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Effect of methionine sulphoximine on brain cyclic nucleotide levels
EFFECT OF METHIONINE SULPHOXIMINE RRAIN CYCLIC NUCLEOTIDE LEVELS
ON
T. HEVORand J. GAYET Lahoratoire de Physiologie Gentrale. Universiti de Nancy 1, CO. 140. 54037 Nancy, Frmce
Methionine sulphoximine administered to rats and mice induced an activation and probably a biosynthesis de noco of the key gluconeogenic enzyme fructose-1,6-biphosphatase (EC 3.1.3.11) in the cerebral cortex, brain stem and cerebellum, during the preconvulsive and convulsive periods and also at the time of recovery from seizures when the animals appear normal (Hesor and Gayet, 197&r, b). These data suggest that the subsequent intracerebral accumulation of glycogen which occurs, within swollen astrocytes, during the period of seizures induced by administration of methionine sulphoximine, and persists up to 72 hr after injection of the convulsant (Folbergrova, Passonneau, Lowry and Schulz, 1969; Phelps, 1975; Berel, Lehr and Gayet, 1977), results from an activation of the gluconeogenic pathway. Since concentrations of adenosine 3’.S’-cyclic monophosphate (cyclic AMP) and guanosine 3’S’-cyclic monophosphate (cyclic GMP) within particular brain regions appear to be dependent upon the type of seizure activity induced by central nervous system stimulants or by electroshock (Lust, Goldberg and Passonneau, 1976; Ferrendelli and Kinscherf, 1977; Lust, Kupferberg. Yonekawa, Penry. Passonneau and Wheaton, 19783, it seems relevant to the study of the relationship between neuronal depolarization and giial glycogenesis, both induced by methioninc sulphoximine, to follow the regional changes of the levels of cyclic AMP and cyclic GMP in the mouse and rat brain. Male Swiss mice (OF1 strain) and Wistar rats (laboratory inbred strain) were used throughout the experiments. L-Methionine-DL-sulphoximine (Sigma, St Louis, MO.) (100 mg/kg) dissolved in 0.2 ml (mice) or 1.0 ml (rats) of 0.9”,, NaCl was injected intraperitoneally; control animals received the same volume of 0.9”,, NaCI. The mice were frozen intact in liquid nitrogen and the rats were decapitated and their heads instantaneously immersed in liquid nitrogen, at different periods following injection of meth~onil~e sulphoximine. The frozen animals and heads were stored at -3o“C until the brain was removed. The chilled brain regions were dissected out in a refrigerated chamber and rapidly weighed. Each brain region was homogenized in 6 vol (w/v) of lo”, trichloroacetic acid, and the homogenate was centrifuged (18,000 g, 15 min) at 4-C. The supernatant was washed 5 times with 4 vol of water-saturated diethyl ether; diethyl ether remaining in the solution was evaporated at
8o’C for 1 min. The content of cyclic nucleotides was estimated in this brain tissue fraction. Cyclic AMP was measured by the competitive protein binding assay (Gilman, 1970) modified (Weller, Rodnight and Carrera, 1972), with an adsorption of the cyclic nucleotide on charcoal (Albano, Barnes, Maudsley, Brown and Etkins, 1974; Tovey. Oldham and Whelan, 1974). Cyclic GMP was estimated by the radioimmunoassay (Steiner. Parker and Kipnis, 1972). The materials for the cyclic nucleotide determinations were purchased from the Radiochemical Centre, Amersham. Values were determined using standards of cyclic AMP and cyclic GMP. Cyclic nucleotide phosphodiesterase (Boehringer Mannheim France, Meylan) hydrolysed 100‘, of the reactive cyclic AMP and cyclic GMP in extracts from brain tissue both from control and methionine sulphoxime-injected mice. The mean yield of cyclic-AMP and cyclic GMP from the biological samples was 94”,,, based on the recovery of known amounts of standard cyclic AMP and cyclic GMP added to tissue