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THE VASOCONSTRICTOR ACTION OF ANGIOTENSIN IN RELATION TO CATECHOLAMINE
THE
VASOCONSTRICTOR RELATION
TSUTOMU
ACTION TO
SAKURAI
OF
ANGIOTENSIN
IN
CATECHOLAMINE
AND YUICHI
HASHIMOTO
Departmentof Pharmacology,Facultyof Medicine,Osaka University,Kita-ku,Osaka Received for publication January 4, 1965
In study of the mechanism of renal hypertension, attention has been concentrated on investigation of the role of neurogenic component. Several reports (1-3) have sug gested that there are some relations between action of angiotensin and function of the sympathetic nerve endings. Lewis (3) suggested that the action of angiotensin on the vessel wall may result from local release of catecholamine from the sympathetic nerve endings. McCubbin and Page (2) found that infusion of angiotensin caused marked enhancement of the cardiovascular response to agents and procedures causing the release of endogenous noradrenaline. The present experiments were undertaken primarily to examine whether the adre nergic mechanism is involved in the process of the vasoconstrictor action of angiotensin. In these experiments isolated rabbit ear vessels were perfused. The second object of the experiments was to test whether release of noradrenaline from sympathetic nerve endings could be augmented during the infusion of angiotensin which causes an eleva tion of the blood pressure. For this study, urinary catecholamine and their ortho methylated products were measured after the infusion of angiotensin. Tyramine which causes release of noradrenaline from sympathetic nerve endings was used to study the functional state of the sympathetic nerve endings. METHODS Perfusion experiments : Rabbit ear vessels were perfused with Locke-Ringer solution (pH 7.3) at a perfusion pressure of 60 to 80 cm H2O by the method of Krawkow-Pissemski (4). The perfusion pressure was maintained at the initial level throughout the experi ment although the flow rate changed. Solutions of drugs were injected into the arterial perfusion cannula at the rate of 0.1 ml per 10 sec, and the total amount given was 0.3 ml. The drugs used in this study were va15-angiotensin II asp-8-amide (Hypertensin, CIBA*), L-noradrenaline, tyramine hydrochloride, 2-benzylimidaline hydrochloride, atro pine sulfate and acetylcholine hydrochloride. 桜井
勗 ・橋 本
裕一
* Kindly supplied by Ciba Products Limited , Osaka, Japan,
The flow rate after injection of the drugs was compared to the flow rate during the control period immediately preceding it, and the rate of decrease was calculated. Procedurefor prolongedinfusion of angiotensinand tyramine: Male albino rabbits (2.2 to 2.5 kg) which had been kept in individual metabolic cages for a week were used. Rab bits were anesthetized by intravenous administration of 25 mg/kg of Nembutal (Pento barbital sodium). A Nelaton catheter was inserted into the urinary bladder and all the urine was collected. Then the bladder was washed out with 5 ml of water and the catheter was drawn out. Each animal was placed in a metal cage small enough to prevent it turning round easily. The solution of drug was infused into the central vein of the left at the rate of 15 ml/kg/hr. During the period of infusion urine was usually not voided. Immediately after the 2 hour infusion period the catheter was again insert ed into the urinary bladder and the urine was collected. The urinary specimens col lected both before and after infusion were immediately acidified to pH 1.0 with 6 N hydrochloric acid and were stored at 0"C for chemical analysis. Stock solutions of both synthetic angiotensin II and tyramine were diluted with physiological saline prior to infusion. The doses of angiotensin and tyramine were 0.04 ieg/kg/min and 200 beg/kg/min respectively. When a solution was infused containing both drugs, a full dose of tyramine and a half dose (0.02 ,t(,,/kg/min) of angiotensin were used. Determinationof catecholamines and their metabolites : Total noradrenaline and adrenaline were estimated by the trihydroxyindole method as improved by Itoh et al. (5), Nukada et al. (6) and Matsuoka (7). The principle of this procedure was as follow. After selec tive absorption of catecholamine on aluminum hydroxide, a Duolite C-25 resin column was used to separate total noradrenaline and adrenaline from other catechol compounds. Determination of these two substances was made by the trihydroxyindole method. The total normetanephrine and metanephrine were determined by the method of Crout et al. (8) with some modifications. The total noradrenaline and adrenaline, and total nor metanephrine and metanephrine were expressed in micrograms of noradrenaline, and normetanephrine per milligram of creatinine, being excreted fairly constantly through the experimental period. Measurementof arterial bloodpressure: Another group of rabbits was used for measure ments of the blood pressure. This was recorded upon an ink-writing kymograph with a mercury manometer connected to the right common carotid artery. The student t-test was applied to test the significance of the results. RESULTS _1. The Mechanismof the Vasoconstrictcr Acitionof Angiotensin 1) Vasoconstrictor action of noradrenaline Fig. 1 shows the vasoconstrictor action of noradrenaline on isolated perfused rabbit ear. Doses of noradrenaline between 0.025 /1g/ml and 0.8 ag/ml were administered. The drug was successively given to the same preparation at increasing doses. Less than 0.025 ,ug/ml of noradrenaline caused no apparent vasoconstriction, whereas more than 0.1 ag/ml
FIG.
1. Constriction
FIG.
2.
of
Constriction
rabbit
of
ear
rabbit
ear
of noradrenaline
reduced the flow to about
of noradrenaline
stopped the flow completely
2)
by
vessels
noradrenaline.
by
angiotensin.
50% of the control
value.
Over 0.8 ug/ml
for several minutes.
Vasoconstrictoraction of angiotensin Fig. 2 shows that the action
adrenaline.
With
reduced to about required
of angiotensin
0.1 ,ng/ml of noradrenaline, 50% of the control
value,
3 times as much as angiotensin,
caused by over 0.8,ug/ml
of noradrenaline
whereas even 5 ug/ml of angiotensin 3)
vessels
was weak compared
with that of nor
as mentioned
above, the flow rate was
while a similar
decrease in the flow rate
namely
0.2 to 0.4 ,ug/ml.
The vasoconstriction
was so great that the flow stopped completely,
did not have so great an effect.
