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EFFECTS OF 10-METHOXYDESERPIDINE AND RESERPINE ON THE SKIN AND RECTAL TEMPERATURE IN RABBITS
EFFECTS
OF
10-METHOXYDESERPIDINE
ON THE SKIN AND RECTAL
AND
TEMPERATURE.
RESERPINE IN RABBITS
NOBORU TODA, KIRO SHIMAMOTO ANDMOTOHATSU FUJIWARA Department of Pharmacology, Faculty of Medicine, KyotoUniversity, Sakyo-ku, .Kyoto Receivedfor publicationFebruary16,1964
Although the peripheral effects of reserpine are generally accepted,there is evidence to support the central action on the adrenal medulla (1-5). No depletion of tissue nor adrenaline by 10-methoxydeserpidine(10-MD)has been reported in mice by Leroy and Schaepdryver (6). However, Higuchi et al. (7) have shown that the intravenous injec tion of 10-MDin rabbits depletes considerably the content of noradrenaline in the brain cortex, brain stem and heart, and of adrenaline in the adrenal medulla without mani festing sedation or diarrhea. 10-MD has recently been used clinically for the essential hypertension. Episode of the side effect that hyperemia and warm sensation of the face and neck manifest in some patients several hours after the oral administration has been reported (8). No such side effect has hitherto been reported in the case of reserpine. Therefore, the current report deals firstly with elucidation of action mechanism of the hyperemia and warm sensation in the face and neck caused by 10-MD, and secondarily with the phar macological difference between reserpine and 10-MD. The results in the present ex periments have shown that though reserpine produces a marked elevation of the ear temperature followed by a steady fall of the rectal temperature , 10-MD elicits a similar elevation of the ear temperature attended with no change in the rectal temperature in rabbits. METHODS The effects of the intravenous injection of reserpine and 10-MD on the ear, thigh and rectal temperature, the spontaneous electroencephalogram (EEG), the electrocardio gram (ECG), the respiration and the content of adrenaline, noradrenaline and serotonin of the various tissues were investigated in albino rabbits, weighing 2.0 to 2.5 kg of either sex, at the room temperature of 24±0.3°C. The animals anesthetized at the stage III (plane 2 or 3) with ether were fixed on the stereotaxic instrument in an abdominal position. The surgical procedures were performed during the anesthesia. The full awaking on the EEG pattern was usually obtained about one hour after the termina tion of ether inhalation. The experiments were commenced after the stabilization of 戸田
昇 ・島 本
暉 朗 ・藤 原
元始
the skin and rectal temperature. During the experiments the external stimuli were excluded as far, as possible. Measurementof the skin and rectal temperature: The surface of the left ear at the middle of the longitudinal axis 1 to 1.5 cm below the superior margin was shaved, and the thermistor (5 x 5 mm plate for the recording of the skin temperature) was attached to the shaved part by holding it with the spring clip, avoiding disturbance of the local circulation. The other surface of the thermistor exposed to the air was covered with a cotton wool and the thermal radiation was prevented. The same type of thermistor was similarly attached to the shaved part at the flexor root of the right thigh. The. thermistor end for the rectal temperature was inserted into the rectum 15 cm` from the anal ring and the mobilization of the thermistor was prevented by fixation with a sticking plaster. The changes of the skin and rectal temperature were transmitted to the automatic temperature recording apparatus (Electro-thermometer, Type MR-5, Nippon Thermistor Seiki) for the reading of the temperature. Though the rectal temperature , was usually stabilized within two hours after the fixation of the animal, 3 to 5 hours were sometimes necessary for the full stabilization of the ear and thigh temperature. The temperature was measured every 5 minutes within 30 minutes after the injection of the test drugs, and thereafter every 15 or 30 minutes. The slight motor movements caused by the injection did scarcely affect the skin and rectal temperature. The normal ear temperature in resting state was 28.6°C (27.1 to 30.2) in average, the thigh tempera ture 28.8°C (26.6 to 30.0) and the rectal temperature 37.8°C (37.5 to 39.0). The skin and rectal temperature subjected to much individual variations, but in a resting state the spontaneous change of it was minimal (--0.5°C in the skin temperature and -±-0.2°Cin the rectal temperature). Recordingof the spontaneousEEG: Daring the anesthesia the recording electrode was located at the site of the cortical surface 2 mm rostral from the coronary suture and 2 mm left from the sagital suture, and the indifferent electrode was placed on the skull 1 to 1.5 cm rostral from the recording electrode. The electrical activity of the brain cortex was recorded by use of the universal recorder (Type EG-129, San'ei Sokki). Recordingof the ECG and the respirationrate : For the recording of the ECG the second lead was used. The rate and the regularity of the heart beat were mainly observed in detail. The rate of the respiration was observed by recording the elastic movement. of the gummi band attached to the costal arc via a mechano-electric transducer. In addi tion, the behavioral changes of the animals kept in the cage during the experiment were macroscopically observed. In some of the animals the right carotid pressure was recorded by means of mercury manometer without anesthesia. Drugs tested: Five hundred milligrams of 10-methoxydeserpidine (Decaserpine, Roussel Co.) were dissolved in the mixed solution of parts of propylene glycol and 1 part of glacial acetic acid, and this was used as stock solution, the pH being 3 to 4. The volume of the injection was always adjusted to 0.4 ml/kg, if necessary, by diluting the stock solution with propylene glycol. Reserpine taken from the commercial stock solution
(Daiichi Seiyaku Co.) was used in the volume of 0.4 ml/kg. The other drugs used were dl noradrenaline hydrochloride, 1-adrenaline hydrochloride, acetylcholine chloride, phenyl cyclopropylamine
maleate
(SKF-385) and hexamethonium
bromide.
Chemical assay of the tissue catecholamine and serotonin : The isolated stem, atria
and adrenal
adrenaline
and noradrenaline
photofluorometrically in the homogenates resin, and eluted
glands were homogenized
brain cortex, brain
in 0.4 N perchloric
in the solution were adsorbed,
eluted
acid solution,
and
(9, 10) and assayed
following the method of von Euler and Floding (11). Serotonin extracted by the same way was adsorbed to the Amberlite XE-64 with 1.2 N HCl according
to the method
in the eluates
was assayed spectrophotofluorometrically
mp fluorescent
wave-length.
of Bertler
(12).
at 315 m,a activating
Serotonin and 550
RESULTS
I. Effects of 10-methoxydeserpidine (10-MD) The intravenous injection of 10 mg/kg of 10-MD produced an abrupt elevation of the ear temperature by 3.0 to 5.5°Cat the latency of 20 to 40 minutes in 9 of 11 rabbits. The peak level of elevated temperature was 32.1 to 35.0°C. The duration of the tem perature elevation subjected to much individual variations. That is, the duration was more than 6 hours in 2 rabbits, 1.5 to 6 hours in 5 and 1 to 1.5 hours in 2. Fig. 1 shows the time course of the temperature elevation in 3 rabbits considerably different in extent and duration of the elevation of the ear temperature. During the tempera ture elevation the ears showed dilatation of the vessels and hyperemia. Some motor movements observed for a few minutes immediately after the injection did not affect the temperature elevation. During the elevation of the ear temperature, the thigh and rectal temperature was not affected or sometimes the thigh temperature was lowered by 0.5 to 1.0°C. The dose of 10-MD produced a moderate degree of bradycardia and the peak effect was observed about 30 minutes after the injection (Fig. 1). The extent of the reduction of the heart rate was 100/oat the largest. Two hours after the injection the heart rate recovered completely. Regularity of the cardiac rhythm was always maintained during the manifestation of bradycardia. The respiratory rate began to decrease 10 minutes after the injection and the reduced rate lasting for 4 hours or more was accompanied with the increased depth. Many rabbits showing an electrical activity of an arousal pattern in the spontaneous EEG responded to 10 mg/kg of 10-MD with a manifesta tion of resting waves intermingled with spindle bursts and high amplitude slow waves from 5 or 10 minutes to 40 minutes after the injection. Sometimes, an abundant mani festation of the spindle bursts was observed again about 60 minutes after the injection. Irrespective of the manifestation of either resting or arousal waves, no behavioral sign of sedation was observed. All rabbits received 10-MD showed no sign of diarrhea. The mean carotid blood pressure was slightly elevated in accordance with the motor excite ment caused by the injection, but, thereafter, it turned to a fall below the original
FIG. 1. Effect of 10 mg/kg of 10-MD on the ear, thigh and rectal temperature, and heart and respiration rates. Ordinates : °C i n temperature change and percent decrease in rates. Abscissae : time in min. In the heart and respiration rates the circles and the solid line show the effect of 10-MD, and the crosses and the broken line show the solvent effect.
level and gradually recovered 7 to 10 minutes after the injection . The injection of solvent alone resulted in the slight and transient manifestation of motor excitement , respiratory stimulation and transient rise of blood pressure without modifying the ear temperature. The intravenous injection of 3 mg/kg of 10-MD also produced a moderate elevation of the ear temperature in 3 of 4 rabbits. The elevation of the ear temperature , the peak effect of which was 1.5 to 4.0°C, manifested at latency of 20 to 50 minutes and lasted for 1.5 to 5 hours. Neither thigh nor rectal temperature was also affected by the dose. Though the spontaneous EEG of the rabbits received 3 mg/kg of 10-MD showed
the resting waves as that of the animals and respiration
rates was much
effect except the transient
less.
received
10 mg/kg, the reduction
The administration
of the heart
of 1 mg/kg did not show any
bradycardia.
