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Guanylate cyclase activity increases after kainic acid lesion of rat striatum
Brain Research, 171 (1979) 567-572 '9 Elsevier/North-Holland Biomedical Press
567
Guanylate cyclase activity increases after kainic acid lesion of rat striatum
MARIE-LOUISE TJORNHAMMAR, ROBERT SCHWARCZ, TAMAS BARTFAI and KJELL FUXE* Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, 106 91 Stockholm and ( R.S. and K.F.) Department of Histology, Karolinska Institute, 104 O1 Stockholm (Sweden)
(Accepted April 12th, 1979)
Cyclic guanosine 3',5'-monophosphate (cGMP) has been implicated in synaptic processes as a second messenger for several putative neurotransmitters such as acetylcholine acting at muscarinic receptors, histamine at (H') and norepinephrine at a-receptors (cf. reviews1,9,21). Cyclic G M P is biosynthesized by the enzyme guanylate cyclaseg, 12 (E.C. 4.6.1.2) which has been shown to occur in different regions of the rat brain s. In subcellular distribution experiments, highest specific activity of the enzyme could be recovered from synaptosomal fractions indicating its close association with neuronal elements 10. Moreover, guanylate cyclase activity has been detected in both cytosol and membrane preparations from different brain regions3, z0. The two guanylate cyclases associated with the two fractions differ with regard to their catalytic properties z,14,z°. In order to extend the rather scarce information on the cellular localization of guanylate cyclase and to examine the possibility of an enzymatic reaction to denervation, we studied the effects of various lesions on the enzyme activity in striatal homogenates. Lesions of striatal afferents and of neurons intrinsic to the striatum were carried out according to standard procedures. Corticostriatal fibers were interrupted by shallow knife cuts as described elsewhere 27. 6-OH-Dopamine lesions (8 /zg/2 /zl, injected into the substantia nigra; coordinates A 3.0, L 2.0, V 6.8 according to the stereotactic atlas of K6nig and Klippe117) were carried out according to UngerstedtZL The success of the lesion in destroying nerve-endings of the dopaminergic nigrostriatal pathway was evaluated by s.c. administration of 0.1 mg/kg apomorphine 7 days after the lesion z2. Animals used for the experiments described in the present study performed 374 ~ 112 (n = 10) contralateral rotations in 45 min. Lesions with kainic acid (Sigma lot No. 47C 0074) were performed as described previously 26. Shortly, 1 #g kainic acid in 1 /~1 phosphate-buffered saline (pH = 7.4) was infused stereotaxically into the striatum (coordinates A 7.9, L 2.6 and V 4.8) over a period of 1 min. In 5 lesioned and in 5 control animals the activities of choline * To whom reprint requests should be addressed.
568 TABLE I Changes in enzyme activities two days after kainic acid lesh~n
Striatal enzyme activities were measured two (guanylate cyclase, CAT, GAD) or three (adenylate cyclase) days after intrastriatal injection of kainic acid as described in Methods. The contralateral uninjected striatum served as control. Values are means " S.E.M. for the number of ~parate experiments indicated in parentheses. Enzyme
Control
Injected
Guanylate cyclase (pmol cGMP/min/mg protein) Adenylate cyclase* (pmoles/3 min/mg protein) Choline acetyltransferase (nmol/mg wet weight/h) Glutamic acid decarboxylase (nmol/mg wet weight/h)
t5.9 ± 2.3 (10) 34.9 3:6:9 (107 750 ± 70 (5) 140 ~ 40 (5) 19. I ~: 2.3 (5) 7.6 -~ 2.5 (5) 12.5 ~ 1.5 (5) 5.3 ~-: 1.3 (5~
A%
-~120 --81 ---60 --58
* Values from ref. 22.