Effects of reserpine on the action of sympathomimetic amines and angiotensin The effect of reserpine
on 8 ears.
on the vasoconstrictor
First the perfusion
experiments
action
of noradrenaline
was examined
were made on the one ear of a normal
bit to obtain the dose-response curve to noradrenaline.
Then
the rabbit
rab
was pretreated
FIG.
3.
FIG. 4.
with reserpine
Effect
Effect
of
reserpine
of
on
reserpine
constriction
on
of
constriction
rabbit
of
ear
rabbit
by giving 10 mg/kg body weight
ear
vessels
by
vessels
intraperitoneally
noradrenaline.
by
angiotensin.
immediately
after re
section of the one ear. The following day the same perfusion experiments were made on the other ear. The results are shown in Fig. 3. After reserpine treatment, the dose response curve to noradrenaline
was shifted to the left. Thus reserpine treatment
the sensitivity to noradrenaline. after reserpine
On the contrary
was reduced
treatment.
The effect of reserpine on the vasoconstrictor on 8 ears.
the response to tyramine
increased
The action of angiotensin
action of angiotensin
was enhanced
by reserpine
was then examined
treatment.
in Fig. 4, the dose-response curve to angiotensin after reserpine treatment to the left, and this shift was larger than that caused by noradrenaline. suggests that angiotensin
exerts its vasoconstrictor
action by a different
As shown was shifted This result
mechanism
from
tyramine. 4)
Effects of imidaline on the action of sympathomimetic amines and angiotensin The effects of the adrenergic
chloride) examined
on the vasoconstrictor on 6 ears.
blocking action
agent,
imidaline
of angiotensin
Doses of 0.3 ml of concentrations
(2-benzyl imidaline
and symathomimetic of 20, 200 and
hydro
amines was 2,000 i g/ml of
FIG. 5. Effect of imidaline (Im) on constriction of rabbit ear vessels by noradrenaline (NA), tyramine (Tyr) and angiotensin (A). * Imidaline (200 pg/ml , 0.3 ml) was administered before NA, Tyr and A administration.
imidaline were given before administration of the drugs. Even a single injection of over 200 fig/ml of imidaline caused vasoconstriction. Therefore, when this dose was given, subsequent infusions of drugs were made after the flow rate had returned to about the initial value. Fig. 5 shows that the vasoconstrictor reduced On
action 5)
by 20 i g/ml of imidaline,
the other
hand,
of angiotensin,
action
and almost
even 2,000 ag/ml
of noradrenaline prevented
of imidaline
and in fact rather
enhanced
and tryramine
were
by 200 ag/ml of imidaline.
did not reduce the vasoconstrictor its action.
Effects of atropine on the vasoconstrictoraction of noradrenaline and angiotensin The effects of the cholinergic
of the drugs was also examined. constrictor
action
blocking
It was found that atropine
of either noradrenaline
FIG. 6, Effect of angiotensin
agent, atropine
(A) on constriction
on the vasoconstrictor
action
had no effect on the vaso
or angiotensin.
of rabbit ear vessels by noradrenaline
(NA).
6) Effect of angiotensinon the vasoconstrictor action of noradrenaline Less than 0.005 pg/ml of angiotensin, which had no vasoconstrictor action in itself, enhanced the vasoconstrictor action of noradrenaline. One of the 7 experiments is shown in Fig. 6, which indicates that the vasoconstrictor actions of 0.25 ,ug/ml and 0.4 ,ug/ml of noradrenaline are both nearly doubled by angiotensin. This is not simple summation of the vasoconstrictor actions of the two drugs, but represents a potentiating action of angiotensin on the vasoconstrictor action of noradrenaline. 7) Effect of angiotensinon the vasoconstrictor action of tyramine A similar potentiating effect of angiotensin on the vasoconstrictor action of tyramine is shown in Fig. 7. Experiments were done on 11 ears and in all cases a tachyphylaxis phenomenon was observed, but the vasoconstrictor action of tyramine was also aug mented by subthreshold amounts of angiotensin.
FiG. 7. Effect of angiotensin (A) on constriction of rabbit ear vessels by tyramine (Tyr). Numbers (1, 2, 3 and 4) represent the order of drug administration. TABLE
1.
Effect
tyramine
The blood pressure
of on
prolonged the
was recorded
infusion
arterial
blood
of
angiotensin
pressure
and
of
rabbit.
during the infusion of angiotensin
pg/kg/min), tyramine (200 teg/kg/min) and a combination teg/kg/min) and angiotensin (0.02 t g/kg/min).
of tyramine
(0.04 (200
II. Effectsof Infusionof Angiotensin,Tyramineand Tyramine with Angiotensinon the Arterial Blood Pressure,and on the Urinary Excretionof Catecholamines and Their Metabolites These results are presented in Tables 1 and 2, and Fig. 8.
1) Physiological saline infusion In twelve rabbits infused with physiological saline there was almost no change in the arterial blood pressure.
During the period of saline infu
sion, there was a slight decrease in the urinary excretion of total noradrenaline and a slight increase phrine 2)
and adrenaline,
in the total normetane
and metanephrine
excretion.
Angiotensin infusion In ten rabbits the infusion of 0.04 ,ug/kg/min
of angiotensin
constantly
of the arterial
blood pressure by 20 to 30 mmHg,
and in some animals mmHg was found.
an initial rise of 60 to 70
During
the period
was
no increase
there
and adrenaline
produced an elevation
of angiotensin
infusion
in total noradrenaline
excretion,
but total normetane
phrine and metanephrine excretion increased by 30% (0.31 ,ug/mg Cr). This increase was not statistically 3)
significant
(.10>p>.05).
Tyra-nine infusion The extent of the elevation
of arterial blood
pressure during the first thirty minutes after infusion of tyramine was very variable in four rabbits. Total
noradrenaline
tion increased the period
and adrenaline
excre
by 29% (0.129 ag/mg Cr) during
of infusion.
During
saline and an
giotesin infusion, however, the urinary excretion of these substances decreased slightly. Therefore this increase
was
statistically
significant
(p
.005). Total normetanephrine and metaneph rine excretion increased by 117% (1.073 pg/mg Cr) and this was statistically 4)
significant
compared
with the control
group (p<.001).
