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STUDIES ON SYMPATHOMIMETIC ACTION OF ACETALDEHYDE
STUDIES
ON
II.
SYMPATHOMIMETIC
SECRETORY
ACTION
OF ACETALDEHYDE
RESPONSE OF THE ADRENAL
ACETALDEHYDE :
EXPERIMENT
THE PERFUSED
MEDULLA
TO
WITH
CAT ADRENALS
JIRO AKABANE, SUEHIRO NAKANISHI, HIROSHI KOHEI, SUSUMU ASAKAWA, RIICHIRO MATSUMURA, IROSHI OGATA* AND TATSUHIKO H MIYAZAWA Department of Pharmacology, Facultyof Medicine, ShinshuUniversity, Matsumoto Receivedfor publicationJanuary 3, 1965
In the from
previous
store
medulla,
sites
and
the action
hexamethonium In
nor
the present
to acetaldehyde into
paper
located
(1), we have in
or
near
shown
the
of acetaldehyde
that
nerve
acetaldehyde
terminals
released
as well
on the adrenal
medulla
catecholamines
as from
the
was blocked
adrenal
by neither
atropine. paper,
in the
we have perfused
dealt
cat
with the
adrenals,
secretory
in
the
response
hope
of adrenal
of gaining
medulla
further
insight
its mechanism. METHODS Cats weighing
chloralose (2) was
given employed
was to perfuse grade fused
approximately intravenously. with
the
minor
adrenal
3 kg were The
7 at
Hg
35°C.
about
6 ml
mental
(Fig. 1). The
fication 赤羽
outflow
reported
The
through
the
main'
abdominal
the
dose of 90 mg/kg
by Douglas
modification aorta
in the
in contrast
fluid
through
was phosphate-buffered
a cannula
A 20-minute
inserted
period
into
Locke's
the
of perfusion
inferior
was
run
technique
with
a retro We per of about
solution vena
before
of
and Rubin
veins as described by Douglas and Rubin. the abdominal aorta at a constant pressure
perfusion
per minute.
of pH
cava the
was
experi
zero time.
Epinephrine mined
The
with
procedure
modification.
glands
fashion through the adrenal the adrenal glands through
70 mm
anesthetized
perfusion
and
according after
the
治郎 ・中西
* Department
of
norepinephrine
to the
in the perfusion
trihydroxyindole
separation
through
頴 央 ・公 平
宏 ・浅川
Psychiatry
, Faculty
of
method a column 湊 ・松 村
Medicine,
fluids
of Euler of cation
理 一 郎 ・小 片 Shinshu
were and
fluorometrically Flooding
exchanger 寛 ・宮 沢
University,
(3) with
Amberleite 辰彦
Matsumoto.
deter modi CG-50.
FIG.
1.
Schematic
divisions through lected
and the through
presentation the adrenal
distribution glands
a cannula
of
the of
via which
anatomy blood
the
of
the
vessels.
abdominal
is inserted
cat
adrenal,
Locke's aorta into
the
solution and
the
inferior
showing
its
is perfused exflow vena
is col cava.
RESULTS 1) Spontaneousoutput of catecholamines Spontaneous output of catecholamines during perfusion with Locke's solution varied from animal to animal. The output of epinephrine ranged from 2.9 to 7.6 ,ug per 5 minutes with an average of 5 ,cagper 5 minutes and that of norepinephrine ranged from 1.1 to 3.7 jag per 5 minutes with an average of 2.4 ,ug per 5 minutes (10 animals). Thirty minutes later, the output of epinephrine decreased to 2.3 peg per 5 minutes (Fig. 2). In the perfusion with Ca-free Locke's solution a striking reduction in the output of catecholamines was observed ; the output of catecholamines was as low as 0.5 ,ug per 5 minutes at zero time. 2) Secretoryresponseto acetaldehyde Acetaldehyde in a dose of 50 mg injected into the perfusion circuit markedly in tensified the secretion of both epinephrine and norepinephrine. A significant increase in output of catecholamines was also observed in the preparations perfused with Ca free Locke's solution (Fig. 3). When 250 ieg of tyramine was introduced into the per fusion fluid, the output of catecholamine remained at an initial level (Fig. 3). The dose of 100,«g of acetylcholine injected into the perfusion circuit significantly
FIG.