II. Effects of rescrpine The intravenous injection of 1.0 mg/kg of reserpine produced a marked elevation of the ear temperature in 7 of 8 rabbits. The peak of the abrupt and steep elevation of the ear temperature at latency of 10 to 40 minutes was 4.5 to 6.5°C. The elevation lasted for more than 6 hours showing sometimes unduration and sometimes plateau, and turned to a gradual decrease (Fig. 2). The thigh temperaiure was slightly elevated
FiG. 2. Effect of 1 mg/kg of reserpine on the ear, rectal and thigh temperature, and heart and respiration rates.
in 2 animals, while in other 6 animals it showed a fall by 2.0 to 3.0°C. In addition, the rectal temperature always showed a fall which appeared 30 to 60 minutes after the injection, and the fall at 4 hours was 1.5 to 2.5°C. No further lowering was observed, thereafter. The extent of the lowering depended on the initial level of the rectal tem perature. As illustrated in Fig. 2, the rectal temperature in the rabbits, the ear tem perature of which was elevated at shorter latency in response to reserpine (• ... •, x ... x), began to fall relatively earier than in the rabbit, the ear temperature of which 'was elevated at longer latency (o ... o) . This evidence may show that the fall of the rectal temperature relates with the increased heat loss caused by elevation of the ear temperature. The gradual decrease in the heart rate reached to a plateau 2 hours after the injec tion of reserpine and the decrease lasted for more than 6 hours. The average reduction
in the rate at 3 to 4 hours was 35%. The respiratory rate was also gradually reduced accompanied with the increased depth of, respiration. Irrespective of the progressive manifestation of the behavioral sedation, the spontaneous EEG showed a resting pattern intermingled with the spindle bursts and high amplitude slow waves from 5 or 10 minutes to 35 or 75 minutes after the injection. All the animals received the. dose of reserpine showed a profuse diarrhea and a behavioral sedation with ptosis and rounding of their backbones. However, the external stimuli still produced a fairly prompt alert reaction. III. Effects of 10-MD and reserpineon the rabbits treated with 10-MD The effects of 10-MD and reserpine were studied in the rabbits after the full re covery of the elevated ear temperature caused by the first injection of 10 mg/kg of 10 MD. The second injection of the dose of 10-MD elevated the ear temperature by 2.0 to 5.4°C at latency of 10 to 40 minutes and the elevation lasted for only 45 to 90 minutes. The elevation of the ear temperature caused by the second injection was smaller in extent and shorter in latency and duration than that caused by the first injection. However, one rabbit which had not responded to the first injection with elevation of the ear temperature showed a marked elevation in response to the second injection. In all case, no change of the thigh and rectal temperature was observed. The brady cardia caused by the drug did not differ between both injections. The reduction of the respiration rate caused by the first injection was not facilitated by the second injection. The resting pattern of the spontaneous EEG produced by the second injection was almost the same as that by the first injection. The injection of reserpine to the rabbits treated previously with 10 mg/kg of 10 MD produced a marked and prolonged elevation of the ear temperature (Fig. 3). The elevation of the ear temperature was 4.2 to 6.0°C in extent, 20 to 30 minutes in latency
FIG. 3.
Typical
effect
temperature 10-MD, 10-MD
of
of
10-MD
animals
respectively. and
broken
and
Solid line
reserpine
pretreated
shows
with
line
shows
that
of
on
the
reserpine the
effect
reserpine,
ear and of
and more than 4 hours in duration. Therefore, the pretreatment of the animals with 10-MD did not affect the reserpine effect on the ear temperature. Similarly, the ad ministration of reserpine lowered the rectal temperature by 1.5 to 2.0°C and the thigh temperature by 1.2 to 3.0°C at 4 to 6 hours after the injection. The reduction of the heart and respiration rate was observed in response to reserpine in the rabbits treated with 10-MD. The manifestation of the resting waves in EEG by reserpine was not prevented by the treatment of the animals with 10-MD. IV.
Effects of 10-MD and reserpine on the rabbits treated with reserpine The effects of 10 mg/kg of 10-MD and
rabbits pretreated
of the ear, thigh and rectal intact
and treated rabbits
treatment
temperature,
1.
Comparison
rates values
in
of
the
ear
untreated
rabbits
were
measured
2 to
Room temperature
and heart
of the rabbits
the
and
and respiration
4 hours
: 24.0--0.3-C, Reserpine
those after
temperature, in
the
setting
heart reserpinized
up
of
the
The differences
rates
to twenty
between
the
hours after the pre
was 32.TC in average
rectal with
were studied in the
16 to 20 hours previously.
are shown in Table 1. Sixteen
the ear temperature TABLE
1.0 mg/kg of reserpine
with 1.0 mg/kg of reserpine
and
and
3.6°C higher
respiration
rabbits.
These
preparations.
1.0 mg/kg i.v. 16-20 hours before.
than that of the control animals, while the rectal temperature was 2.5-C lower than that of the controls. The heart and respiration rates in the treated animals decreased 35 and 44% respectively, comparing with those of the control animals. The full reco very of the EEG to manifest the arousal waves in the intact rabbits was obtained 30 to 60 minutes after the termination of ether anesthesia, while the time length for the recovery from ether anesthesia in the treated animals was 180 minutes or more. The reserpinized animals showed a sign of sedation, as described above. The effect of 10-MD on the ear temperature was markedly affected by the reserpine treatment (Fig. 4). Two of four rabbits showed a more prompt but slight elevation of the ear temperature, 1.5 and 1.0°C, lasting about 30 minutes. The second injection of the same dose of 10-MD 4 and 6 hours later resulted in the transient fall of the ear temperature by 1.3 and 0.5°C, respectively. Remaining 2 rabbits showed the fall of ear temperature, 0.5 and l.5`C, even by the first injection of 10-MD. However, the thigh temperature was affected by neither injection of 10-MD. On the other hand, the rectal temperature gradually fell and reached a plateau, showing a fall by 0.5 to 2.0°C, 4 hours after 10 mg/kg of 10-MD. The reduction of the heart and respiration rates by 10-MD was decreased by the treatment.
FIG. 4. Effect of 10 mg/kg of 10-MD on the ear and temperature of the reserpinized rabbits. In the figure the solid and broken lines of the same illustrate the effects of the first and the second tions, respectively. See text.
rectal upper mark injec
The previous treatment of the animals with reserpine markedly depressed or blocked the elevation of the ear tempera ture caused by the injection of reserpine (Fig. 5). However, the thigh temperature was lowered by reserpine injection and the fall was 1.0 to l.5°C 3 to 4 hours after the injection. The fall of the rectal tempera ture was abolished or unaffected by the injection of reserpine. The effect of reser pine on the rectal temperature depended on the extent of the fall produced by reserpine pretreatment. The transient manifestation of the resting waves in EEG in response to 10 mg/kg of 10-MD or 1 FiG. 5. Effect of 1 mg/kg of reserpine on the ear, thigh and rectal temperature mg/kg of reserpine was not modified by of the reserpinized rabbits. the previous reserpinization (Fig. 6). The resting waves intermingled with spindle bursts were observed for 30 to 60 minutes at the latency of 5 to 10 minutes. In some cases, 3 hours after the injection the spindles were repeatedly seen at the intervals of about 10 minutes. V. Effects of 10-MD and reserpineon the rabbits pretreatedwith SKF-385 The effects of 10 mg/kg of 10-MD and 1 mg/kg of reserpine were studied in the rabbits treated with 5 mg/kg of SKF-385 2 hours previously. The ear and thigh tem
FIG. 6.