Infusion of tyramine together with angiotensin When both drugs were infused together,
when tyramine
was infused alone, even
the blood pressure was elevated
though a half
min) was given, which alone had no effect on the arterial a slight increase in the first 10 to 20 minutes
dose of angiotensin
more than (0.02 ,ug/kg/
blood pressure except to cause
after infusion.
When , both drugs
were
FIG. 8. Excretion of catecholamines and their metabolites before (B) and during (D) infusion of angiotensin and tyramine. Urinary excretion of catecholamines and their metabolites before and during 2 hour periods of infusion of saline, angiotensin (0.04 pg/kg/min), tyramine (200 ug/kg/min) and a combination of tyramine (200 pg/kg/min) and angio tensin (0.02 pg/kg/min). * SEM : standard error of the mean.
given, the elevation shortly after the beginning one hour there were no significant difference tyramine
alone and tyramine
and angiotensin
of infusion was very marked, but after in the effects on the blood pressure of together.
With both drugs the increases in total noradrenaline also of total normetanephrine the group
infused with tyramine
was increased metanephrine
Total
(p---1.005). The very great
excretion,
the increase
normetanephrine
alone.
and adrenaline
excretion
were greater
noradrenaline
and
by 38% (0.193 ag/mg Cr), and this was statistically
to the control group neither
and metanephrine
and metanephrine
the group infused with tyramine
and
adrenaline
were statistically alone (.40>p>.30,
adrenaline
excretion
significant
compared
(p>.001).
excretion
significant
and
those in
increase in total normetanephrine
161% (2.34 beg/mg Cr) was also significant
in total noradrenalme
excretion than
However,
nor that
compared
and
of total
to those in
.20>p>.10).
DISCUSSION It is well known that angiotensin causes marked vasoconstriction. Finnerty (9) report ed that the effect of angiotensin on the arterial blood pressure was about 10 times as great as that of noradrenaline. Similar results were presented by Mendlowitz (10), namely the potency of angiotensin in constricting digital blood vessels was 10 times greater than that of noradrenaline in both normotensive and hypertensive subjects. However, in our experiments on isolated rabbit ear vessels the vasoconstrictor action of angiotensin was
weak compared with that of noradrenaline. The vasoconstrictor response to noradre naline was so great (Fig. 1) that over 0.8 ,ug/ml of noradrenaline stopped the flow completely. On the other hand, the response to angiotensin was less than that to nor adrenaline (Fig. 2). With regard to the vasoconstrictor action of angiotensin, there is much evidence suggesting, besides that, to have a direct action [Furchgott (11); Hurwits et al. (12); Haddy et al. (13)], angiotensin may have an indirect vasoconstrictor action on blood vessels [Laverty (14); Bickerton and Buckley (15); Buckley et al. (16)]. Therefore , in our experiments the indirect action of angiotensin may have been diminished due to abolition of sympathetic tone by isolation of the ear, and so there was less response to angiotensin. Concerning the indirect action of angiotensin on blood vessels, Lewis (3) suggested that the action of angiotensin is not a direct effect on smooth muscle, but it is as the result of a local release of catecholamines from the sympathetic nerve end ings in the vascular wall. If so, the indirect vasoconstrictor action of angiotensin may be similar to that of catecholamine releasing agents, such as tyramine, which are generally considered to act mainly by causing release of endogenous noradrenaline [Burn and Rand (17, 18)]. If so the action of angiotensin, like that of tyramine, should be diminished by reserpine. In the present experiments the action of tyramine on the ear vessels of rabbits pretreated with reserpine was diminised. However, the vasoconstrictor action of angiotensin on the same preparation was enhanced. The action of noradrenaline was enhanced as expected (Figs. 3, 4). These results suggest that angiotensin does not act like tyramine, and that a catecholamine releasing action is not involved in the major action of angiotensin in these experiments. Khairallah and Page (19) showed that adrenergic blocking agents inhibit the response of intestinal smooth muscle to angiotensin, and suggested that this inhibition might be a direct effect on the cell membrane. In our perfusion experiments on isolated rabbit ear vessels, however, imidaline reduced the response to both noradrenaline and tyra mine, but the response to angiotensin was not affected (Fig 5). If an adrenergic mecha nism is involved in the action of angiotensin on vascular smooth muscle, the response to angiotensin should be reduced by this agent. These results indicate that the direct vasoconstrictor action of angiotensin on blood vessels may have nothing to do with the so-called "noradrenaline-receptors". Khairallah and Page (20) reported that the response of intestinal smooth muscle to angiotensin was blocked by atropine. In our experiments, however, doses of atropine sufficient to affect the action of acetylcholine, had no effect on 'the direct action of angiotensin on isolated perfused rabbit ear vessels. From the above results a neurotransmitter appears to have no relation with the mechanism of the direct vasoconstrictor action of angiotensin. In other words , it appears that the vascular receptors on which angiotensin exerts a direct action differ from those concerned with neurotransmission. However, a very interesting effect of angiotensin on the action of noradrenaline was found in our experiments (Fig. 6). Very small amounts of angiotensin , which had no effect on the perfused vessels, greatly enhanced the vasoconstrictor action of noradre
naline.
This
in the
suggests
vasoconstrictor
tensin
which
marked However,
in combination
The
effective
vasoconstrictor (Fig.
had
and
angiotensin
Heller
no significant
and noradrenaline
(21) found
that
effect on isolated
doses of noradrenaline.
action 7).
adrenaline-receptors" noradrenaline
a crude
rabbit
It is considered
was also
and
(2) reported results
in
of endogenous
enhanced
is partly also
in the presence
Page
which
the release
tyramine
augmentation
angiotensin,
tyramine
and
system,
of
This
by
by
McCubbin
cause
Mylon
in itself
between
angio
ear vessels, of noradre were used
that
angiotensin
the "noradrenaline-receptors".
of angiotensin
nervous
is an interaction
vasoconstriction when mixed with subthreshold amounts in our experiments, subthreshold amounts of angiotensin with
sensitize
there
mechanism.
preparation,
produced naline.
may
that
due
may
be due
of angiotensin
that
angiotensin
an enhanced
amounts of "nor
to the increased
has an effect
They
small
release
of
(see below).
response
noradrenaline.
by very
to the sensitization
to agents
suggested
on the sympathetic and
that
procedures
the
that
effect of angio
tensin on the sympathetic nervous system did not appear to involve sensitization "noradrenaline -receptors" , because there was little or no effect of angiotensin on response of the blood pressure to exogenous noradrenaline. But in our experiments enhanced
by
systemic cular
angiotensin.
responsiveness
beds,
some
However,
McCubbin to study
of noradrenaline
in the
and
This
and the
may
administered
be
noradrenaline
due
and
innervated
or
pressure that
and
and
nerve
noradrenaline
to a difference
the
response
denervated
by
sion of angiotensin.
