adrenin is present. On the other hand, substances producing
relaxation of smooth muscle are few, and are unusual in blood.[41]
We used, therefore, the strip of intestinal muscle as an indicator.
Later Hoskins[42] modified our procedure by taking, instead of the
strip, a short segment of the rabbit intestine. The segment is not
subjected to danger of injury during its preparation, and when fresh
it is almost incredibly sensitive. It may be noticeably inhibited
by adrenin, 1 part in 200 millions!
The strip, or the intestinal segment, was suspended between minute
wire pincers (_serres fines_) in a cylindrical chamber 8 millimeters
in diameter and 5 centimeters deep. By a thread attached to the
lower serre fine the preparation was drawn into the chamber, and was
held firmly; by the upper one it was attached to the short end of
a writing lever (see Fig. 2). When not exposed to blood, the strip
was immersed in a normal solution of the blood salts (Ringer’s).
The blood or the salt solution could be quickly withdrawn from or
introduced into the chamber, without disturbing the muscle, by
means of a fine pipette passed down along the inner surface. The
chamber and its contents, the stock of Ringer’s solution, and the
samples of “quiet” and “excited” blood were all surrounded by a
large volume of water kept approximately at body temperature (37°
C.). Through the blood or the salt solution in the chamber oxygen
was passed in a slow but steady stream of bubbles. Under these
circumstances the strip will live for hours, and will contract
and relax in a beautifully regular rhythm, which may be recorded
graphically by the writing lever.
[Illustration: Figure 2.--Diagram of the arrangements for recording
contractions of the intestinal muscle.]
The first effect of surrounding the muscle with blood, whether
“quiet” or “excited,” was to send it into a strong contraction which
might persist, sometimes with slight oscillations, for a minute or
two (see Figs. 4 and 5). After the initial shortening, the strip,
if in quiet blood soon began to contract and relax rhythmically
and with each relaxation to lengthen more, until a fairly even base
line appeared in the written record. At this stage the addition of
fresh “quiet” blood usually had no effect, even though the strip
were washed once with Ringer’s solution before the second portion
of the blood was added. For comparison of the effects of “quiet”
and “excited” blood on the contracting strip, the two samples were
each added to the muscle immediately after the Ringer’s solution
had been removed, or they were applied to the muscle alternately
and the differences in effect then noted. The results obtained by
these methods are next to be presented.
REFERENCES
[Footnote 29: Jacobi: Archiv für experimentelle Pathologie und
Pharmakologie, 1891, xxix, p. 185.]
[Footnote 30: Biedl: Archiv für die gesammte Physiologie, 1897,
lxvii, pp. 456, 481.]
[Footnote 31: Dreyer: American Journal of Physiology, 1898-99, ii,
p. 219.]
[Footnote 32: Tscheboksaroff: Archiv für die gesammte Physiologie,
1910, cxxxvii, p. 103.]
[Footnote 33: Zeitschrift für Biologie, 1912, lviii, p. 274.]
[Footnote 34: Meltzer and Joseph: American Journal of Physiology,
1912, xxix, p. xxxiv.]
[Footnote 35: Elliott: Journal of Physiology, 1912, xliv, p. 400.]
[Footnote 36: Cannon and Lyman: American Journal of Physiology,
1913, xxxi, p. 377.]
[Footnote 37: Elliott: Journal of Physiology, 1912, xliv, p. 400.]
[Footnote 38: Folin, Cannon and Denis: Journal of Biological
Chemistry, 1913, xiii, p. 477.]
[Footnote 39: Fraenkel: Archiv für experimentelle Pathologie und
Pharmakologie, 1909, lx, p. 399.]
[Footnote 40: See O’Connor: Archiv für die experimentelle Pathologie
und Pharmakologie, 1912, lxvii, p. 206.]
[Footnote 41: Grutzner: Ergebnisse der Physiologie, 1904, iii²,
p. 66; Magnus: _Loc. cit._, p. 69.]
[Footnote 42: Hoskins: Journal of Pharmacology and Experimental
Therapeutics, 1911, iii, p. 95.]