Effects
of
10-MD
and
reserpine
on
the
cortical
EEG
of
the
reserpinized
rabbits.
perature was hardly affected by SKF-385 within 4 hours after the injection. On the other hand, the rectal temperature was gradually elevated until the elevation of 0.5 to 1.0-C showed a plateau 2 hours after the injection. The heart and respiration rates were usually increased about 10%, respectively.
FIG. 7. Effect of 10 mg/kg of 10-MD on the ear temperature of the animals treated with SKF-385, 5 mg/kg.
The
previous
the elevation ear
treatment
temperature
minutes. animals
rates nor
In the treated However, Though
spiration
spontaneous
animals
the
previous
rate and manifestation
the procedure
animals.
the administration
produced
animals
treatment
lasted , for 90 to 120 by , 10-MD in the treated Effects of the drug on, neither heart
was not affected
EEG were modified by the previous
of the ear temperature
in some treated
mg/kg of SKF-385 , dit u,not,.,modify The elevation of the
of 20 to 60 minutes
temperature
as well as in the non-treated
duce the elevation tion.
with.5
caused by 10-MD (Fig. 7).
by 3.1 to 5.8°C at latency
The thigh and rectal
and respiration
8).
of the animals
of the ear temperature
of 1 mg/kg of reserpine
did neither
nor the fall of the. rectal: temperature,
the thigh significantly
temperature depressed
instead
pro (Fig.
' showed some late eleva the reduction of the re
of the resting waves in EEG in response
slight tachycardia
treatment.
to reserpine,
of bradycardia.
FiG. 8. Effect of 1 mg/kg of reserpine on the ear, thigh and rectal temperature of the animals treated with 5 mg/kg of SKF-385.
VI. Effects of adrenaline,noradrenaline,acetylcholineand hexamethonium The intravenous injection of 3 pg/kg of adrenaline or 6 ug/kg of noradrenaline produced no significant change in the ear and rectal temperature. The injection of 10 pg/kg of adrenaline but not 20 beg/kg of noradrenaline, however, resulted in the slight and transient elevation of the ear temperature without changing the thigh and rectal temperature (Fig. 9). The heart rate was markedly decreased. The injection of 10 lug/kg of adrenaline or 20 beg/kg of noradrenaline to the rabbits , the ear temperature of which was elevated 3.6°C in average by the previous treatment with 1 mg/kg of reserpine, pro duced a marked ventricular rhythm. In these reserpinized animals the ear temperature responded to adrenaline as well as noradrenaline with slight elevation preceded by a
moderate fall. However, the duration of the response was 20 to 30 minutes. The effects of noradrenaline were somewhat less than those of adrenaline. Neither thigh npr rectal temperature was affected by the doses of the amines. Though the ear temperature was not affected by 5 and 10 fig/kg of acetylcholine, 10 mg/kg of hexamethonium elevated the ear temperature at latency of a few minutes. The peak elevation, 4.1 to 6.3 C, was observed 5 to 10 minutes after the injection and the elevated temperature fell to the normal level 30 to 40 minutes after the injection. During the elevation of the ear temperature the thigh and rectal tem perature was not affected. VII
Effects of 10-MD on the content of tissue catecholamines and serotonin The contents
of noradrenaline
brain cortex, brain renaline
in the
stem and atria, of ad
in the adrenals
and of serotonin
in the brain cortex, brain stem and ileum
FiG. 9. Effects of adrenaline (.-.l and nor adrenaline 'x-x) on the ear temperature and heart rate. Upper figure shows the effects of 3 beg/kg of adrenaline and 6 ug/ kg of noradrenaline, while the lower two illustrate the effects of 10 ,ug/kg of adrena line and 20,ug/kg of noradrenaline.
TABLE 2. Effects of 10 mg/kg of 10-MD on the catecholamine and serotonin content in the various tissues 3 and 16 hours after the injection. Catecholamine content in the brain and atria is calculated as noradrenaline ug/g wet tissue, while that in the adrenal glands is calculated as adrenaline ug/ two glands. Serotonin content is illustrated as ug/g wet tissue. Catecholamine
Serotonin
content
content
were studied in rabbits 3 and 16 hours after the intravenous injection of 10 mg/kg of 10-MD (Table 2). The isolated terminal of the ileum (2 g weight of wet tissue) was used for serotonin assay. The reduction of noradrenaline content in the brain cortex, brain stem and atria were 54, 46%; 68, 55%; and 77, 62% at 3 and 16 hours after 10-MD, respectively. Though the reduction was more marked at 3 hours than at 16 hours after the injection, the differences were statistically insignificant. The content of adrenaline in the adrenal glands was reduced by 22% at 3 hours after the injection, but the content at 16 hours was found to be almost normal. The content of serotonin in the brain cortex and brain stem showed significant decrease, 40, 30% and 57, 71% at 3 and 16 hours after the injection, respectively. However, the serotonin content in the ileum was not significantly affected by 10-MD. DISCUSSION The therapeutic indication of 10-MD for the clinical hypertension has been present ed by Mir and Lewis (13) because of mild and gradual fall of blood pressure with minor side effects. Thereafter, the compound was subjected to wide clinical application. The moderately prolonged hyperemia and warm sensation in the face and neck in response to the oral application of the compound, encountered in some patients, have facilitated the present experiments. The sensitive elevation of the ear or tail temperature in rabbits or rats in response to environmental temperature rise has been regarded to be the radiation mechanism of the body temperature (14, 15). Further, it is reasonable to consider that the warm sensation or hyperemia caused by the increased blood flow reflects to the elevation of the local temperature. Therefore, the effects of 10-MD on the ear temperature were mainly studied in the present experiments in order to analyse the mode of side actions described above. From the results of the present experiments the marked differences of the effects between 10-MD and reserpine are as follows 1) Unlike reserpine, 10-MD did not lower the rectal temperature in spite of the marked elevation of the ear temperature. 2) The pretreatment with 10-MD did not change the effects of both compounds, while the reserpinization modified the effects. 3) The pretreatment with SKF-385 prevented the temperature effects of reserpine but not of 10-MD. Although the ear, thigh and rectal temperatures in the restrained animals were considerably different individually, they were fairly constant in one animal for many hours at the room temperature of 24--0.3°C and under the exclusion of the external stimuli as far as possible. The administration of 10 mg/kg of 10-MD elevated the ear temperature by 3.0 to 5.5°C at latency of 20 to 40 minutes for 1.5 to 6 hours without changing the thigh or rectal temperature. Though the administration of 1 mg/kg of reserpine resulted in the similar elevation of the ear temperature lasting for more than 6 hours, the thigh temperature showed a tendency of slight fall and the rectal tempera
ture showed a steady fall, of 1.0 to 2.0°C. Some accordance` of the progression and maintenance of the elevated ear temperature with the phase of the progressive deple= tion of the tissue catecholamine by either compound, especially reserpine, suggests that the elevated ear temperature is a reflection of the increased blood flow elicited by the decreased sympathetic tone, of peripheral vascular bed. It is generally considered that the elevation of the ear temperature reflects the increased radiation of the body tem perature and, therefore, it should usually be followed by the fall of the rectal tempera ture. This is the case of reserpine in which the time length of the latency for the elevation of the ear temperature is proportional to that for the fall of the. rectal tem perature. However, since the fall of the rectal temperature .was also observed in the rabbits the ear temperature of which did not respond to reserpine, some depression of the heat regulating center might be involved in the fall 'of rectal, temperature by reser pine. On the contrary, in the case of 10-MD no fall of the rectal temperature was observed despite the marked elevation of the ear temperature. Accordingly, no affec tion of the rectal temperature by 10-MD was likely to derive from lack of, such central effect. Shorter duration of elevation of ear temperature by 10-MD than by reserpinee is also indicative for the lack of the depression of heat regulating center. The admi nistration of 10 mg/kg of hexamethonium did not affect the rectal temperature, although striking but short-lasting elevation of the ear temperature was always observed. Again, this may be due to the lack of central depression of hexamethonium. During the manifestation of the marked sedation in rabbits produced by 1 mg/kg of reserpine the content of noradrenaline or serotonin in the brain has been reported to be deprived by 90% or more (10, 16, 17). . When the content of both amines was depleted more than 50% at 3 and 16 hours after the administration of 10-MD, the rabbits did not show any sign of sedation. The evidence is suggestive for denying the causal relationship between the development of the sedation and the depletion of the brain amines. Nevertheless, it is accepted that reserpine, but not 10-MD, has the effect of central nervous depression which may or may not have some relations to the marked depletion of amine content in the brain. The effects of 10-MD and reserpine on the ear and rectal temperature were not modified by the prior treatment of the rabbits with the effective dose of 10-MD. On the other hand, the reserpirl}~ation of the animals depressed or blocked the responses of the ear and rectal temperature to 10-MD as well as reserpine. This evidence indicates that the elevation of the ear temperature in response to both compounds relates with the tissue catecholamine and/or serotonin. Three hours after 10 mg/kg of 10-MD the noradrenaline content was reduced by 54% in the brain cortex, by 68% in the brain stem and by 77% in the atria, while the serotonin content was reduced by 40% in the brain cortex and by 57% in the brain stern. The similar reduction on the tissue cate cholamine byo 10-MD has been shown by Higuchi et al. (7). The lesser depletion of noradrenaline or serotonin by 10-MD than by reserpine (10, 16, 17) may be responsible for the less susceptibility of the ear and rectal temperature to 10-MD or"reserpine by
the
pretreatment
of the
the
pretreatment
with
the
amines, The
making
long-lasting
accumulation
animals 10-MD
the
with may
subsequent
inhibition
of noradrenaline
10-MD.
