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THE EFFECT OF CARBON TETRACHLORIDE ON PLASMA CORTICOSTERONE LEVEL
THE
EFFECT
OF
PLASMA
CARBON
TETRACHLORIDE
CORTICOSTERONE
ON
LEVEL
TATSUO FURUKAWA Department of Pharmacology, Wakayama MedicalCollege,Wakayama Receivedfor publicationSeptember24, 1965
Carbon
tetrachloride
hydrocarbon,
has been
used
as an
(CCl4) well
anesthetic.
has a narcotic
as
known
well
CCl4 likewise
effect
as chloroform
to induce acts
liver
directly
even
though
the potency
that
CHCl3
induced
(CHCl3),
damage. on
which
CHCl3
the
central
is considerably
is chlorinated
however
nervous
weaker
has been system
than
that
and
of CHCl3
(1). It
was
reported
glands (2). by CHCl3. On
This
the
the adrenal of CCl4
this
hand, and
these
stimulates
adrenocorticotropic concentration
So they
catecholamine
phenomenon From
and
other glands
(3, 4).
stimulates
observation
Brody
imply
et al.
an increase
observed
that
from the
be involved
a release
of corticotropin
hormone of adrenocortical
(ACTH)
and
nerve
mechanism that
CCl4
releasing
MATERIAL
might
acid
factor
following on
directly from
adrenal axis
contents
in
administration
the hypothalamus
and the adrenal
gland.
the
hypophyseo-adrenal
of CCl4-induced acts
in
of catecholamine urine
CCl4 acts directly
from the pituitary
hormone
of the
a decrease
plasma
sympathetic
in the
it can be presumed
of ascorbic
an activation
in the
have assumed release
might facts,
might
a reduction
and
glands,
and
toxicity.
on the
hypothalamus
the hypothalamus Consequently
and
of
the plasma
be increased.
AND METHODS
The experiments were carried out using female Sprague-Dawley or albino Holtzman rats which ranged in weight from 180 to 220 g. CCl4 was administered orally by a stomach tube with the same volume of peanut oil. The animals were anesthetized by the intraperitoneal injection of hexobarbital in a dose of 100 mg/kg in the rat cage, and, immediately, the blood was collected by syringe from the vena cava inferior just below the diaphragm at 10 A.M. All these experimental procedures were undertaken in a quiet animal room unless otherwise specially indicated. Corticosterone in the plasma was assayed by the method of Zenker and Bernstein (5). Surgical procedures, which have been proposed as being effective in preventing CCl4 toxicity (6, 7), were performed under diethyl ether anesthesia. The adrenal glands were 古川 達雄
removed by dorsal approach, then the rats were supplied with 1% saline until sacrifice. The pituitary gland was removed by suction with a syringe and needle through the tympanic bulla. The spinal cord was transected at the level of C,, and the animals were fed crumbled food pellets and given water by periodic subcutaneous injections. The animals which were operated on were used for experiments from 5 days to 10 days after adrenalectomy or hypophysectomy and 2 days after cord-section. RESULTS Corticosterone level in the plasma was elevated as early as 1 hour following the stress of insertion of stomach tube and oral administration of peanut oil. This elevation was maintained for 5 hours although gradually diminishing over the period. It finally returned to normal 10 hours after the stress. CCI,, given in a dose of 1.5 ml/kg, induced an increase of plasma corticosterone throughout the period observed and, in using 2.5 ml/kg, a similar result was obtained though the response appeared to be a little stronger (Table 1). TABLE
1.
Effect
sacrificed
of in
pg/dl of plasma, mean+standard
CCI,
on
a quiet
plasma
corticosterone
animal
quarter
at
level
in
rats
10 A.M.
error
The hypophyseo-adrenal function has diurnal variation and is very susceptible to external stimuli such as the emotional stress of a strange environment. So the corticoid level elevates progressively in day time though it is rather lower in the morning (8). It likewise rises when the animals are moved from the animal quarter to the laboratory. In the following case, the corticosterone levels in the control rats were nearly 2 times higher than that in the rats which were sacrificed in the quiet animal quarter at 10 A.M. The rats were brought up to the laboratory just before the administration of CC1, or peanut oil and then the experimental procedures were undertaken. They had been left in the laboratory until collecting the blood under hexobarbital anesthesia at 4 P.M. The control rats were moved before sacrifice. Even in this case, CC1, induced an eleva tion of corticosterone levels and the high level was maintained for 10 hour period. After that the values showed lower levels than that of the control rats during 20 and 40 hour period (Table 2). Assay of plasma corticosterone revealed that adrenalectomy as well as hypophysec tomy markedly lowered the levels and completely eliminated the corticoid elevation normally observed after CC1, (Table 3).