acetyltransferase and glutamic acid decarboxylase, markers for the striatal cholinergic and GABAergic neurons were measured according to the methods of Bull and Oderfeld-Novak n and Wilson et al. aa, respectively. Homogenates of lesioned and contralateral striata were prepared in 50 mM Tris.HC1 buffer, pH 7.4 by sonification of the tissue with Sonifier Cell Disruptor, setting 7 for 30 sec to yield a 10 ~ w/v homogenate. Guanylate cyclase activity was measured in an incubation mixture of 200 #1 composed of 50 #1 of the homogenate, 150/~1 triethanol amine-C1 buffer 50 raM, pH ---- 7.6. The final concentration of the reactants was GTP 1 mM, MnCI2 2 mM, SQ 20009 (cyclic nucleotide phosphodiesterase inhibitor; Squibb and Sons, N.J:) 0.5 raM. The incubation was carried out for 5 min at 37 °C and stopped by the addition of 200 pl ice-cold 0.4 M zinc acetate and 200 #1 ice-cold 0.4 M Na2COa to the reaction mixture. The tubes were shaken vigorously and placed into a dry ice-acetone bath. The samples were thawed while centrifuged at 4 °C for 30 min at 2000g, The formed precipitate (ZnCO3) contained 92-95 ~ ofthe G T P present in the mixture and less than 5 ~ of the c G M P formed. The c G M P content of the clear supernatant was immediately assayed by radioimmunoassay according to Steiner et al. 29. The results were evaluated by means of a standard curve (0-10 pmol cGMP) containing aliquots of a similarly treated reaction mixture which was devoid of protein or contained previously denatured protein. The incubations were carried out in duplicates and the radioimmunoassay was performed in triplicates. As demonstrated previously, intrastriatal injections of kainic acid lead to rapid loss of neuronal cell bodies which is paralleled by substantial decreases in striated choline acetyl-transferase and glutamic acid decarboxylase activities as well as a 60°°80 ~ reduction of adenylate cyclase activity two days after the lesion 2a. In contrast, guanylate cyclase activity at this time-point is increased by 120--140~ when compared to the contralateral side or to striata of untreated control animals (Table I). Ten days after the kainic acid lesion this increase is less prominent but still significant (Fig. 1). Lesions of striatal afferents by intranigral injection of 6-OH-dopamine and mechanical interruption of corticostriatal fibers,
569 6-OH DA
Kainic acid 2 doys
10 doys
10 doys
100-
0
10 doys [~controt ~,njected controaterol
- ; 200
o
Cortica[ oblation
F
9 10 10
6 10 10
6 10 10
6 10 10
Fig. 1. Guanylate cyclase activity in homogenates of lesioned, contralateral and control striata of rats. The lesions were carried out as described in the text. 100 ~ activity corresponds to 15.9 pmol cGMP/ rain/rag protein. The values are given as means ± S.D., and were compared to the control by the Wilcoxon test; (* 0.05 < P <5 0.1 and **P < 0.02). respectively, failed to produce any significant change in striatal guanylate cyclase activity when measured 10 days after lesioning (Fig. 1). Various types of lesions have been applied previously to delineate the localization of small neuroactive substances, enzymes or receptor proteins in cerebral tissue; and long-lasting decreases after selective neuronal lesions are generally interpreted as an indication for their association with neuronal elements. The neuronal network of the corpus striatum consists of several major components: two prominent input systems deriving from the substantia nigra and the cerebral cortex respectively; small Golgi ll-type interneurons and efferents projecting primarily to globus pallidus and substantia nigra 7. While the striatal cell bodies degenerate after intrastriatal application of nanomole quantities of kainic acid 26, chemical lesions with 6-OH-dopamine or mechanical cortical ablations selectively deplete the striatum from its nigral and cortical afferents, respectively27, 3x. In the present study, these lesions did not produce any significant decrease in striatal guanylate cyclase activity ten days after the respective operations while cortical ablation, similar to intrastriatal kainic acid in injection, is likely to lead to glial proliferation in the internal capsule fibre