produce reserpine
of monoamine and
serotonin
Otherwise, some
change
or 10-MD oxidase in the rat's
the
reserpinization
other
than
the
but
not
depletion
of
non-effective. by SKF-385 brain
and
in vivo, the increased' the elevation
of the
catecholaminelevel in the brain, atria and adrenal glands of the rabbits have alreaty been shown by several investigators (18-20). In addition, the pressor response of the dogs to SKF-385 has been shown to relate with the endogenous noradrenaline (21). Despite the possible manifestation of the sympathetic effects the administration of SKF 385 did not affect the ear temperature, but elevated the rectal temperature moderately. The prior treatment of the rabbits with SKF-385 blocked the effects of reserpine on the ear and rectal temperature, though it did not affect the effects of 10-MD. The blocking action of SKF-385 may be attributable to the prevention of the catecholamine depletion due to reserpine, as shown by Shimamoto et al. (20) in the rabbits. On the other hand, the amine depletion by 10-MD may be not prevented by the prior treat ment with SKF-385. Otherwise, the elevation of the ear temperature by 10-MD may be due to the direct dilator effect on the ear vessels independent of the amine depletion. The relatively large doses of adrenaline or noradrenaline did not elicit a significant change in the ear and rectal temperature. However, this evidence does not exclude the possibility that the elevation of the ear temperature by reserpine or 10-MD derives from the decrease of noradrenaline content in the peripheral tissues, because the ear temperature elevated by reserpine was somewhat lowered by the doses of the amines. The administration of 10-MD produced a resting pattern of waves in the EEG ir respective of the prior treatment of the animals with 10-MD or reserpine. Therefore, the manifestation of the resting waves could not be ascribed to the release and deple tion of catecholamine or serotonin in the tissues including the central nervous system. The resting waves are likely to relate with the effect other than the amine-depleting effect of the compound on some central nervous structures. The depletion of adrenaline in the adrenals observed in the present experiments was 22% at 3 hours after the injection of 10 mg/kg of 10-MD, and 16 hours later the level of adrenaline returned to normal. von Euler and Purkhold (22), Goodall and Kirshner (23) and von Euler and Ryd (24) have suggested that noradrenaline and adrenaline are stored at different sites, noticing that"while noradrenaline almost disappears in the organs after denervation, the adrenaline content, although low, is well maintained. The marked and prolonged depletion of noradrenaline in contrast to the less and short lasting depletion of adrenaline is suggestive for the action mechanism of 10-MD. The blockade by reserpine of the pressor response of rabbit to stimulation of the splanchnic nerve was shown by Shimamoto and Torii (25), while the same response of cat was reported to be not affected by 10-MD (13). The lack of diarrhea, by the way, may be attributable to no,-change in the intestinal serotonin content by 10-MD,
SUMMARY Effects
of 10-methoxydeserpidine
and rectal 1. and
temperature,
10-MD
rectal
by the
Although with
3. MD
but 4.
brain
rectal
From
temperature
especially
on
the
temperature
on
the ear
tested
ear
skin,
thigh
in the restrained
without
the
any
change
temperature
effects
of reserpine
the
due
with
skin
rabbits.
in the thigh
usually
on the
moderately
stem
atria,
content
that
to the different
regulating
center
ear
not
and
followed
scarcely
of
in the ileum the different
mechanism
modify
on
the
the treat
and reserpine.
the
effects
of 10
temperature.
the content
serotonin
change
temperature,
rectal
of adrenaline
of serotonin
the possibility
did
decreased
and
in the content
did
rectal
the effects of 10-MD
SKF-385
of 10-MD and
10-MD and
or abolished
animals
results, are
with thigh
of the
brain
the heat
animals
on the ear,
depressed
the decrease
further these
reserpine were
elevated
of the
reserpine
markedly
cortex,
less and 4.
and
administration
while
of ear
reserpine
treatment
treatment
brain stem,
much
the 10-MD
abolished The
in the
while
and
respiration
temperature.
reserpine
The
(10-MD)
ECG and
an elevation
of rectal
effects of both ment
caused
temperature,
fall
2.
EEG,
in the in the
of noradrenaline brain
cortex
and
adrenal
glands
was
10-MD
and
was not affected. effects
between
the central
nervous
system,
is discussed.
REFERENCES 1) KRONEBERG,G. AND SCHUMANN,H.J.: Arch. exp. Path. Pharmak. 231, 349 (1957) 2) ERANKO, O. AND Hopsu, V. : Endocrinology 62, 15 (1958) 3) STJARNE,L. AND SCHAPIRO,S. : Nature, Lond. 182, 1450 (1958) 4) HILLARP,N.A. : Ibid. 187, 1032 (1960) 5) MIRKIN, B.L. : J. Pharmacol. 133, 34 (1961) 6) LEROY, J.G. AND SCHAEPDRYVER, A.F.DE. : Arch. int. Pharmacodyn. 130, 231 (1961) 7) HIGUCHI,H., MATSUO, T., NAKATANI,G. AND SHIMAMOTO,K. : THIS JOURNAL 13, 36 (1963) 8) SHIMIZU,I.: Personal communication 9) BERTLER,A., CARLSON,A. AND ROSENGREN,E. : Acta physiol. scand. 44, 273 (1958) 10) HIGUCHI, H. : THIS JOURNAL 12, 34 (1962) 11) EULER, U.S. VON AND FLODING,I.: Acta physiol. scand. 33, Suppl. 118, 45 (1955) 12) BERTLER,A. : Ibid. 51, 75 (1961) 13) MIR, B.J. AND LEwIS, J.J. : J. Pharmacol. 12, 677 (1960) 14) JOHANSEN,K.: Acta physiol. scand. 55, 160 (1962) 15) GRANT, R.T. : J. Physiol. 167, 311 (1963) 16) CARLSSON,A., ROSENGREN,'E., BERTLER, A. AND NILSSON, J. : Psychotropic Drugs Edited GARATTINI,S. AND GHETTI, V., p. 363, Elsevier Publishing Co., Amsterdam (1958)
by
17) BRODIE, B. B. : 5-Hydroxytryptamine Edited by LEWIS, G. P., p. 64, Pergamon Press, New Nork (1958) 18) MAASS, A.R. AND NIMMO, M.J.: Nature, Lond. 184, 57 (1959) 19) GREEN, H. AND ERICKSON,R.W. : J. Pharmacol. 129, 237 (1960) 20) SHIMAMOTO,K., MATSUO, T., HATTORI, K. AND HONJO, T. : THIS JOURNAL (in press)
21) TODA, N., YAMAWAKI,T. AND Mlsu, Y. : Ibid. 12, 166 (1962) 22) EULER, U.S. VON AND PURKHOLD,A.: Acta physiol. scand. 24, 212 (1951) 23) GOODALL,McC. AND KIRSHNER,N. : J. clin. Invest. 35, 649 (1956) 24) EULER, U.S. VON AND RYD, G.: Acta physiol. scand. 59, 62 (1963) 25) SHIMAMOTO,K. AND TORII, H.: THIS JOURNAL 10, 126 (1961)
OF
10-METHOXYDESERPIDINE
ON THE SKIN AND RECTAL
AND
TEMPERATURE.