TABLE
2.
Effect
sacrificed
Morphine
of
CCI,
in
the
has been proposed
pituitary-adrenal
hormonal
on
plasma
laboratory
corticosterone
level
in
rats
at 4 P.M.
as inhibiting
response
system (9-11), so different
to additional
administrations
stimuli
in the
of morphine
were
previously used as suggested by theses authors, and yet CCI, induced a similar elevation of corticosterone level as seen in the normal rats (Table 4). Morphine, ministration
reserpine
and
cortisone
were administered
of these drugs for 2 days was effective in depressing
induced by CCI, (Table 5), while the administration TABLE
3. or
repeatedly.
Effect of CCI, hypophysectomized
in
The repeated
the corticoid
of morphine
ad
elevation
in twice did not affect
the effect of CC14 (Table 4).
the adrenalectomized rats.
Surgical
trauma
likewise
stimu
lates the hypophyseo-adrenal
system.
The values of corticosterone
levels in
the
were
78.6±5.4
,ug/dl (n=;4) at 1 hour and
42.9±2.3
plasma
assayed
teg/dl (n=4) at 24 hour period after spinal cord transection. CCI, admi nistered The blood rats 5 hours hours
after
was collected from the adrenalectomized and from the hypophysectomized rats the
administration TABLE
4. of
Influence plasma
of
peanut of
the
corticosterone
oil
after
bring about
2
corticosterone
or CCI,.
pretreatment
with
levels
induced
morphine by
on
the operation the elevation
did not in plasma
levels (Table 6). the
elevation
CCI,.
1) CCI, or peanut oil was administered 10 minutes following the injection of morphine (20 mg/kg, i.p.) and, 2 hours after that , the blood was collected. 2) CCI, or peanut oil was given 1 hour following the injection of morphine (20 mg/kg, s.c.) and the blood was taken 1 hour later. 3) CCI4 or peanut oil was given 10 minutes following the admi nistration of morphine (40 mg/kg, i.p.) and the blood was ob tained 2 hours later. 4) Morphine (4 mg/kg, i.p.) was injected 11 hours and 5 hours prior to the administration of peanut oil or CC14 and the blood was collected 2 hours after CCI,.
On
the
ministration
other
hand,
of CCI,
was
the
previous
found
effective
treatment
for
elevation
of
plasma
corticosterone
morphine
or histamine
induced
by
to
ad
TABLE
be an
preventing
the
5.
Effects
of
morphine,
cortisone
administered
elevation
of plasma
induced
by
reserpine
and
repeatedly
on
corticosterone
the
levels
CCI,.
levels (Table
7).
DISCUSSION It has been reported that morphine administered in once is shown to inhibit the activation of ACTH release induced by several drugs or surgical trauma (9-11), by determining ascorbic acid contents in the adrenal glands. However, after one or two administration of morphine which stimu lates the hypophyseo-adrenal hormonal system by itself, CC14 likewise showed an increase of the plasma corticoid, though the hormone level is already fairly high by morphine. Montanari et al. observed that an increase of plasma corticosterone did not always accompany a reduction of adrenal ascorbic acid (12). This finding casts doubt on the value of adrenal ascorbic acid deter mination as an index of ACTH secretion
Morphine (50 mg/kg s.c.) 3 times a day, reserpine (5 mg/kg s.c.) or cortisone acetate mg/kg s.c.) once a day was administered 2 days. The blood was collected 2 hours after administration of peanut oil or CC14. TABLE
6.
The
blood
TABLE
(15, 16). Smelik however gical concentration
They therefore
proposed
opposed the theory and
of the corticoids
CCI4
of
7.
on
CC14
of
which
the
cortico had
been
on.