2.
The
time
continuous
course perfusion
E : epinephrine,
NE
of
spontaneous of
both
output
adrenal
of
glands
catecholamines with,
Locke's
during solution.
: norepinephrine.
FIG. 3. Secretory response to tyramine and acetaldehyde. Left : perfusion with Locke's solution. Right : perfusion with Ca-free Locke's solution. A single dose of tyramine (250 pg) and acetaldehyde (50 mg) is injected into the perfusion circuit. Tyr : tyramine, AA : acetaldehyde, E : epinephrine, NE : norepine phrine. intensified noticed
3)
the with
secretion Ca-free
of both Locke's
epinephrine
solution
and
norepinephrine,
but
this effect
was not
(Fig. 4).
Effect of hexamethonium on the secretory action of acetaldehyde When Locke's solution containing
was employed, cantly
increased
choline induced
introduction
hexamethonium
of 50 mg of acetaldehyde
the output of catetholamines,
in a concentration
of 10
into the perfusion
fluid signifi
whereas
no changes in the output of catecholamine
injection
g/ml
of 100 ,ug of acetyl
(Fig. 5).
FIG. 4. Secretory response to acetylcholine and acetaldehyde. Left : perfusion with Locke's solution. Right : perfusion with Ca-free Locke's solution. A single dose of acetylcholine (100 pg) and acetaldehyde (50 mg) is injected into the perfusion circuit. ACh : acetylcholine, AA : acetaldehyde, E : epinephrine, NE: nor epinephrine.
FIG. 5. Effect of hexamethonium on the secretory action of acetylcholine and acetaldehyde. Left : perfusion with Locke's solution containing hexamethonium (10-5 g/ml). Right : perfusion with Ca-free Locke's solution containing hexame thonium (10-5 g/ml). A single dose of acetylcholine (100 pg) and acetaldehyde (50 mg) is injected into the perfusion circuit. C5 : hexamethonium, ACh : acetylcholine, AA : acetaldehyde, E: epinephrine, NE : norepinephrine.
4) Effect of atropineon the secretoryaction of acetaldehyde When Locke's solution containing atro
FIG. 6. Effect of atropine on the secretory action of acetaldehyde. Perfusion with Locke's solution containing atropine (10-4 g/ml) is employed, and then a single dose of acetaldehyde (50 mg) is injected into the perfusion circuit. AA : acetaldehyde, E : epinephrine, NE : norepinephrine.
