Received for publication October 16, 1928
G. R. SUCKOW AND G. E. BURGET
From the Department of Physiology, University of Oregon
It has been shown that the response of the vascular system of the pithed
cat to adrenalin varies with the pH of the blood (Burget and Visscher,
1927). This difference in response of the vascular system to adrenalin
when the pH of the blood is altered is due to a change in irritability at the
site of action of the drug (Burget and Crisler, 1927). These findings apply
to the vasoconstrictor mechanism only. The present work was undertaken
to determine whether or not the inhibitory innervation to the intestine
undergoes similar changes in irritability when the pH of the bath is altered.
Manyinvestigators have studied the differences in activity of intestine and
uterine segments upon varying the hydrogen ion concentration of the
bath in which they were suspended (Evans and Underhill, 1923; Acton and
Chopra, 1925; Thienes, 1926) but no attempt has been made to determine
the influence of the hydrogen ion concentration of the bath upon the
irritability of the inhibitory mechanism of the intestine. Adrenalin was
used because it lent itself well for this purpose.
Thirty-five young adult female rabbits were used in this series. The
animals were killed at approximately one-thirty o'clock in the afternoon of
the day of the experiment by being struck upon the occiput. It was
noticed that in every case the animal's stomach was distended with food
when the evisceration was made.. The diet of the animals consisted of
barley and carrots. This diet factor was especially watched for the effect
it might have upon the activity of the tissue. Approximately five minutes
after the death of the animal the following different methods were used in
preparing the tissues: a, the tissues to be used were washed carefully
inside and outside with 0.9 per cent saline solution and then mounted
immediately in the contraction vessel; b, mounted immediately without
washing; c, washed as above, placed for two hours in the refrigerator in a
closed vessel at about 100C. and then mounted; d, immediately put in the
refrigerator, left there for two hours at 100C. in a closed vessel, and then
mounted without any preliminary washing, and e, the unwashed tissues,
having been kept for two hours in the refrigerator at 100C., were washed
143
Reprinted from THE AMERICAN JOURNAL OF PHYSIOLOGY
VoL 88, No.1, February, 1929
THE RESPONSE OF ISOLATED SEGMENTS OF INTESTINE AND
UTERUS TO ADRENALIN AT DIFFERENT pH LEVELS
OF THE BATH
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with 0.9 per cent saline and then mounted. These preliminary segrega-
tions were made in an attempt to explain a "drug action fading phenome-
non" to be discussed later in this paper. Tissues over two hours old in
most cases proved unsatisfactory for the purposes of this experiment.
Segments of proximal duodenum, proximal jejunum, lower ileum,
scybalar colon, and uterus were employed. Gut segments 2 em. long, and
cornate segments of uterus 2 em. long, were used. Since only one horn of
the uterus could be used at a time in each experiment, the other horn was
left intact and freely suspended in the bath.
The bath consisted of Locke's mammalian solution, kept at 37.0°C., with
a variation of 1.0°C. either way which occurred not faster than 1.0°C. per
half-hour. Air was bubbled through from an immersed glass tube of
3 mm. internal diameter at a constant rate of approximately 300 bubbles
per minute. The volume of the tissue bath was 250 cc. In order to keep
the temperature constant, this bath was surrounded by another bath of
1000 cc. volume, the latter bath being heated by a carefully regulated
micro-burner.
Estimations of pH on the bath were made by withdrawing 5.0 cc. with
a calibrated pipette, adding 0.25 cc. phenol red solution in alcohol, and
comparing colorimetrically to a phenol red set of standards (McCrudden,
1922; Dale and Evans, 1920). This method was found to be accurate
enough for the purpose of these experiments. Changes in the pH could be
made at will by the addition of N/lO NaOH and N/lO HCl in drop frac-
tions. To allow for a more thorough mixture in the solvent of the acid or
alkali, the drug was added about two minutes after the addition of the
acid or alkali. pH estimations were made immediately after adding the
drug.
Bath pH variations were also effected and studied by exchanging an
acid bath for a basic bath, and vice versa, instead of altering the pH of a
single bath.
Adrenalin chloride (Parke, Davis & Co.) in 1/1000 solution was used.
Fresh dilutions of 1/10,000 were made before each experiment. It was
assumed that the chloretone content would be negligible, since this as a
factor has been shown by previous investigators to playa small role in the
action of the drug on the tissues employed. The drug was added to the
bath by a drop method from a standard pipette kept for this purpose only.
The concentrations of the drug in the bath ranged from 1/8,000,000 to
1/25,000,000 (however, a constant concentration was used through each
experiment). The minimal amount that would cause a depression that
could be compared quantitatively to a depression caused by a similar dose
at a different pH, was employed. Experiments, were also done using
adrenalin hydrochloride tablets, which were dissolved in 0.9 per cent
saline to make a 1/10,000 solution.