1
or
of
peanut
prior on
plasma
levels
plasma
rats
collected CCI,
Effect
with
or
in
operated
was
administration
physeo-adrenal activity under conditions of stress, Yates et al. conducted experiments on animals into which they had injected corticosteroid in varying doses in order to elevate the plasma concentration before animal was exposed to experimental stimuli, and they found that the hormonal system lies under proportional closed-loop control set point.
of level
previously
(12, 13). So morphine does not seem to in hibit the CC14-induced pituitary-adrenal activation, and this result might agree with the observation of Rerup (14). Concerning regulation of the hypo
with variable
Effect
sterone
and (60 for
the
hour
after
the
oil.
treatment elevation
corticosterone
induced
by
morphine
histamine.
Morphine (30 mg/kg i.p.) or his tamine (60 mg/kg i.p.) was injected 19 hours after the administration of peanut oil or CC14i and the blood was obtained 1 hour later.
the variable proposed
had an inhibitory
set point
that
control theory
only a supraphysiolo
effect on acute release of cortico
tropin (17). The results obtained by the administration of CCI, to morphine-pretreated rats showed that the CCl4-induced high level was similar to that observed normally and did not depend on the initial level of the hormone. These results suggest that the variable set point control mechanism play a part in regulating the hypophyseo-adrenal system under the conditions of drugs-induced stimuli. The repeated administration of morphine, reserpine and cortisone for 2 days, or sur gical trauma, was found to eliminate the elevation of plasma corticosterone levels normally induced by CC14,and CC14 likewise inhibited the morphine or histamine-induced rise in the circulating hormone. These treatments are well-known to evoke hypersecretion of ACTH, so a reduction of ACTH in the hypophysis caused by a large dose or prolonged treatment of the drugs has been reported (18-21). Brodie et al. have proposed that the rate of ACTH discharge following reserpine is greater than the rate of its synthesis and the discharge results in a condition in which animals can not respond to an additional pituitary stimulus (22). Although only the prolonged treatment by morphine inhibits an elevation of the corticoid, ACTH contents in the pituitary following morphine have not been reported in detail. Corticosteroid administered daily likewise has been shown to induce a fall of pituitary ACTH concentration (20, 23, 24). However Vernikos Danellis demonstrated that a single blocking dose of hydrocortisone prevented both release and increase in pituitary ACTH contents after stress but had no effect on the resting level in the blood and gland (25). The facts therefore appear to be that the synthesis and secretion of ACTH in the gland occur differently in resting stage and in response to stress, and that the corticoid inhibit the stress-induced ACTH release, not by depressing pituitary ACTH stores but by interfering with ability of the gland to synthesize new ACTH (26-30). Further studies are needed to clarify the mechanism by which CCI, inhibits the additional hypophyseo-adrenal activation. There still remains the question whether CC14 acts directly on the hypothalamus and accelerates the pituitary-adrenal function, or whether the activation of adrenocortical hormone secretion caused by CCI, is simply a physiological response to CC14-induced injury, or whether it is further involved in the mechanism of its toxicity. Although it is almost impossible to know exactly the time course or the onset of biological alteration induced by CC14,the apparent elevation of plasma corticosterone level was observed as early as 1 hour after CC14,and electronmicroscopical observation revealed that a patho logical alteration of the liver occurred in the endoplasmic reticulum within 1 or 2 hour period (31, 32). There is no evidence, of course, that the hypophyseo-adrenal activation occurs before the appearance of the other biological alterations, such as the liver damage. On the other hand, reports, proposing that ACTH release was accelerated by narcotics or sedatives such as ether (33), CHC13 (2), Rauwolfia alkaloids and phenothiazine, have been presented. Besides, of a number of Rauwolf a alkaloids, only those compounds which produce sedation also evoke ACTH hypersecretion (22), and phenothiazine deriva tives likewise cause ACTH hypersecretion but do so only in sedative compounds (34). The reason why such central depressants induce the hypophyseo-adrenal activation is