pine in a concentration of 10-4 g/ml was employed, introduction of 50 mg of acet aldehyde into the perfusion circuit signi ficantly increased the output of catechol amines (Fig. 6). Injection of 100 ,ug of acetylcholine also increased significantly the output of catecholamines. In conclusion, acetaldehyde induced a significant release of catecholamines from the adrenal medulla perfused with ordi nary as well as Ca-free Locke's solution, and in addition, this secretory action was blocked by neither hexamethonium nor atropine. DISCUSSION
Recently Douglas and Rubin (2) have studied how acetylcholine causes the medullary cells to secrete. They have found that the omission of calcium practically abolished the secretory response of the adrenal medulla to acetylcholine. Thus, they have suggested that the role of acetylcholine as a trans mitter at the adrenal medulla is to cause some brief changes in medullary cells which allow calcium ions to penetrate and trigger the catecholamine release process. In our previous paper (1) it was reported that intraarterial injection of acetaldehyde close to the adrenal gland induced a pronounced pressor response and a marked nictitating membrane response. We have, therefore, concluded that acetaldehyde has an action, ganglion-stimulating action in a broad sense and that this effect, however, is not blocked by hexamethonium and atropine. In the present study we have confirmed that acetaldehyde induces a striking increase in secretion of epinephrine and norepinephrine from the adrenal medulla , and that this effect is observed even in the preparations perfused with Ca-free Locke's solution and is not blocked by hexamethonium and atropine. Therefore, acetaldehyde clearly evokes adrenal medullary secretion through a calcium-independent process. This find ing suggests that acetaldehyde may exert directly upon the meduallary cells and trigger the catecholamine release process as contrasted with acetylcholine. The effect of acetal dehyde on the medullary cells seems closely correlated to a membrane phenomenon. Douglas and Rubin (2) have discussed that although calcium is obviously to evoke cate cholamine secretion in the whole gland, the addition of calcium 1 mm does not increase the release of catecholamines from the isolated adrenal medullary granules suspended in
sucrose (4). Therefore, there are grounds for believing that the process of secretion oc curring in the whole cell does not involve the intracellular disruption of amine-containing granules or indeed the leakage of catecholamines from them. On the contrary, Schumann and Philippu (5) have reported that in incubation experiments with isolated chromaffin granules of adrenal medulla of cattle, calcium produces a dose dependent release of catecholamines and ATP at 0 and 37°C. It might be speculated that acetaldehyde acts on the membrane of structures which store catecholamines, and subsequently releases catecholamines. Regarding the action of tyramine on the adrenal secretion Burn (6) suggested that tyramine causes some release of epinephrine from the adrenal medulla, although it was not very much. Philippu and Schumann (7) observed that tyramine and phenylethyl amine induced an increase in release of catecholamines from the cattle adrenal medulla in perfusion experiments with normal and Ca-free Tyrode solution. On the contrary, Stroemblad (8) perfused the cat adrenal gland with tyramine solution and he could not obtain any evidence for releasing action. Schumann and Philippu (9) incubated chrom affin granules from adrenal medulla of cattle in isotonic sucrose-phosphate buffer of pH 6.8 at 37°C in air and observed that the amount of the released catecholamines under the influence of tyramine was replaced stoichiometrically by an uptake of tyramine. In the present study tyramine failed to show significant action on the adrenal secretion, distinctly differing from acetaldehyde. SUMMARY Investigations were made to elucidate the mechanism involved in the secretory response of the adrenal medulla to acetaldehyde with the perfused cat adrenals. 1. Acetaldehyde injected into the perfusion circuit markedly intensified the secretion of both epinephrine and norepinephrine from the adrenal medulla. When tyramine was introduced into the perfusion fluid, output of catecholamine remained at the control level. 2. A significant increase in output of catecholamines by injection of acetaldehyde was also observed in the preparations perfused with Ca-free Locke's solution and this action of acetaldehyde was blocked by neither hexamethonium or atropine. REFERENCES 1) AKABANE, J., NAKANISHI, S., KO)HEI, H., MATSUMURA, R. ANDOGATA, H. : THISJOURNAL 14,.295 (1964) 2) DOUGLAS, W.W.ANDRUBIN,R.P.: J. Physiol.159, 40 (1961) 3) V. EULER, U.S. ANDFLOODING, I.: Scand.J. clin. Lab.Invest.7, 1 (1955) 4) HILLARP, N.A.: Actaphysiol.stand.43, 292 (1958) 5) SCHUMANN, H.J. ANDPHILIPPU, A. : Arch.exp.Path.Pharmak.244, 466 (1963) 6) BURN, J.H. : Adrenergic Mechanisms p. 491, Churchill, London (1960) 7) PHILIPPU, A. ANDSCHUMANN, H.J. : Experientia 18, 138 (1962) 8) STROEMBLAD, B.C.R.: Brit.J. Pharmacol. 15, 328 (1960) 9) SCHUMANN, H.J. ANDPHILIPPU, A.: Arch.exp.Path. Pharrrak.241 (1961)
ON
II.
