- Email: [email protected]
Effect of heat and cold exposure on the rat brain monoamine oxidase and antioxidative enzyme activities
ARTICLE IN PRESS
Journal of Thermal Biology 29 (2004) 861–864 www.elsevier.com/locate/jtherbio
Effect of heat and cold exposure on the rat brain monoamine oxidase and antioxidative enzyme activities Jelena Djordjevic´, Gordana Cvijic´, Tamara Vucˇkovic´, Vukosava Davidovic´ Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia and Montenegro
Abstract 1. Changes in MAO and antioxidative enzymes copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) activities were examined in the hypothalamus and the hippocampus of Wistar rats exposed to cold stress (6 1C) for 180 min and heat stress (38 1C) for 60 min. 2. Extreme environmental temperatures caused stressor-specific changes in the hypothalamic and hippocampal MAO and antioxidative enzyme activities, being dependent on the stressor applied (cold or heat) but not on the brain region studied (the hypothalamus or hippocampus). r 2004 Elsevier Ltd. All rights reserved. Keywords: Catalase; Cold stress; Heat stress; Hippocampus; Hypothalamus; Monoamine oxidase; Superoxide dismutase
1. Introduction The maintenance of homeostasis requires precise coordination of autonomic, neuroendocrine and behavioral responses to the stressor perceived. Although the entire central nervous system is involved in the maintenance of homeostasis and participates in the organization of stress response, some areas may have specific roles in these regulatory mechanisms. Higher centers, such as the neuroendocrine hypothalamus, the limbic system and the cerebral cortex are the modulatory centers of the stress response exerting their effects through actions on the brain stem and spinal neurons. The hypothalamus has a special neuroendocrine output route, the hypothalamo–pituitary–adrenocortical (HPA) system, which is involved, in a prominent fashion, in Corresponding author. Tel.: +381-11-639882; fax: +38111-639064. E-mail address: [email protected] (J. Djordjevic´).
stress response. It is proved that brain catecholaminergic neurons are involved in the central processing of stress response. Neurons in the ventrolateral and dorsomedial medulla oblongata are the major sources of noradrenergic nerve terminals in the hypothalamus and the limbic system (Cunningham and Sawchenko, 1988). Their activation is stressor specific, certain stressors activate them rapidly while others may have only minor influences (Pacak and Palkovits, 2001). The important factor in the regulation of noradrenaline concentration in the neurons is, among others, its degrading enzyme monoamine oxidase (MAO), which deaminates biogenic amines and during that activity produces hydrogen peroxyde (H2O2), which along with superoxide and hydroxyl radicals is highly toxic for the neurons (Cohen, 1985). Thus, as a result of very intense catecholamine metabolism, free radicals may be generated and, consequently, antioxidative enzymes activated in the brain regions. Maintenance of normal temperature requires the integrity of the hypothalamus but other
0306-4565/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2004.08.070
ARTICLE IN PRESS J. Djordjevic´ et al. / Journal of Thermal Biology 29 (2004) 861–864
2. Material and methods 2.1. Animals and experimental design Male rats of Wistar strain (Rattus norvegicus), 60–90 days old, weighing 180–220 g, were used for the experiments. The animals were acclimated to 2271 1C, kept at a 12:12 h light–dark cycle and given commercial rat food and tap water ad libitum. The rats were divided into three groups, each consisting of six animals. The first group represented intact controls. The rats from the second and third group were exposed to the extreme ambient temperatures 6 1C for 180 min and 38 1C for 60 min, respectively, in temperature controlled chambers. Animals were killed immediately after stress exposure, their brains were removed, the hypothalamus and the hippocampus excised, dissected and used for the measurements of enzyme activities. 