bundles. No significant increase in striatal guanylate cyclase activity was found as a result of this lesion. A possible explanation for this is that the volume of the internal capsule is rather small as compared to that of the whole striatum13, 3z and an increase in activity in this area may not be detected with the whole striatum as background, c G M P has previously been shown to be localized in C6 glioma cells 25 and it seems conceivable in view of the present findings that, in the rat striatum, its biosynthetic enzyme, too, is largely associated with glial or other nonneuronal structures. Under in vivo conditions or in slice preparations narcotic analgesics 23 and cholinomimetic drugs 11 have been reported to produce short-lasting accumulations of striatal cGMP. These responses are lost in cell-free homogenates. Therefore our data cannot exclude the existence of a small neuronal striatal guanylate
570 cyclase pool, the measurement of which may have been obscured by the bulk of nonneuronal enzyme. In contrast to the basal guanylate cyclase activity measured in our preparations, basal adenylate cyclase activity is decreased 60-80 ~ as soon as two days after an intrastriatal kainic acid injection indicating a differential localization of the two cyclic nucleotide synthesizing enzymesS,lZ, TM. As shown in this paper, guanylate cyclase activity is increased by more than 120% two days after intrastriatal kainate. This substantial change is partly reversible with time indicating a temporary functional reaction of the enzyme activity to the lesion. Increased c G M P levels in cell division processes have previously been noted 9 and proliferation of glia is one of the major tissue reactions to kainate lesions as assessed at the light microscopic level. In addition, original glial elements, which do not undergo cell division in response to kainic acid, may react to loss of neighbouring neurons by increasing their guanylate cyclase activity. In slices of rat cerebellum, doses of kainic acid, which may well correspond to the concentrations present after intracerebral infusions markedly increase the levels of c G M P and cAMP 24. Intracerebellar injection of kainic acid caused transient increase (at 6-12 h) followed by a permanent decrease in cerebellar c G M P levels 4. Conflicting reports have been published on the respective kainate effects also in other brain areas like striatum and cerebral cortex 24,2s. It remains to be investigated if these short-term effects observed in brain slices should be compared to the guanylate cyclase activity increases reported in this study. A recent finding by Minneman et al. 2°, indicating a substantial loss of striatal cGMP-phosphodiesterase activity after neuronal lesions, supports the view that the entire system responsible for c G M P metabolism is imbalanced as a direct or indirect consequence of kainate injections. Finally, it should be noted that depolarizing concentrations of potassium and glutamate 15, the latter probably playing an important role in the neurodegenerative action of kainic acidZ, 16,19, have also been demonstrated to raise c G M P levels in a number of brain regions, while other endogenous and exogenous compounds were found to be inactive lz. Further elucidation of the effects of kainate lesions on the guanosine nucleotide systems are therefore necessary and may contribute to a better understanding of the mechanism of neurotoxicity elicited by neuro-excitatory amino acids. This work was supported by grants (04X-715, 04X-05415) from the Swedish Medical Research Council, a grant (MH-25504) from the National Institute of Mental Health, and by a grant from Magn. Bergvalls Stiftelse.
1 Bartfai, T., Cyclicnucleotides in the central nervous system, Trends Biochem. Sci., 3 (1978) 121-t24. 2 Bartfai, T., Breakefield,X. O. and Greengard, P., Regulation of synthesis of guanosine 3',5'-cyclic monophosphate in neuroblastoma cells, Biochem. J., 176 (1978) 119-127. 3 Bartfai, T., Study, R. E. and Greengard, P., Muscarinic stimulation and cGMP synthesis in the nervous system. In D. J. Jenden (Ed.), Chotinergic Mechanisms andPsychopharmacology, Plenum Press, N.Y., 1977, pp. 285-295.
