JOURNAL
OF
Vol.
/iPPLIEr,
PHYSIOLOGY
38, No. 2, February
1975.
Printed
in U.S.A.
Nature
of induction
in cold
exposure
V. SITARAMAM AND Department of Biochemdry,
of tryptophan
RAMASARMA Indian Institute of Science, Bangdora
T.
SITARAMAM, V., AND T . RAMASARMA. Nature of induction of trypto/ban ~yrrolase in cold exposure. J. Appl. Physiol. 38(Z) : 245-249. 1975 -The activity of hepatic tryptophan pyrrolase in rats exposed to cold increased rapidly and reached a maximum of threefold at 8 h. On continued exposure up to 48 h stress, the activity partly decreased but remained at a level higher than the initial. Withdrawal from the cold stress reversed the change. Adrenalectomy or treatment with inhibitors of protein synthesis abolished the increase in the enzyme activity during cold stress indicating a possible involvement of corticosteroids and de nova protein synthesis. Treatment with drugs known to block autonomic nervous system failed to inhibit the cold-mediated increase in enzyme activity. The results suggest that the increase in enzyme activity obtained on cold exposure is mediated by corticosteroids and not by either indolealkylamines or autonomic nervous system. The changes in the enzyme obtained under cold stress with respect to the overshoot phenomenon, relationship to the degree of stress and reversibility on withdrawal from the stress indicate the CCadaptate” nature of the response.
enzyme
induction;
cortisol;
autonomic
pyrrolase
nervous
system
TRYPTOPHAN PYRROLASE (EC. 1.13 q 1.12, tryptophan-2,3dioxygenase) in the liver catalyzes the first reaction in the catabolism of tryptophan and is known to be induced by a variety of agents such as cortisol ( 19), tryptophan ( 17), certain drugs (32) and toxins (38), and a number of psychopharmacological compounds (27), as well as by stress conditions of immobilization (4), cold exposure (27), and hypobaric hypoxia ( 14) but not hypoxia per se (37). Essentially these diverse agents and conditions fall into three modes of induction. The ,first mode of induction is mediated by cortisol by a process implicating increase in mRNA and protein synthesis de novo (30). The second mode of induction is mediated by tryptophan by a combination of increased polysomal aggregation (7), increased tryptophanyl tRNA (25), stabilization of preformed enzyme from degradation (35), and increased partition of the heme cofactor to the apoenzyme (20). The third mode of induction involves autonomic nervous system as supported by the evidence of increased enzyme activity on spinal section at C7--8 in adrenalectomized animals (39), and on stimulation of selected areas in hypothalamus (34). It is of interest to identify the causal factor involved in the rapid induction of tryptophan pyrrolase obtained during cold exposure wherein it is known that in a matter of hours adrenocortical and autonomic nervous systems are acti-
560012,
India
vated (15, 22). While it is generally considered that cortisol is responsible for this induction (8, 3O), it had been reported that adrenalectomy failed to abolish the increased enzyme activity in cold-exposed animals (27). We have reexamined the issue with respect to the three modes of induction using the experimental approaches of adrenalectomy and treatment with specific inhibitors. The data recorded here indicate that during the initial phase of cold stress tryptophan pyrrolase showed an overshoot phenomenon that can be classified as “adaptate response” as described by Adolph (l), and that the increase was due to protein synthesis de novo and was mediated by corticosteroids and not by indolealkylamines or autonomic nervous system. MATERIALS
AND
METHODS
Animals. Male albino rats of Wistar strain of the weight range 150-200 g were obtained from the Central Animal Facility of the Institute. They were fed Hindustan Lever pellet diet consisting of 24 % protein, 4 G/ fat, 50 % carbohydrate, and other nutrients. Water was given ad libitum. Animals were kept in ventilated rooms maintaining the light-darkness cycles at 25&2”C ambient temperature. The animals were exposed to cold at specified temperature in a ventilated chamber with regular cycles of light (8 A.M.-~ P.M.) and darkness (5 P.M.-~ A.M.), comparable with the control animals. They were normally killed between 10 and 11 A.M. by cervical dislocation and the livers were removed. Preparation of liver extracts. The livers were homogenized in 2 vol (v/w) of cold 1.15 % aq KC1 in a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 36,000 X g for 40 min in a refrigerated Sorval RC 2B centrifuge. The clear supernatant consisting of cytosol and light microsomal fractions was aspirated with a needle avoiding the top lipid layer. This extract was used for the assay of the enzyme. M&o& of assay. The method of Knox and Auerbach (18) was modified in that a) the supernatant fraction contained microsomes, presence of which is known to yield a higher activity (1 l), 6) preincubation of the enzyme with tryptophan was carried out for 30 min prior to incubation (6), as described below. The assays were performed in Erlenmeyer flasks kept in a metabolic shaker at 37°C. The preincubation mixture consists of 2 pmol L-tryptophan, 50 pg hematin, 200 pmol potassium phosphate bufier, pH 7.0, and 25-30 mg super-