On
cant
increase
urinary
even
great
increase
in
that
endings.
in the urinary and
prolonged
the
attracted
infusion release
was
between
the
in specialized vascular
attention. on
vas
beds,
or to
experiments to clarify
of either
of angiotensin.
of
total
of tyramine
noradrenaline
normetanephrine
and
the
elevation
these
the period resulted
and
urinary
noradrenaline
of the
Therefore, during
it
or release
this question.
total
in spite
of noradrenaline
administration
Therefore, storage
In these
excretion
excretion
the
was estimated
retanephrine,
contrary,
of total
our
of angiotensin
metabolites
normetanephrine
was no increased
in the
suggestions
of infusion
their
increase
total
caused
there
Page's effect
sympathetic
was no significant adrenaline,
suggest
discrepancy
between
of catecholamines
of blood
to exogenously
the
difference.
was of interest
There
response
to exogenous
or to a difference
other
excretion
the
of
results of infu
in a signifi
adrenaline,
metanephrine.
and Thus
an this
phenomenon apparently reflects a catecholamine releasing action of tyramine. From these observations, it is also suggested that angiotensin does not seem to manifest its action
in the same
When excretion This
of these
increment
cholamine from
tyramine
as catecholamine
was infused substances
action studies.
releasing
together
with
was greater
than
was not statistically
releasing
physiological
way
significant,
of tyramine,
agents
such
angiotensin,
the
that but
as McCubbin
when
increase
tyramine
angiotensin and
as tyramine.
Page
in the
alone
was
urinary infused.
may
enhance
the
cate
have
already
suggested
SUMMARY 1.
In
of angiotensin
perfusion was
experiments very
weak
on
isolated
compared
rabbit
with
that
ear
vessels,
the vasoconstrictor
action
of noradrenaline.
2. This action of angiotensin was not affected by either by in vivo pretreatment with reserpine or in vitro pretreatment with imidaline, or atropine. 3. Subthreshold amounts of angiotensin greatly enhanced the response of rabbit ear vessels to noradrenaline, and also to tyramine. 4. The intravenous infusion of angiotensin into rabbits did not produce a signifi cant increase in the urinary catecholamines and their metabolites, but a marked increase was caused by the infusion of tyramine. 5. When a combination of tyramine and angiotensin was infused, a more remarkable increase in urinary catecholamines and their metabolites was observed. These results were discussed, and it was concluded that (i) the vasoconstrictor action of angiotensin is not mediated by neurotransmitters, (ii) angiotensin potentiates the vaso constrictor response to noradrenaline and tyramine, and (iii) in the presence of angio tensin, the catecholamine releasing action of tyramine might be enhanced. Acknowledgements : The authors wish to express their appreciation to ProfessorR. Imaizumi and Assistant ProfessorH. Yoshida for their kind guidanceand helpful suggestionsduring this study and preparation of the manuscript. REFERENCES 1) ZIMMERMAN,B.H. : Circulation Res. 11, 780 (1962) 2) MCCUBBIN,J.W. AND PAGE, I.H. : Ibid. 12, 553 (1963) 3) LEwis, G.P. : Canad. msd. Ass. J. 90, 302 (1964) 4) KRAWKOW-PISSEMSKI : Handb. biol. Arbth. (1923) 5) ITOH, T., MATSUOKA,M., NAKAZIMA,K., TAGAWA,K. AND IMAIZUMI,R.: THIS JOURNAL 12, 130 (1962) 6) NUKADA, T., MATSUOKA,M. AND IMAIZUMI,R. : Ibid. 12, 57 (1962) 7) MATSUOKA,M. : Ibid. 14, 181 (1964) 8) GROUT, J.R., PISANO,J.J. AND SJOERDSMA,A. : Amer. Heart J. 61, 377 (1961) 9) FINNER'rY,F.A. : Circulation 25, 255 (1962) 10) MENDLOWITZ,M., WOLF, R.L., GITLOW, S.E. AND NAFTCHI, N.E. : Ibid. 25, 231 (1962) 11) FURCHGOTT,R.F. : Pharmacol. Rev. 7, 183 (1955) 12) HURWITZ, R., CAMPBELL,R.W., GORDON, P. AND HADDY, F.J.: J. Pharmacol. 133, 57 (1961) 13) HADDY,F.J., MOLNAR, J.I., BORDEN,C.W. AND TEXTER, E.G.: Circulation 25, 239 (1962) 14) LAVERTY,R. : J. Pharm. Pharmacol. 15, 63 (1963) 15) BICKERTON,R.K. AND BUCKLEY,J.P. : Proc. Soc. exp. Biol., N.Y. 106, 834 (1964) 16) BUCKLEY,J.P., BICKERTON,R.K., HALLIDAY,R.P. ANDKATO, H.: Ann. N. Y. Acad. Sci. 104, 299 (1963) 17) BURN, J.H. AND RAND, M.J.: J. Physiol. 144, 314 (1958) 18) BURN, J.H. AND RAND, M.J.: Brit. J. Pharmacol. 15, 56 (1961) 19) KHAIRALLAH,P.A. AND PAGE, I.H.: Amer. J. Physiol. 202, 841 (1962) 20) KHAIRALLAH,P.A. AND PAGE, I.H.: Ibid. 200, 51 (1961) 21) MYLON, E. AND HELLER, J.H.: Pror. Soc. exp. Biol., N.Y. 67, 62 (1948)
VASOCONSTRICTOR RELATION
TSUTOMU
ACTION TO
SAKURAI
OF
ANGIOTENSIN
IN
CATECHOLAMINE
AND YUICHI
HASHIMOTO
Departmentof Pharmacology,Facultyof Medicine,Osaka University,Kita-ku,Osaka Received for publication January 4, 1965
