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Seasonal changes in phenylethanolamine-N-methyl transferase and monoamine oxidase activity in the ground squirrel (Citellus citellus)
Camp. Biochem. Physiol., 1974, Vol. 48A, pp. 127 to 131. Pergamon Press. Printed in Great Britain
SEASONAL CHANGES IN PHENYLETHANOLAMINE-NMETHYL TRANSFERASE AND MONOAMINE OXIDASE ACTIVITY IN THE GROUND SQUIRREL (CITELLUS CITELLUS) V. M. PETROVICl, VERA JANI@, D. GRIPOIS2 and with the technical collaboration of LJ. JELI~
J. ROFF12
lInstitute of Physiology, Faculty of Sciences, Laboratory of Endocrinology, Institute for Biological Research, Belgrade (Yugoslavia); and 2Laboratory of Endocrinology, University of Paris XI, 91405-Orsay (France) (Received
5 June
1973)
Abstract-l. In the active, normothermic, male ground squirrel (Citellus citellus) phenylethanolamine-N-methyl transferase (PNMT) and monoamine oxidase (MAO) activity was studied during different seasons: in September, February and April. 2. The adrenal weight and PNMT activity (counts/min per pair of adrenals) were significantly higher in animals kept at a room temperature of 20-25°C and examined in early spring (April) than in those kept at the same room temperature but examined in autumn, before the period of hibernation (September). 3. MAO activity (counts/min per mg) in the liver, heart, kidney and hypothalamus was significantly lower in animals examined in April compared with those examined in September. 4. In animals kept at a room temperature of 30°C (so that hibernation could be prevented) and examined in February the adrenal weight and PNMT activity were significantly higher when compared with animals examined in autumn. MAO activity in the liver, heart, kidney and hypothalamus of these animals was significantly lower than in those examined in September.
INTRODUCTION MANY RESULTSindicate
that in different seasons the amount of catecholamines in the adrenals, heart, hypothalamus and some other organs of hibernators changes. In autumn, before hibernation, in a normothermic hedgehog an accumulation of these amines in the adrenals occurs (see Kayser, 1961). PetroviC & Davidovic (1965, 1968) found in autumn an accumulation of catecholamines in the adrenals of the normothermic ground squirrel as well as a fresh increase at the end of hibernation. The increased adrenocortical activity, as evaluated by the augmentation of plasma 17-OHCS and decreased adrenal ascorbic acid levels, was also found in the ground squirrel examined in autumn before hibernation, whereas extremely high adrenocortical activity in these animals was found in spring (PetroviC & Janid, 1964a). All these data suggest the possibility of an interrelationship between adrenocortical and medular activity in hibernators during different seasons. 5
127
128
V. M. PETROVIC, V. JANIC, D. GRIPOIS AND J. ROFFI
The work reported here was initiated while investigating catecholamines degradation in some tissues of the ground squirrel exposed to cold in early autumn (PetroviC et al., 1973). The aim of the present investigation is to determine the phenylethanolamine-N-methyl transferase (PNMT) and monoamine oxidase (MAO) activity in animals kept at the same room temperature and examined in different seasons. It was also interesting to find out the level of activity of these enzymes in normothermic animals in winter. In order to maintain their normothermic state it was necessary to keep them at a room temperature of 30°C during the autumn and winter.