245 Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
246 natant protein in a total volume of 2.0 ml. Preincubation was started with the addition of the enzyme and carried out for 30 min at 37°C without shaking, in two identical sets of flasks. Into one set, serving as the blank, 1.0 ml of 10 5; trichloroacetic acid (TCA) was added after 30 min. Then into both sets of flasks were added 10 pm01 L-tryptophan in 2 ml. The reaction flasks (including the blanks) were further incubated for a period of 20 min with shaking at 37°C. Reaction was stopped with 1.0 ml of 10% TCA as in the case of blanks. The protein-free supernatant was adjusted to pH 7.0 with 1 N NaOH. Kynurenine formed was measured by its absorption at 365 nm in a Beckman Protein in the enzyme extract was IX spectrophotometer. measured by biuret method (9). Specific activity is expressed as nanomoles of kynurenine formed per hour per milligram of protein. Absorbance at 365 nm measured as the difference between the reaction flask minus the blank, corresponds to the product formed in the later 20 min of incubation. This method of assay was standardized after obtaining the following information on the properties: a) there was no lag phase as shown by time study; 6) preincubation for 30 min yielded maximum activity; c) ascorbate addition did not enhance the activity; d) addition of heme before or after preincubation did not affect the activity; and e) presence of microsomes in the supernatant enhanced the activity though microsomes per se had no activity. Assay performed in the presence and absence of exogenous haematin in animals treated with cortisol and tryptophan showed an ratio and therefore this identical holoenzyrne : apoenzyme assay system does not distinguish between the two types of induction . Adrenalec~oq. Adrenalectomy was done by dorsal approach under Nembutal (50 mg/kg) anesthesia and these rats were maintained on saline for drinking water. Animals were kept for 5-7 days and then used for experiments. The completeness of adrenalectomy was checked by examination of the retroperitoneal tissues postmortem and by the inability of randomly selected operated animals to survive in cold beyond 4-6 h on exposure to cold at 0-5°C. Eualuntion of data. The data are statistically analyzed by the Student t-test. P values 0.05; 4-12 h, P < 0.001; 24 hr, P < 0.002; 48 h, P < 0.05; the decrease during 8-48 h, P < 0.001. TABLE 1. Reversal of cold-mediated induction of he)atic tryfltofhan pyrrolase on withdrawal of stress -Period Withdrawal,
Specific
of h
Activity*
Control
29.6
Initial 4 8
exposed
zt
2.5
71.5
(1~) 56.6 &
7.9
97.6
+ 7.0t _I (6) + 10.0:
47.6
(6) zt 3.8
49.8
+
5.0
(6)
.- -.70 of Control
Cold
(6)
240 173 96
(6)
Groups of rats were exposed to cold (0-2°C) for 4 h and withdrawn from the stress and restored to room temperature for specified periods. Control group of animals were killed at each time interval and the results are compared. * Values are means =t SE, with no. of animals in each group given in parentheses. t Levels of significance: P < 0.001. $ P < 0.01. 2. llegree of cold stress and induction of hej9atic tryptophan pyrrolase -“---
TABLE
D if?erence Temp,
Nil RESULTS
SITARAMAM
from Ambient (AT), “C
(control) 7-9 13 -15 20-25
No. of Animals
Specific
12 4 4 4
26.1 44.2 45.0 57.7
Activity*
zt + zt 41
7,f 7.4t 5.ot 8,9x
y. of Control
100 170 173 220
Groups of rats were exposed for a period of 4 h in cold chambers set at different temperatures. The control animals were kept at room temperature (about 25°C) * Values are means & SE. t Levels of significance compared to controls: P < 0.05. $P< 0.02. l
reached a maximum of threefold by about 8 h. The activity decreased thereafter but continued to be higher than the initial level even at 48 h. It is noted that the increase at the short interval of 3 h compared to initial and the decrease at
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
TRYPTOPHAN
PYRROLASE
IN
COLD
EXPOSURE
247
TABLE 3. Effect of treatment with #role&-synthesis inhibitors cold-mediated induction of hepatic tryptophan ;byrrolase -~--------. -- -Specific _~
---
Treatment
Control.