2.2. Tissue preparation and assays The brain tissues were homogenized in a buffer, containing 0.25 M sucrose, 0.05 M Tris and 0.1 mM EDTA, pH 7.4. The homogenates were sonicated (three times at 100 W for 20 s with a 10 s pause in a Bronson model B-12 sonicator), as described by Takada et al. (1982) to release MnSOD. The homogenates were then centrifugated at 100 000 g in ultracentrifuge (Ti 50 rotor) for 90 min and used for determinations of CAT, CuZnSOD and MnSOD activities. SOD activity was determined by the adrenaline method of Misra and Fridovich (1972), based on the spectrophotometrical measurement of the degree of adrenaline autooxidation inhibition by SOD, contained in the examined samples in 50 mM sodium-carbonate buffer, pH 10.2, within a linear range of the autooxidation curve. One SOD unit was defined as the amount of enzyme inhibiting the oxidation of adrenaline by 50% under fixed reaction conditions of the assay. Total specific SOD and MnSOD activities, after CuZnSOD inhibition with 4 mM KCN, were measured, and then the CuZnSOD activity was calculated. CAT activity was measured by the method of Beutler (1982) based on the rate of H2O2 degradation by the action of CAT, contained in the examined samples followed spectrophotometrically at 230 nm in 5 mM
EDTA, Tris–HCl solution, pH 8.0. MAO activity was determined by method of Wurtman and Axelrod (1963) based on measurement of radioactivity of 14C-indol-3acetic acid, which is generated during incubation of 14Ctriptamine bissuccinate with hypothalamus and hippocampus homogenate. Brain tissues were homogenized at 0–4 1C in 100 volumes of 0.9% KCl and MAO was assayed in tubes containing 0.2 ml potassium phosphate buffer pH 7.4, 0.1 ml tissue homogenate and 0.05 ml solution of 14C-triptamine bissuccinate (0.005 mCi isotope, specific activity 59 mCi/mmol, NEN UK). After incubating at 37 1C for 20 min, the reaction was stopped by adding 0.2 ml 2 N HCl. The 14C-indol-3-acetic acid was extracted in 6 ml toluole by mixing on a horizontal shaker for 20 min. After centrifuging for 3 min at 3000 g, 5 ml toluole fraction was separated and 14C-indol-3acetic acid activity measured in scintillation counter. 2.3. Data analysis The values are expressed as pmol/mg tissue/min, and U/mg tissue (the hypothalamus or hippocampus). Anova one way test was used to detect differences among the groups. The values are presented as means7SE of six animals and the level of significance was set at po0:05:
3. Results As illustrated in Fig. 1, the exposure of animals to cold stress for 180 min did not change either hypothalamic MAO or CAT activity as compared to controls maintained at room temperature, whereas CuZnSOD
2
HYPOTHALAMUS Controls Cold (180 min) Heat (60 min)
**
1.5
0.15
*** *
1
0.1
U/mg tissue
brain areas (e.g. limbic system) are considered to be important in thermoregulation as well (Bligh, 1966). Bearing in mind the facts mentioned above, we examined changes in the hypothalamic and hippocampal MAO activity as well as the activities of antioxidative enzymes copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) under the influence of physical-environmental stressors, cold and heat.
pmol/mg tissue/min
862
0.05
0.5
** 0
MAO
CuZnSOD
MnSOD
Catalase
Fig 1. The effect of cold (6 1C) for 180 min and heat (38 1C) for 60 min on MAO and antioxidative enzyme activities in the rat hypothalamus. The values represent the means7SE of six animals and are expressed in pmol/mg tissue/min and in U/mg tissue, respectively. po0:05; po0:01; po0:001:
ARTICLE IN PRESS J. Djordjevic´ et al. / Journal of Thermal Biology 29 (2004) 861–864 2
0.2
HIPPOCAMPUS Controls Cold (180 min) Heat (60 min)
*
0.15
1
0.1
*
*
U/mg tissue
pmol/mg tissue/min
1.5
0.05
0.5
*** 0
MAO
CuZnSOD
MnSOD
**
Catalase
Fig 2. The effect of cold (6 1C) for 180 min and heat (38 1C) for 60 min on MAO and antioxidative enzyme activities in the rat hippocampus. The values represent the means7SE of six animals and are expressed in pmol/mg tissue/min and in U/mg tissue, respectively. po0:05; po0:01; po0:001:
( po0:05) and MnSOD activities ( po0:01) were increased. After a 60 min heat exposure MAO activity decreased ( po0:01), MnSOD and CAT activities remained unchanged, whereas CuZnSOD activity was significantly increased in this brain region ( po0:001) in respect to control values. Under the same experimental conditions the changes in the hippocampal MAO, CuZnSOD and MnSOD activities were similar to those obtained in the hypothalamus (Fig. 2). Besides, cold stress did not affect the hippocampal CAT activity whereas the heat stress resulted in the decrement of CAT activity ( po0:01) in this brain region.