not clear. It is however possible that the secretion of the humoral agent is controlled by both inhibitory and stimulatory pathways in the hypothalamus (35). Therefore the activation of stimulatory pathways which is probably caused by stress, or the depression of inhibitory pathways which is presumably induced by central depressants, might result in the acceleration of ACTH discharge. Although it is difficult to say whether CCI, acts primarily on the hypothalamus, or whether the stimulation of the hypophyseo-adrenal axis is physiological response to the CCI,-induced injury, it is tempting to speculate that CCI, acts directly on the central nervous system and stimulates a release of corticotropin releasing factor from the hypothalamus through its narcotic effect like CHC13. If so, it is likely that the CCI,-induced injury is related to the pituitary-adrenal activation as non-specific stress in later period. Experiments done by this author show that corticoids hormone is necessary for full development of the CC14-induced toxicity presumably by affecting the hepatic functions and sensitizing them to attack with CCI„ and that certain contents of the hormone is necessary for that (36). Although the adrenocortical hormone modifies the effect of CCI, in its toxicity, the increase of the corticoid secretion by CCI, might not be involved es sentially in the mechanism of its toxicity. SUMMARY CCI, induced an elevation of plasma corticosterone level in rats. This phenomenon was observed even in rats in which the corticoid level was fairly high, resulting from emotional and diurnal variations. Although the administrations of morphine in once or twice did not inhibit the elevation of the corticoid level induced by CCI,, the repeated administrations of morphine, reserpine or cortisone for 2 days almost inhibited the eleva tion normally induced by CC1,. Surgical trauma likewise prevented this elevation. The administration of CCI, was effective in depressing the elevation of corticosterone level induced by morphine or histamine. The experiment is discussed with regard to possible mechanism involved in the CCI, toxicity. Acknowledgement : My sincereappreciationis due to Dr. T.M. Brody. REFERENCES 1) GOODMAN, L.S. ANDGILMAN, A. : The Pharmacological Basisof Therapeutics, p. 1140.MacmillanCo., New York (1958) 2) SAYERS, G. ANDSAYERS, M.A.: Ann.N.Y. Acad . Sci.50, 521 (1949) 3) BRODY, T.M. ANDCALVERT, D.N.: Amer.J. Physiol.198, 682 (1960) 4) STERN, P.H. ANDBRODY, T.M.: J. Pharmacol. 141, 65 (1963) 5) ZENKER, N. ANDBERNSTEIN, D.E.: J. biol.Chem.231, 695 (1958) 6) CALVERT, D.N. ANDBRODY, T.M.: Amer . J. Physiol.198, 669 (1960) 7) BRODY, T.M., CALVERT, D.N. ANDSCHNEIDER, A.F. : J. Pharmacol. 131, 341 (1961) 8) SLUSHER, M.A. ANDBROWING, B.: Amer.J. Physiol . 200, 1032(1961) 9) BRIGGS, F.N. ANDMUNSON, P.L. : Endocrinology 57, 205 (1955)
10) OHLER, F.A. AND SEVY, R.W. : Ibid. 59, 347 (1956) 11) BURDETTE,B.H., LEEMAN,S. AND MUNSON, P.L. : J. Pharmacol. 132, 323 (1961) 12) MONTANARI,R. AND STOCKHAM,M.A.: Brit. J. Pharmacol. 18, 337 (1962) 13) SLUSHER,M.A. AND ROBERTS,S. : Endocrinology 61, 98 (1957) 14) RERUP, K. : Acta endocr., Kbh. 49, 387 (1964) 15) YATES, F.E., LEEMAN, S.E., GLENISTER,D.W. AND DALLMAN,M.F. : Endocrinology 69, 67 (1961) 16) YATES, F.E. AND URQUHART,J. : Physiol. Rev. 42, 359 (1962) 17) SMELIK, P.G.: Acta endocr., Kbh. 44, 36 (1963) 18) KITAY, J.I., HOLUB, D.A. AND JAILER, J.W. : Endocrinology 65, 548 (1959) 19) SAFFRAN,M. AND VOGT, M.: Brit. J. Pharmacol. 15, 165 (1960) 20) FORTIER, C.: Proc. Soc. exp. Biol., N.Y. 100, 16 (1959) 21) HOLUB, D.A., KITAY, J.I. AND JAILER, J.W. : J. clin. Invest. 38, 291 (1959) 22) MAICKEL,R.P., WESTERMANN,E.O. AND BRODIE,B.B. : J. Pharmacol. 