57l 4 Biggio, G., Corda, M. G., Casu, M., Salis, M. and Gessa, G. L., Disappearance of cerebellar cyclic GMP induced by kainic acid, Brain Research, 154 (,1978) 203-208. 5 Biziere, K. and Coyle, J. T., Influence ofcorticostriatal afferents on striatal kainic acid neurotoxicity, Neurosci, Lett., 8 (1978) 303-310. 6 Bull, G. and Oderfeld-Novak, B., Standardization of a radiochemicaI assay of choline-acetyltransferase and a study of the activation of the enzyme in rabbit brain, J. Neurochem., 19 (1971) 935 947. 7 Carpenter, M. B., Anatomical organization of the corpus striatum and related nuclei. In M. D. Yahr (Ed.), The Basal Ganglia, Raven Press, New York, (1976) pp. 1 35. 8 Chiara, G. D., Porceddu, M. L., Spano, P. F. and Gessa, G. L., Haloperidol increases and apomorphine decreases striatal dopamine metabolism after destruction of striatal dopamine sensitive adenylate cyclase by kainic acid, Brain Research, 130 (1977) 374-382. 9 Goldberg, N. D. and Haddox, M. K., Cyclic GMP metabolism and involvement in biological regulation, Ann. Rev. Biochem., 46 (1978) 823 896. 10 Goridis, C., Morgan, 1. G., Guanyl cyclase in rat brain subcellular fractions, FEBS Lett., 34 (1973) 71 73. 11 Hanley, M. R. and Iversen, L. L., Muscarinic cholinergic receptors in rat corpus striatum and regulation of guanosine cyclic 3',5'-monophosphate, Molee. Pharmacol., 14 (1978) 246-255. 12 Hardman, J. G. and Sutherland, E. W., Guanyl cyclase, an enzyme catalyzing the formation of guanosine 3',5'-monophosphate from guanosine triphosphate, J. biol. Chem., 244 (1969) 6363 6370. 13 Kemp, J. M., Observations on the caudate nucleus of the cat impregnated with the Golgi method, Brain Research, 11 (1968) 467-470. 14 Kimura, H. and Murad, F., Subcellular localization of guanylate cyclase, Life Sci., 17 (1975) 837-844. 15 Kinscherf, D. A., Chang, M. M., Rubin, E. H., Schneider, D. R. and Ferrendelli, J. A., Comparison of the effects of depolarizing agents and neurotransmitters on regional CNS cyclic GMP levels in various animals, J. Neurochem., 26 (1976) 527-530. 16 K6hler, C., Schwarcz, R. and Fuxe, K,, Perforant pass transections protect hippocampal granule cells from kainate lesion, Neurosci. Lett., 10 (1978) 241 246. 17 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963. 18 McGeer, E. G., lnnanen, V. T. and McGeer, P. L., Evidence on the cellular localization of adenyl cyclase in the neostriatum, Brain Research, 118 (I 976) 356-358. 19 McGeer, E. G., McGeer, P. L. and Singh, K., Kainate-induced degeneration of neostriatal neurons: dependency upon corticostriatal tract, Braht Research, 139 (1978) 381-383. 20 Minneman, K. P., Quik, M. and Emson, P. C., Receptor-linked cyclic AMP systems in rat neostriaturn : differential localization revealed by kainic acid injection, Brain Research, 151 (1978) 507-521. 21 Nathanson, J. A., Cyclic nucleotides and central nervous system function, Physiol. Rev., 57 (1977) 157--256. 22 Von Namba, M., Cytoarkitechtonische Untersuchung an Striatum, J. Hirlforsch., 3 (1957) 24-48. 23 Racagni, G., Zsilla, G., Guidotti, A. and Costa, E., Accumulation of cGMP in striatum of rats injected with narcotic analgesics: antagonism by naltrexone, J. Pharm. Pharmacol., 28 (1976) 258-260. 24 Schmidt, M. J., Ryan, J. J. and Molloy, B. B., Effects of kainic acid, on cyclic nucleotide accumulation in slices of rat cerebellum, Brain Research, 112 (1976) 113-126. 25 Schwartz, J. P., Catecholamine-mediated elevation of cyclic GMP in the rat C-6 glioma cells, J. Cyclic Nucleotide Res., 2 (1976) 287 296. 26 Schwarcz, R. and Coyle, J. T., Striatal lesions with kainic acid: neurochemical characteristics, Brain Research, 127 (1977) 235-249. 27 Schwarcz, R., Creese, [., Coyle, J. T. and Snyder, S. H., Dopamine receptors localised on cerebral cortical afferents to rat corpus striatum, Nature (Lond.), 271 (1978) 766 768. 28 Shimizu, H., lchishita, H. and Umeda, l., Inhibition of glutamate-elicited accumulation of adenosine cyclic 3',5'-monophosphate in brain slices by ~,eJ-diaminocarboxylic acids, Molec. Pharmacol., II (1975) 866 873. 29 Steiner, A. L., Parker, G. W. and Kipnis, D. M., Radioimmunoassay for cyclic nucleotides, J. biol. Chem., 247 (1972) 1106-1113. 30 Troyo, E. W., Hall, I. A. and Ferrendelli, J. A., Guanylate cyclases in CNS enzymatic characteristics of soluble and particulate enzymes from mouse cerebellum and retina, J. Neurochem., (1978) 825834.