samples prior to purification. After the intraperitoneal injection of methionine sulphoximine (10 mgikg), no change in the cyclic AMP content was noticeable in the mouse cerebral cortex and in the rat cerebral cortex, striatum. hypothalamus and cerebellum too, during the convulsive and postconvulsive periods, respectively about 8 and 24 hr after administration of methionine sulphoximine. During the preconvulsive period, 4 hr after administration of methionine sulphoximine. the concentration of cyclic GMP did not change signi~cantly in the mouse cerebral cortex, brain stem and cerebellum (Fig. 1). From 4 to 7 hr after injection of the drug, when the animals exhibited an increasing syndrome of ataxia, the concentration of cyclic GMP was augmented by 40”” in the brain stem. When the mice were sacrificed just after the onset of the first spontaneous clonic seizure, the cyclic GMP content in the cerebral cortex and brain stem reached respectively 87 and 82 pmolig tissue wet wt; 8 hr after ~tdministration of methionine sulphoximine, during the period of intense tonic and clonic seizure activity, the concentration of cyclic GMP increased approximately by 44 and lOOu;, respectively in the cerebral cortex and brain stem, without any change of this concentration in the cerebellum (Fig. 1). At the end of the period of seizure activity, about 9 hr after adn~inistration of nlethionine sulphoximiIle. the cyclic GMP content in
T. HWOR
103
and J. GAYET
1
80. 60.
Fig. 1. Ellect of methionine sulphoximine on cyclic GMP content in mouse cerebral cortex (CC), brain stem (S) and cerebellum (C). The animals were sacrificed in the preconvulsive (A). convulsive (B) and postconvulsive (C)periods, respectiv,ely about 4. 8 and 24 hr after an intraperitoneal injection of t.-methionine-DL-sulpboximine (100 mg;‘kg) dissolved in 0.2 ml of 0.9”,, NaCl; control animals received the same volume of O.Y,, NaCI. Open columns represent control (saline-treated) and hatched columns represent methronine s~llphoximine-treated mice. The values are the mean levels of cyclic GMP expressed as picomolesig tissue wet wt i_ SD for 3 10 animals. Statistical comparisons are est~~blishcd using Student’s i-test: *P < 0.05 **P < 0.005.
the brain stem was still 56”,, higher than that determined in the control mice. During the postconvulsive period at the time of recovery from seizures, 24 hr after injection of the convulsant, the concentration of cyclic GMP in the mouse brain stem was only increased by 27”,, (Fig. t ). It has been suggested that changes in cyclic GMP levels in all regions of the brain directly reflect altcrations in neuronal activity (Ferrendelh. 1978). The elevated concentration of cyclic GMP observed in the bram during seizure activity originates from depolarization of neurons following the action of a specific neurotr~nsmitter. However, in the present results. it seems unlikely that cyclic GMP levels increased only in association with neuronal depolarization. since an increase in cyclic GMP content occurred at the time of recovery from methionine sulphoximine-induced seizures. The cyclic GMP-mediated metabolic events in the brain are compartmentalized and it is possible that the increase of the level of the cyclic nucleotide may develop sirnult~~n~~~~lsiy in neuronai and glial cells, during
some periods
followinp
administration
of
The brain stem appears to be a region of rat and mouse brain in which methionine suiphoximine or its metabolite(s) primarily exert their stimulating effect on some specific neuronal cells, followed by an induction of gluconeopenesis in glial cells. the convulsant.
A(~i,itol~ir~iiyerncitt\ from
the Centre
This research was supported by grants National de la Recherche Scienti~qlic
(E.R.A. 331) and from Medicale.