134, 167 (1961) 23) FARRELL,G.L. AND LEQUEUR, G. : Endocrinology 56, 471 (1955) 24) VERNIKOS-DANELLIS, J. AND MARKS, B.H. : Proc. Soc. exp. Biol., N.Y. 109, 10 (1962) 25) VERNIKOS-DANELLIS, J. : Endocrinology 72, 574 (1963) 26) HODGES,J.R. AND VERNIKOS-DANELLIS, J.: J. Physiol. 150, 683 (1960) 27) HODGES,J.R. AND VERNIKOS-DANELLIS, J.: Acta endocr., Kbh. 39, 79 (1962) 28) MARKS, B.H. AND VERNIKOS-DANELLIS, J.: Endocrinology 72, 582 (1963) 29) SCHAPIRO,S. : Ibid. 71, 986 (1962) 30) JACOBOWITZ,D., MARKS, B.H. AND VERNIKOS-DANELLIS, J.: Ibid. 72, 592 (1963) 31) BASSI, M. : Exp. Cell Res. 20, 313 (1960) 32) SMUCKLER,E.A., ISERI, O.A. AND BENDITT,E.P.: J. exp. Med. 116, 55 (1962) 33) SAYERS,G. AND BURKS, R.: Fed. Proc. 14, 131 (1955) 34) SMITH, R.L., MAICKEL, R.P. AND BRODIE,B.B.: J. Pharmacol. 139, 185 (1963) 35) EGDAHL,R.H.: Endocrinology 66, 200 (1960) 36) FURUKAWA,T.: Wakayama Med. Rep. 9, 203 (1965)
EFFECT
OF
PLASMA
CARBON
TETRACHLORIDE
CORTICOSTERONE
ON
LEVEL
TATSUO FURUKAWA Department of Pharmacology, Wakayama MedicalCollege,Wakayama Receivedfor publicationSeptember24, 1965
Carbon
tetrachloride
hydrocarbon,
has been
used
as an
(CCl4) well
anesthetic.
has a narcotic
as
known
well
CCl4 likewise
effect
as chloroform
to induce acts
liver
directly
even
though
the potency
that
CHCl3
induced
(CHCl3),
damage. on
which
CHCl3
the
central
is considerably
is chlorinated
however
nervous
weaker
has been system
than
that
and
of CHCl3
(1). It
was
reported
glands (2). by CHCl3. On
This
the
the adrenal of CCl4
this
hand, and
these
stimulates
adrenocorticotropic concentration
So they
catecholamine
phenomenon From
and
other glands
(3, 4).
stimulates
observation
Brody
imply
et al.
an increase
observed
that
from the
be involved
a release
of corticotropin
hormone of adrenocortical
(ACTH)
and
nerve
mechanism that
CCl4
releasing
MATERIAL
might
acid
factor
following on
directly from
adrenal axis
contents
in
administration
the hypothalamus
and the adrenal
gland.
the
hypophyseo-adrenal
of CCl4-induced acts
in
of catecholamine urine
CCl4 acts directly
from the pituitary
hormone
of the
a decrease
plasma
sympathetic
in the
it can be presumed
of ascorbic
an activation
in the
have assumed release
might facts,
might
a reduction
and
glands,
and
toxicity.
on the
hypothalamus
the hypothalamus Consequently
and
of
the plasma
be increased.
AND METHODS
The experiments were carried out using female Sprague-Dawley or albino Holtzman rats which ranged in weight from 180 to 220 g. CCl4 was administered orally by a stomach tube with the same volume of peanut oil. The animals were anesthetized by the intraperitoneal injection of hexobarbital in a dose of 100 mg/kg in the rat cage, and, immediately, the blood was collected by syringe from the vena cava inferior just below the diaphragm at 10 A.M. All these experimental procedures were undertaken in a quiet animal room unless otherwise specially indicated. Corticosterone in the plasma was assayed by the method of Zenker and Bernstein (5). Surgical procedures, which have been proposed as being effective in preventing CCl4 toxicity (6, 7), were performed under diethyl ether anesthesia. The adrenal glands were 古川 達雄
removed by dorsal approach, then the rats were supplied with 1% saline until sacrifice. The pituitary gland was removed by suction with a syringe and needle through the tympanic bulla. The spinal cord was transected at the level of C,, and the animals were fed crumbled food pellets and given water by periodic subcutaneous injections. The animals which were operated on were used for experiments from 5 days to 10 days after adrenalectomy or hypophysectomy and 2 days after cord-section. RESULTS Corticosterone level in the plasma was elevated as early as 1 hour following the stress of insertion of stomach tube and oral administration of peanut oil. This elevation was maintained for 5 hours although gradually diminishing over the period. It finally returned to normal 10 hours after the stress. CCI,, given in a dose of 1.5 ml/kg, induced an increase of plasma corticosterone throughout the period observed and, in using 2.5 ml/kg, a similar result was obtained though the response appeared to be a little stronger (Table 1). TABLE
1.