572 31 Ungerstedt, U., 6-Hydroxydopamine induced degeneration of central monoamine neurons, Europ. J. PharmacoL, 5 (1968) 107-110. 32 Ungerstedt, U. andArbuthnott, G. W.,Quantitativerecordingofrotational behaviourin rats after 6-hydroxydopamine lesions of the nigro-striatal dopamine system, Brai;l Researrh, 24 (1970) 485 493. 33 Wilson, S. H., Schrier, B. K., Farber, J. L., Thompson, E. J., Rosenberg, R. N., 131ume, A. J. and Nirenberg, M, W., Markers for gene expression in cultured cells from nervous systcm, ,I. bioL Chem., 247 (1972) 3159-3169.
567
Guanylate cyclase activity increases after kainic acid lesion of rat striatum
MARIE-LOUISE TJORNHAMMAR, ROBERT SCHWARCZ, TAMAS BARTFAI and KJELL FUXE* Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, 106 91 Stockholm and ( R.S. and K.F.) Department of Histology, Karolinska Institute, 104 O1 Stockholm (Sweden)
(Accepted April 12th, 1979)
Cyclic guanosine 3',5'-monophosphate (cGMP) has been implicated in synaptic processes as a second messenger for several putative neurotransmitters such as acetylcholine acting at muscarinic receptors, histamine at (H') and norepinephrine at a-receptors (cf. reviews1,9,21). Cyclic G M P is biosynthesized by the enzyme guanylate cyclaseg, 12 (E.C. 4.6.1.2) which has been shown to occur in different regions of the rat brain s. In subcellular distribution experiments, highest specific activity of the enzyme could be recovered from synaptosomal fractions indicating its close association with neuronal elements 10. Moreover, guanylate cyclase activity has been detected in both cytosol and membrane preparations from different brain regions3, z0. The two guanylate cyclases associated with the two fractions differ with regard to their catalytic properties z,14,z°. In order to extend the rather scarce information on the cellular localization of guanylate cyclase and to examine the possibility of an enzymatic reaction to denervation, we studied the effects of various lesions on the enzyme activity in striatal homogenates. Lesions of striatal afferents and of neurons intrinsic to the striatum were carried out according to standard procedures. Corticostriatal fibers were interrupted by shallow knife cuts as described elsewhere 27. 6-OH-Dopamine lesions (8 /zg/2 /zl, injected into the substantia nigra; coordinates A 3.0, L 2.0, V 6.8 according to the stereotactic atlas of K6nig and Klippe117) were carried out according to UngerstedtZL The success of the lesion in destroying nerve-endings of the dopaminergic nigrostriatal pathway was evaluated by s.c. administration of 0.1 mg/kg apomorphine 7 days after the lesion z2. Animals used for the experiments described in the present study performed 374 ~ 112 (n = 10) contralateral rotations in 45 min. Lesions with kainic acid (Sigma lot No. 47C 0074) were performed as described previously 26. Shortly, 1 #g kainic acid in 1 /~1 phosphate-buffered saline (pH = 7.4) was infused stereotaxically into the striatum (coordinates A 7.9, L 2.6 and V 4.8) over a period of 1 min. In 5 lesioned and in 5 control animals the activities of choline * To whom reprint requests should be addressed.