4. Discussion Our previous results showed that animal exposure to physical-environmental stressors (heat and cold) appears to be the strongest stressor activating the HPA system in comparison to metabolic and psychosocial stressors (Djordjevic et al., 2003). Stressors differ not only by their evoked responses, but also by their neuronal circuits (Pacak and Palkovits, 2001). The hypothalamic neurons, which synthesize and release CRH and vasopressin, represent the origin of final common pathways for neurohormonal regulation of ACTH–corticosterone release. Along with neuronal projections to the hypothalamus, the limbic system may also influence the activity of neuroendocrine hypothalamo-pituitary system. Our present results show that exposure of rats to extreme environmental temperature of 6 1C for 180 min and 38 1C for 60 min affected hypothalamic and hippocampal MAO and antioxidative enzyme activities.
863
The exposure of animals to cold stress did not alter hypothalamic MAO activity but after the heat exposure MAO activity was decreased. It has already been shown that concentration of catecholamines in the hypothalamic preoptic area increased and that of catecholamines metabolites decreased during heat exposure (Lin, 1994; Kendrick et al., 1989). It is evident that during the exposure to physical-environmental stressors, catecholamine secretion is increased while its degradation is decreased in the rat brain, probably due to the inhibition of the activity of the catecholamine degrading enzyme— MAO. Bhattacharya et al. (1988) have shown that exposure of rats to acute cold restrain stress resulted in the increment of the endogenous MAO inhibitor— tribuline, which is probably competing for the active site of the enzyme (Clow et al., 1989). Endogenous generation of MAO inhibitors is a normal homeostatic regulatory mechanism, which serves for maintaining the optimal concentration of catecholamines—the signal transmitters in the brain and regulators of the HPA axis activity (Clow et al., 1997). Bearing in mind that MAO activity was not affected and even diminished under the influence of cold and heat stress respectively, there was probably no change in H2O2 production. Thus, for this reason the activity of CAT, H2O2 scavengened enzyme, remained at the control level, for sustaining the optimal H2O2 concentration in the hypothalamic and hippocampal neurons. It was predictable since the CAT activity is directly correlated with the substrate concentration (Shull et al., 1991). As we mentioned above there was obvious increase in catecholamine secretion from the catecholaminergic projection into the hypothalamus and the limbic system. There is strong evidence that the oxidative metabolic pathway of catecholamines, dopamine and noradrenaline, via their quinines, occur in vivo in the brain. Catecholamines and their metabolites, containing two free hydroxyl residues in their benzene ring, exert neurotoxic effect mainly due to the generation of highly reactive quinones and superoxide radicals (Haque et al., 2003). Neurotoxicity might be prevented by superoxide dismutase. Therefore, it may be the reason why both SODs activities were elevated under the influence of heat and cold stress exposure. Haque et al. (2003) have also shown that SOD helps in preventing quinone formation by eliminating the superoxide radical and acting as a superoxide: semiquinone oxidoreductase. Besides, it was reported that the administration of an irreversible MAO B inhibitor, L-deprenyl, increases striatal cytosolic CuZnSOD and mitochondrial MnSOD activities (Thiffault et al., 1997). Stress-associated increased oxidative damage contributes to age-related diseases (Liu and Mori, 1999). However, there is some concern that a higher catecholamine concentration, i.e. their oxidation products e.g. toxic quinones, might contribute to the
ARTICLE IN PRESS 864
J. Djordjevic´ et al. / Journal of Thermal Biology 29 (2004) 861–864
pathogenesis of Parkinson‘s disease and schizophrenia (Smythies, 2000).
5. Summary Extreme environmental temperatures caused changes in the hypothalamic and hippocampal MAO and antioxidative enzyme activities, being dependent on the stressor applied (cold or heat) but not on the brain region studied (the hypothalamus or hippocampus).
Acknowledgements This paper is supported by the Serbian Ministry of Science and Technology, Grant No. 1550.