SYMPATHOMIMETIC
SECRETORY
ACTION
OF ACETALDEHYDE
RESPONSE OF THE ADRENAL
ACETALDEHYDE :
EXPERIMENT
THE PERFUSED
MEDULLA
TO
WITH
CAT ADRENALS
JIRO AKABANE, SUEHIRO NAKANISHI, HIROSHI KOHEI, SUSUMU ASAKAWA, RIICHIRO MATSUMURA, IROSHI OGATA* AND TATSUHIKO H MIYAZAWA Department of Pharmacology, Facultyof Medicine, ShinshuUniversity, Matsumoto Receivedfor publicationJanuary 3, 1965
In the from
previous
store
medulla,
sites
and
the action
hexamethonium In
nor
the present
to acetaldehyde into
paper
located
(1), we have in
or
near
shown
the
of acetaldehyde
that
nerve
acetaldehyde
terminals
released
as well
on the adrenal
medulla
catecholamines
as from
the
was blocked
adrenal
by neither
atropine. paper,
in the
we have perfused
dealt
cat
with the
adrenals,
secretory
in
the
response
hope
of adrenal
of gaining
medulla
further
insight
its mechanism. METHODS Cats weighing
chloralose (2) was
given employed
was to perfuse grade fused
approximately intravenously. with
the
minor
adrenal
3 kg were The
7 at
Hg
35°C.
about
6 ml
mental
(Fig. 1). The
fication 赤羽
outflow
reported
The
through
the
main'
abdominal
the
dose of 90 mg/kg
by Douglas
modification aorta
in the
in contrast
fluid
through
was phosphate-buffered
a cannula
A 20-minute
inserted
period
into
Locke's
the
of perfusion
inferior
was
run
technique
with
a retro We per of about
solution vena
before
of
and Rubin
veins as described by Douglas and Rubin. the abdominal aorta at a constant pressure
perfusion
per minute.
of pH
cava the
was
experi
zero time.
Epinephrine mined
The
with
procedure
modification.
glands
fashion through the adrenal the adrenal glands through
70 mm
anesthetized
perfusion
and
according after
the
治郎 ・中西
* Department
of
norepinephrine
to the
in the perfusion
trihydroxyindole
separation
through
頴 央 ・公 平
宏 ・浅川
Psychiatry
, Faculty
of
method a column 湊 ・松 村
Medicine,
fluids
of Euler of cation
理 一 郎 ・小 片 Shinshu
were and
fluorometrically Flooding
exchanger 寛 ・宮 沢
University,
(3) with
Amberleite 辰彦
Matsumoto.
deter modi CG-50.
FIG.
1.
Schematic
divisions through lected
and the through
presentation the adrenal
distribution glands
a cannula
of
the of
via which
anatomy blood
the
of
the
vessels.
abdominal
is inserted
cat
adrenal,
Locke's aorta into
the
solution and
the
inferior
showing
its
is perfused exflow vena
is col cava.
RESULTS 1) Spontaneousoutput of catecholamines Spontaneous output of catecholamines during perfusion with Locke's solution varied from animal to animal. The output of epinephrine ranged from 2.9 to 7.6 ,ug per 5 minutes with an average of 5 ,cagper 5 minutes and that of norepinephrine ranged from 1.1 to 3.7 jag per 5 minutes with an average of 2.4 ,ug per 5 minutes (10 animals). Thirty minutes later, the output of epinephrine decreased to 2.3 peg per 5 minutes (Fig. 2). In the perfusion with Ca-free Locke's solution a striking reduction in the output of catecholamines was observed ; the output of catecholamines was as low as 0.5 ,ug per 5 minutes at zero time. 2) Secretoryresponseto acetaldehyde Acetaldehyde in a dose of 50 mg injected into the perfusion circuit markedly in tensified the secretion of both epinephrine and norepinephrine. A significant increase in output of catecholamines was also observed in the preparations perfused with Ca free Locke's solution (Fig. 3). When 250 ieg of tyramine was introduced into the per fusion fluid, the output of catecholamine remained at an initial level (Fig. 3). The dose of 100,«g of acetylcholine injected into the perfusion circuit significantly
FIG.