In study of the mechanism of renal hypertension, attention has been concentrated on investigation of the role of neurogenic component. Several reports (1-3) have sug gested that there are some relations between action of angiotensin and function of the sympathetic nerve endings. Lewis (3) suggested that the action of angiotensin on the vessel wall may result from local release of catecholamine from the sympathetic nerve endings. McCubbin and Page (2) found that infusion of angiotensin caused marked enhancement of the cardiovascular response to agents and procedures causing the release of endogenous noradrenaline. The present experiments were undertaken primarily to examine whether the adre nergic mechanism is involved in the process of the vasoconstrictor action of angiotensin. In these experiments isolated rabbit ear vessels were perfused. The second object of the experiments was to test whether release of noradrenaline from sympathetic nerve endings could be augmented during the infusion of angiotensin which causes an eleva tion of the blood pressure. For this study, urinary catecholamine and their ortho methylated products were measured after the infusion of angiotensin. Tyramine which causes release of noradrenaline from sympathetic nerve endings was used to study the functional state of the sympathetic nerve endings. METHODS Perfusion experiments : Rabbit ear vessels were perfused with Locke-Ringer solution (pH 7.3) at a perfusion pressure of 60 to 80 cm H2O by the method of Krawkow-Pissemski (4). The perfusion pressure was maintained at the initial level throughout the experi ment although the flow rate changed. Solutions of drugs were injected into the arterial perfusion cannula at the rate of 0.1 ml per 10 sec, and the total amount given was 0.3 ml. The drugs used in this study were va15-angiotensin II asp-8-amide (Hypertensin, CIBA*), L-noradrenaline, tyramine hydrochloride, 2-benzylimidaline hydrochloride, atro pine sulfate and acetylcholine hydrochloride. 桜井
勗 ・橋 本
裕一
* Kindly supplied by Ciba Products Limited , Osaka, Japan,
The flow rate after injection of the drugs was compared to the flow rate during the control period immediately preceding it, and the rate of decrease was calculated. Procedurefor prolongedinfusion of angiotensinand tyramine: Male albino rabbits (2.2 to 2.5 kg) which had been kept in individual metabolic cages for a week were used. Rab bits were anesthetized by intravenous administration of 25 mg/kg of Nembutal (Pento barbital sodium). A Nelaton catheter was inserted into the urinary bladder and all the urine was collected. Then the bladder was washed out with 5 ml of water and the catheter was drawn out. Each animal was placed in a metal cage small enough to prevent it turning round easily. The solution of drug was infused into the central vein of the left at the rate of 15 ml/kg/hr. During the period of infusion urine was usually not voided. Immediately after the 2 hour infusion period the catheter was again insert ed into the urinary bladder and the urine was collected. The urinary specimens col lected both before and after infusion were immediately acidified to pH 1.0 with 6 N hydrochloric acid and were stored at 0"C for chemical analysis. Stock solutions of both synthetic angiotensin II and tyramine were diluted with physiological saline prior to infusion. The doses of angiotensin and tyramine were 0.04 ieg/kg/min and 200 beg/kg/min respectively. When a solution was infused containing both drugs, a full dose of tyramine and a half dose (0.02 ,t(,,/kg/min) of angiotensin were used. Determinationof catecholamines and their metabolites : Total noradrenaline and adrenaline were estimated by the trihydroxyindole method as improved by Itoh et al. (5), Nukada et al. (6) and Matsuoka (7). The principle of this procedure was as follow. After selec tive absorption of catecholamine on aluminum hydroxide, a Duolite C-25 resin column was used to separate total noradrenaline and adrenaline from other catechol compounds. Determination of these two substances was made by the trihydroxyindole method. The total normetanephrine and metanephrine were determined by the method of Crout et al. (8) with some modifications. The total noradrenaline and adrenaline, and total nor metanephrine and metanephrine were expressed in micrograms of noradrenaline, and normetanephrine per milligram of creatinine, being excreted fairly constantly through the experimental period. Measurementof arterial bloodpressure: Another group of rabbits was used for measure ments of the blood pressure. This was recorded upon an ink-writing kymograph with a mercury manometer connected to the right common carotid artery. The student t-test was applied to test the significance of the results. RESULTS _1. The Mechanismof the Vasoconstrictcr Acitionof Angiotensin 1) Vasoconstrictor action of noradrenaline Fig. 1 shows the vasoconstrictor action of noradrenaline on isolated perfused rabbit ear. Doses of noradrenaline between 0.025 /1g/ml and 0.8 ag/ml were administered. The drug was successively given to the same preparation at increasing doses. Less than 0.025 ,ug/ml of noradrenaline caused no apparent vasoconstriction, whereas more than 0.1 ag/ml
FIG.
1. Constriction
FIG.
2.
of
Constriction
rabbit
of
ear
rabbit
ear
of noradrenaline
reduced the flow to about
of noradrenaline
stopped the flow completely
2)
by
vessels
noradrenaline.
by
angiotensin.
50% of the control
value.
Over 0.8 ug/ml
for several minutes.
Vasoconstrictoraction of angiotensin Fig. 2 shows that the action
adrenaline.
With
reduced to about required
of angiotensin
0.1 ,ng/ml of noradrenaline, 50% of the control
value,
3 times as much as angiotensin,
caused by over 0.8,ug/ml
of noradrenaline
whereas even 5 ug/ml of angiotensin 3)
vessels
was weak compared
with that of nor
as mentioned
above, the flow rate was
while a similar
decrease in the flow rate
namely
0.2 to 0.4 ,ug/ml.
The vasoconstriction
was so great that the flow stopped completely,
did not have so great an effect.