MATERIALS
AND
METHODS
Adult male ground squirrels (Citellus citellus) were housed in individual plastic cages with wood-chip bedding, in a room at 20-25°C or at 30°C. They were maintained on daily illumination and given pelleted rat diet and water ad lib. The experiment was performed in three groups of animals, as follows: one group was kept at 20-25°C and examined in September; the second was kept at 20-25°C and examined in April; the third one was kept from the beginning of autumn in the warm room (the temperature of which was 30°C in order to prevent hibernation) and was examined in February. Phenylethanolamine-Nmethyl transferase activity was determined by the method of Wurtman & Axelrod (1965) as modified by Gripois & Parvez (1972). This method consists of the incubation of adrenal extracts with S-adenosyl methionine- 14C, in the presence of normethanephrine, and of the measurement of the radioactivity of epinephrine formed during this process. Determination of monoamine-oxidase activity was made according to the method of Wurtman & Axelrod (1963). Tryptamine- 14C bisuccinate was used as a radioactive substrate. Results are expressed in counts/min per pair of adrenals or per mg of tissue and are presented in the Tables 1 and 2. TABLE l-PHENYLETHANOLAMINE-N-METHYL TRANSFERASE ACTIVITYIN ADRENALS Season and experimental conditions
Body temperature (“C)
No. of ground squirrels
Weight of pairs of adrenals (mg)
Counts/min per pair of adrenals
Counts/min per mg of adrenals
Septemberanimals kept at 20-25°C
46.53 f 5.1 (A,)
2886k109 (B,)
6497 f 596
6
87.99 f 11.31 (A,)
4122f285 (B,)
4884+419
6
120.44 f 11.29 (A,)
3813+179 (B3)
3404+ 367
36-37
8
36-37
36-37
Aprilanimals kept at 20-25°C Februaryanimals kept at 30°C
MeanfS.E. of the mean. PcO~OO5; B1-B,, P
A,-A2,
P
Al-AS,
P
B1-B2,
SEASONAL CHANGES IN GROUND SQUIRREL METABOLISM TABLE 2--MONOAMINE
OXIDASE ACTIVITY IN THE LIVER, HEART, KIDNEY AND HYPOTHALAMUS Counts/min
Organs
129
per mg tissue
September-animals kept at 20-25°C
April-animals kept at 20-25°C
February-animals kept at 30°C 2840 + 97 (Aa)
Liver
4923 f 268 (A,)
2832 + 312 (A,)
Heart
2174 + 45 (B,)
1364 f 87 (B,)
1203 + 52 (Ba)
Kidney
32442 128
2561+ 166
2434 + 110 (C,)
Hypothalamus
96815 364 (DJ
Mean+S.E.
of
the
P-cO.01; D,-D,, P
(C,)
mean.
A,-A,,
(C,)
8802 f 247 (D,) Al-AZ,
P
P
B,-B,,
7075 f 474 (DJ P
C,-C2,
P
RESULTS
Phenylethanolamine-N-methyl
transferase activity
The adrenal weight and PNMT activity, expressed in counts/min per pair of adrenals, were significantly higher in animals examined in early spring than in those kept at the same room temperature (20-25°C) and studied in September. The adrenal PNMT activity in animals which were prevented from hibernating by keeping them in the warm room at 30°C and which were examined in February was at about the same level as that of animals kept at 20-25°C and examined in early spring, but the activity was significantly higher compared with that of animals studied in the autumn. The adrenal weight of these animals, however, was higher than that of animals examined in the spring. Monoamine oxidase activity The activity of MAO was significantly lower in all organs studied (liver, heart, kidney and hypothalamus) in animals examined in April compared with those examined in September. The activity of this enzyme in animals examined in February was also significantly lower than that in animals of the first group. The values in the second and third groups were at the same level. Some differences between the values obtained in animals which were prevented from hibernating and which were studied in February and those studied in April are not statistically significant, except for the hypothalamus. DISCUSSION
The highest PNMT activity found in the adrenals of animals examined in early spring indicates an intense methylation of noradrenaline into adrenaline. The weight of adrenals in this group was also significantly higher than in the first one. An extremely high adrenocortical activity, evaluated by a high level of circulating glucocorticoids in early spring, has also been found previously by PetroviC & Janid (1964b). Wurtman & Axelrod (1966) s h owed for the rat that glycocorticoids
130
V. M. PETROVII~, V. JANI& D. GRIPOISANDJ. ROFFI