Saline
34.3
Cycloheximide Actinomycin -.- _- -- ----
24.7 D
36.4
+ c (3) + (8) + (8)
-
Activity* Cold
5.9 2.9 8.1
on
68.6 28.3 34.7
exposed
+
3.9t
(4) + 3.01 (8) + 2.7$ (7)
Groups of rats were in.jected intraperitoneally cycloheximide fig/rat) or actinomycin D (160 pg/rat) in 1 ml 0.9% NaCl and after 30 min at room temperature they were transferred to a cold chamber (O-2 “C) and kept for 3 h. Control animals at room temperature were also treated similarly with the inhibitors. *Values are means + SE, with no. of animals in each group given in parentheses. t Levels of significance compared to controls: $ Not significant. P < 0.01. (250
48 h, compared to the peak at 8 h as well as initial, are statistically significant (Fig. 1). Reversal after withdrawal from stress. Rats were exposed to cold (O-5°C) f or 4 h which stimulated the activity over twofold. They were then withdrawn from the stress and kept at room temperature for the time intervals of 4 and 8 h. At the end of cold exposure (4 h) and at the end of withdrawal periods (8 and 12 h, from the starting time), control animals were also killed along with the experiin view of the known increase mental groups for comparison in the enzyme activity due to circadian rhythm. The initial increase of 240 % was reduced to 173 c/o at 4 h and to the control level by 8 h of withdrawal from stress (Table l)Thus withdrawal from the stress causes a reversal though at a slower rate than the increase. Degree of cold dress. It was of interest to see whether any correlation exists between the degree of stress (as measured by the difference from control temperature) and the change in the enzyme activity. The results in Table 2 show that a difference from ambient temperature of 7-9°C was enough to obtain the response. With increase in the degree of stress, increased stimulation of the enzyme activity was obtained at the time interval tested, though without exact proportionality. Assay performed in the absence of exogenous heme also showed a similar pattern. Inhibitors of;brotein y)nthesis. To determine whether or not the stimulation of enzyme activity was by increasing the synthesis of protein was tested by treating the rats simultaneously with cycloheximide or actimonycin D, the wellknown inhibitors of protein synthesis. Rats were given intraperitoneally, cycloheximide (250 pg/rat) or actinomycin D (160 pg/rat) at the specified dosages known to prevent protein synthesis for at least 4 h. After 30 min at the rats were transferred to the cold room temperature, chamber (0-5°C) for a further period of 3 h. The rats in the control group were also given the same dosages of the inhibitors and left at room temperature for the entire experimental period. The enzyme activity was measured at the end of the experimental period. It can be seen from results in Table 3 that both inhibitors totally abolished the induction of the enzvme.