References Beutler, E., 1982. Catalase. In: Beutler, E. (Ed.), Red Cell Metabolism, a Manual of Biochemical Methods. Grune and Stratton, New York, pp. 105–106. Bligh, J., 1966. The thermosensitivity of the hypothalamus and thermoregulation in mammals. Biol. Rev. 41, 317–367. Bhattacharya, S.K., Glover, V., McIntyre, I., Oxenkrug, G., Sandler, M., 1988. Stress causes an increase in endogenous monoamine oxidase inhibitor tribulin in the rat brain. Neurosci.Lett. 92, 218–221. Clow, A., Glover, V., Oxenkrug, G.F., Sandler, M., 1989. Stress reduces in vivo inhibition of monoamine oxidase by phenelzine in rat brain. Neurosci.Lett. 107, 331–334. Clow, A., Patel, S., Najafi, M., Evans, P.D., Hucklebridge, F., 1997. The cortisol response to psychological challenge is preceeded by a transient rise in endogenous inhibitor of monoamine oxidase. Life Sci. 61, 567–575. Cohen, G., 1985. Oxidative stress in the nervous system. In: Sies, H. (Ed.), Oxidative Stress. Academic Press, New York, pp. 383–401. Cunningham, E.T., Sawchenko, P.E., 1988. Anatomical specificity of noradrenergic inputs to the paraventricular and
supraoptic nuclei of the rat hypothalamus. J. Comp. Neurol. 274, 60–76. Djordjevic, J., Cvijic, G., Davidovic, V., 2003. Different activation of ACTH and corticosterone release in response to various stressors in rats. Physiol.Res. 52, 67–72. Haque, M.E., Asanuma, M., Higashi, Y., Miyazaki, I., Tanaka, K., Ogawa, N., 2003. Apoptosis-inducing neurotoxicity of dopamine and its metabolites via reactive quinine generation in neuroblastoma cells. Bioch. Biophys. Acta. 1619, 39–52. Kendrick, K.M., Riva, D.L., Hinton, M., Baldwin, B.A., 1989. Microdialysis measurement of monoamine and aminoacid release from the medial preoptic region of the sheep in response to heat exposure. Brain Res. 32, 541–544. Lin, M.T., 1994. Brain monoamine and body temperature regulation. Asia Pacific J. Pharmacol. 9, 49–65. Liu, J., Mori, A., 1999. Stress, aging and brain oxidative damage. Neurochem. Res. 24, 1479–1497. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170–3175. Pacak, K., Palkovits, M., 2001. Stressor specificity of central neuroendocrine responses: implications for stress-related disorders. Endo. Rev. 22, 502–548. Shull, S., Heintz, N.H., Periasamy, M., Manohar, M., Janssen, Y.M., Marsh, J.P., Mossman, B.T., 1991. Differential regulation of antioxidant enzymes in response to oxidants. J.Biol.Chem. 266, 24398–24403. Smythies, J., 2000. Redox aspects of signaling by catecholamines and their metabolites. Antioxidant Redox Cycling 2, 575–583. Takada, Y., Noguchit, T., Kayiama, M., 1982. Superoxide dismutase in various tissues from rabbit bearing the Vx-2 carcinoma in the maxillary sinus. Cancer.Res. 42, 4233–4235. Thiffault, C., Quirion, R., Poirier, J., 1997. The effect of Ldeprenyl, D-deprenyl and MDL 72974 on mitochondrial respiration: a possible mechanism leading to an adaptive increase in superoxidase dismutase activity. Mol. Brain Res. 49, 127–136. Wurtman, R.J., Axelrod, J., 1963. A sensitive and specific assay for the estimation of monoamine oxidase. Biochemical Pharmacology 12, 1439–1441.
Journal of Thermal Biology 29 (2004) 861–864 www.elsevier.com/locate/jtherbio
Effect of heat and cold exposure on the rat brain monoamine oxidase and antioxidative enzyme activities Jelena Djordjevic´, Gordana Cvijic´, Tamara Vucˇkovic´, Vukosava Davidovic´ Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia and Montenegro
Abstract 1. Changes in MAO and antioxidative enzymes copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) activities were examined in the hypothalamus and the hippocampus of Wistar rats exposed to cold stress (6 1C) for 180 min and heat stress (38 1C) for 60 min. 2. Extreme environmental temperatures caused stressor-specific changes in the hypothalamic and hippocampal MAO and antioxidative enzyme activities, being dependent on the stressor applied (cold or heat) but not on the brain region studied (the hypothalamus or hippocampus). r 2004 Elsevier Ltd. All rights reserved. Keywords: Catalase; Cold stress; Heat stress; Hippocampus; Hypothalamus; Monoamine oxidase; Superoxide dismutase
1. Introduction The maintenance of homeostasis requires precise coordination of autonomic, neuroendocrine and behavioral responses to the stressor perceived. Although the entire central nervous system is involved in the maintenance of homeostasis and participates in the organization of stress response, some areas may have specific roles in these regulatory mechanisms. Higher centers, such as the neuroendocrine hypothalamus, the limbic system and the cerebral cortex are the modulatory centers of the stress response exerting their effects through actions on the brain stem and spinal neurons. The hypothalamus has a special neuroendocrine output route, the hypothalamo–pituitary–adrenocortical (HPA) system, which is involved, in a prominent fashion, in Corresponding author. Tel.: +381-11-639882; fax: +38111-639064. E-mail address: [email protected] (J. Djordjevic´).