the Fondation
pour
la Rccherche
REFERENC‘ES Albano, J. D. M.. Barnes. G. D.. Maudsley. D, V.. Brown, B. L. and Etkins. R. P. (1974). Factors affectmg the saturation assay of cyclic AMP in biological systems. 4rrr/it_i. Bj~~~,~f[~~~. 50: 130.14I Berel. A.. Lehr, P. R. and Gayei. J. (19771. Inhibition by metyrapone of convulsions and storage of brain glycogen in mice induced by methionine sulfoximtne (MSO). Bruirt Ras. 128: 193 196. Ferrendelli. J. A. (1978). Distribution and regulatton of cyclic GMP in the central nervous system. In: .I~lr.~nc~c~\rr~ Ctz.lic Nrrclroritlc Rr~.s~~it (George. W. J. and Ignarro L. J., Eds), Vol. 9, pp. 453 464. Raven Press. New Yt~rh. Ferrmdelli, J. A. and Kinscherf. D. A. (1977). C‘ychc nucleotides in epileptic brain : Effects 01‘ pcntyienctetrarol on regional cyclic AMP and cyclic
Effect of methionine
sulphoximine
phosphate and guanosine 3’,5’ monophosphate following maximal electroshock or decapitation. .I. Neurochem. 26: 5510. Lust, W. D., Kupferberg. H. J., Yonekawa, W. D., Penry, J. K., Passonneau, J. V. and Wheaton, A. B. (1978). Changes in brain metabolites induced by convulsants or electroshock: effects ol anticonvulsant agents. Mol. Pharmat. 14: 3477356. Phelps, C. G. (1975). An ultrastructural study of methionine sulphoximine-induced glycogen accumulation in astrocytes of the mouse cerebral cortex. J. Neuroc)tol. 4: 479490.
: on brain
cyclic nucleotide
levels
1031
Steiner, A. L., Parker, C. W. and Kipnis, D. M. (1972). Radioimmunoassay for cyclic nucleotides. 1. Preparations of antibodies and iodinated cyclic nucleotides. J. hid. Chem. 247: 1106-l 113. Tovey, K. C., Oldham, K. G. and Whelan, J. A. M. (1974). A simple assay for cyclic AMP in plasma and other biological samples using an improved competitive protein binding system. C/in. Chim. Acta 56: 221-234. Weller, M.. Rodnight. R. and Carrera. D. (1972). Determination of adenosine 3’.5’ cyclic monophosphate in cerebral tissues by saturation analysis. Biochern. J. 129: 113~121.
ON
T. HEVORand J. GAYET Lahoratoire de Physiologie Gentrale. Universiti de Nancy 1, CO. 140. 54037 Nancy, Frmce
Methionine sulphoximine administered to rats and mice induced an activation and probably a biosynthesis de noco of the key gluconeogenic enzyme fructose-1,6-biphosphatase (EC 3.1.3.11) in the cerebral cortex, brain stem and cerebellum, during the preconvulsive and convulsive periods and also at the time of recovery from seizures when the animals appear normal (Hesor and Gayet, 197&r, b). These data suggest that the subsequent intracerebral accumulation of glycogen which occurs, within swollen astrocytes, during the period of seizures induced by administration of methionine sulphoximine, and persists up to 72 hr after injection of the convulsant (Folbergrova, Passonneau, Lowry and Schulz, 1969; Phelps, 1975; Berel, Lehr and Gayet, 1977), results from an activation of the gluconeogenic pathway. Since concentrations of adenosine 3’.S’-cyclic monophosphate (cyclic AMP) and guanosine 3’S’-cyclic monophosphate (cyclic GMP) within particular brain regions appear to be dependent upon the type of seizure activity induced by central nervous system stimulants or by electroshock (Lust, Goldberg and Passonneau, 1976; Ferrendelli and Kinscherf, 1977; Lust, Kupferberg. Yonekawa, Penry. Passonneau and Wheaton, 19783, it seems relevant to the study of the relationship between neuronal depolarization and giial glycogenesis, both induced by methioninc sulphoximine, to follow the regional changes of the levels of cyclic AMP and cyclic GMP in the mouse and rat brain. Male Swiss mice (OF1 strain) and Wistar rats (laboratory inbred strain) were used throughout the experiments. L-Methionine-DL-sulphoximine (Sigma, St Louis, MO.) (100 mg/kg) dissolved in 0.2 ml (mice) or 1.0 ml (rats) of 0.9”,, NaCl was injected intraperitoneally; control animals received the same volume of 0.9”,, NaCI. The