RESERPINE IN RABBITS
NOBORU TODA, KIRO SHIMAMOTO ANDMOTOHATSU FUJIWARA Department of Pharmacology, Faculty of Medicine, KyotoUniversity, Sakyo-ku, .Kyoto Receivedfor publicationFebruary16,1964
Although the peripheral effects of reserpine are generally accepted,there is evidence to support the central action on the adrenal medulla (1-5). No depletion of tissue nor adrenaline by 10-methoxydeserpidine(10-MD)has been reported in mice by Leroy and Schaepdryver (6). However, Higuchi et al. (7) have shown that the intravenous injec tion of 10-MDin rabbits depletes considerably the content of noradrenaline in the brain cortex, brain stem and heart, and of adrenaline in the adrenal medulla without mani festing sedation or diarrhea. 10-MD has recently been used clinically for the essential hypertension. Episode of the side effect that hyperemia and warm sensation of the face and neck manifest in some patients several hours after the oral administration has been reported (8). No such side effect has hitherto been reported in the case of reserpine. Therefore, the current report deals firstly with elucidation of action mechanism of the hyperemia and warm sensation in the face and neck caused by 10-MD, and secondarily with the phar macological difference between reserpine and 10-MD. The results in the present ex periments have shown that though reserpine produces a marked elevation of the ear temperature followed by a steady fall of the rectal temperature , 10-MD elicits a similar elevation of the ear temperature attended with no change in the rectal temperature in rabbits. METHODS The effects of the intravenous injection of reserpine and 10-MD on the ear, thigh and rectal temperature, the spontaneous electroencephalogram (EEG), the electrocardio gram (ECG), the respiration and the content of adrenaline, noradrenaline and serotonin of the various tissues were investigated in albino rabbits, weighing 2.0 to 2.5 kg of either sex, at the room temperature of 24±0.3°C. The animals anesthetized at the stage III (plane 2 or 3) with ether were fixed on the stereotaxic instrument in an abdominal position. The surgical procedures were performed during the anesthesia. The full awaking on the EEG pattern was usually obtained about one hour after the termina tion of ether inhalation. The experiments were commenced after the stabilization of 戸田
昇 ・島 本
暉 朗 ・藤 原
元始
the skin and rectal temperature. During the experiments the external stimuli were excluded as far, as possible. Measurementof the skin and rectal temperature: The surface of the left ear at the middle of the longitudinal axis 1 to 1.5 cm below the superior margin was shaved, and the thermistor (5 x 5 mm plate for the recording of the skin temperature) was attached to the shaved part by holding it with the spring clip, avoiding disturbance of the local circulation. The other surface of the thermistor exposed to the air was covered with a cotton wool and the thermal radiation was prevented. The same type of thermistor was similarly attached to the shaved part at the flexor root of the right thigh. The. thermistor end for the rectal temperature was inserted into the rectum 15 cm` from the anal ring and the mobilization of the thermistor was prevented by fixation with a sticking plaster. The changes of the skin and rectal temperature were transmitted to the automatic temperature recording apparatus (Electro-thermometer, Type MR-5, Nippon Thermistor Seiki) for the reading of the temperature. Though the rectal temperature , was usually stabilized within two hours after the fixation of the animal, 3 to 5 hours were sometimes necessary for the full stabilization of the ear and thigh temperature. The temperature was measured every 5 minutes within 30 minutes after the injection of the test drugs, and thereafter every 15 or 30 minutes. The slight motor movements caused by the injection did scarcely affect the skin and rectal temperature. The normal ear temperature in resting state was 28.6°C (27.1 to 30.2) in average, the thigh tempera ture 28.8°C (26.6 to 30.0) and the rectal temperature 37.8°C (37.5 to 39.0). The skin and rectal temperature subjected to much individual variations, but in a resting state the spontaneous change of it was minimal (--0.5°C in the skin temperature and -±-0.2°Cin the rectal temperature). Recordingof the spontaneousEEG: Daring the anesthesia the recording electrode was located at the site of the cortical surface 2 mm rostral from the coronary suture and 2 mm left from the sagital suture, and the indifferent electrode was placed on the skull 1 to 1.5 cm rostral from the recording electrode. The electrical activity of the brain cortex was recorded by use of the universal recorder (Type EG-129, San'ei Sokki). Recordingof the ECG and the respirationrate : For the recording of the ECG the second lead was used. The rate and the regularity of the heart beat were mainly observed in detail. The rate of the respiration was observed by recording the elastic movement. of the gummi band attached to the costal arc via a mechano-electric transducer. In addi tion, the behavioral changes of the animals kept in the cage during the experiment were macroscopically observed. In some of the animals the right carotid pressure was recorded by means of mercury manometer without anesthesia. Drugs tested: Five hundred milligrams of 10-methoxydeserpidine (Decaserpine, Roussel Co.) were dissolved in the mixed solution of parts of propylene glycol and 1 part of glacial acetic acid, and this was used as stock solution, the pH being 3 to 4. The volume of the injection was always adjusted to 0.4 ml/kg, if necessary, by diluting the stock solution with propylene glycol. Reserpine taken from the commercial stock solution
(Daiichi Seiyaku Co.) was used in the volume of 0.4 ml/kg. The other drugs used were dl noradrenaline hydrochloride, 1-adrenaline hydrochloride, acetylcholine chloride, phenyl cyclopropylamine
maleate
(SKF-385) and hexamethonium
bromide.
Chemical assay of the tissue catecholamine and serotonin : The isolated stem, atria
and adrenal
adrenaline
and noradrenaline
photofluorometrically in the homogenates resin, and eluted
glands were homogenized
brain cortex, brain
in 0.4 N perchloric
in the solution were adsorbed,
eluted
acid solution,
and
(9, 10) and assayed
following the method of von Euler and Floding (11). Serotonin extracted by the same way was adsorbed to the Amberlite XE-64 with 1.2 N HCl according
to the method
in the eluates
was assayed spectrophotofluorometrically
mp fluorescent
wave-length.
of Bertler
(12).
at 315 m,a activating
Serotonin and 550
RESULTS
I. Effects of 10-methoxydeserpidine (10-MD) The intravenous injection of 10 mg/kg of 10-MD produced an abrupt elevation of the ear temperature by 3.0 to 5.5°Cat the latency of 20 to 40 minutes in 9 of 11 rabbits. The peak level of elevated temperature was 32.1 to 35.0°C. The duration of the tem perature elevation subjected to much individual variations. That is, the duration was more than 6 hours in 2 rabbits, 1.5 to 6 hours in 5 and 1 to 1.5 hours in 2. Fig. 1 shows the time course of the temperature elevation in 3 rabbits considerably different in extent and duration of the elevation of the ear temperature. During the tempera ture elevation the ears showed dilatation of the vessels and hyperemia. Some motor movements observed for a few minutes immediately after the injection did not affect the temperature elevation. During the elevation of the ear temperature, the thigh and rectal temperature was not affected or sometimes the thigh temperature was lowered by 0.5 to 1.0°C. The dose of 10-MD produced a moderate degree of bradycardia and the peak effect was observed about 30 minutes after the injection (Fig. 1). The extent of the reduction of the heart rate was 100/oat the largest. Two hours after the injection the heart rate recovered completely. Regularity of the cardiac rhythm was always maintained during the manifestation of bradycardia. The respiratory rate began to decrease 10 minutes after the injection and the reduced rate lasting for 4 hours or more was accompanied with the increased depth. Many rabbits showing an electrical activity of an arousal pattern in the spontaneous EEG responded to 10 mg/kg of 10-MD with a manifesta tion of resting waves intermingled with spindle bursts and high amplitude slow waves from 5 or 10 minutes to 40 minutes after the injection. Sometimes, an abundant mani festation of the spindle bursts was observed again about 60 minutes after the injection. Irrespective of the manifestation of either resting or arousal waves, no behavioral sign of sedation was observed. All rabbits received 10-MD showed no sign of diarrhea. The mean carotid blood pressure was slightly elevated in accordance with the motor excite ment caused by the injection, but, thereafter, it turned to a fall below the original
FIG. 1. Effect of 10 mg/kg of 10-MD on the ear, thigh and rectal temperature, and heart and respiration rates. Ordinates : °C i n temperature change and percent decrease in rates. Abscissae : time in min. In the heart and respiration rates the circles and the solid line show the effect of 10-MD, and the crosses and the broken line show the solvent effect.
level and gradually recovered 7 to 10 minutes after the injection . The injection of solvent alone resulted in the slight and transient manifestation of motor excitement , respiratory stimulation and transient rise of blood pressure without modifying the ear temperature. The intravenous injection of 3 mg/kg of 10-MD also produced a moderate elevation of the ear temperature in 3 of 4 rabbits. The elevation of the ear temperature , the peak effect of which was 1.5 to 4.0°C, manifested at latency of 20 to 50 minutes and lasted for 1.5 to 5 hours. Neither thigh nor rectal temperature was also affected by the dose. Though the spontaneous EEG of the rabbits received 3 mg/kg of 10-MD showed
the resting waves as that of the animals and respiration
rates was much
effect except the transient
less.
received
10 mg/kg, the reduction
The administration
of the heart
of 1 mg/kg did not show any
bradycardia.