are involved in the activation of adrenal PNMT. These data suggest that high PNMT activity, found in the active ground squirrels in early spring, may be the consequence of an extremely high adrenocortical activity observed at this season. High PNMT activity in the adrenals of animals examined in February as well as an increased adrenal weight suggest also an elevated adrenocortical activity in these animals. Adrenocortical activity in these experimental conditions, however, has yet to be elucidated. It was shown by Avakian & Callingham (1968) and by Parvez & Parvez (1972) for the rat and by Petrovid & Janid (1973) for the ground squirrel that glycocorticoids had an inhibitory effect on MAO activity. A decreased MAO activity, found in our present experiment, in the liver, heart, kidney and hypothalamus of animals examined in early spring could be due to an inhibitory effect of glycocorticoids on this enzyme. The depressed MAO activity found in the liver, heart, kidney and hypothalamus of animals which were prevented from sleeping and which were studied in February may be also the consequence of increased adrenocortical activity in these animals, but this fact, however, has still be to elucidated. Summarizing the results reported here: in addition to our previous findings we may conclude that not only in autumn, before hibernation in the normothermic ground squirrel, but also in early spring, in animals from a natural habitat, an intensification of the methylation of noradrenaline into adrenaline occurs in the adrenals. In the same season as well as under the same experimental conditions a depression of MAO activity in the liver, heart, kidney and hypothalamus was found. Since in animals examined in spring an extremely high adrenocortical activity was previously found, it seems likely that glycocorticoids are responsible for the increased PNMT and depressed MAO activity in these conditions. It has still to be elucidated whether the increased adrenal weight, increased PNMT and decreased MAO activity, found in winter in animals which were prevented from hibernating and kept at 30°C were the consequence of an increased adrenocortical activity in these experimental conditions.
REFERENCES AVAKIANV. M. & CALLINGHAMB. A. (1968) An effect of adrenalectomy upon catecholamines metabolism BY. J. Pharmac. Chemother. 33, 211. KAYSER, CH. (1961) The Physiology of Natural Hibernation, pp. 241-247. Pergamon, Oxford. GRIPOIS D. & PARVEZM. (1972) Simplification du dosage de la PNMT; inutilite d’une ultracentrifugation. Biochinaie 54, 413-414. PAFWEZH. and PARVEZS. (1972) Regulation de l’activite de la monoramine oxydase par les glucocorticoids. C. R. Acad. Sci. Paris 274, 919-921. PETROVI~V. M. & DAVIDOVIC V. (1965) Le taux des catecholamines surrenalienes chez le spermophile au tours des differentes saisons de l’annee. J. Physiol., Paris 57, 678-679. PETROVICV. M. & DAVIDOVIC V. (1968) Le rapport adr6naline/noradrCnaline dans l’hypothalamus, le coeur et la surrenale chez le spermophile en hibernation et en &at de veille. ‘j. Physiol., Paris 60, 514.
SEASONAL CHANGES IN GROUND SQUIRRELMETABOLISM
131
PBTROVICV. M., GRIPOISD., JANIC VERA& ROFFI J. (1973) The effect of cold exposure or arousal from hibernation on monoamine oxidase activity in the ground squirrel (Cite& citellus). Iugoslav. physiol. pharmacol. Acta. 9, No. 2. PETROVI~~ V. M. & JANI~ V. (1964a) ActivitC corticosurrenalienne chez le spermophile en hibernation et en Ctat de veille. J. Physiol., Paris 56, 421. PETROVICV. M. & JANI~ V. (196413) Comparative study of the effect of cold on the plasma concentration of 17,21-hydroxy, 20-ketosteroids and adrenal ascorbic acid of rat and ground squirrel. Arch. Biol. Sci. 16, 53-59. PETROVICV. M. & JANIC V. (1973) The effect of hydrocortisone and ACTH on monoamine oxidase activity in the ground squirrel (Citellus cite&s). Gen. & compar. Endocr. 20. WURTMANR. J. & AXELRODJ. (1963) A sensitive and specific assay for the estimation of monoamine oxidase. Biochem. Pharmac. 12, 1439-1440. WURTMANR. J. & AXELRODJ. (1965) Adrenaline synthesis: control by the pituitary gland and adrenal glucocorticoids. Science, N. Y. 150, 1464-1465. WURTMANR. J. & AXELRODJ. (1966) Control of enzymatic synthesis of adrenaline in the adrenal medulla by adrenal cortical steroids. ‘j. biol. Chem. 241, 2301-2305. Key Word Index-Hibernation; amine-N methyl transferase.