mice were frozen intact in liquid nitrogen and the rats were decapitated and their heads instantaneously immersed in liquid nitrogen, at different periods following injection of meth~onil~e sulphoximine. The frozen animals and heads were stored at -3o“C until the brain was removed. The chilled brain regions were dissected out in a refrigerated chamber and rapidly weighed. Each brain region was homogenized in 6 vol (w/v) of lo”, trichloroacetic acid, and the homogenate was centrifuged (18,000 g, 15 min) at 4-C. The supernatant was washed 5 times with 4 vol of water-saturated diethyl ether; diethyl ether remaining in the solution was evaporated at
8o’C for 1 min. The content of cyclic nucleotides was estimated in this brain tissue fraction. Cyclic AMP was measured by the competitive protein binding assay (Gilman, 1970) modified (Weller, Rodnight and Carrera, 1972), with an adsorption of the cyclic nucleotide on charcoal (Albano, Barnes, Maudsley, Brown and Etkins, 1974; Tovey. Oldham and Whelan, 1974). Cyclic GMP was estimated by the radioimmunoassay (Steiner. Parker and Kipnis, 1972). The materials for the cyclic nucleotide determinations were purchased from the Radiochemical Centre, Amersham. Values were determined using standards of cyclic AMP and cyclic GMP. Cyclic nucleotide phosphodiesterase (Boehringer Mannheim France, Meylan) hydrolysed 100‘, of the reactive cyclic AMP and cyclic GMP in extracts from brain tissue both from control and methionine sulphoxime-injected mice. The mean yield of cyclic-AMP and cyclic GMP from the biological samples was 94”,,, based on the recovery of known amounts of standard cyclic AMP and cyclic GMP added to tissue samples prior to purification. After the intraperitoneal injection of methionine sulphoximine (10 mgikg), no change in the cyclic AMP content was noticeable in the mouse cerebral cortex and in the rat cerebral cortex, striatum. hypothalamus and cerebellum too, during the convulsive and postconvulsive periods, respectively about 8 and 24 hr after administration of methionine sulphoximine. During the preconvulsive period, 4 hr after administration of methionine sulphoximine. the concentration of cyclic GMP did not change signi~cantly in the mouse cerebral cortex, brain stem and cerebellum (Fig. 1). From 4 to 7 hr after injection of the drug, when the animals exhibited an increasing syndrome of ataxia, the concentration of cyclic GMP was augmented by 40”” in the brain stem. When the mice were sacrificed just after the onset of the first spontaneous clonic seizure, the cyclic GMP content in the cerebral cortex and brain stem reached respectively 87 and 82 pmolig tissue wet wt; 8 hr after ~tdministration of methionine sulphoximine, during the period of intense tonic and clonic seizure activity, the concentration of cyclic GMP increased approximately by 44 and lOOu;, respectively in the cerebral cortex and brain stem, without any change of this concentration in the cerebellum (Fig. 1). At the end of the period of seizure activity, about 9 hr after adn~inistration of nlethionine sulphoximiIle. the cyclic GMP content in
T. HWOR
103
and J. GAYET
1
80. 60.
Fig. 1. Ellect of methionine sulphoximine on cyclic GMP content in mouse cerebral cortex (CC), brain stem (S) and cerebellum (C). The animals were sacrificed in the preconvulsive (A). convulsive (B) and postconvulsive (C)periods, respectiv,ely about 4. 8 and 24 hr after an intraperitoneal injection of t.-methionine-DL-sulpboximine (100 mg;‘kg) dissolved in 0.2 ml of 0.9”,, NaCl; control animals received the same volume of O.Y,, NaCI. Open columns represent control (saline-treated) and hatched columns represent methronine s~llphoximine-treated mice. The values are the mean levels of cyclic GMP expressed as picomolesig tissue wet wt i_ SD for 3 10 animals. Statistical comparisons are est~~blishcd using Student’s i-test: *P < 0.05 **P < 0.005.
