Noradrenaline-Sensitive Cyclic AMP-Generating System of Rat Cerebral Cortex with Iron- Induced Epileptiform Activity

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1 Short Communication Japanese Journal of Physiology, 37, , 1987 Noradrenaline-Sensitive Cyclic AMP-Generating System of Rat Cerebral Cortex with Iron- Induced Epileptiform Activity Yukio HATTORI, Akiyoshi MoRIwAKI, Hiromichi YASUHARA, and Yasuo HORI Department of Physiology, Okayama University Medical School, Okayama, 700 Japan Summary Noradrenaline-elicited accumulation of cyclic AMP and effects of an a-, f3-adrenoceptor, or adenosine receptor antagonist on the accumulation were examined in slices of different areas of rat cerebral cortex in which ferrous chloride solution was injected unilaterally into the sensorimotor cortex to induce epileptiform activity. The cyclic AMP accumulation was altered regionally in relation to both lateral dominance of electrographic isolated spike activity and variance of the epileptic process. Involvement of a fl-adrenergic, and possibly a-adrenergic, mechanism in the alterations in the cyclic AMP accumulation was indicated. Key words : epileptic cortex, cyclic AMP, noradrenaline. Elevated levels of cyclic AMP or cyclic GMP have been reported in cortex with epileptic activity induced by ethyl chloride (WALKER et al:, 1973) or penicillin (KItIVANEK and MARES, 1977; RAABE et al., 1978), suggesting that the regulatory function of cyclic nucleotides plays a role in abnormal neuronal activity. Since accumulation of cyclic nucleotides, especially of cyclic AMP, has been shown to be elicited by putative neurotransmitters or neuromodulators in slice preparations of several brain structures, alterations in the elicitation mechanisms are presumed to be involved in the process of epilepsy. WILLMORE et al. (1978) found that an intracortical injection of ferrous or ferric chloride solution induced the appearance of focal epileptiform activity in rats and cats. The chronic nature of the epileptiform activity has led to the speculation that generation systems of cyclic nucleotides in the cortex may be persistently altered in iron-induced epilepsy. In this study, we examined the responsiveness of the noradrenaline-sensitive cyclic AMP-generating system in slices of different areas of the iron-induced epileptic cortex of rats. Received for publication December 11,

2 162 Y. HATTORI et al. Male Wistar rats weighing g were used. For experimental rats, 5p1 of 100 mm FeC12 solution was injected into the left sensorimotor cortex employing the method of WILLMORE et al. (1978). For control rats, an equal volume of physiological saline was injected in the same way. Then, stainless steel electrodes for recording electrocorticograms (ECoGs) were implanted in the cranial bone. The ECoG recordings were made and rats were killed by decapitation 8 to 10 days (early stage) or 31 to 60 days (late stage) after the injection. The cerebrum was quickly excised and dissected into four parts: the left anterior, right anterior, left posterior, and right posterior quadrants. The injection site was in the left anterior quadrant. Cross-chopped cortical slices of 400 µm thickness were prepared from each quadrant with a McIlwain tissue chopper. The cortical slices (about 3 mg protein) were preincubated for 30 min in 5 ml of Krebs-Ringer bicarbonate-glucose buffer. After the preincubation, the slices were further incubated in 5 ml of fresh buffer with or without 0.1 mm noradrenaline, 0.1 mm phentolamine, 0.1 mm propranolol, and 10 J M 8-phenyltheophylline. The preincubation and incubation of the slice suspensions were performed at 37 C with constant aerating with 95 c 02-5 o C02. At the end of the incubation, 2.5 ml of cold 7 o trichloroacetic acid was added to the slices, and the mixture was homogenized in an ice bath. After the homogenization, [3H] cyclic AMP (0.05 pmol, about 3,000 dpm, for recovery check) and 0.2 ml of 1 N HC1 were added to 2.0 ml of the homogenate. The precipitated protein was removed by centrifugation (2,000 x g, 15 min) at 4 C. The supernatant was treated four times with water-saturated ether. After the remaining aqueous phase was applied to a column of Dowex 50W-X8 (OTTEN et al., 1972), cyclic AMP was assayed by a cyclic AMP assay kit (Amersham International) based on the protein binding method of GILMAN (1970). Protein content of the homogenate was determined by the method of LoWRY et al. (1951) using bovine serum albumin as the standard. Phentolamine hydrochloride was kindly supplied by Ciba-Geigy. All other materials were purchased from commercial sources. In ECoGs of FeCl2 solution-injected rats, isolated spikes and spike and wave complexes appeared mainly at the frontal leads. Such epileptiform discharges were not observed in control rats. In the experimental rats, the isolated spike activity increased up to about 30 days after the injection, and then was almost constant. The appearance of isolated spikes was observed both ipsilaterally and contralaterally to the injection site. In this study, therefore, rats showing only isolated spikes were classified into three groups according to ratios of ECoG spike frequency on one side of the cortex to that on the other: Fe-I, the spike frequency on the left side was twofold or more than that on the right; Fe-II, the spike frequency on the dominant side was less than twofold that on the other; Fe-III, the spike frequency on the right was twofold or more than that on the left. No appreciable change in the spike frequency ratios was detected in each rat of these groups up to at least 90 days after the injection. Behavioral manifestations of the epileptic activity were restricted to intermittent twitching of the face or neck muscles at the most and were practically unchanged in the FeCl2 solution-induced epileptic process. Most of the spike and Japanese Journal of Physiology