Citellus
cite&s;
monoamine oxidase; phenylethanol-
SEASONAL CHANGES IN PHENYLETHANOLAMINE-NMETHYL TRANSFERASE AND MONOAMINE OXIDASE ACTIVITY IN THE GROUND SQUIRREL (CITELLUS CITELLUS) V. M. PETROVICl, VERA JANI@, D. GRIPOIS2 and with the technical collaboration of LJ. JELI~
J. ROFF12
lInstitute of Physiology, Faculty of Sciences, Laboratory of Endocrinology, Institute for Biological Research, Belgrade (Yugoslavia); and 2Laboratory of Endocrinology, University of Paris XI, 91405-Orsay (France) (Received
5 June
1973)
Abstract-l. In the active, normothermic, male ground squirrel (Citellus citellus) phenylethanolamine-N-methyl transferase (PNMT) and monoamine oxidase (MAO) activity was studied during different seasons: in September, February and April. 2. The adrenal weight and PNMT activity (counts/min per pair of adrenals) were significantly higher in animals kept at a room temperature of 20-25°C and examined in early spring (April) than in those kept at the same room temperature but examined in autumn, before the period of hibernation (September). 3. MAO activity (counts/min per mg) in the liver, heart, kidney and hypothalamus was significantly lower in animals examined in April compared with those examined in September. 4. In animals kept at a room temperature of 30°C (so that hibernation could be prevented) and examined in February the adrenal weight and PNMT activity were significantly higher when compared with animals examined in autumn. MAO activity in the liver, heart, kidney and hypothalamus of these animals was significantly lower than in those examined in September.
INTRODUCTION MANY RESULTSindicate
that in different seasons the amount of catecholamines in the adrenals, heart, hypothalamus and some other organs of hibernators changes. In autumn, before hibernation, in a normothermic hedgehog an accumulation of these amines in the adrenals occurs (see Kayser, 1961). PetroviC & Davidovic (1965, 1968) found in autumn an accumulation of catecholamines in the adrenals of the normothermic ground squirrel as well as a fresh increase at the end of hibernation. The increased adrenocortical activity, as evaluated by the augmentation of plasma 17-OHCS and decreased adrenal ascorbic acid levels, was also found in the ground squirrel examined in autumn before hibernation, whereas extremely high adrenocortical activity in these animals was found in spring (PetroviC & Janid, 1964a). All these data suggest the possibility of an interrelationship between adrenocortical and medular activity in hibernators during different seasons. 5
127
128
V. M. PETROVIC, V. JANIC, D. GRIPOIS AND J. ROFFI
The work reported here was initiated while investigating catecholamines degradation in some tissues of the ground squirrel exposed to cold in early autumn (PetroviC et al., 1973). The aim of the present investigation is to determine the phenylethanolamine-N-methyl transferase (PNMT) and monoamine oxidase (MAO) activity in animals kept at the same room temperature and examined in different seasons. It was also interesting to find out the level of activity of these enzymes in normothermic animals in winter. In order to maintain their normothermic state it was necessary to keep them at a room temperature of 30°C during the autumn and winter.