the brain stem was still 56”,, higher than that determined in the control mice. During the postconvulsive period at the time of recovery from seizures, 24 hr after injection of the convulsant, the concentration of cyclic GMP in the mouse brain stem was only increased by 27”,, (Fig. t ). It has been suggested that changes in cyclic GMP levels in all regions of the brain directly reflect altcrations in neuronal activity (Ferrendelh. 1978). The elevated concentration of cyclic GMP observed in the bram during seizure activity originates from depolarization of neurons following the action of a specific neurotr~nsmitter. However, in the present results. it seems unlikely that cyclic GMP levels increased only in association with neuronal depolarization. since an increase in cyclic GMP content occurred at the time of recovery from methionine sulphoximine-induced seizures. The cyclic GMP-mediated metabolic events in the brain are compartmentalized and it is possible that the increase of the level of the cyclic nucleotide may develop sirnult~~n~~~~lsiy in neuronai and glial cells, during
some periods
followinp
administration
of
The brain stem appears to be a region of rat and mouse brain in which methionine suiphoximine or its metabolite(s) primarily exert their stimulating effect on some specific neuronal cells, followed by an induction of gluconeopenesis in glial cells. the convulsant.
A(~i,itol~ir~iiyerncitt\ from
the Centre
This research was supported by grants National de la Recherche Scienti~qlic
(E.R.A. 331) and from Medicale.
the Fondation
pour
la Rccherche
REFERENC‘ES Albano, J. D. M.. Barnes. G. D.. Maudsley. D, V.. Brown, B. L. and Etkins. R. P. (1974). Factors affectmg the saturation assay of cyclic AMP in biological systems. 4rrr/it_i. Bj~~~,~f[~~~. 50: 130.14I Berel. A.. Lehr, P. R. and Gayei. J. (19771. Inhibition by metyrapone of convulsions and storage of brain glycogen in mice induced by methionine sulfoximtne (MSO). Bruirt Ras. 128: 193 196. Ferrendelli. J. A. (1978). Distribution and regulatton of cyclic GMP in the central nervous system. In: .I~lr.~nc~c~\rr~ Ctz.lic Nrrclroritlc Rr~.s~~it (George. W. J. and Ignarro L. J., Eds), Vol. 9, pp. 453 464. Raven Press. New Yt~rh. Ferrmdelli, J. A. and Kinscherf. D. A. (1977). C‘ychc nucleotides in epileptic brain : Effects 01‘ pcntyienctetrarol on regional cyclic AMP and cyclic
Effect of methionine
sulphoximine
phosphate and guanosine 3’,5’ monophosphate following maximal electroshock or decapitation. .I. Neurochem. 26: 5510. Lust, W. D., Kupferberg. H. J., Yonekawa, W. D., Penry, J. K., Passonneau, J. V. and Wheaton, A. B. (1978). Changes in brain metabolites induced by convulsants or electroshock: effects ol anticonvulsant agents. Mol. Pharmat. 14: 3477356. Phelps, C. G. (1975). An ultrastructural study of methionine sulphoximine-induced glycogen accumulation in astrocytes of the mouse cerebral cortex. J. Neuroc)tol. 4: 479490.
: on brain
cyclic nucleotide
levels
1031
Steiner, A. L., Parker, C. W. and Kipnis, D. M. (1972). Radioimmunoassay for cyclic nucleotides. 1. Preparations of antibodies and iodinated cyclic nucleotides. J. hid. Chem. 247: 1106-l 113. Tovey, K. C., Oldham, K. G. and Whelan, J. A. M. (1974). A simple assay for cyclic AMP in plasma and other biological samples using an improved competitive protein binding system. C/in. Chim. Acta 56: 221-234. Weller, M.. Rodnight. R. and Carrera. D. (1972). Determination of adenosine 3’.5’ cyclic monophosphate in cerebral tissues by saturation analysis. Biochern. J. 129: 113~121.