3 CYCLIC AMP SYSTEM IN EPILEPTIC CORTEX 163 Fig. 1. Cyclic AMP contents of incubated slices of anterior cortex of rats 8 to 10 days after injection of FeC12 solution or saline. Cortical slices were prepared from rats in three groups (Fe-I, Fe-II, and Fe-III) classified according to ECoG isolated spike frequency and from those in the control group (Saline), and incubated with no additon (0), 0.1 mm noradrenaline ( ), 0.1 mm noradrenaline and 0.1 mm phentolamine ( ), 0.1 mm noradrenaline and 0.1 mm propranolol (~ ), 0.1 mr t noradrenaline, 0.1 mm phentolamine, and 10 JLM 8-phenyltheophylline ( ), or 0.1 mm noradrenaline, 0.1 mm propranolol, and 10,uM 8-phenyltheophylline ( ). L and R indicate the left anterior and right anterior cortical areas, respectively. Each value represents the mean + S.E.M. of 10 to 13 different experiments. Significantly higher than the value of the contralateral cortical area determined by Student's t-test: *p<0.005, **p< wave complexes appeared 30 days or more after the injection in rats in groups Fe-I and Fe-II. Cyclic AMP contents in incubated slices of anterior cortical areas of rats in groups Fe-I, Fe-II, and Fe-III and control rats at the early stage are shown in Fig. 1. The cyclic AMP contents after incubation with no additions did not vary between the left and right cortical areas. The cyclic AMP contents were increased 4- to 7-fold by addition of noradrenaline. In the noradrenaline-elicited accumulation of cyclic AMP in the anterior half of the cortex, there were regional differences in relation to the patterns of the isolated spikes. In group Fe-I, the cyclic AMP contents were significantly higher on the left side than on the right, while in group Fe-III they were significantly higher on the right than on the left. Phentolamine and propranolol inhibited the elicitation of cyclic AMP accumulation by noradrenaline by 30 to 50 and by 65 to 80%, respectively. There were also regional differences in the anterior cortex in the levels of cyclic AMP when accumulation was antagonized by Vol. 37, No. 1, 1987