MATERIALS
AND
METHODS
Adult male ground squirrels (Citellus citellus) were housed in individual plastic cages with wood-chip bedding, in a room at 20-25°C or at 30°C. They were maintained on daily illumination and given pelleted rat diet and water ad lib. The experiment was performed in three groups of animals, as follows: one group was kept at 20-25°C and examined in September; the second was kept at 20-25°C and examined in April; the third one was kept from the beginning of autumn in the warm room (the temperature of which was 30°C in order to prevent hibernation) and was examined in February. Phenylethanolamine-Nmethyl transferase activity was determined by the method of Wurtman & Axelrod (1965) as modified by Gripois & Parvez (1972). This method consists of the incubation of adrenal extracts with S-adenosyl methionine- 14C, in the presence of normethanephrine, and of the measurement of the radioactivity of epinephrine formed during this process. Determination of monoamine-oxidase activity was made according to the method of Wurtman & Axelrod (1963). Tryptamine- 14C bisuccinate was used as a radioactive substrate. Results are expressed in counts/min per pair of adrenals or per mg of tissue and are presented in the Tables 1 and 2. TABLE l-PHENYLETHANOLAMINE-N-METHYL TRANSFERASE ACTIVITYIN ADRENALS Season and experimental conditions
Body temperature (“C)
No. of ground squirrels
Weight of pairs of adrenals (mg)
Counts/min per pair of adrenals
Counts/min per mg of adrenals
Septemberanimals kept at 20-25°C
46.53 f 5.1 (A,)
2886k109 (B,)
6497 f 596
6
87.99 f 11.31 (A,)
4122f285 (B,)
4884+419
6
120.44 f 11.29 (A,)
3813+179 (B3)
3404+ 367
36-37
8
36-37
36-37
Aprilanimals kept at 20-25°C Februaryanimals kept at 30°C
MeanfS.E. of the mean. PcO~OO5; B1-B,, P
A,-A2,
P
Al-AS,
P
B1-B2,
SEASONAL CHANGES IN GROUND SQUIRREL METABOLISM TABLE 2--MONOAMINE
OXIDASE ACTIVITY IN THE LIVER, HEART, KIDNEY AND HYPOTHALAMUS Counts/min
Organs
129
per mg tissue
September-animals kept at 20-25°C
April-animals kept at 20-25°C
February-animals kept at 30°C 2840 + 97 (Aa)
Liver
4923 f 268 (A,)
2832 + 312 (A,)
Heart
2174 + 45 (B,)
1364 f 87 (B,)
1203 + 52 (Ba)
Kidney
32442 128
2561+ 166
2434 + 110 (C,)
Hypothalamus
96815 364 (DJ
Mean+S.E.
of
the
P-cO.01; D,-D,, P
(C,)
mean.
A,-A,,
(C,)
8802 f 247 (D,) Al-AZ,
P
P
B,-B,,
7075 f 474 (DJ P
C,-C2,
P
RESULTS
Phenylethanolamine-N-methyl
transferase activity
The adrenal weight and PNMT activity, expressed in counts/min per pair of adrenals, were significantly higher in animals examined in early spring than in those kept at the same room temperature (20-25°C) and studied in September. The adrenal PNMT activity in animals which were prevented from hibernating by keeping them in the warm room at 30°C and which were examined in February was at about the same level as that of animals kept at 20-25°C and examined in early spring, but the activity was significantly higher compared with that of animals studied in the autumn. The adrenal weight of these animals, however, was higher than that of animals examined in the spring. Monoamine oxidase activity The activity of MAO was significantly lower in all organs studied (liver, heart, kidney and hypothalamus) in animals examined in April compared with those examined in September. The activity of this enzyme in animals examined in February was also significantly lower than that in animals of the first group. The values in the second and third groups were at the same level. Some differences between the values obtained in animals which were prevented from hibernating and which were studied in February and those studied in April are not statistically significant, except for the hypothalamus. DISCUSSION
The highest PNMT activity found in the adrenals of animals examined in early spring indicates an intense methylation of noradrenaline into adrenaline. The weight of adrenals in this group was also significantly higher than in the first one. An extremely high adrenocortical activity, evaluated by a high level of circulating glucocorticoids in early spring, has also been found previously by PetroviC & Janid (1964b). Wurtman & Axelrod (1966) s h owed for the rat that glycocorticoids
130
V. M. PETROVII~, V. JANI& D. GRIPOISANDJ. ROFFI
are involved in the activation of adrenal PNMT. These data suggest that high PNMT activity, found in the active ground squirrels in early spring, may be the consequence of an extremely high adrenocortical activity observed at this season. High PNMT activity in the adrenals of animals examined in February as well as an increased adrenal weight suggest also an elevated adrenocortical activity in these animals. Adrenocortical activity in these experimental conditions, however, has yet to be elucidated. It was shown by Avakian & Callingham (1968) and by Parvez & Parvez (1972) for the rat and by Petrovid & Janid (1973) for the ground squirrel that glycocorticoids had an inhibitory effect on MAO activity. A decreased MAO activity, found in our present experiment, in the liver, heart, kidney and hypothalamus of animals examined in early spring could be due to an inhibitory effect of glycocorticoids on this enzyme. The depressed MAO activity found in the liver, heart, kidney and hypothalamus of animals which were prevented from sleeping and which were studied in February may be also the consequence of increased adrenocortical activity in these animals, but this fact, however, has still be to elucidated. Summarizing the results reported here: in addition to our previous findings we may conclude that not only in autumn, before hibernation in the normothermic ground squirrel, but also in early spring, in animals from a natural habitat, an intensification of the methylation of noradrenaline into adrenaline occurs in the adrenals. In the same season as well as under the same experimental conditions a depression of MAO activity in the liver, heart, kidney and hypothalamus was found. Since in animals examined in spring an extremely high adrenocortical activity was previously found, it seems likely that glycocorticoids are responsible for the increased PNMT and depressed MAO activity in these conditions. It has still to be elucidated whether the increased adrenal weight, increased PNMT and decreased MAO activity, found in winter in animals which were prevented from hibernating and kept at 30°C were the consequence of an increased adrenocortical activity in these experimental conditions.