II. Effects of rescrpine The intravenous injection of 1.0 mg/kg of reserpine produced a marked elevation of the ear temperature in 7 of 8 rabbits. The peak of the abrupt and steep elevation of the ear temperature at latency of 10 to 40 minutes was 4.5 to 6.5°C. The elevation lasted for more than 6 hours showing sometimes unduration and sometimes plateau, and turned to a gradual decrease (Fig. 2). The thigh temperaiure was slightly elevated
FiG. 2. Effect of 1 mg/kg of reserpine on the ear, rectal and thigh temperature, and heart and respiration rates.
in 2 animals, while in other 6 animals it showed a fall by 2.0 to 3.0°C. In addition, the rectal temperature always showed a fall which appeared 30 to 60 minutes after the injection, and the fall at 4 hours was 1.5 to 2.5°C. No further lowering was observed, thereafter. The extent of the lowering depended on the initial level of the rectal tem perature. As illustrated in Fig. 2, the rectal temperature in the rabbits, the ear tem perature of which was elevated at shorter latency in response to reserpine (• ... •, x ... x), began to fall relatively earier than in the rabbit, the ear temperature of which 'was elevated at longer latency (o ... o) . This evidence may show that the fall of the rectal temperature relates with the increased heat loss caused by elevation of the ear temperature. The gradual decrease in the heart rate reached to a plateau 2 hours after the injec tion of reserpine and the decrease lasted for more than 6 hours. The average reduction
in the rate at 3 to 4 hours was 35%. The respiratory rate was also gradually reduced accompanied with the increased depth of, respiration. Irrespective of the progressive manifestation of the behavioral sedation, the spontaneous EEG showed a resting pattern intermingled with the spindle bursts and high amplitude slow waves from 5 or 10 minutes to 35 or 75 minutes after the injection. All the animals received the. dose of reserpine showed a profuse diarrhea and a behavioral sedation with ptosis and rounding of their backbones. However, the external stimuli still produced a fairly prompt alert reaction. III. Effects of 10-MD and reserpineon the rabbits treated with 10-MD The effects of 10-MD and reserpine were studied in the rabbits after the full re covery of the elevated ear temperature caused by the first injection of 10 mg/kg of 10 MD. The second injection of the dose of 10-MD elevated the ear temperature by 2.0 to 5.4°C at latency of 10 to 40 minutes and the elevation lasted for only 45 to 90 minutes. The elevation of the ear temperature caused by the second injection was smaller in extent and shorter in latency and duration than that caused by the first injection. However, one rabbit which had not responded to the first injection with elevation of the ear temperature showed a marked elevation in response to the second injection. In all case, no change of the thigh and rectal temperature was observed. The brady cardia caused by the drug did not differ between both injections. The reduction of the respiration rate caused by the first injection was not facilitated by the second injection. The resting pattern of the spontaneous EEG produced by the second injection was almost the same as that by the first injection. The injection of reserpine to the rabbits treated previously with 10 mg/kg of 10 MD produced a marked and prolonged elevation of the ear temperature (Fig. 3). The elevation of the ear temperature was 4.2 to 6.0°C in extent, 20 to 30 minutes in latency
FIG. 3.
Typical
effect
temperature 10-MD, 10-MD
of
of
10-MD
animals
respectively. and
broken
and
Solid line
reserpine
pretreated
shows
with
line
shows
that
of
on
the
reserpine the
effect
reserpine,
ear and of
and more than 4 hours in duration. Therefore, the pretreatment of the animals with 10-MD did not affect the reserpine effect on the ear temperature. Similarly, the ad ministration of reserpine lowered the rectal temperature by 1.5 to 2.0°C and the thigh temperature by 1.2 to 3.0°C at 4 to 6 hours after the injection. The reduction of the heart and respiration rate was observed in response to reserpine in the rabbits treated with 10-MD. The manifestation of the resting waves in EEG by reserpine was not prevented by the treatment of the animals with 10-MD. IV.
Effects of 10-MD and reserpine on the rabbits treated with reserpine The effects of 10 mg/kg of 10-MD and
rabbits pretreated
of the ear, thigh and rectal intact
and treated rabbits
treatment
temperature,
1.
Comparison
rates values
in
of
the
ear
untreated
rabbits
were
measured
2 to
Room temperature
and heart
of the rabbits
the
and
and respiration
4 hours
: 24.0--0.3-C, Reserpine
those after
temperature, in
the
setting
heart reserpinized
up
of
the
The differences
rates
to twenty
between
the
hours after the pre
was 32.TC in average
rectal with
were studied in the
16 to 20 hours previously.
are shown in Table 1. Sixteen
the ear temperature TABLE
1.0 mg/kg of reserpine
with 1.0 mg/kg of reserpine
and
and
3.6°C higher
respiration
rabbits.
These
preparations.
1.0 mg/kg i.v. 16-20 hours before.
than that of the control animals, while the rectal temperature was 2.5-C lower than that of the controls. The heart and respiration rates in the treated animals decreased 35 and 44% respectively, comparing with those of the control animals. The full reco very of the EEG to manifest the arousal waves in the intact rabbits was obtained 30 to 60 minutes after the termination of ether anesthesia, while the time length for the recovery from ether anesthesia in the treated animals was 180 minutes or more. The reserpinized animals showed a sign of sedation, as described above. The effect of 10-MD on the ear temperature was markedly affected by the reserpine treatment (Fig. 4). Two of four rabbits showed a more prompt but slight elevation of the ear temperature, 1.5 and 1.0°C, lasting about 30 minutes. The second injection of the same dose of 10-MD 4 and 6 hours later resulted in the transient fall of the ear temperature by 1.3 and 0.5°C, respectively. Remaining 2 rabbits showed the fall of ear temperature, 0.5 and l.5`C, even by the first injection of 10-MD. However, the thigh temperature was affected by neither injection of 10-MD. On the other hand, the rectal temperature gradually fell and reached a plateau, showing a fall by 0.5 to 2.0°C, 4 hours after 10 mg/kg of 10-MD. The reduction of the heart and respiration rates by 10-MD was decreased by the treatment.
FIG. 4. Effect of 10 mg/kg of 10-MD on the ear and temperature of the reserpinized rabbits. In the figure the solid and broken lines of the same illustrate the effects of the first and the second tions, respectively. See text.
rectal upper mark injec
The previous treatment of the animals with reserpine markedly depressed or blocked the elevation of the ear tempera ture caused by the injection of reserpine (Fig. 5). However, the thigh temperature was lowered by reserpine injection and the fall was 1.0 to l.5°C 3 to 4 hours after the injection. The fall of the rectal tempera ture was abolished or unaffected by the injection of reserpine. The effect of reser pine on the rectal temperature depended on the extent of the fall produced by reserpine pretreatment. The transient manifestation of the resting waves in EEG in response to 10 mg/kg of 10-MD or 1 FiG. 5. Effect of 1 mg/kg of reserpine on the ear, thigh and rectal temperature mg/kg of reserpine was not modified by of the reserpinized rabbits. the previous reserpinization (Fig. 6). The resting waves intermingled with spindle bursts were observed for 30 to 60 minutes at the latency of 5 to 10 minutes. In some cases, 3 hours after the injection the spindles were repeatedly seen at the intervals of about 10 minutes. V. Effects of 10-MD and reserpineon the rabbits pretreatedwith SKF-385 The effects of 10 mg/kg of 10-MD and 1 mg/kg of reserpine were studied in the rabbits treated with 5 mg/kg of SKF-385 2 hours previously. The ear and thigh tem
FIG. 6.