145INFLUENCE OF H-IONS ON INTESTINAL STRIPS
The pH of the bath was varied within the limits 6.5 and 8.3, but most of
the observations were made within the limits 7.3 to 8.3. It was deemed
necessary to vary this much past the physiological limits of blood in order
to try the wider pH range which might exist in the living tissues as well as
to amplify the gradations in response which might be produced, without
any great change in muscle tone.
RESULTS. The results of Evans and Underhill (1923) and Sollmann,
von Oettingen and Ishikawa (1928) on the activity of segments of small
intestine and uterus when the pH of the bath is altered were confirmed.
With segments of small intestine, sudden drops of pH down to 6.5 or even
to 6.8 stopped the contractions and produced relaxation, with a permanent
loss of tonus, unless the media pH was again raised. A change to the acid
side of neutrality caused a decrease in the amplitude of the rhythmical
contractions. A sudden change in the opposite direction, e.g., to a pH
8.0 produced an increase of tonus, usually with an increase of amplitude.
Sometimes with a high pH value the amplitude of the rhythmical contrac-
tions was reduced. Sudden increases of pH caused sudden increases of
tonus. Gradual increases of pH caused less alteration in tone but more
marked changes in the rhythmical contractions (causing them to increase in
rate and amplitude). The rhythmical contractions appeared best at about
pH 7.4 and were definitely depressed at pH 6.5; the rate of contraction
was decreased in acid and increased in alkaline media. The segments of
uterus showed very slight changes in activity due to pH changes from
6.8 to 8.0. The uterus of the rabbit was noticed to be less sensitive to
pH changes than that of the guinea pig; when placed in a bath with a
constant pH between 7.3 and 7.5 the rabbit uterus exhibted a more con-
stant tonus than the guinea pig uterus. A decrease in pH caused a relaxa-
.tion of the tonus of the rabbit uterus, with a decreased rate and increased
amplitude of the rhythmical contractions.
The duodenum showed in approximately one segment out of every four
an atypical response to successive equivalent doses of adrenalin (fig. 1, a).
This response was noticed to a lesser extent in the jejunum (one segment
out of every six), to a still lesser extent in the ileum than in the jejunum
(one segment out of every eight), and least of all intestinal segments in the
colon. It occurred in the uterus in about one segment out of every eight.
This atypical response manifested itself in that repeated doses of equal
amounts of the drug produced successively smaller depressions until, after
six to eight doses, no response to the drug could be elicited (even when the
dose was doubled or tripled). Care was taken that the tissue had fully
recovered from the last dose before another dose was given. This could
not be eliminated by changing from the fresh chloretone-drug solution to
the tablet solution for a source of the drug, although it seemed to occur
more often when using the latter. It also occurred regardless of the
G. R. SUCKOW AND G. E. BURGET144
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147INFLUENCE OF H-IONS ON INTESTiNAL STRIPS
methods employed in preparing the segments (see methods above). It
was noticed that it could not be eliminated by using Ringer's mammalian
solution or Tyrode's solution instead of Locke's solution. A possible diet
factor was eliminated because rabbits which showed no such phenomena
were receiving the same diet at the same hour as those in which it occurred.
No difference in the response according to sex could be seen. Repeatedly
renewing the bath or substituting an acid bath for a basic bath would not
eliminate it. This phenomenon seemed to be a characteristic of the tissue
rather than of the animal, in that, e.g., the duodenum from a certain animal
would show it while the uterus from this same animal would fail to do so.
In the absence of it the depressions caused by equivalent successive doses on
a given segment were equal (Le., when the pH of the bath was constant,
etc.), and could be duplicated again and again until 20 to 30 doses had
been given, when the tissues would begin to be sluggish and inactive.
As far as quantitative and qualitative comparisons of the action of
adrenalin on the tissues at different pH levels are concerned, only those in
which there was an absence of the "drug action fading phenomenon" will
be mentioned.
DUODENUM. On this tissue equivalent doses of adrenalin produced a
greater and more rapid depression of tonus with slower recovery at pH
8.2 than at pH 7.3 (fig. 1, b, c, d, e). Especially was this observed when'
using tissues which were very sensitive to the drug (e.g., showing a marked
depression by 1/25,000,000 adrenalin). The amplitude of the rythmical
contractions after a depression returned more slowly to normal at pH 8.2
than at pH 7.3. The rate of the rhythmical contractions was not affected
by the drug at any pH. A marked contrast could be seen between the
effect of the drug on the tissue at pH 8.2 and at pH 7.3 (fig. 1, e). In a
minority of cases equivalent doses of adrenalin in acid bath would cause a
depression of tonus to a lower absolute level than in basic bath, but since
the tonus in the basic bath was the higher before the drug was added, the
total depression in the basic bath was the more marked. Some segments
of duodenum showed periodic variations in amplitude much better than
others, and a few segments showed no variations at all. The amplitude
variations could be augmented by alkalinization and diminished (but not
removed) by acidification (within the pH limits used). The less active
phase in amplitude in acid media would often be seen as nearly a straight
line (fig. 1, c, d). However, such segments showed a more marked initial
depression in the basic than in the acid bath (fig. 1, c, d). Such strips were
also very erratic in their tonus levels.