stress response. It is proved that brain catecholaminergic neurons are involved in the central processing of stress response. Neurons in the ventrolateral and dorsomedial medulla oblongata are the major sources of noradrenergic nerve terminals in the hypothalamus and the limbic system (Cunningham and Sawchenko, 1988). Their activation is stressor specific, certain stressors activate them rapidly while others may have only minor influences (Pacak and Palkovits, 2001). The important factor in the regulation of noradrenaline concentration in the neurons is, among others, its degrading enzyme monoamine oxidase (MAO), which deaminates biogenic amines and during that activity produces hydrogen peroxyde (H2O2), which along with superoxide and hydroxyl radicals is highly toxic for the neurons (Cohen, 1985). Thus, as a result of very intense catecholamine metabolism, free radicals may be generated and, consequently, antioxidative enzymes activated in the brain regions. Maintenance of normal temperature requires the integrity of the hypothalamus but other
0306-4565/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2004.08.070
ARTICLE IN PRESS J. Djordjevic´ et al. / Journal of Thermal Biology 29 (2004) 861–864
2. Material and methods 2.1. Animals and experimental design Male rats of Wistar strain (Rattus norvegicus), 60–90 days old, weighing 180–220 g, were used for the experiments. The animals were acclimated to 2271 1C, kept at a 12:12 h light–dark cycle and given commercial rat food and tap water ad libitum. The rats were divided into three groups, each consisting of six animals. The first group represented intact controls. The rats from the second and third group were exposed to the extreme ambient temperatures 6 1C for 180 min and 38 1C for 60 min, respectively, in temperature controlled chambers. Animals were killed immediately after stress exposure, their brains were removed, the hypothalamus and the hippocampus excised, dissected and used for the measurements of enzyme activities. 2.2. Tissue preparation and assays The brain tissues were homogenized in a buffer, containing 0.25 M sucrose, 0.05 M Tris and 0.1 mM EDTA, pH 7.4. The homogenates were sonicated (three times at 100 W for 20 s with a 10 s pause in a Bronson model B-12 sonicator), as described by Takada et al. (1982) to release MnSOD. The homogenates were then centrifugated at 100 000 g in ultracentrifuge (Ti 50 rotor) for 90 min and used for determinations of CAT, CuZnSOD and MnSOD activities. SOD activity was determined by the adrenaline method of Misra and Fridovich (1972), based on the spectrophotometrical measurement of the degree of adrenaline autooxidation inhibition by SOD, contained in the examined samples in 50 mM sodium-carbonate buffer, pH 10.2, within a linear range of the autooxidation curve. One SOD unit was defined as the amount of enzyme inhibiting the oxidation of adrenaline by 50% under fixed reaction conditions of the assay. Total specific SOD and MnSOD activities, after CuZnSOD inhibition with 4 mM KCN, were measured, and then the CuZnSOD activity was calculated. CAT activity was measured by the method of Beutler (1982) based on the rate of H2O2 degradation by the action of CAT, contained in the examined samples followed spectrophotometrically at 230 nm in 5 mM