Effect
sacrificed
of in
pg/dl of plasma, mean+standard
CCI,
on
a quiet
plasma
corticosterone
animal
quarter
at
level
in
rats
10 A.M.
error
The hypophyseo-adrenal function has diurnal variation and is very susceptible to external stimuli such as the emotional stress of a strange environment. So the corticoid level elevates progressively in day time though it is rather lower in the morning (8). It likewise rises when the animals are moved from the animal quarter to the laboratory. In the following case, the corticosterone levels in the control rats were nearly 2 times higher than that in the rats which were sacrificed in the quiet animal quarter at 10 A.M. The rats were brought up to the laboratory just before the administration of CC1, or peanut oil and then the experimental procedures were undertaken. They had been left in the laboratory until collecting the blood under hexobarbital anesthesia at 4 P.M. The control rats were moved before sacrifice. Even in this case, CC1, induced an eleva tion of corticosterone levels and the high level was maintained for 10 hour period. After that the values showed lower levels than that of the control rats during 20 and 40 hour period (Table 2). Assay of plasma corticosterone revealed that adrenalectomy as well as hypophysec tomy markedly lowered the levels and completely eliminated the corticoid elevation normally observed after CC1, (Table 3).
TABLE
2.
Effect
sacrificed
Morphine
of
CCI,
in
the
has been proposed
pituitary-adrenal
hormonal
on
plasma
laboratory
corticosterone
level
in
rats
at 4 P.M.
as inhibiting
response
system (9-11), so different
to additional
administrations
stimuli
in the
of morphine
were
previously used as suggested by theses authors, and yet CCI, induced a similar elevation of corticosterone level as seen in the normal rats (Table 4). Morphine, ministration
reserpine
and
cortisone
were administered
of these drugs for 2 days was effective in depressing
induced by CCI, (Table 5), while the administration TABLE
3. or
repeatedly.
Effect of CCI, hypophysectomized
in
The repeated
the corticoid
of morphine
ad
elevation
in twice did not affect
the effect of CC14 (Table 4).
the adrenalectomized rats.
Surgical
trauma
likewise
stimu
lates the hypophyseo-adrenal
system.
The values of corticosterone
levels in
the
were
78.6±5.4
,ug/dl (n=;4) at 1 hour and
42.9±2.3
plasma
assayed
teg/dl (n=4) at 24 hour period after spinal cord transection. CCI, admi nistered The blood rats 5 hours hours
after
was collected from the adrenalectomized and from the hypophysectomized rats the
administration TABLE
4. of
Influence plasma
of
peanut of
the
corticosterone
oil
after
bring about
2
corticosterone
or CCI,.
pretreatment
with
levels
induced
morphine by
on
the operation the elevation
did not in plasma
levels (Table 6). the
elevation
CCI,.
1) CCI, or peanut oil was administered 10 minutes following the injection of morphine (20 mg/kg, i.p.) and, 2 hours after that , the blood was collected. 2) CCI, or peanut oil was given 1 hour following the injection of morphine (20 mg/kg, s.c.) and the blood was taken 1 hour later. 3) CCI4 or peanut oil was given 10 minutes following the admi nistration of morphine (40 mg/kg, i.p.) and the blood was ob tained 2 hours later. 4) Morphine (4 mg/kg, i.p.) was injected 11 hours and 5 hours prior to the administration of peanut oil or CC14 and the blood was collected 2 hours after CCI,.