Effects of reserpine on the action of sympathomimetic amines and angiotensin The effect of reserpine
on 8 ears.
on the vasoconstrictor
First the perfusion
experiments
action
of noradrenaline
was examined
were made on the one ear of a normal
bit to obtain the dose-response curve to noradrenaline.
Then
the rabbit
rab
was pretreated
FIG.
3.
FIG. 4.
with reserpine
Effect
Effect
of
reserpine
of
on
reserpine
constriction
on
of
constriction
rabbit
of
ear
rabbit
by giving 10 mg/kg body weight
ear
vessels
by
vessels
intraperitoneally
noradrenaline.
by
angiotensin.
immediately
after re
section of the one ear. The following day the same perfusion experiments were made on the other ear. The results are shown in Fig. 3. After reserpine treatment, the dose response curve to noradrenaline
was shifted to the left. Thus reserpine treatment
the sensitivity to noradrenaline. after reserpine
On the contrary
was reduced
treatment.
The effect of reserpine on the vasoconstrictor on 8 ears.
the response to tyramine
increased
The action of angiotensin
action of angiotensin
was enhanced
by reserpine
was then examined
treatment.
in Fig. 4, the dose-response curve to angiotensin after reserpine treatment to the left, and this shift was larger than that caused by noradrenaline. suggests that angiotensin
exerts its vasoconstrictor
action by a different
As shown was shifted This result
mechanism
from
tyramine. 4)
Effects of imidaline on the action of sympathomimetic amines and angiotensin The effects of the adrenergic
chloride) examined
on the vasoconstrictor on 6 ears.
blocking action
agent,
imidaline
of angiotensin
Doses of 0.3 ml of concentrations
(2-benzyl imidaline
and symathomimetic of 20, 200 and
hydro
amines was 2,000 i g/ml of
FIG. 5. Effect of imidaline (Im) on constriction of rabbit ear vessels by noradrenaline (NA), tyramine (Tyr) and angiotensin (A). * Imidaline (200 pg/ml , 0.3 ml) was administered before NA, Tyr and A administration.
imidaline were given before administration of the drugs. Even a single injection of over 200 fig/ml of imidaline caused vasoconstriction. Therefore, when this dose was given, subsequent infusions of drugs were made after the flow rate had returned to about the initial value. Fig. 5 shows that the vasoconstrictor reduced On
action 5)
by 20 i g/ml of imidaline,
the other
hand,
of angiotensin,
action
and almost
even 2,000 ag/ml
of noradrenaline prevented
of imidaline
and in fact rather
enhanced
and tryramine
were
by 200 ag/ml of imidaline.
did not reduce the vasoconstrictor its action.
Effects of atropine on the vasoconstrictoraction of noradrenaline and angiotensin The effects of the cholinergic
of the drugs was also examined. constrictor
action
blocking
It was found that atropine
of either noradrenaline
FIG. 6, Effect of angiotensin
agent, atropine
(A) on constriction
on the vasoconstrictor
action
had no effect on the vaso
or angiotensin.
of rabbit ear vessels by noradrenaline
(NA).
6) Effect of angiotensinon the vasoconstrictor action of noradrenaline Less than 0.005 pg/ml of angiotensin, which had no vasoconstrictor action in itself, enhanced the vasoconstrictor action of noradrenaline. One of the 7 experiments is shown in Fig. 6, which indicates that the vasoconstrictor actions of 0.25 ,ug/ml and 0.4 ,ug/ml of noradrenaline are both nearly doubled by angiotensin. This is not simple summation of the vasoconstrictor actions of the two drugs, but represents a potentiating action of angiotensin on the vasoconstrictor action of noradrenaline. 7) Effect of angiotensinon the vasoconstrictor action of tyramine A similar potentiating effect of angiotensin on the vasoconstrictor action of tyramine is shown in Fig. 7. Experiments were done on 11 ears and in all cases a tachyphylaxis phenomenon was observed, but the vasoconstrictor action of tyramine was also aug mented by subthreshold amounts of angiotensin.
FiG. 7. Effect of angiotensin (A) on constriction of rabbit ear vessels by tyramine (Tyr). Numbers (1, 2, 3 and 4) represent the order of drug administration. TABLE
1.
Effect
tyramine
The blood pressure
of on
prolonged the
was recorded
infusion
arterial
blood
of
angiotensin
pressure
and
of
rabbit.
during the infusion of angiotensin
pg/kg/min), tyramine (200 teg/kg/min) and a combination teg/kg/min) and angiotensin (0.02 t g/kg/min).
of tyramine
(0.04 (200
II. Effectsof Infusionof Angiotensin,Tyramineand Tyramine with Angiotensinon the Arterial Blood Pressure,and on the Urinary Excretionof Catecholamines and Their Metabolites These results are presented in Tables 1 and 2, and Fig. 8.
1) Physiological saline infusion In twelve rabbits infused with physiological saline there was almost no change in the arterial blood pressure.
During the period of saline infu
sion, there was a slight decrease in the urinary excretion of total noradrenaline and a slight increase phrine 2)
and adrenaline,
in the total normetane
and metanephrine
excretion.
Angiotensin infusion In ten rabbits the infusion of 0.04 ,ug/kg/min
of angiotensin
constantly
of the arterial
blood pressure by 20 to 30 mmHg,
and in some animals mmHg was found.
an initial rise of 60 to 70
During
the period
was
no increase
there
and adrenaline
produced an elevation
of angiotensin
infusion
in total noradrenaline
excretion,
but total normetane
phrine and metanephrine excretion increased by 30% (0.31 ,ug/mg Cr). This increase was not statistically 3)
significant
(.10>p>.05).
Tyra-nine infusion The extent of the elevation
of arterial blood
pressure during the first thirty minutes after infusion of tyramine was very variable in four rabbits. Total
noradrenaline
tion increased the period
and adrenaline
excre
by 29% (0.129 ag/mg Cr) during
of infusion.
During
saline and an
giotesin infusion, however, the urinary excretion of these substances decreased slightly. Therefore this increase
was
statistically
significant
(p
.005). Total normetanephrine and metaneph rine excretion increased by 117% (1.073 pg/mg Cr) and this was statistically 4)
significant
compared
with the control
group (p<.001).