2.
The
time
continuous
course perfusion
E : epinephrine,
NE
of
spontaneous of
both
output
adrenal
of
glands
catecholamines with,
Locke's
during solution.
: norepinephrine.
FIG. 3. Secretory response to tyramine and acetaldehyde. Left : perfusion with Locke's solution. Right : perfusion with Ca-free Locke's solution. A single dose of tyramine (250 pg) and acetaldehyde (50 mg) is injected into the perfusion circuit. Tyr : tyramine, AA : acetaldehyde, E : epinephrine, NE : norepine phrine. intensified noticed
3)
the with
secretion Ca-free
of both Locke's
epinephrine
solution
and
norepinephrine,
but
this effect
was not
(Fig. 4).
Effect of hexamethonium on the secretory action of acetaldehyde When Locke's solution containing
was employed, cantly
increased
choline induced
introduction
hexamethonium
of 50 mg of acetaldehyde
the output of catetholamines,
in a concentration
of 10
into the perfusion
fluid signifi
whereas
no changes in the output of catecholamine
injection
g/ml
of 100 ,ug of acetyl
(Fig. 5).
FIG. 4. Secretory response to acetylcholine and acetaldehyde. Left : perfusion with Locke's solution. Right : perfusion with Ca-free Locke's solution. A single dose of acetylcholine (100 pg) and acetaldehyde (50 mg) is injected into the perfusion circuit. ACh : acetylcholine, AA : acetaldehyde, E : epinephrine, NE: nor epinephrine.
FIG. 5. Effect of hexamethonium on the secretory action of acetylcholine and acetaldehyde. Left : perfusion with Locke's solution containing hexamethonium (10-5 g/ml). Right : perfusion with Ca-free Locke's solution containing hexame thonium (10-5 g/ml). A single dose of acetylcholine (100 pg) and acetaldehyde (50 mg) is injected into the perfusion circuit. C5 : hexamethonium, ACh : acetylcholine, AA : acetaldehyde, E: epinephrine, NE : norepinephrine.
4) Effect of atropineon the secretoryaction of acetaldehyde When Locke's solution containing atro
FIG. 6. Effect of atropine on the secretory action of acetaldehyde. Perfusion with Locke's solution containing atropine (10-4 g/ml) is employed, and then a single dose of acetaldehyde (50 mg) is injected into the perfusion circuit. AA : acetaldehyde, E : epinephrine, NE : norepinephrine.