On
the
ministration
other
hand,
of CCI,
was
the
previous
found
effective
treatment
for
elevation
of
plasma
corticosterone
morphine
or histamine
induced
by
to
ad
TABLE
be an
preventing
the
5.
Effects
of
morphine,
cortisone
administered
elevation
of plasma
induced
by
reserpine
and
repeatedly
on
corticosterone
the
levels
CCI,.
levels (Table
7).
DISCUSSION It has been reported that morphine administered in once is shown to inhibit the activation of ACTH release induced by several drugs or surgical trauma (9-11), by determining ascorbic acid contents in the adrenal glands. However, after one or two administration of morphine which stimu lates the hypophyseo-adrenal hormonal system by itself, CC14 likewise showed an increase of the plasma corticoid, though the hormone level is already fairly high by morphine. Montanari et al. observed that an increase of plasma corticosterone did not always accompany a reduction of adrenal ascorbic acid (12). This finding casts doubt on the value of adrenal ascorbic acid deter mination as an index of ACTH secretion
Morphine (50 mg/kg s.c.) 3 times a day, reserpine (5 mg/kg s.c.) or cortisone acetate mg/kg s.c.) once a day was administered 2 days. The blood was collected 2 hours after administration of peanut oil or CC14. TABLE
6.
The
blood
TABLE
(15, 16). Smelik however gical concentration
They therefore
proposed
opposed the theory and
of the corticoids
CCI4
of
7.
on
CC14
of
which
the
cortico had
been
on.
1
or
of
peanut
prior on
plasma
levels
plasma
rats
collected CCI,
Effect
with
or
in
operated
was
administration
physeo-adrenal activity under conditions of stress, Yates et al. conducted experiments on animals into which they had injected corticosteroid in varying doses in order to elevate the plasma concentration before animal was exposed to experimental stimuli, and they found that the hormonal system lies under proportional closed-loop control set point.
of level
previously
(12, 13). So morphine does not seem to in hibit the CC14-induced pituitary-adrenal activation, and this result might agree with the observation of Rerup (14). Concerning regulation of the hypo
with variable
Effect
sterone
and (60 for
the
hour
after
the
oil.
treatment elevation
corticosterone
induced
by
morphine
histamine.
Morphine (30 mg/kg i.p.) or his tamine (60 mg/kg i.p.) was injected 19 hours after the administration of peanut oil or CC14i and the blood was obtained 1 hour later.
the variable proposed
had an inhibitory
set point
that
control theory
only a supraphysiolo
effect on acute release of cortico
tropin (17). The results obtained by the administration of CCI, to morphine-pretreated rats showed that the CCl4-induced high level was similar to that observed normally and did not depend on the initial level of the hormone. These results suggest that the variable set point control mechanism play a part in regulating the hypophyseo-adrenal system under the conditions of drugs-induced stimuli. The repeated administration of morphine, reserpine and cortisone for 2 days, or sur gical trauma, was found to eliminate the elevation of plasma corticosterone levels normally induced by CC14,and CC14 likewise inhibited the morphine or histamine-induced rise in the circulating hormone. These treatments are well-known to evoke hypersecretion of ACTH, so a reduction of ACTH in the hypophysis caused by a large dose or prolonged treatment of the drugs has been reported (18-21). Brodie et al. have proposed that the rate of ACTH discharge following reserpine is greater than the rate of its synthesis and the discharge results in a condition in which animals can not respond to an additional pituitary stimulus (22). Although only the prolonged treatment by morphine inhibits an elevation of the corticoid, ACTH contents in the pituitary following morphine have not been reported in detail. Corticosteroid administered daily likewise has been shown to induce a fall of pituitary ACTH concentration (20, 23, 24). However Vernikos Danellis demonstrated that a single blocking dose of hydrocortisone prevented both release and increase in pituitary ACTH contents after stress but had no effect on the resting level in the blood and gland (25). The facts therefore appear to be that the synthesis and secretion of ACTH in the gland occur differently in resting stage and in response to stress, and that the corticoid inhibit the stress-induced ACTH release, not by depressing pituitary ACTH stores but by interfering with