Infusion of tyramine together with angiotensin When both drugs were infused together,
when tyramine
was infused alone, even
the blood pressure was elevated
though a half
min) was given, which alone had no effect on the arterial a slight increase in the first 10 to 20 minutes
dose of angiotensin
more than (0.02 ,ug/kg/
blood pressure except to cause
after infusion.
When , both drugs
were
FIG. 8. Excretion of catecholamines and their metabolites before (B) and during (D) infusion of angiotensin and tyramine. Urinary excretion of catecholamines and their metabolites before and during 2 hour periods of infusion of saline, angiotensin (0.04 pg/kg/min), tyramine (200 ug/kg/min) and a combination of tyramine (200 pg/kg/min) and angio tensin (0.02 pg/kg/min). * SEM : standard error of the mean.
given, the elevation shortly after the beginning one hour there were no significant difference tyramine
alone and tyramine
and angiotensin
of infusion was very marked, but after in the effects on the blood pressure of together.
With both drugs the increases in total noradrenaline also of total normetanephrine the group
infused with tyramine
was increased metanephrine
Total
(p---1.005). The very great
excretion,
the increase
normetanephrine
alone.
and adrenaline
excretion
were greater
noradrenaline
and
by 38% (0.193 ag/mg Cr), and this was statistically
to the control group neither
and metanephrine
and metanephrine
the group infused with tyramine
and
adrenaline
were statistically alone (.40>p>.30,
adrenaline
excretion
significant
compared
(p>.001).
excretion
significant
and
those in
increase in total normetanephrine
161% (2.34 beg/mg Cr) was also significant
in total noradrenalme
excretion than
However,
nor that
compared
and
of total
to those in
.20>p>.10).
DISCUSSION It is well known that angiotensin causes marked vasoconstriction. Finnerty (9) report ed that the effect of angiotensin on the arterial blood pressure was about 10 times as great as that of noradrenaline. Similar results were presented by Mendlowitz (10), namely the potency of angiotensin in constricting digital blood vessels was 10 times greater than that of noradrenaline in both normotensive and hypertensive subjects. However, in our experiments on isolated rabbit ear vessels the vasoconstrictor action of angiotensin was
weak compared with that of noradrenaline. The vasoconstrictor response to noradre naline was so great (Fig. 1) that over 0.8 ,ug/ml of noradrenaline stopped the flow completely. On the other hand, the response to angiotensin was less than that to nor adrenaline (Fig. 2). With regard to the vasoconstrictor action of angiotensin, there is much evidence suggesting, besides that, to have a direct action [Furchgott (11); Hurwits et al. (12); Haddy et al. (13)], angiotensin may have an indirect vasoconstrictor action on blood vessels [Laverty (14); Bickerton and Buckley (15); Buckley et al. (16)]. Therefore , in our experiments the indirect action of angiotensin may have been diminished due to abolition of sympathetic tone by isolation of the ear, and so there was less response to angiotensin. Concerning the indirect action of angiotensin on blood vessels, Lewis (3) suggested that the action of angiotensin is not a direct effect on smooth muscle, but it is as the result of a local release of catecholamines from the sympathetic nerve end ings in the vascular wall. If so, the indirect vasoconstrictor action of angiotensin may be similar to that of catecholamine releasing agents, such as tyramine, which are generally considered to act mainly by causing release of endogenous noradrenaline [Burn and Rand (17, 18)]. If so the action of angiotensin, like that of tyramine, should be diminished by reserpine. In the present experiments the action of tyramine on the ear vessels of rabbits pretreated with reserpine was diminised. However, the vasoconstrictor action of angiotensin on the same preparation was enhanced. The action of noradrenaline was enhanced as expected (Figs. 3, 4). These results suggest that angiotensin does not act like tyramine, and that a catecholamine releasing action is not involved in the major action of angiotensin in these experiments. Khairallah and Page (19) showed that adrenergic blocking agents inhibit the response of intestinal smooth muscle to angiotensin, and suggested that this inhibition might be a direct effect on the cell membrane. In our perfusion experiments on isolated rabbit ear vessels, however, imidaline reduced the response to both noradrenaline and tyra mine, but the response to angiotensin was not affected (Fig 5). If an adrenergic mecha nism is involved in the action of angiotensin on vascular smooth muscle, the response to angiotensin should be reduced by this agent. These results indicate that the direct vasoconstrictor action of angiotensin on blood vessels may have nothing to do with the so-called "noradrenaline-receptors". Khairallah and Page (20) reported that the response of intestinal smooth muscle to angiotensin was blocked by atropine. In our experiments, however, doses of atropine sufficient to affect the action of acetylcholine, had no effect on 'the direct action of angiotensin on isolated perfused rabbit ear vessels. From the above results a neurotransmitter appears to have no relation with the mechanism of the direct vasoconstrictor action of angiotensin. In other words , it appears that the vascular receptors on which angiotensin exerts a direct action differ from those concerned with neurotransmission. However, a very interesting effect of angiotensin on the action of noradrenaline was found in our experiments (Fig. 6). Very small amounts of angiotensin , which had no effect on the perfused vessels, greatly enhanced the vasoconstrictor action of noradre
naline.
This
in the
suggests
vasoconstrictor
tensin
which
marked However,
in combination
The
effective
vasoconstrictor (Fig.
had
and
angiotensin
Heller
no significant
and noradrenaline
(21) found
that
effect on isolated
doses of noradrenaline.
action 7).
adrenaline-receptors" noradrenaline
a crude
rabbit
It is considered
was also
and
(2) reported results
in
of endogenous
enhanced
is partly also
in the presence
Page
which
the release
tyramine
augmentation
angiotensin,
tyramine
and
system,
of
This
by
by
McCubbin
cause
Mylon
in itself
between
angio
ear vessels, of noradre were used
that
angiotensin
the "noradrenaline-receptors".
of angiotensin
nervous
is an interaction
vasoconstriction when mixed with subthreshold amounts in our experiments, subthreshold amounts of angiotensin with
sensitize
there
mechanism.
preparation,
produced naline.
may
that
due
may
be due
of angiotensin
that
angiotensin
an enhanced
amounts of "nor
to the increased
has an effect
They
small
release
of
(see below).
response
noradrenaline.
by very
to the sensitization
to agents
suggested
on the sympathetic and
that
procedures
the
that
effect of angio
tensin on the sympathetic nervous system did not appear to involve sensitization "noradrenaline -receptors" , because there was little or no effect of angiotensin on response of the blood pressure to exogenous noradrenaline. But in our experiments enhanced
by
systemic cular
angiotensin.
responsiveness
beds,
some
However,
McCubbin to study
of noradrenaline
in the
and
This
and the
may
administered
be
noradrenaline
due
and
innervated
or
pressure that
and
and
nerve
noradrenaline
to a difference
the
response
denervated
by
sion of angiotensin.