4 164 Y. HATTORI et al. Fig. 2. Cyclic AMP contents of incubated slices of anterior cortex of rats 31 to 60 days after injection of FeCl2 solution or saline. Symbols are the same as in Fig. 1. Each value represents the mean + S.E.M. of 10 to 14 different experiments. Significantly higher than the value of the contralateral cortical area determined by Student's t- test: *p<002., **p< phentolamine. In groups Fe-I and Fe-III, the cyclic AMP accumulation elicited by a noradrenaline-phentolamine combination was significantly higher on the side of dominant spike activity than on the other. In these groups, the accumulation elicited by a noradrenaline-propranolol combination did not vary between the two cortical areas. In group Fe-II and the control group, a regional difference in the cyclic AMP content was not observed under these incubation conditions. Cyclic AMP contents in incubated slices of anterior cortical areas of rats at the late stage are shown in Fig. 2. At this stage, the cyclic AMP contents after incubation with no additions did not vary between the two anterior cortical areas as at the early stage. The cyclic AMP contents were increased 4.5- to 7.5-fold by addition of noradrenaline, and phentolamine and propranolol inhibited the elicitation by 35 to 60% and by 65 to 80%, respectively. There were regional differences in the anterior cortex in the cyclic AMP contents under these incubation conditions. In groups Fe-I and Fe-III, the noradrenaline-elicited accumulation of cyclic AMP was significantly lower at the late stage on the side of dominant spike activity than on the other. In these groups, the cyclic AMP accumulations elicited by combinations of noradrenaline and phentolamine or propranolol did not vary between the cortical areas. In group Fe-II at the late stage, there were regional differences in the cyclic AMP contents: Cyclic AMP accumulations elicited by noradrenaline and a noradrenaline-phentolamine combination were significantly higher on the left side Japanese Journal of Physiology

5 CYCLIC AMP SYSTEM IN EPILEPTIC CORTEX 165 than on the right. In the control group, no regional difference was observed as at the early stage. Figures 1 and 2 show that the correlation of noradrenaline-elicited accumulation of cyclic AMP with the lateral dominance of spike activity at the early stage was reversed at the late stage. In separate experiments performed between the early and late stages, it was confirmed that there was no regional difference in noradrenaline-elicited accumulation of cyclic AMP about 20 days after the injection in groups Fe-I and Fe-III, and that the cyclic AMP accumulation was higher on the left side than on the right in group Fe-II. Figures 1 and 2 also show that 8- phenyltheophylline almost completely inhibited the elicitation of cyclic AMP accumulation by a noradrenaline-propranolol combination, but not by a noradrenaline-phentolamine combination. In the posterior half of the cortex, no regional difference was observed in cyclic AMP contents under these incubation conditions in groups Fe-I, Fe-II, and Fe-III, and the control group (data not shown). The results of this study clearly show that the noradrenaline-elicited accumulation of cyclic AMP varied regionally in the iron-induced epileptic cortex. The elevated levels of cortical cyclic nucleotides have been reported in other experimental models of epilepsy (WALKER et al., 1973; KRIVANEK and MARES, 1977; RAABE et al., 1978), but the reason for the elevation remains obscure. Furthermore, little is known about the cyclic AMP accumulation in response to certain neurotransmitters or neuromodulators in relation to the process of epilepsy. The results presented here suggest that the elevation in cyclic AMP levels in the epileptic cortex is due, at least in part, to the altered responsiveness of the cortical cyclic AMP system. The results also suggest that some noradrenergic mechanisms are involved in the iron-induced epileptic process, as has been implied in reports of epileptic cortex induced with cobalt (TROTTIER et al., 1981) or penicillin (KOBAYASHI et al., 1976), in which decreases in noradrenergic terminals or noradrenaline contents occur. In slice preparations of rat cerebral cortex, cyclic AMP accumulation in response to noradrenaline has been reported to be mediated by both x- and (- adrenergic mechanisms (PERKINS and MOORE, 1973; SCHULTZ and DALY, 1973). Mediation by both x- and f3-adrenergic mechanisms was also shown in this study, i.e., phentolamine or propranolol partially inhibited the elicitation of cyclic AMP accumulation by noradrenaline. /3-Adrenergic activation of adenylate cyclase has been shown in cell-free preparations of rat cerebral cortex (BOCKAERT et al., 1977; PARTINGTON and DALY, 1979), but no convincing evidence is available for direct x- adrenergic activation of the enzyme in such brain preparations. Concerning the functional role of x-adrenergic mechanisms in cyclic AMP generation, DALY et al. (1980) have proposed that in slice preparations, a-adrenergic mechanisms potentiate the activation of adenylate cyclase coupled to such receptors as /3-adrenoceptors or adenosine receptors. In view of this concept, the noradrenaline-elicited accumulation of cyclic AMP can be divided into two components: cyclic AMP generation through the /3-adrenoceptor-coupled adenylate cyclase system, and cyclic AMP Vol. 37, No. 1, 1987