Efect of adrenalectoml:. In view of the known induction of I this enzyme by cortisol, it was of interest to test whether increased concentration of corticosteroids known to occur in cold exposure was responsible for the observed efiects. Results in Table 4 show that adrenalectomized control animals have lower enzyme activity than the control intact animals suggesting that corticosteroids also contribute to the normal levels of the enzyme. Further, adrenalectomy totally abolished the induction due to cold stress contrary to the earlier report (27). In the same set of experiments four other groups of adrenalectomized rats were supplemented with small doses of cortisol known to induce tyrosine aminotransferase in such animals (29) and therefore may also normalize other corticosteroid-dependent functions. As expected even these small doses of cortisol induced tryptophan pyrrolase in adrenalectomized animals (1’ < 0.01). On simultaneous exposure of such animals to cold, however, significantly lower enzyme levels compared to corresponding controls at room temperature, were obtained (Table 4). These results point out that additional factors are involved in the control of the enzyme levels in cold exposure. Drugs aJecting autonomic nervous vstem. In view of the known increase in the activity of autonomic nervous system in cold exposure, its role in the cold-induced stimulation of the enzyme activity was studied. The following drugs affecting this system at different levels were employed for this purpose : bretylium-an agent which decreases the discharge of norepinephrine from nerve endings; atropine-a blocker of the muscarinic receptors of acetylcholine; RO44602-an L-aromatic amino acid decarboxylase inhibitor. The drugs, atropine and bretylium, were without effect in 5-100 lug range/assay when tested in vitro. R04-4602 was without effect in 50-1,000 pg range/assay in vitro. TABLE 4. Effeci of adrenalectomy and autonomic drugs on cold-mediated induction of hepatic tryp tufihan pyrrolase .---- - - .- _ .__~__ ^-_------- -- - -. ,.. ____ Specific
Activity*
Treatment Control
Normal IIdrenalectomized
40.5
It
27.1
(8) +
3.1 2.2
exposed
72.9
+
22.3
(6) zt 2.2
(12) Adrenalectomized cortisol 0.1 mg/rat
~I
Cold
12.0t
W)
plus 68,7
+
4.0
43.0
h
75.7
(5) + 12.4t
9.6t
139.6
(6) + 21.6
htropine
52.2
(5) + 4.5
118.7
(5) + 15.35:
Bretylium
45.7
(7) zk 3.4
101.5
(7) + 6.81
79.3
(9> & 9.65
132.0
(6) + 8.41
0.3
mg/rat
Atropine
plus
-Groups of adrenalectomized with cortisol. peritoneally mg/rat) 0.5 * Values are in parentheses.
bertylium
(7)
--_.
controls: mal-
P
OF
Vol.
/iPPLIEr,
PHYSIOLOGY
38, No. 2, February
1975.
Printed
in U.S.A.
Nature
of induction
in cold
exposure
V. SITARAMAM AND Department of Biochemdry,
of tryptophan
RAMASARMA Indian Institute of Science, Bangdora
T.
SITARAMAM, V., AND T . RAMASARMA. Nature of induction of trypto/ban ~yrrolase in cold exposure. J. Appl. Physiol. 38(Z) : 245-249. 1975 -The activity of hepatic tryptophan pyrrolase in rats exposed to cold increased rapidly and reached a maximum of threefold at 8 h. On continued exposure up to 48 h stress, the activity partly decreased but remained at a level higher than the initial. Withdrawal from the cold stress reversed the change. Adrenalectomy or treatment with inhibitors of protein synthesis abolished the increase in the enzyme activity during cold stress indicating a possible involvement of corticosteroids and de nova protein synthesis. Treatment with drugs known to block autonomic nervous system failed to inhibit the cold-mediated increase in enzyme activity. The results suggest that the increase in enzyme activity obtained on cold exposure is mediated by corticosteroids and not by either indolealkylamines or autonomic nervous system. The changes in the enzyme obtained under cold stress with respect to the overshoot phenomenon, relationship to the degree of stress and reversibility on withdrawal from the stress indicate the CCadaptate” nature of the response.