EDTA, Tris–HCl solution, pH 8.0. MAO activity was determined by method of Wurtman and Axelrod (1963) based on measurement of radioactivity of 14C-indol-3acetic acid, which is generated during incubation of 14Ctriptamine bissuccinate with hypothalamus and hippocampus homogenate. Brain tissues were homogenized at 0–4 1C in 100 volumes of 0.9% KCl and MAO was assayed in tubes containing 0.2 ml potassium phosphate buffer pH 7.4, 0.1 ml tissue homogenate and 0.05 ml solution of 14C-triptamine bissuccinate (0.005 mCi isotope, specific activity 59 mCi/mmol, NEN UK). After incubating at 37 1C for 20 min, the reaction was stopped by adding 0.2 ml 2 N HCl. The 14C-indol-3-acetic acid was extracted in 6 ml toluole by mixing on a horizontal shaker for 20 min. After centrifuging for 3 min at 3000 g, 5 ml toluole fraction was separated and 14C-indol-3acetic acid activity measured in scintillation counter. 2.3. Data analysis The values are expressed as pmol/mg tissue/min, and U/mg tissue (the hypothalamus or hippocampus). Anova one way test was used to detect differences among the groups. The values are presented as means7SE of six animals and the level of significance was set at po0:05:
3. Results As illustrated in Fig. 1, the exposure of animals to cold stress for 180 min did not change either hypothalamic MAO or CAT activity as compared to controls maintained at room temperature, whereas CuZnSOD
2
HYPOTHALAMUS Controls Cold (180 min) Heat (60 min)
**
1.5
0.15
*** *
1
0.1
U/mg tissue
brain areas (e.g. limbic system) are considered to be important in thermoregulation as well (Bligh, 1966). Bearing in mind the facts mentioned above, we examined changes in the hypothalamic and hippocampal MAO activity as well as the activities of antioxidative enzymes copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) under the influence of physical-environmental stressors, cold and heat.
pmol/mg tissue/min
862
0.05
0.5
** 0
MAO
CuZnSOD
MnSOD
Catalase
Fig 1. The effect of cold (6 1C) for 180 min and heat (38 1C) for 60 min on MAO and antioxidative enzyme activities in the rat hypothalamus. The values represent the means7SE of six animals and are expressed in pmol/mg tissue/min and in U/mg tissue, respectively. po0:05; po0:01; po0:001:
ARTICLE IN PRESS J. Djordjevic´ et al. / Journal of Thermal Biology 29 (2004) 861–864 2
0.2
HIPPOCAMPUS Controls Cold (180 min) Heat (60 min)
*
0.15
1
0.1
*
*
U/mg tissue
pmol/mg tissue/min
1.5
0.05
0.5
*** 0
MAO
CuZnSOD
MnSOD
**
Catalase
Fig 2. The effect of cold (6 1C) for 180 min and heat (38 1C) for 60 min on MAO and antioxidative enzyme activities in the rat hippocampus. The values represent the means7SE of six animals and are expressed in pmol/mg tissue/min and in U/mg tissue, respectively. po0:05; po0:01; po0:001:
( po0:05) and MnSOD activities ( po0:01) were increased. After a 60 min heat exposure MAO activity decreased ( po0:01), MnSOD and CAT activities remained unchanged, whereas CuZnSOD activity was significantly increased in this brain region ( po0:001) in respect to control values. Under the same experimental conditions the changes in the hippocampal MAO, CuZnSOD and MnSOD activities were similar to those obtained in the hypothalamus (Fig. 2). Besides, cold stress did not affect the hippocampal CAT activity whereas the heat stress resulted in the decrement of CAT activity ( po0:01) in this brain region.
4. Discussion Our previous results showed that animal exposure to physical-environmental stressors (heat and cold) appears to be the strongest stressor activating the HPA system in comparison to metabolic and psychosocial stressors (Djordjevic et al., 2003). Stressors differ not only by their evoked responses, but also by their neuronal circuits (Pacak and Palkovits, 2001). The hypothalamic neurons, which synthesize and release CRH and vasopressin, represent the origin of final common pathways for neurohormonal regulation of ACTH–corticosterone release. Along with neuronal projections to the hypothalamus, the limbic system may also influence the activity of neuroendocrine hypothalamo-pituitary system. Our present results show that exposure of rats to extreme environmental temperature of 6 1C for 180 min and 38 1C for 60 min affected hypothalamic and hippocampal MAO and antioxidative enzyme activities.