166 Y. HATTORI et al. generation through a-adrenergic activation of some systems such as the adenosine receptor- or /3-adrenoceptor-coupled adenylate cyclase system.

6 166 Y. HATTORI et al. generation through a-adrenergic activation of some systems such as the adenosine receptor- or /3-adrenoceptor-coupled adenylate cyclase system. In the present study, cyclic AMP generation through the /3-adrenoceptor-coupled adenylate cyclase system corresponds to the cyclic AMP levels after incubation with a noradrenalinephentolamine combination. The regional difference in this cyclic AMP generation was detected in rats in groups Fe-I and Fe-III at the early stage and in those in group Fe-II at the late stage. In the present study, cyclic AMP accumulation involving the a-adrenoceptor, which is elicited possibly by endogenous adenosine, corresponds to the cyclic AMP levels after incubation with a noradrenalinepropranolol combination, in which the elicitation was almost completely blocked by the adenosine receptor antagonist 8-phenyltheophylline. As for this cyclic AMP generation, no regional difference was observed in rats showing only isolated spike activity, as presented here. In rats of groups Fe-I and Fe-III at the late stage, there was no regional difference in cyclic AMP accumulation through both the f3- adrenoceptor-coupled adenylate cyclase system and x-adrenergic activation of the adenosine receptor-coupled adenylate cyclase system. Further, the elicitation of cyclic AMP accumulation by noradrenaline was completely blocked by simultaneous addition of phentolamine and propranolol (data not shown). In these rats, therefore, the regional difference in the noradrenaline-elicited accumulation of cyclic AMP appears to be due to alterations in cyclic AMP generation through a- adrenergic activation of the f3-adrenoceptor-coupled adenylate cyclase system. The present results on the epileptiform discharges and cyclic AMP accumulation lead to the proposal that periods up to and exceeding about 30 days after the injection are regarded as the transient and sustained processes, respectively. It is conceivable, therefore, that noradrenaline-elicited accumulation of cyclic AMP is high on the side of dominant spike activity in the transient process and then the regional difference disappears gradually, and that the cyclic AMP accumulation is low on the dominant side in the sustained process. The results also show that some components of the noradrenaline-elicited accumulation of cyclic AMP participate in its regional difference in various ways at the two stages. In this context, in rats in group Fe-II at the early stage, noradrenaline-elicited accumulation of cyclic AMP did not vary regionally since lateral dominance was not observed in the isolated spike activity. In these rats, however, the cyclic AMP accumulation elicited by noradrenaline and the cyclic AMP generation through the f3-adrenoceptor-coupled adenylate cyclase system were high on the side of injection site at the late stage. Similar regional differences were detected in rats showing spike and wave complexes in separate experiments. Therefore, the regional difference in rats in group Fe-II appears to be related to the development into the spike and wave complexes at the stage. Taken together, the results of this study demonstrate that the noradrenalineelicited accumulation of cyclic AMP and its components are altered regionally in the epileptic cortex as a function of both the epileptiform ECoG pattern and elapsed time after the injection of FeC12 solution. Thus, it seems likely that these alterations Japanese Journal of Physiology