enzyme
induction;
cortisol;
autonomic
pyrrolase
nervous
system
TRYPTOPHAN PYRROLASE (EC. 1.13 q 1.12, tryptophan-2,3dioxygenase) in the liver catalyzes the first reaction in the catabolism of tryptophan and is known to be induced by a variety of agents such as cortisol ( 19), tryptophan ( 17), certain drugs (32) and toxins (38), and a number of psychopharmacological compounds (27), as well as by stress conditions of immobilization (4), cold exposure (27), and hypobaric hypoxia ( 14) but not hypoxia per se (37). Essentially these diverse agents and conditions fall into three modes of induction. The ,first mode of induction is mediated by cortisol by a process implicating increase in mRNA and protein synthesis de novo (30). The second mode of induction is mediated by tryptophan by a combination of increased polysomal aggregation (7), increased tryptophanyl tRNA (25), stabilization of preformed enzyme from degradation (35), and increased partition of the heme cofactor to the apoenzyme (20). The third mode of induction involves autonomic nervous system as supported by the evidence of increased enzyme activity on spinal section at C7--8 in adrenalectomized animals (39), and on stimulation of selected areas in hypothalamus (34). It is of interest to identify the causal factor involved in the rapid induction of tryptophan pyrrolase obtained during cold exposure wherein it is known that in a matter of hours adrenocortical and autonomic nervous systems are acti-
560012,
India
vated (15, 22). While it is generally considered that cortisol is responsible for this induction (8, 3O), it had been reported that adrenalectomy failed to abolish the increased enzyme activity in cold-exposed animals (27). We have reexamined the issue with respect to the three modes of induction using the experimental approaches of adrenalectomy and treatment with specific inhibitors. The data recorded here indicate that during the initial phase of cold stress tryptophan pyrrolase showed an overshoot phenomenon that can be classified as “adaptate response” as described by Adolph (l), and that the increase was due to protein synthesis de novo and was mediated by corticosteroids and not by indolealkylamines or autonomic nervous system. MATERIALS
AND
METHODS
Animals. Male albino rats of Wistar strain of the weight range 150-200 g were obtained from the Central Animal Facility of the Institute. They were fed Hindustan Lever pellet diet consisting of 24 % protein, 4 G/ fat, 50 % carbohydrate, and other nutrients. Water was given ad libitum. Animals were kept in ventilated rooms maintaining the light-darkness cycles at 25&2”C ambient temperature. The animals were exposed to cold at specified temperature in a ventilated chamber with regular cycles of light (8 A.M.-~ P.M.) and darkness (5 P.M.-~ A.M.), comparable with the control animals. They were normally killed between 10 and 11 A.M. by cervical dislocation and the livers were removed. Preparation of liver extracts. The livers were homogenized in 2 vol (v/w) of cold 1.15 % aq KC1 in a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 36,000 X g for 40 min in a refrigerated Sorval RC 2B centrifuge. The clear supernatant consisting of cytosol and light microsomal fractions was aspirated with a needle avoiding the top lipid layer. This extract was used for the assay of the enzyme. M&o& of assay. The method of Knox and Auerbach (18) was modified in that a) the supernatant fraction contained microsomes, presence of which is known to yield a higher activity (1 l), 6) preincubation of the enzyme with tryptophan was carried out for 30 min prior to incubation (6), as described below. The assays were performed in Erlenmeyer flasks kept in a metabolic shaker at 37°C. The preincubation mixture consists of 2 pmol L-tryptophan, 50 pg hematin, 200 pmol potassium phosphate bufier, pH 7.0, and 25-30 mg super-
245 Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