568 TABLE I Changes in enzyme activities two days after kainic acid lesh~n
Striatal enzyme activities were measured two (guanylate cyclase, CAT, GAD) or three (adenylate cyclase) days after intrastriatal injection of kainic acid as described in Methods. The contralateral uninjected striatum served as control. Values are means " S.E.M. for the number of ~parate experiments indicated in parentheses. Enzyme
Control
Injected
Guanylate cyclase (pmol cGMP/min/mg protein) Adenylate cyclase* (pmoles/3 min/mg protein) Choline acetyltransferase (nmol/mg wet weight/h) Glutamic acid decarboxylase (nmol/mg wet weight/h)
t5.9 ± 2.3 (10) 34.9 3:6:9 (107 750 ± 70 (5) 140 ~ 40 (5) 19. I ~: 2.3 (5) 7.6 -~ 2.5 (5) 12.5 ~ 1.5 (5) 5.3 ~-: 1.3 (5~
A%
-~120 --81 ---60 --58
* Values from ref. 22.
acetyltransferase and glutamic acid decarboxylase, markers for the striatal cholinergic and GABAergic neurons were measured according to the methods of Bull and Oderfeld-Novak n and Wilson et al. aa, respectively. Homogenates of lesioned and contralateral striata were prepared in 50 mM Tris.HC1 buffer, pH 7.4 by sonification of the tissue with Sonifier Cell Disruptor, setting 7 for 30 sec to yield a 10 ~ w/v homogenate. Guanylate cyclase activity was measured in an incubation mixture of 200 #1 composed of 50 #1 of the homogenate, 150/~1 triethanol amine-C1 buffer 50 raM, pH ---- 7.6. The final concentration of the reactants was GTP 1 mM, MnCI2 2 mM, SQ 20009 (cyclic nucleotide phosphodiesterase inhibitor; Squibb and Sons, N.J:) 0.5 raM. The incubation was carried out for 5 min at 37 °C and stopped by the addition of 200 pl ice-cold 0.4 M zinc acetate and 200 #1 ice-cold 0.4 M Na2COa to the reaction mixture. The tubes were shaken vigorously and placed into a dry ice-acetone bath. The samples were thawed while centrifuged at 4 °C for 30 min at 2000g, The formed precipitate (ZnCO3) contained 92-95 ~ ofthe G T P present in the mixture and less than 5 ~ of the c G M P formed. The c G M P content of the clear supernatant was immediately assayed by radioimmunoassay according to Steiner et al. 29. The results were evaluated by means of a standard curve (0-10 pmol cGMP) containing aliquots of a similarly treated reaction mixture which was devoid of protein or contained previously denatured protein. The incubations were carried out in duplicates and the radioimmunoassay was performed in triplicates. As demonstrated previously, intrastriatal injections of kainic acid lead to rapid loss of neuronal cell bodies which is paralleled by substantial decreases in striated choline acetyl-transferase and glutamic acid decarboxylase activities as well as a 60°°80 ~ reduction of adenylate cyclase activity two days after the lesion 2a. In contrast, guanylate cyclase activity at this time-point is increased by 120--140~ when compared to the contralateral side or to striata of untreated control animals (Table I). Ten days after the kainic acid lesion this increase is less prominent but still significant (Fig. 1). Lesions of striatal afferents by intranigral injection of 6-OH-dopamine and mechanical interruption of corticostriatal fibers,
569 6-OH DA
Kainic acid 2 doys
10 doys
10 doys
100-
0
10 doys [~controt ~,njected controaterol
- ; 200
o
Cortica[ oblation
F
9 10 10
6 10 10
6 10 10
6 10 10
Fig. 1. Guanylate cyclase activity in homogenates of lesioned, contralateral and control striata of rats. The lesions were carried out as described in the text. 100 ~ activity corresponds to 15.9 pmol cGMP/ rain/rag protein. The values are given as means ± S.D., and were compared to the control by the Wilcoxon test; (* 0.05 < P <5 0.1 and **P < 0.02). respectively, failed to produce any significant change in striatal guanylate cyclase activity when measured 10 days after lesioning (Fig. 1). Various types of lesions have been applied previously to delineate the localization of small neuroactive substances, enzymes or receptor proteins in cerebral tissue; and long-lasting decreases after selective neuronal lesions are generally interpreted as an indication for their association with neuronal elements. The neuronal network of the corpus striatum consists of several major components: two prominent input systems deriving from the substantia nigra and the cerebral cortex respectively; small Golgi ll-type interneurons and efferents projecting primarily to globus pallidus and substantia nigra 7. While the striatal cell bodies degenerate after intrastriatal application of nanomole quantities of kainic acid 26, chemical lesions with 6-OH-dopamine or mechanical cortical ablations selectively deplete the striatum