Effects
of
10-MD
and
reserpine
on
the
cortical
EEG
of
the
reserpinized
rabbits.
perature was hardly affected by SKF-385 within 4 hours after the injection. On the other hand, the rectal temperature was gradually elevated until the elevation of 0.5 to 1.0-C showed a plateau 2 hours after the injection. The heart and respiration rates were usually increased about 10%, respectively.
FIG. 7. Effect of 10 mg/kg of 10-MD on the ear temperature of the animals treated with SKF-385, 5 mg/kg.
The
previous
the elevation ear
treatment
temperature
minutes. animals
rates nor
In the treated However, Though
spiration
spontaneous
animals
the
previous
rate and manifestation
the procedure
animals.
the administration
produced
animals
treatment
lasted , for 90 to 120 by , 10-MD in the treated Effects of the drug on, neither heart
was not affected
EEG were modified by the previous
of the ear temperature
in some treated
mg/kg of SKF-385 , dit u,not,.,modify The elevation of the
of 20 to 60 minutes
temperature
as well as in the non-treated
duce the elevation tion.
with.5
caused by 10-MD (Fig. 7).
by 3.1 to 5.8°C at latency
The thigh and rectal
and respiration
8).
of the animals
of the ear temperature
of 1 mg/kg of reserpine
did neither
nor the fall of the. rectal: temperature,
the thigh significantly
temperature depressed
instead
pro (Fig.
' showed some late eleva the reduction of the re
of the resting waves in EEG in response
slight tachycardia
treatment.
to reserpine,
of bradycardia.
FiG. 8. Effect of 1 mg/kg of reserpine on the ear, thigh and rectal temperature of the animals treated with 5 mg/kg of SKF-385.
VI. Effects of adrenaline,noradrenaline,acetylcholineand hexamethonium The intravenous injection of 3 pg/kg of adrenaline or 6 ug/kg of noradrenaline produced no significant change in the ear and rectal temperature. The injection of 10 pg/kg of adrenaline but not 20 beg/kg of noradrenaline, however, resulted in the slight and transient elevation of the ear temperature without changing the thigh and rectal temperature (Fig. 9). The heart rate was markedly decreased. The injection of 10 lug/kg of adrenaline or 20 beg/kg of noradrenaline to the rabbits , the ear temperature of which was elevated 3.6°C in average by the previous treatment with 1 mg/kg of reserpine, pro duced a marked ventricular rhythm. In these reserpinized animals the ear temperature responded to adrenaline as well as noradrenaline with slight elevation preceded by a
moderate fall. However, the duration of the response was 20 to 30 minutes. The effects of noradrenaline were somewhat less than those of adrenaline. Neither thigh npr rectal temperature was affected by the doses of the amines. Though the ear temperature was not affected by 5 and 10 fig/kg of acetylcholine, 10 mg/kg of hexamethonium elevated the ear temperature at latency of a few minutes. The peak elevation, 4.1 to 6.3 C, was observed 5 to 10 minutes after the injection and the elevated temperature fell to the normal level 30 to 40 minutes after the injection. During the elevation of the ear temperature the thigh and rectal tem perature was not affected. VII
Effects of 10-MD on the content of tissue catecholamines and serotonin The contents
of noradrenaline
brain cortex, brain renaline
in the
stem and atria, of ad
in the adrenals
and of serotonin
in the brain cortex, brain stem and ileum
FiG. 9. Effects of adrenaline (.-.l and nor adrenaline 'x-x) on the ear temperature and heart rate. Upper figure shows the effects of 3 beg/kg of adrenaline and 6 ug/ kg of noradrenaline, while the lower two illustrate the effects of 10 ,ug/kg of adrena line and 20,ug/kg of noradrenaline.
TABLE 2. Effects of 10 mg/kg of 10-MD on the catecholamine and serotonin content in the various tissues 3 and 16 hours after the injection. Catecholamine content in the brain and atria is calculated as noradrenaline ug/g wet tissue, while that in the adrenal glands is calculated as adrenaline ug/ two glands. Serotonin content is illustrated as ug/g wet tissue. Catecholamine
Serotonin
content
content
were studied in rabbits 3 and 16 hours after the intravenous injection of 10 mg/kg of 10-MD (Table 2). The isolated terminal of the ileum (2 g weight of wet tissue) was used for serotonin assay. The reduction of noradrenaline content in the brain cortex, brain stem and atria were 54, 46%; 68, 55%; and 77, 62% at 3 and 16 hours after 10-MD, respectively. Though the reduction was more marked at 3 hours than at 16 hours after the injection, the differences were statistically insignificant. The content of adrenaline in the adrenal glands was reduced by 22% at 3 hours after the injection, but the content at 16 hours was found to be almost normal. The content of serotonin in the brain cortex and brain stem showed significant decrease, 40, 30% and 57, 71% at 3 and 16 hours after the injection, respectively. However, the serotonin content in the ileum was not significantly affected by 10-MD. DISCUSSION The therapeutic indication of 10-MD for the clinical hypertension has been present ed by Mir and Lewis (13) because of mild and gradual fall of blood pressure with minor side effects. Thereafter, the compound was subjected to wide clinical application. The moderately prolonged hyperemia and warm sensation in the face and neck in response to the oral application of the compound, encountered in some patients, have facilitated the present experiments. The sensitive elevation of the ear or tail temperature in rabbits or rats in response to environmental temperature rise has been regarded to be the radiation mechanism of the body temperature (14, 15). Further, it is reasonable to consider that the warm sensation or hyperemia caused by the increased blood flow reflects to the elevation of the local temperature. Therefore, the effects of 10-MD on the ear temperature were mainly studied in the present experiments in order to analyse the mode of side actions described above. From the results of the present experiments the marked differences of the effects between 10-MD and reserpine are as follows 1) Unlike reserpine, 10-MD did not lower the rectal temperature in spite of the marked elevation of the ear temperature. 2) The pretreatment with 10-MD did not change the effects of both compounds, while the reserpinization modified the effects. 3) The pretreatment with SKF-385 prevented the temperature effects of reserpine but not of 10-MD. Although the ear, thigh and rectal temperatures in the restrained animals were considerably different individually, they were fairly constant in one animal for many hours at the room temperature of 24--0.3°C and under the exclusion of the external stimuli as far as possible. The administration of 10 mg/kg of 10-MD elevated the ear temperature by 3.0 to 5.5°C at latency of 20 to 40 minutes for 1.5 to 6 hours without changing the thigh or rectal temperature. Though the administration of 1 mg/kg of reserpine resulted in the similar elevation of the ear temperature lasting for more than 6 hours, the thigh temperature showed a tendency of slight fall and the rectal tempera
ture showed a steady fall, of 1.0 to 2.0°C. Some accordance` of the progression and maintenance of the elevated ear temperature with the phase of the progressive deple= tion of the tissue catecholamine by either compound, especially reserpine, suggests that the elevated ear temperature is a reflection of the increased blood flow elicited by the decreased sympathetic tone, of peripheral vascular bed. It is generally considered that the elevation of the ear temperature reflects the increased radiation of the body tem perature and, therefore, it should usually be followed by the fall of the rectal tempera ture. This is the case of reserpine in which the time length of the latency for the elevation of the ear temperature is proportional to that for the fall of the. rectal tem perature. However, since the fall of the rectal temperature .was also observed in the rabbits the ear temperature of which did not respond to reserpine, some depression of the heat regulating center might be involved in the fall 'of rectal, temperature by reser pine. On the contrary, in the case of 10-MD no fall of the rectal temperature was observed despite the marked elevation of the ear temperature. Accordingly, no affec tion of the rectal temperature by 10-MD was likely to derive from lack of, such central effect. Shorter duration of elevation of ear temperature by 10-MD than by reserpinee is also indicative for the lack of the depression of heat regulating center. The admi nistration of 10 mg/kg of hexamethonium did not affect the rectal temperature, although striking but short-lasting elevation of the ear temperature was always observed. Again, this may be due to the lack of central depression of hexamethonium. During the manifestation of the marked sedation in rabbits produced by 1 mg/kg of reserpine the content of noradrenaline or serotonin in the brain has been reported to be deprived by 90% or more (10, 16, 17). . When the content of both amines was depleted more than 50% at 3 and 16 hours after the administration of 10-MD, the rabbits did not show any sign of sedation. The evidence is suggestive for denying the causal relationship between the development of the sedation and the depletion of the brain amines. Nevertheless, it is accepted that reserpine, but not 10-MD, has the effect of central nervous depression which may or may not have some relations to the marked depletion of amine content in the brain. The effects of 10-MD and reserpine on the ear and rectal temperature were not modified by the prior treatment of the rabbits with the effective dose of 10-MD. On the other hand, the reserpirl}~ation of the animals depressed or blocked the responses of the ear and rectal temperature to 10-MD as well as reserpine. This evidence indicates that the elevation of the ear temperature in response to both compounds relates with the tissue catecholamine and/or serotonin. Three hours after 10 mg/kg of 10-MD the noradrenaline content was reduced by 54% in the brain cortex, by 68% in the brain stem and by 77% in the atria, while the serotonin content was reduced by 40% in the brain cortex and by 57% in the brain stern. The similar reduction on the tissue cate cholamine byo 10-MD has been shown by Higuchi et al. (7). The lesser depletion of noradrenaline or serotonin by 10-MD than by reserpine (10, 16, 17) may be responsible for the less susceptibility of the ear and rectal temperature to 10-MD or"reserpine by
the
pretreatment
of the
the
pretreatment
with
the
amines, The
making
long-lasting
accumulation
animals 10-MD
the
with may
subsequent
inhibition
of noradrenaline
10-MD.