pine in a concentration of 10-4 g/ml was employed, introduction of 50 mg of acet aldehyde into the perfusion circuit signi ficantly increased the output of catechol amines (Fig. 6). Injection of 100 ,ug of acetylcholine also increased significantly the output of catecholamines. In conclusion, acetaldehyde induced a significant release of catecholamines from the adrenal medulla perfused with ordi nary as well as Ca-free Locke's solution, and in addition, this secretory action was blocked by neither hexamethonium nor atropine. DISCUSSION
Recently Douglas and Rubin (2) have studied how acetylcholine causes the medullary cells to secrete. They have found that the omission of calcium practically abolished the secretory response of the adrenal medulla to acetylcholine. Thus, they have suggested that the role of acetylcholine as a trans mitter at the adrenal medulla is to cause some brief changes in medullary cells which allow calcium ions to penetrate and trigger the catecholamine release process. In our previous paper (1) it was reported that intraarterial injection of acetaldehyde close to the adrenal gland induced a pronounced pressor response and a marked nictitating membrane response. We have, therefore, concluded that acetaldehyde has an action, ganglion-stimulating action in a broad sense and that this effect, however, is not blocked by hexamethonium and atropine. In the present study we have confirmed that acetaldehyde induces a striking increase in secretion of epinephrine and norepinephrine from the adrenal medulla , and that this effect is observed even in the preparations perfused with Ca-free Locke's solution and is not blocked by hexamethonium and atropine. Therefore, acetaldehyde clearly evokes adrenal medullary secretion through a calcium-independent process. This find ing suggests that acetaldehyde may exert directly upon the meduallary cells and trigger the catecholamine release process as contrasted with acetylcholine. The effect of acetal dehyde on the medullary cells seems closely correlated to a membrane phenomenon. Douglas and Rubin (2) have discussed that although calcium is obviously to evoke cate cholamine secretion in the whole gland, the addition of calcium 1 mm does not increase the release of catecholamines from the isolated adrenal medullary granules suspended in
sucrose (4). Therefore, there are grounds for believing that the process of secretion oc curring in the whole cell does not involve the intracellular disruption of amine-containing granules or indeed the leakage of catecholamines from them. On the contrary, Schumann and Philippu (5) have reported that in incubation experiments with isolated chromaffin granules of adrenal medulla of cattle, calcium produces a dose dependent release of catecholamines and ATP at 0 and 37°C. It might be speculated that acetaldehyde acts on the membrane of structures which store catecholamines, and subsequently releases catecholamines. Regarding the action of tyramine on the adrenal secretion Burn (6) suggested that tyramine causes some release of epinephrine from the adrenal medulla, although it was not very much. Philippu and Schumann (7) observed that tyramine and phenylethyl amine induced an increase in release of catecholamines from the cattle adrenal medulla in perfusion experiments with normal and Ca-free Tyrode solution. On the contrary, Stroemblad (8) perfused the cat adrenal gland with tyramine solution and he could not obtain any evidence for releasing action. Schumann and Philippu (9) incubated chrom affin granules from adrenal medulla of cattle in isotonic sucrose-phosphate buffer of pH 6.8 at 37°C in air and observed that the amount of the released catecholamines under the influence of tyramine was replaced stoichiometrically by an uptake of tyramine. In the present study tyramine failed to show significant action on the adrenal secretion, distinctly differing from acetaldehyde. SUMMARY Investigations were made to elucidate the mechanism involved in the secretory response of the adrenal medulla to acetaldehyde with the perfused cat adrenals. 1. Acetaldehyde injected into the perfusion circuit markedly intensified the secretion of both epinephrine and norepinephrine from the adrenal medulla. When tyramine was introduced into the perfusion fluid, output of catecholamine remained at the control level. 2. A significant increase in output of catecholamines by injection of acetaldehyde was also observed in the preparations perfused with Ca-free Locke's solution and this action of acetaldehyde was blocked by neither hexamethonium or atropine. REFERENCES 1) AKABANE, J., NAKANISHI, S., KO)HEI, H., MATSUMURA, R. ANDOGATA, H. : THISJOURNAL 14,.295 (1964) 2) DOUGLAS, W.W.ANDRUBIN,R.P.: J. Physiol.159, 40 (1961) 3) V. EULER, U.S. ANDFLOODING, I.: Scand.J. clin. Lab.Invest.7, 1 (1955) 4) HILLARP, N.A.: Actaphysiol.stand.43, 292 (1958) 5) SCHUMANN, H.J. ANDPHILIPPU, A. : Arch.exp.Path.Pharmak.244, 466 (1963) 6) BURN, J.H. : Adrenergic Mechanisms p. 491, Churchill, London (1960) 7) PHILIPPU, A. ANDSCHUMANN, H.J. : Experientia 18, 138 (1962) 8) STROEMBLAD, B.C.R.: Brit.J. Pharmacol. 15, 328 (1960) 9) SCHUMANN, H.J. ANDPHILIPPU, A.: Arch.exp.Path. Pharrrak.241 (1961)