On
cant
increase
urinary
even
great
increase
in
that
endings.
in the urinary and
prolonged
the
attracted
infusion release
was
between
the
in specialized vascular
attention. on
vas
beds,
or to
experiments to clarify
of either
of angiotensin.
of
total
of tyramine
noradrenaline
normetanephrine
and
the
elevation
these
the period resulted
and
urinary
noradrenaline
of the
Therefore, during
it
or release
this question.
total
in spite
of noradrenaline
administration
Therefore, storage
In these
excretion
excretion
the
was estimated
retanephrine,
contrary,
of total
our
of angiotensin
metabolites
normetanephrine
was no increased
in the
suggestions
of infusion
their
increase
total
caused
there
Page's effect
sympathetic
was no significant adrenaline,
suggest
discrepancy
between
of catecholamines
of blood
to exogenously
the
difference.
was of interest
There
response
to exogenous
or to a difference
other
excretion
the
of
results of infu
in a signifi
adrenaline,
metanephrine.
and Thus
an this
phenomenon apparently reflects a catecholamine releasing action of tyramine. From these observations, it is also suggested that angiotensin does not seem to manifest its action
in the same
When excretion This
of these
increment
cholamine from
tyramine
as catecholamine
was infused substances
action studies.
releasing
together
with
was greater
than
was not statistically
releasing
physiological
way
significant,
of tyramine,
agents
such
angiotensin,
the
that but
as McCubbin
when
increase
tyramine
angiotensin and
as tyramine.
Page
in the
alone
was
urinary infused.
may
enhance
the
cate
have
already
suggested
SUMMARY 1.
In
of angiotensin
perfusion was
experiments very
weak
on
isolated
compared
rabbit
with
that
ear
vessels,
the vasoconstrictor
action
of noradrenaline.
2. This action of angiotensin was not affected by either by in vivo pretreatment with reserpine or in vitro pretreatment with imidaline, or atropine. 3. Subthreshold amounts of angiotensin greatly enhanced the response of rabbit ear vessels to noradrenaline, and also to tyramine. 4. The intravenous infusion of angiotensin into rabbits did not produce a signifi cant increase in the urinary catecholamines and their metabolites, but a marked increase was caused by the infusion of tyramine. 5. When a combination of tyramine and angiotensin was infused, a more remarkable increase in urinary catecholamines and their metabolites was observed. These results were discussed, and it was concluded that (i) the vasoconstrictor action of angiotensin is not mediated by neurotransmitters, (ii) angiotensin potentiates the vaso constrictor response to noradrenaline and tyramine, and (iii) in the presence of angio tensin, the catecholamine releasing action of tyramine might be enhanced. Acknowledgements : The authors wish to express their appreciation to ProfessorR. Imaizumi and Assistant ProfessorH. Yoshida for their kind guidanceand helpful suggestionsduring this study and preparation of the manuscript. REFERENCES 1) ZIMMERMAN,B.H. : Circulation Res. 11, 780 (1962) 2) MCCUBBIN,J.W. AND PAGE, I.H. : Ibid. 12, 553 (1963) 3) LEwis, G.P. : Canad. msd. Ass. J. 90, 302 (1964) 4) KRAWKOW-PISSEMSKI : Handb. biol. Arbth. (1923) 5) ITOH, T., MATSUOKA,M., NAKAZIMA,K., TAGAWA,K. AND IMAIZUMI,R.: THIS JOURNAL 12, 130 (1962) 6) NUKADA, T., MATSUOKA,M. AND IMAIZUMI,R. : Ibid. 12, 57 (1962) 7) MATSUOKA,M. : Ibid. 14, 181 (1964) 8) GROUT, J.R., PISANO,J.J. AND SJOERDSMA,A. : Amer. Heart J. 61, 377 (1961) 9) FINNER'rY,F.A. : Circulation 25, 255 (1962) 10) MENDLOWITZ,M., WOLF, R.L., GITLOW, S.E. AND NAFTCHI, N.E. : Ibid. 25, 231 (1962) 11) FURCHGOTT,R.F. : Pharmacol. Rev. 7, 183 (1955) 12) HURWITZ, R., CAMPBELL,R.W., GORDON, P. AND HADDY, F.J.: J. Pharmacol. 133, 57 (1961) 13) HADDY,F.J., MOLNAR, J.I., BORDEN,C.W. AND TEXTER, E.G.: Circulation 25, 239 (1962) 14) LAVERTY,R. : J. Pharm. Pharmacol. 15, 63 (1963) 15) BICKERTON,R.K. AND BUCKLEY,J.P. : Proc. Soc. exp. Biol., N.Y. 106, 834 (1964) 16) BUCKLEY,J.P., BICKERTON,R.K., HALLIDAY,R.P. ANDKATO, H.: Ann. N. Y. Acad. Sci. 104, 299 (1963) 17) BURN, J.H. AND RAND, M.J.: J. Physiol. 144, 314 (1958) 18) BURN, J.H. AND RAND, M.J.: Brit. J. Pharmacol. 15, 56 (1961) 19) KHAIRALLAH,P.A. AND PAGE, I.H.: Amer. J. Physiol. 202, 841 (1962) 20) KHAIRALLAH,P.A. AND PAGE, I.H.: Ibid. 200, 51 (1961) 21) MYLON, E. AND HELLER, J.H.: Pror. Soc. exp. Biol., N.Y. 67, 62 (1948)