ability of the gland to synthesize new ACTH (26-30). Further studies are needed to clarify the mechanism by which CCI, inhibits the additional hypophyseo-adrenal activation. There still remains the question whether CC14 acts directly on the hypothalamus and accelerates the pituitary-adrenal function, or whether the activation of adrenocortical hormone secretion caused by CCI, is simply a physiological response to CC14-induced injury, or whether it is further involved in the mechanism of its toxicity. Although it is almost impossible to know exactly the time course or the onset of biological alteration induced by CC14,the apparent elevation of plasma corticosterone level was observed as early as 1 hour after CC14,and electronmicroscopical observation revealed that a patho logical alteration of the liver occurred in the endoplasmic reticulum within 1 or 2 hour period (31, 32). There is no evidence, of course, that the hypophyseo-adrenal activation occurs before the appearance of the other biological alterations, such as the liver damage. On the other hand, reports, proposing that ACTH release was accelerated by narcotics or sedatives such as ether (33), CHC13 (2), Rauwolfia alkaloids and phenothiazine, have been presented. Besides, of a number of Rauwolf a alkaloids, only those compounds which produce sedation also evoke ACTH hypersecretion (22), and phenothiazine deriva tives likewise cause ACTH hypersecretion but do so only in sedative compounds (34). The reason why such central depressants induce the hypophyseo-adrenal activation is
not clear. It is however possible that the secretion of the humoral agent is controlled by both inhibitory and stimulatory pathways in the hypothalamus (35). Therefore the activation of stimulatory pathways which is probably caused by stress, or the depression of inhibitory pathways which is presumably induced by central depressants, might result in the acceleration of ACTH discharge. Although it is difficult to say whether CCI, acts primarily on the hypothalamus, or whether the stimulation of the hypophyseo-adrenal axis is physiological response to the CCI,-induced injury, it is tempting to speculate that CCI, acts directly on the central nervous system and stimulates a release of corticotropin releasing factor from the hypothalamus through its narcotic effect like CHC13. If so, it is likely that the CCI,-induced injury is related to the pituitary-adrenal activation as non-specific stress in later period. Experiments done by this author show that corticoids hormone is necessary for full development of the CC14-induced toxicity presumably by affecting the hepatic functions and sensitizing them to attack with CCI„ and that certain contents of the hormone is necessary for that (36). Although the adrenocortical hormone modifies the effect of CCI, in its toxicity, the increase of the corticoid secretion by CCI, might not be involved es sentially in the mechanism of its toxicity. SUMMARY CCI, induced an elevation of plasma corticosterone level in rats. This phenomenon was observed even in rats in which the corticoid level was fairly high, resulting from emotional and diurnal variations. Although the administrations of morphine in once or twice did not inhibit the elevation of the corticoid level induced by CCI,, the repeated administrations of morphine, reserpine or cortisone for 2 days almost inhibited the eleva tion normally induced by CC1,. Surgical trauma likewise prevented this elevation. The administration of CCI, was effective in depressing the elevation of corticosterone level induced by morphine or histamine. The experiment is discussed with regard to possible mechanism involved in the CCI, toxicity. Acknowledgement : My sincereappreciationis due to Dr. T.M. Brody. REFERENCES 1) GOODMAN, L.S. ANDGILMAN, A. : The Pharmacological Basisof Therapeutics, p. 1140.MacmillanCo., New York (1958) 2) SAYERS, G. ANDSAYERS, M.A.: Ann.N.Y. Acad . Sci.50, 521 (1949) 3) BRODY, T.M. ANDCALVERT, D.N.: Amer.J. Physiol.198, 682 (1960) 4) STERN, P.H. ANDBRODY, T.M.: J. Pharmacol. 141, 65 (1963) 5) ZENKER, N. ANDBERNSTEIN, D.E.: J. biol.Chem.231, 695 (1958) 6) CALVERT, D.N. ANDBRODY, T.M.: Amer . J. Physiol.198, 669 (1960) 7) BRODY, T.M., CALVERT, D.N. ANDSCHNEIDER, A.F. : J. Pharmacol. 131, 341 (1961) 8) SLUSHER, M.A. ANDBROWING, B.: Amer.J. Physiol . 200, 1032(1961) 9) BRIGGS, F.N. ANDMUNSON, P.L. : Endocrinology 57, 205 (1955)
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