produce reserpine
of monoamine and
serotonin
Otherwise, some
change
or 10-MD oxidase in the rat's
the
reserpinization
other
than
the
but
not
depletion
of
non-effective. by SKF-385 brain
and
in vivo, the increased' the elevation
of the
catecholaminelevel in the brain, atria and adrenal glands of the rabbits have alreaty been shown by several investigators (18-20). In addition, the pressor response of the dogs to SKF-385 has been shown to relate with the endogenous noradrenaline (21). Despite the possible manifestation of the sympathetic effects the administration of SKF 385 did not affect the ear temperature, but elevated the rectal temperature moderately. The prior treatment of the rabbits with SKF-385 blocked the effects of reserpine on the ear and rectal temperature, though it did not affect the effects of 10-MD. The blocking action of SKF-385 may be attributable to the prevention of the catecholamine depletion due to reserpine, as shown by Shimamoto et al. (20) in the rabbits. On the other hand, the amine depletion by 10-MD may be not prevented by the prior treat ment with SKF-385. Otherwise, the elevation of the ear temperature by 10-MD may be due to the direct dilator effect on the ear vessels independent of the amine depletion. The relatively large doses of adrenaline or noradrenaline did not elicit a significant change in the ear and rectal temperature. However, this evidence does not exclude the possibility that the elevation of the ear temperature by reserpine or 10-MD derives from the decrease of noradrenaline content in the peripheral tissues, because the ear temperature elevated by reserpine was somewhat lowered by the doses of the amines. The administration of 10-MD produced a resting pattern of waves in the EEG ir respective of the prior treatment of the animals with 10-MD or reserpine. Therefore, the manifestation of the resting waves could not be ascribed to the release and deple tion of catecholamine or serotonin in the tissues including the central nervous system. The resting waves are likely to relate with the effect other than the amine-depleting effect of the compound on some central nervous structures. The depletion of adrenaline in the adrenals observed in the present experiments was 22% at 3 hours after the injection of 10 mg/kg of 10-MD, and 16 hours later the level of adrenaline returned to normal. von Euler and Purkhold (22), Goodall and Kirshner (23) and von Euler and Ryd (24) have suggested that noradrenaline and adrenaline are stored at different sites, noticing that"while noradrenaline almost disappears in the organs after denervation, the adrenaline content, although low, is well maintained. The marked and prolonged depletion of noradrenaline in contrast to the less and short lasting depletion of adrenaline is suggestive for the action mechanism of 10-MD. The blockade by reserpine of the pressor response of rabbit to stimulation of the splanchnic nerve was shown by Shimamoto and Torii (25), while the same response of cat was reported to be not affected by 10-MD (13). The lack of diarrhea, by the way, may be attributable to no,-change in the intestinal serotonin content by 10-MD,
SUMMARY Effects
of 10-methoxydeserpidine
and rectal 1. and
temperature,
10-MD
rectal
by the
Although with
3. MD
but 4.
brain
rectal
From
temperature
especially
on
the
temperature
on
the ear
tested
ear
skin,
thigh
in the restrained
without
the
any
change
temperature
effects
of reserpine
the
due
with
skin
rabbits.
in the thigh
usually
on the
moderately
stem
atria,
content
that
to the different
regulating
center
ear
not
and
followed
scarcely
of
in the ileum the different
mechanism
modify
on
the
the treat
and reserpine.
the
effects
of 10
temperature.
the content
serotonin
change
temperature,
rectal
of adrenaline
of serotonin
the possibility
did
decreased
and
in the content
did
rectal
the effects of 10-MD
SKF-385
of 10-MD and
10-MD and
or abolished
animals
results, are
with thigh
of the
brain
the heat
animals
on the ear,
depressed
the decrease
further these
reserpine were
elevated
of the
reserpine
markedly
cortex,
less and 4.
and
administration
while
of ear
reserpine
treatment
treatment
brain stem,
much
the 10-MD
abolished The
in the
while
and
respiration
temperature.
reserpine
The
(10-MD)
ECG and
an elevation
of rectal
effects of both ment
caused
temperature,
fall
2.
EEG,
in the in the
of noradrenaline brain
cortex
and
adrenal
glands
was
10-MD
and
was not affected. effects
between
the central
nervous
system,
is discussed.
REFERENCES 1) KRONEBERG,G. AND SCHUMANN,H.J.: Arch. exp. Path. Pharmak. 231, 349 (1957) 2) ERANKO, O. AND Hopsu, V. : Endocrinology 62, 15 (1958) 3) STJARNE,L. AND SCHAPIRO,S. : Nature, Lond. 182, 1450 (1958) 4) HILLARP,N.A. : Ibid. 187, 1032 (1960) 5) MIRKIN, B.L. : J. Pharmacol. 133, 34 (1961) 6) LEROY, J.G. AND SCHAEPDRYVER, A.F.DE. : Arch. int. Pharmacodyn. 130, 231 (1961) 7) HIGUCHI,H., MATSUO, T., NAKATANI,G. AND SHIMAMOTO,K. : THIS JOURNAL 13, 36 (1963) 8) SHIMIZU,I.: Personal communication 9) BERTLER,A., CARLSON,A. AND ROSENGREN,E. : Acta physiol. scand. 44, 273 (1958) 10) HIGUCHI, H. : THIS JOURNAL 12, 34 (1962) 11) EULER, U.S. VON AND FLODING,I.: Acta physiol. scand. 33, Suppl. 118, 45 (1955) 12) BERTLER,A. : Ibid. 51, 75 (1961) 13) MIR, B.J. AND LEwIS, J.J. : J. Pharmacol. 12, 677 (1960) 14) JOHANSEN,K.: Acta physiol. scand. 55, 160 (1962) 15) GRANT, R.T. : J. Physiol. 167, 311 (1963) 16) CARLSSON,A., ROSENGREN,'E., BERTLER, A. AND NILSSON, J. : Psychotropic Drugs Edited GARATTINI,S. AND GHETTI, V., p. 363, Elsevier Publishing Co., Amsterdam (1958)
by
17) BRODIE, B. B. : 5-Hydroxytryptamine Edited by LEWIS, G. P., p. 64, Pergamon Press, New Nork (1958) 18) MAASS, A.R. AND NIMMO, M.J.: Nature, Lond. 184, 57 (1959) 19) GREEN, H. AND ERICKSON,R.W. : J. Pharmacol. 129, 237 (1960) 20) SHIMAMOTO,K., MATSUO, T., HATTORI, K. AND HONJO, T. : THIS JOURNAL (in press)
21) TODA, N., YAMAWAKI,T. AND Mlsu, Y. : Ibid. 12, 166 (1962) 22) EULER, U.S. VON AND PURKHOLD,A.: Acta physiol. scand. 24, 212 (1951) 23) GOODALL,McC. AND KIRSHNER,N. : J. clin. Invest. 35, 649 (1956) 24) EULER, U.S. VON AND RYD, G.: Acta physiol. scand. 59, 62 (1963) 25) SHIMAMOTO,K. AND TORII, H.: THIS JOURNAL 10, 126 (1961)