REFERENCES AVAKIANV. M. & CALLINGHAMB. A. (1968) An effect of adrenalectomy upon catecholamines metabolism BY. J. Pharmac. Chemother. 33, 211. KAYSER, CH. (1961) The Physiology of Natural Hibernation, pp. 241-247. Pergamon, Oxford. GRIPOIS D. & PARVEZM. (1972) Simplification du dosage de la PNMT; inutilite d’une ultracentrifugation. Biochinaie 54, 413-414. PAFWEZH. and PARVEZS. (1972) Regulation de l’activite de la monoramine oxydase par les glucocorticoids. C. R. Acad. Sci. Paris 274, 919-921. PETROVI~V. M. & DAVIDOVIC V. (1965) Le taux des catecholamines surrenalienes chez le spermophile au tours des differentes saisons de l’annee. J. Physiol., Paris 57, 678-679. PETROVICV. M. & DAVIDOVIC V. (1968) Le rapport adr6naline/noradrCnaline dans l’hypothalamus, le coeur et la surrenale chez le spermophile en hibernation et en &at de veille. ‘j. Physiol., Paris 60, 514.
SEASONAL CHANGES IN GROUND SQUIRRELMETABOLISM
131
PBTROVICV. M., GRIPOISD., JANIC VERA& ROFFI J. (1973) The effect of cold exposure or arousal from hibernation on monoamine oxidase activity in the ground squirrel (Cite& citellus). Iugoslav. physiol. pharmacol. Acta. 9, No. 2. PETROVI~~ V. M. & JANI~ V. (1964a) ActivitC corticosurrenalienne chez le spermophile en hibernation et en Ctat de veille. J. Physiol., Paris 56, 421. PETROVICV. M. & JANI~ V. (196413) Comparative study of the effect of cold on the plasma concentration of 17,21-hydroxy, 20-ketosteroids and adrenal ascorbic acid of rat and ground squirrel. Arch. Biol. Sci. 16, 53-59. PETROVICV. M. & JANIC V. (1973) The effect of hydrocortisone and ACTH on monoamine oxidase activity in the ground squirrel (Citellus cite&s). Gen. & compar. Endocr. 20. WURTMANR. J. & AXELRODJ. (1963) A sensitive and specific assay for the estimation of monoamine oxidase. Biochem. Pharmac. 12, 1439-1440. WURTMANR. J. & AXELRODJ. (1965) Adrenaline synthesis: control by the pituitary gland and adrenal glucocorticoids. Science, N. Y. 150, 1464-1465. WURTMANR. J. & AXELRODJ. (1966) Control of enzymatic synthesis of adrenaline in the adrenal medulla by adrenal cortical steroids. ‘j. biol. Chem. 241, 2301-2305. Key Word Index-Hibernation; amine-N methyl transferase.
Citellus
cite&s;
monoamine oxidase; phenylethanol-