246 natant protein in a total volume of 2.0 ml. Preincubation was started with the addition of the enzyme and carried out for 30 min at 37°C without shaking, in two identical sets of flasks. Into one set, serving as the blank, 1.0 ml of 10 5; trichloroacetic acid (TCA) was added after 30 min. Then into both sets of flasks were added 10 pm01 L-tryptophan in 2 ml. The reaction flasks (including the blanks) were further incubated for a period of 20 min with shaking at 37°C. Reaction was stopped with 1.0 ml of 10% TCA as in the case of blanks. The protein-free supernatant was adjusted to pH 7.0 with 1 N NaOH. Kynurenine formed was measured by its absorption at 365 nm in a Beckman Protein in the enzyme extract was IX spectrophotometer. measured by biuret method (9). Specific activity is expressed as nanomoles of kynurenine formed per hour per milligram of protein. Absorbance at 365 nm measured as the difference between the reaction flask minus the blank, corresponds to the product formed in the later 20 min of incubation. This method of assay was standardized after obtaining the following information on the properties: a) there was no lag phase as shown by time study; 6) preincubation for 30 min yielded maximum activity; c) ascorbate addition did not enhance the activity; d) addition of heme before or after preincubation did not affect the activity; and e) presence of microsomes in the supernatant enhanced the activity though microsomes per se had no activity. Assay performed in the presence and absence of exogenous haematin in animals treated with cortisol and tryptophan showed an ratio and therefore this identical holoenzyrne : apoenzyme assay system does not distinguish between the two types of induction . Adrenalec~oq. Adrenalectomy was done by dorsal approach under Nembutal (50 mg/kg) anesthesia and these rats were maintained on saline for drinking water. Animals were kept for 5-7 days and then used for experiments. The completeness of adrenalectomy was checked by examination of the retroperitoneal tissues postmortem and by the inability of randomly selected operated animals to survive in cold beyond 4-6 h on exposure to cold at 0-5°C. Eualuntion of data. The data are statistically analyzed by the Student t-test. P values 0.05; 4-12 h, P < 0.001; 24 hr, P < 0.002; 48 h, P < 0.05; the decrease during 8-48 h, P < 0.001. TABLE 1. Reversal of cold-mediated induction of he)atic tryfltofhan pyrrolase on withdrawal of stress -Period Withdrawal,
Specific
of h
Activity*
Control
29.6
Initial 4 8
exposed
zt
2.5
71.5
(1~) 56.6 &
7.9
97.6
+ 7.0t _I (6) + 10.0:
47.6
(6) zt 3.8
49.8
+
5.0
(6)
.- -.70 of Control
Cold
(6)
240 173 96
(6)
Groups of rats were exposed to cold (0-2°C) for 4 h and withdrawn from the stress and restored to room temperature for specified periods. Control group of animals were killed at each time interval and the results are compared. * Values are means =t SE, with no. of animals in each group given in parentheses. t Levels of significance: P < 0.001. $ P < 0.01. 2. llegree of cold stress and induction of hej9atic tryptophan pyrrolase -“---
TABLE
D if?erence Temp,
Nil RESULTS
SITARAMAM
from Ambient (AT), “C
(control) 7-9 13 -15 20-25
No. of Animals
Specific
12 4 4 4
26.1 44.2 45.0 57.7
Activity*
zt + zt 41
7,f 7.4t 5.ot 8,9x
y. of Control
100 170 173 220
Groups of rats were exposed for a period of 4 h in cold chambers set at different temperatures. The control animals were kept at room temperature (about 25°C) * Values are means & SE. t Levels of significance compared to controls: P < 0.05. $P< 0.02. l
reached a maximum of threefold by about 8 h. The activity decreased thereafter but continued to be higher than the initial level even at 48 h. It is noted that the increase at the short interval of 3 h compared to initial and the decrease at
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
TRYPTOPHAN
PYRROLASE
IN
COLD
EXPOSURE
247
TABLE 3. Effect of treatment with #role&-synthesis inhibitors cold-mediated induction of hepatic tryptophan ;byrrolase -~--------. -- -Specific _~
---
Treatment
Control.