from its nigral and cortical afferents, respectively27, 3x. In the present study, these lesions did not produce any significant decrease in striatal guanylate cyclase activity ten days after the respective operations while cortical ablation, similar to intrastriatal kainic acid in injection, is likely to lead to glial proliferation in the internal capsule fibre bundles. No significant increase in striatal guanylate cyclase activity was found as a result of this lesion. A possible explanation for this is that the volume of the internal capsule is rather small as compared to that of the whole striatum13, 3z and an increase in activity in this area may not be detected with the whole striatum as background, c G M P has previously been shown to be localized in C6 glioma cells 25 and it seems conceivable in view of the present findings that, in the rat striatum, its biosynthetic enzyme, too, is largely associated with glial or other nonneuronal structures. Under in vivo conditions or in slice preparations narcotic analgesics 23 and cholinomimetic drugs 11 have been reported to produce short-lasting accumulations of striatal cGMP. These responses are lost in cell-free homogenates. Therefore our data cannot exclude the existence of a small neuronal striatal guanylate
570 cyclase pool, the measurement of which may have been obscured by the bulk of nonneuronal enzyme. In contrast to the basal guanylate cyclase activity measured in our preparations, basal adenylate cyclase activity is decreased 60-80 ~ as soon as two days after an intrastriatal kainic acid injection indicating a differential localization of the two cyclic nucleotide synthesizing enzymesS,lZ, TM. As shown in this paper, guanylate cyclase activity is increased by more than 120% two days after intrastriatal kainate. This substantial change is partly reversible with time indicating a temporary functional reaction of the enzyme activity to the lesion. Increased c G M P levels in cell division processes have previously been noted 9 and proliferation of glia is one of the major tissue reactions to kainate lesions as assessed at the light microscopic level. In addition, original glial elements, which do not undergo cell division in response to kainic acid, may react to loss of neighbouring neurons by increasing their guanylate cyclase activity. In slices of rat cerebellum, doses of kainic acid, which may well correspond to the concentrations present after intracerebral infusions markedly increase the levels of c G M P and cAMP 24. Intracerebellar injection of kainic acid caused transient increase (at 6-12 h) followed by a permanent decrease in cerebellar c G M P levels 4. Conflicting reports have been published on the respective kainate effects also in other brain areas like striatum and cerebral cortex 24,2s. It remains to be investigated if these short-term effects observed in brain slices should be compared to the guanylate cyclase activity increases reported in this study. A recent finding by Minneman et al. 2°, indicating a substantial loss of striatal cGMP-phosphodiesterase activity after neuronal lesions, supports the view that the entire system responsible for c G M P metabolism is imbalanced as a direct or indirect consequence of kainate injections. Finally, it should be noted that depolarizing concentrations of potassium and glutamate 15, the latter probably playing an important role in the neurodegenerative action of kainic acidZ, 16,19, have also been demonstrated to raise c G M P levels in a number of brain regions, while other endogenous and exogenous compounds were found to be inactive lz. Further elucidation of the effects of kainate lesions on the guanosine nucleotide systems are therefore necessary and may contribute to a better understanding of the mechanism of neurotoxicity elicited by neuro-excitatory amino acids. This work was supported by grants (04X-715, 04X-05415) from the Swedish Medical Research Council, a grant (MH-25504) from the National Institute of Mental Health, and by a grant from Magn. Bergvalls Stiftelse.
1 Bartfai, T., Cyclicnucleotides in the central nervous system, Trends Biochem. Sci., 3 (1978) 121-t24. 2 Bartfai, T., Breakefield,X. O. and Greengard, P., Regulation of synthesis of guanosine 3',5'-cyclic monophosphate in neuroblastoma cells, Biochem. J., 176 (1978) 119-127. 3 Bartfai, T., Study, R. E. and Greengard, P., Muscarinic stimulation and cGMP synthesis in the nervous system. In D. J. Jenden (Ed.), Chotinergic Mechanisms andPsychopharmacology, Plenum Press, N.Y., 1977, pp. 285-295.
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