Jejunum. With this tissue the results were similar to those mentioned
above with the duodenum. In dealing with these segments attempts were
made to add the drug during the more active periods in the amplitude
cycles. However, it was found that such precautions were not altogether
146
Fig. L a. Duodenum. To illustrate the "drug action fading phenomenon'"
encountered, in that repeated equivalent doses of adrenalin produce successively
smaller depressions of tonus and amplitude of contractions.
b, c, d, e. Duodenum. To show the greater and more prolonged depression of
tonus and rhythmical contractions in basic than in acid bath as caused by adrena-
lin, as well as individual differences in gut activity.
f. Ileum. Results similar to those found in duodenum.
g. Colon. Showing that the tonus depression caused by adrenalin is more rapid,
and of greater extent but of shorter duration in basic bath and that the depressed
rhythmical contractions and tonus recover more slowly ~at the lower pH level.
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148 G. R. SUCKOW AND G. E. BURGET INFLUENCE OF H-IONS ON INTESTINAL STRIPS 149
SUMMARY
action of the drug seemed to occur at about pH 7.2. Evidently the
recovery of a tonus (depressed by adrenalin) which is low before the addi-
tion of the drug (as in acid media) is slower than when the tonus is high to
begin with (as in basic media). The lower absolute limit of tonus depres-
sion by equivalent doses in a minor number of segments appeared about
the same at pH 7.3 and 8.3. The latent period appeared shorter at a pH
of 8.3 than at a pH of 7.3 (fig. 1, g).
Uterus. The uterus showed- a nearly constant tonus, regardless of the
pH (Le., between pH levels 7.2 and 8.3). A few segments showed an
increase of tonus on decreasing the pH, especially after previous doses of
adrenalin had been given. In practically all of the segments employed no
difference in the action of the drug could be elicited even in changes of pH
from 7.0 to 8.3. A minor number of segments showed a higher stimulatory
rise of tonus at pH of 8.2 than at pH 7.3, but in these cases the durations of
the tonus changes and amplitude inhibitions were similar. It appears that
the pH factor plays a relatively small part here, when one deals with
variations between 7.0 and 8.3.
DISCUSSION. It is well known that adrenalin is oxidized more rapidly in
the oxygenated alkaline than in the oxygenated acid bath. This might be
thought to cause a less prolonged depressant action in alkaline media
(especially if we, had been using large doses of the drug), but this latter
fact was not observed to be the case with the gut segments used. Per-
haps this factor does not enter into the physiological response of the tissues.
Most of the tissues used in these experiments seemed 'to withstand excesses
of alkali better than excesses of acid (between pH levels 6.8 and 8.3 as lower
and upper limits). Possibly the ionized calcium in the bath may playa
part in this latter fact and in the variable responses at different pH levels
especially if we had employed Tyrode's instead of Locke's solution. It
llJay be added that it is quite essential that one take into consideration the
pH of the various tissues in order to more fully appreciate the role of pH
in affecting the action of adrenalin on the "myoneural junction."
Evidently the inhibitory innervation to the intestine responds to adrena-
lin'at various pH levels with similar differences as does the vasoconstrictor
mechanism, while that to the large intestine responds with differences
reverse from those of the small intestine to a certain extent. The loss of
tonus of the colon at a low pH probably accounts for its slower recovery
when the inhibitory mechanism is stimulated in the more acid medium.
The results of Evans and Underhill (1923) on the activity of segments of
small intestine and uterus when the pH of the bath is changed are con-
firmed. Segments of colon were found to show a marked sensitivity to
necessary, since the depressions when' effected during the relatively inac-
tive periods in the amplitude cycles seemed to be just as marked as when
effected during the active stages. On the other hand it was found that such
a precaution is demanded when using uterine segments in order to get
consistent results.
Ileum. With segments of ileum, there was a greater and more prolonged
depression of tonus, and a longer inhibition of the rhyth.mical contractions
at pH 8.2 than at pH 7.3 (fig. 1, f). The rate of the rhythmical contrac-
tions was not affected much in either the acid or basic bath by the addition
of the drug. Segments were found which showed no inhibition of rhythmical
contractions or depression of tonus at a pH of 7.3 and which with an
equivalent dose showed a marked inhibition of rhythmical contractions and
depression of tonus at pH 8.2 even though the gut was contracting actively
in both cases. A minority of segments of ileum showed an increased
tonus on acidification, especially after the drug had been added on previous
occasions to the bath.