Saline
34.3
Cycloheximide Actinomycin -.- _- -- ----
24.7 D
36.4
+ c (3) + (8) + (8)
-
Activity* Cold
5.9 2.9 8.1
on
68.6 28.3 34.7
exposed
+
3.9t
(4) + 3.01 (8) + 2.7$ (7)
Groups of rats were in.jected intraperitoneally cycloheximide fig/rat) or actinomycin D (160 pg/rat) in 1 ml 0.9% NaCl and after 30 min at room temperature they were transferred to a cold chamber (O-2 “C) and kept for 3 h. Control animals at room temperature were also treated similarly with the inhibitors. *Values are means + SE, with no. of animals in each group given in parentheses. t Levels of significance compared to controls: $ Not significant. P < 0.01. (250
48 h, compared to the peak at 8 h as well as initial, are statistically significant (Fig. 1). Reversal after withdrawal from stress. Rats were exposed to cold (O-5°C) f or 4 h which stimulated the activity over twofold. They were then withdrawn from the stress and kept at room temperature for the time intervals of 4 and 8 h. At the end of cold exposure (4 h) and at the end of withdrawal periods (8 and 12 h, from the starting time), control animals were also killed along with the experiin view of the known increase mental groups for comparison in the enzyme activity due to circadian rhythm. The initial increase of 240 % was reduced to 173 c/o at 4 h and to the control level by 8 h of withdrawal from stress (Table l)Thus withdrawal from the stress causes a reversal though at a slower rate than the increase. Degree of cold dress. It was of interest to see whether any correlation exists between the degree of stress (as measured by the difference from control temperature) and the change in the enzyme activity. The results in Table 2 show that a difference from ambient temperature of 7-9°C was enough to obtain the response. With increase in the degree of stress, increased stimulation of the enzyme activity was obtained at the time interval tested, though without exact proportionality. Assay performed in the absence of exogenous heme also showed a similar pattern. Inhibitors of;brotein y)nthesis. To determine whether or not the stimulation of enzyme activity was by increasing the synthesis of protein was tested by treating the rats simultaneously with cycloheximide or actimonycin D, the wellknown inhibitors of protein synthesis. Rats were given intraperitoneally, cycloheximide (250 pg/rat) or actinomycin D (160 pg/rat) at the specified dosages known to prevent protein synthesis for at least 4 h. After 30 min at the rats were transferred to the cold room temperature, chamber (0-5°C) for a further period of 3 h. The rats in the control group were also given the same dosages of the inhibitors and left at room temperature for the entire experimental period. The enzyme activity was measured at the end of the experimental period. It can be seen from results in Table 3 that both inhibitors totally abolished the induction of the enzvme.
Efect of adrenalectoml:. In view of the known induction of I this enzyme by cortisol, it was of interest to test whether increased concentration of corticosteroids known to occur in cold exposure was responsible for the observed efiects. Results in Table 4 show that adrenalectomized control animals have lower enzyme activity than the control intact animals suggesting that corticosteroids also contribute to the normal levels of the enzyme. Further, adrenalectomy totally abolished the induction due to cold stress contrary to the earlier report (27). In the same set of experiments four other groups of adrenalectomized rats were supplemented with small doses of cortisol known to induce tyrosine aminotransferase in such animals (29) and therefore may also normalize other corticosteroid-dependent functions. As expected even these small doses of cortisol induced tryptophan pyrrolase in adrenalectomized animals (1’ < 0.01). On simultaneous exposure of such animals to cold, however, significantly lower enzyme levels compared to corresponding controls at room temperature, were obtained (Table 4). These results point out that additional factors are involved in the control of the enzyme levels in cold exposure. Drugs aJecting autonomic nervous vstem. In view of the known increase in the activity of autonomic nervous system in cold exposure, its role in the cold-induced stimulation of the enzyme activity was studied. The following drugs affecting this system at different levels were employed for this purpose : bretylium-an agent which decreases the discharge of norepinephrine from nerve endings; atropine-a blocker of the muscarinic receptors of acetylcholine; RO44602-an L-aromatic amino acid decarboxylase inhibitor. The drugs, atropine and bretylium, were without effect in 5-100 lug range/assay when tested in vitro. R04-4602 was without effect in 50-1,000 pg range/assay in vitro. TABLE 4. Effeci of adrenalectomy and autonomic drugs on cold-mediated induction of hepatic tryp tufihan pyrrolase .---- - - .- _ .__~__ ^-_------- -- - -. ,.. ____ Specific
Activity*
Treatment Control
Normal IIdrenalectomized
40.5
It
27.1
(8) +
3.1 2.2
exposed
72.9
+
22.3
(6) zt 2.2
(12) Adrenalectomized cortisol 0.1 mg/rat
~I
Cold
12.0t
W)
plus 68,7
+
4.0
43.0
h
75.7
(5) + 12.4t
9.6t
139.6
(6) + 21.6
htropine
52.2
(5) + 4.5
118.7
(5) + 15.35:
Bretylium
45.7
(7) zk 3.4
101.5
(7) + 6.81
79.3
(9> & 9.65
132.0
(6) + 8.41
0.3
mg/rat
Atropine
plus
-Groups of adrenalectomized with cortisol. peritoneally mg/rat) 0.5 * Values are in parentheses.
bertylium
(7)
--_.
controls: mal-
P