7 CYCLIC AMP SYSTEM IN EPILEPTIC CORTEX 167 in the cortical noradrenaline-sensitive cyclic AMP-generating system are associated with the process of iron-induced epilepsy. REFERENCES BOCKAERT, J., TASSIN, J. P., THIERRY, A. M., GLOWINSKI, J., and PREMONT, J. (1977) Characteristics of dopamine and J3-adrenergic sensitive adenylate cyclases in the frontal cerebral cortex of the rat. Comparative effects of neuroleptics on frontal cortex and striatal dopamine sensitive adenylate cyclases. Brain Res., 122: DALY, J. W., PADGETT, W., NIMITKITPAISAN, Y., CREVELING, C. R., CANTACUZENE, D., and KIRK, K. L. (1980) Fluoronorepinephrines: Specific agonists for the activation of alpha and beta adrenergic-sensitive cyclic AMP-generating systems in brain slices. J. Pharmacol. Exp. Ther., 212: GILMAN, A. G. (1970) A protein binding assay for adenosine 3' : 5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A., 67: KOBAYASHI, K., SHIRAKABE, T., KISHIKAWA, H., and MORI, A. (1976) Catecholamine levels in penicillin-induced epileptic focus of the cat cerebral cortex. Acta Neurochir. (Suppl.), 23: KItIVANEK, J. and MARES, P. (1977) Cyclic adenosine 3',5'-monophosphate in epileptogenic foci induced by penicillin. Neurosci. Lett., 6: LoWRY, 0. H., RosEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: OTTEN, J., JOHNSON, G. S., and PASTAN, I. (1972) Regulation of cell growth by cyclic adenosine 3 ',5 '-monophosphate. Effect of cell density and agents which alter cell growth on cyclic adenosine 3',5'-monophosphate levels in fibroblasts. J. Biol. Chem., 247: PARTINGTON, C. R. and DALY, J. W. (1979) Effect of gangliosides on adenylate cyclase activity in rat cerebral cortical membranes. Mol. Pharmacol., 15: PERKINS, J. P, and MOORE, M. M. (1973) Characterization of the adrenergic receptors mediating a rise in cyclic 3',5'-adenosine monophosphate in ra L cerebral cortex. J. Pharmacol. Exp. Ther., 185: RAABE, W., NIcoL, S., GUMNIT, R. J., and GOLDBERG, N. D. (1978) Focal penicillin epilepsy increases cyclic GMP in cerebral cortex. Brain Res., 144: SCHULTZ, J. and DALY, J. W. (1973) Accumulation of cyclic adenosine 3',5'-monophosphate in cerebral cortical slices from rat and mouse: Stimulatory effect of a- and f 3- adrenergic agents and adenosine. J. Neurochem., 21: TROTTIER, S., BERGER, B., CHAUVEL, P., DEDEK, J., and GAY, M. (1981) Alterations of the cortical noradrenergic system in chronic cobalt epileptogenic foci in the rat: A histofluorescent and biochemical study. Neuroscience, 6: WALKER, J. E., LEWIN, E., SHEPPARD, J. R., and CROMWELL, R. (1973) Enzymatic regulation of adenosine 3',5'-monophosphate (cyclic AMP) in the freezing epileptogenic lesion of rat brain and in homologous contralateral cortex. J. Neurochem., 21: WILLMORE, L. J., SYPERT, G. W., MUNSON, J. B., and HURD, R. W. (1978) Chronic focal epileptiform discharges induced by injection of iron into rat and cat cortex. Science, 200: Vol. 37, No. 1, 1987

Synopsis. Received March 2, adrenaline. Mosinger and Kujalova (1964) reported that adrenaline-induced lipolysis

Synopsis. Received March 2, adrenaline. Mosinger and Kujalova (1964) reported that adrenaline-induced lipolysis Studies on Reduction of Lipolysis in Adipose Tissue on Freezing and Thawing YASUSHI SAITO1, NoBUO MATSUOKA1, AKIRA KUMAGAI1, HIROMICHI OKUDA2, AND SETSURO FUJII3 Chiba University, Chiba 280, Japan, 2Department