Colon. Segments of colon were found to show a marked sensitivity to
changes in pH as far as tonus was concerned (fig. 1, g). An increase in
pH caused a marked increase in tonus while a decrease in pH showed just
the reverse. In a minority of segments the tonus remaned fairly constant
as long as the air supply, temperature, and pH were not changed, while
most of the segments were very variable in their tonus levels. When the
segments were removed from the animal and immediately mounted, they
would in most cases begin their rhythmical contractions immediately,
usually with greater amplitude than they would show if allowed to stand in
a refrigerator for 24 hours. Apparently the segments were not absolutely
tonically contracted upon immediate removal from the body. However,
segments which had been contracting in the bath for 4 to 5 hours or which
stood for 24 hours in the refrigerator did have a slightly more relaxed
tonus which coincides with the observation of Thienes (1926), probably
accounting for our observation that the fresh tissues showed, upon adding
the drug, greater drops in tonus than the 24 hour tissues. Evidently the
24 hour tissues (being more relaxed than the fresh ones) are not as capable
of further relaxation bythe drug as the fresh ones. Equivalent additions of
the drug produced a more sudden and greater (since tonus was higher)
drop of tonus in the alkaline than in the acid bath (fig. 1, g) However,
the lowest absolute level of the depressed tonus was lower in the acid bath
even though the fall was not so great. The depressed tonus (Le., depressed
by drug) showed a much slower recovery in the acid media, the effect of the
drug apparently being more prolonged at the lower pH level (fig. 1, g).
The rate of the rhythmical contractions was not affected by the drug at any
pH tried, although the recovery of the depressed rhythmical contractions
was faster in the alkaline bath (fig. 1, g). The maximum tonus depressant
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BIBLIOGRAPHY
ACTON, H. W. AND R. N. CHOPRA. 1925. Indian JOllrn. Med. Res., xii, 443.
BURGET, G. E. AND M. B. VISSCHER. 1927. This Journal, lxxxi, 113.
BURGET, G. E. AND G. CRISLER. 1927. This Journal, lxxxiii, 373.
DALE, H. H. AND C. L. EVANS. 1920. JOllrn. Physiol., !iv, 167.
EVANS, C. L. AND S. W. F. UNDERHILL. 1923. Journ. Physiol., lviii, 1.
MCCRUDDEN, F. H. 1922. Weekly Public Health Repts., xxxvii, 334.
SOLLMANN, T., W. F. VON OETTINGEN AND Y. ISHIKAWA. 1928. This Journal,
lxxxvi, 661.
THIENES, C. H. 1926. Arch. Intern. Pharm. et Therap., xxxi, Fasc. v-vi, 447.
G. R. SUCKOW AND G. E. BURGET
changes in pH as far as tonus is concerned. An increase in pH caused a
marked rise in tonus while a decrease in pH caused the reverse (between
7.0 and 8;-2). The tonus remained constant as long as the air supply,
temperature, and pH were unchanged. It was possible to vary the tonus
of fresh tissues by drug additon more than of tissue 24 hours old. The
segments of uterus showed very slight changes in activity due to changes in
pH from 6.8 to 8.3.
Isolated segments of duodenum (more so than those of jejunum, ileum,
colon, and uterus, in the order of occurrence) show in about one case out of
every four an irregularity in response to adrenalin in that successive doses
produce progressively diminishing responses even though an equivalent
dose of freshly prepared drug is given each time.
In the absence of this drug action fading phenomenon segments of
duodenum, jejunum, and ileum show in most cases after equivalent
additions of the drug to the bath at a pH of 8.2 an earlier, greater, more
rapid, and more prolonged depression of tonus with a slower recovery of
the amplitude of the rhythmical contractions than at a pH of 7.3. The rate
of the rhythmical contractions is not affected by the drug at any pH between
7.0 and 8.3. An optimum pH for the depressant action of a minimal dose
of adrenalin on these tissues is thought to exist at about 7.8.
In the absence of the drug action fading phenomenon, segments of
scybalar colon show in most cases after equivalent additions of the drug to
the bath at a pH of 8.3 a greater fall of tonus than at a pH of 7.2. The
recovery of depressed tonus and depressed rhythmical contractions is more
rapid at a pH of 8.3 than at a pH of 7.2. The rate of the rhythmical
contractions is not affected by the drug at any pH between 7.0 and 8.3.
An optimum pH for the depressant action of a minimal dose of adrenalin
on the colon is thought to exist at about 7.3.
These observations are in agreement with the previous findings :;s
regards the vasoconstrictor mechanism.
Segments of non-pregnant rabbit uterus show no difference in response to
minimal stimulatory doses of adrenalin at various pH levels ranging from
7.0 to 8.3.
150
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