863
The exposure of animals to cold stress did not alter hypothalamic MAO activity but after the heat exposure MAO activity was decreased. It has already been shown that concentration of catecholamines in the hypothalamic preoptic area increased and that of catecholamines metabolites decreased during heat exposure (Lin, 1994; Kendrick et al., 1989). It is evident that during the exposure to physical-environmental stressors, catecholamine secretion is increased while its degradation is decreased in the rat brain, probably due to the inhibition of the activity of the catecholamine degrading enzyme— MAO. Bhattacharya et al. (1988) have shown that exposure of rats to acute cold restrain stress resulted in the increment of the endogenous MAO inhibitor— tribuline, which is probably competing for the active site of the enzyme (Clow et al., 1989). Endogenous generation of MAO inhibitors is a normal homeostatic regulatory mechanism, which serves for maintaining the optimal concentration of catecholamines—the signal transmitters in the brain and regulators of the HPA axis activity (Clow et al., 1997). Bearing in mind that MAO activity was not affected and even diminished under the influence of cold and heat stress respectively, there was probably no change in H2O2 production. Thus, for this reason the activity of CAT, H2O2 scavengened enzyme, remained at the control level, for sustaining the optimal H2O2 concentration in the hypothalamic and hippocampal neurons. It was predictable since the CAT activity is directly correlated with the substrate concentration (Shull et al., 1991). As we mentioned above there was obvious increase in catecholamine secretion from the catecholaminergic projection into the hypothalamus and the limbic system. There is strong evidence that the oxidative metabolic pathway of catecholamines, dopamine and noradrenaline, via their quinines, occur in vivo in the brain. Catecholamines and their metabolites, containing two free hydroxyl residues in their benzene ring, exert neurotoxic effect mainly due to the generation of highly reactive quinones and superoxide radicals (Haque et al., 2003). Neurotoxicity might be prevented by superoxide dismutase. Therefore, it may be the reason why both SODs activities were elevated under the influence of heat and cold stress exposure. Haque et al. (2003) have also shown that SOD helps in preventing quinone formation by eliminating the superoxide radical and acting as a superoxide: semiquinone oxidoreductase. Besides, it was reported that the administration of an irreversible MAO B inhibitor, L-deprenyl, increases striatal cytosolic CuZnSOD and mitochondrial MnSOD activities (Thiffault et al., 1997). Stress-associated increased oxidative damage contributes to age-related diseases (Liu and Mori, 1999). However, there is some concern that a higher catecholamine concentration, i.e. their oxidation products e.g. toxic quinones, might contribute to the
ARTICLE IN PRESS 864
J. Djordjevic´ et al. / Journal of Thermal Biology 29 (2004) 861–864
pathogenesis of Parkinson‘s disease and schizophrenia (Smythies, 2000).
5. Summary Extreme environmental temperatures caused changes in the hypothalamic and hippocampal MAO and antioxidative enzyme activities, being dependent on the stressor applied (cold or heat) but not on the brain region studied (the hypothalamus or hippocampus).
Acknowledgements This paper is supported by the Serbian Ministry of Science and Technology, Grant No. 1550.
References Beutler, E., 1982. Catalase. In: Beutler, E. (Ed.), Red Cell Metabolism, a Manual of Biochemical Methods. Grune and Stratton, New York, pp. 105–106. Bligh, J., 1966. The thermosensitivity of the hypothalamus and thermoregulation in mammals. Biol. Rev. 41, 317–367. Bhattacharya, S.K., Glover, V., McIntyre, I., Oxenkrug, G., Sandler, M., 1988. Stress causes an increase in endogenous monoamine oxidase inhibitor tribulin in the rat brain. Neurosci.Lett. 92, 218–221. Clow, A., Glover, V., Oxenkrug, G.F., Sandler, M., 1989. Stress reduces in vivo inhibition of monoamine oxidase by phenelzine in rat brain. Neurosci.Lett. 107, 331–334. Clow, A., Patel, S., Najafi, M., Evans, P.D., Hucklebridge, F., 1997. The cortisol response to psychological challenge is preceeded by a transient rise in endogenous inhibitor of monoamine oxidase. Life Sci. 61, 567–575. Cohen, G., 1985. Oxidative stress in the nervous system. In: Sies, H. (Ed.), Oxidative Stress. Academic Press, New York, pp. 383–401. Cunningham, E.T., Sawchenko, P.E., 1988. Anatomical specificity of noradrenergic inputs to the paraventricular and
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