EXPERIMENTALNEUROLOGY
115,
297-303.
(1992)
Protein Kinase C Activity and Subcellular Distribution in Rat Brain following Repeated Electroconvulsive Seizures OLIVIER VERNET, KLARA ROSTWOROWSKI, Montreal
neurological
Institute,
McGill
University,
Protein kinase C (PKC) activity was measured in samples of neocortex, cerebellum, and hippoeampus from adult rats receiving a series of 10 electroeonvulsive seizures (ECS). Rats were sacrificed immediately and at various intervals from 16 min to 24 h after the last seizure. From 77 to 84% of total PKC activity was found in the cytosol versus the membrane fraction. PKC activity in cerebellum was significantly higher than in neocortex (IS%, P < 0.05). Repeated ECS treatment-did not affect total PKC activity nor its distribution between membrane and cytosolic fractions when compared with sham ECS controls. This finding is in keeping with reports that adrenergic-stimulated phosphoinositol turnover is not altered 24 h following repeated ECS. o issz Academic
Press,
Inc.
INTRODUCTION Among the many kinases, the calcium/phospholipiddependent kinase or protein kinase C (PKC) plays a fundamental role in the transmembrane signaling of many extracellular agonists (26). This enzyme is especially abundant in brain (18) and has been implicated in neurotransmitter release, ion channel regulation, and neuronal plasticity (26). In the presence of phospholipid and physiological concentrations of calcium, the second messenger diacylglycerol (DAG), produced by receptormediated phosphoinositide (PI) hydrolysis, activates PKC at the plasma membrane level (15). Tumor promoting phorbol esters can substitute for DAG and directly activate PKC (5) causing the translocation of enzyme from cytosol to cellular membrane (9, 17). Activating of PKC by platelet-activating factor, however, does not require the association of cytosolic PKC with membrane (30), while in GH, cells thyrotropin-releasing hormone stimulation merely elicits a brief translocation (22). In hepatocytes activation of PKC in response to Ca” mobilizing hormones involves preexistent membrane-bound enzyme only without affecting the soluble enzyme (7). Gleiter et al. (11) employed [‘Hlphorbol-12,13butyrate ([3H]PDBu) binding as a marker of mem-
AND ALLAN L. SHERWIN
Montreal,
Q&bee,
Cmuda
H3A
2B4
brane-bound PKC in neural tissues from rats subjected to 10 once-daily electroconvulsive seizures (ECS). They observed a 30% diminution in the density (B,,) of 13H]PDBu binding sites in neocortex and cerebellum but not hippocampus 24 h after the last treatment. No change was noted following a single seizure. They suggested that repeated ECS downregulates PI breakdown leading to a reduction in DAG-activated membranebound PKC. Phorbol ester binding, however, does not permit measurements of the subcellular distribution of PKC since it determines only the amount of membranebound PKC protein. Moreover, phorbol ester binding sites may not strictly correlate with enzyme activity (12, 28). ECS alters the biochemical parameters of a variety of neurotransmitter systems (3, 32). Repeated daily ECS significantly increases the density of a-l adrenoceptors (37, 41) which are coupled to PI metabolism (13). This increase which is comprised of the (Y--1a subtype is confined to a band of binding in the outermost lamina of the neocortex (4). Michel et al. (23) reported that a-1B sites mediate PI hydrolysis in rat neocortex. In an early report Neuman et al. (25) observed that single or repeated ECS treatment increased PI hydrolysis in rat neocortex but not in caudate nucleus or hippocampus. Blendy et ~1. (3) and Dubeau et al. (8), however, were not able to demonstrate a change in either basal or norepinephrine-induced PI hydrolysis in rat cortex sampled 24 h after repeated ECS. Pile et al. (31) noted no change in a-l receptor-coupled PI hydrolysis but observed a marked reduction in the CAMP response which involves an interaction between ar and @receptors. Vadnal and Bazan (38) used an in uiuo model to measure the acute effects of ECS on radio-labeled polyphosphoinositides and inositol phosphates. ECS increased the level of inositol trisphosphate (IP,) but diminished the endogenous mass of phosphatidyl4,5-biphosphate (PIP,). They concluded that ECS stimulates the inosito1 lipid cycle in brain possibly due to neurotransmitter release. Pretreatment of animals with lithium did not affect inositol monophosphate levels but did appear to attenuate the ECS-induced changes in PIP, and IP, suggesting that lithium has other sites of action in addi-
297 All
Copyright 0 1992 rights of reproduction
0014.4886192 $3.00 by Academic Press, Inc. in any form reserved.
298
VERNET,
ROSTWOROWSKI,
tion to an inhibition of inositol-1-phosphatase. In contrast, carbamazepine-ECS-treated rats showed enhanced labeling of PIP,, decreased IP, levels, andinhibition of IP, accumulation (39). Both these drugs may elicit differential effects on cell signal transduction perhaps at the G protein phospholipase C level (39). The aim of the present study was to determine if daily repeated ECS treatment reduces membrane-bound PKC activity. The data show that membrane and cytosolic PKC activities remain remarkably consistent when examined at various intervals following the last seizure. MATERIALS
AND
METHODS
Animals. Male Sprague-Dawley rats (Charles River, St. Constant, Quebec) weighing between 250 and 300 g were used in all experiments. Rats were housed in constant temperature animal quarters with a light/dark cycle and allowed access to food and water ad libitum. Electroconuulsiue seizures. Maximal ECS were induced by delivering current (100 mA, 60 Hz, 200 ms) through saline-moistened soft earclip electrodes (37). Each stimulus induced a generalized tonic-clonic seizure lasting 20 to 30 s. Test animals received a series of 10 daily treatments. Matched control rats were handled in the same manner daily but no current was delivered (sham ECS). Animals were sacrificed by decapitation at fixed intervals just at the end of the seizure (30 s after the shock) or 15 min, 60 min, 240 min, and 24 h after the last ECS. Sham ECS animals were sacrificed at 30 s, 240 min, or 24 h. Brains were rapidly removed and dissected free from blood vessels on an ice-chilled ceramic tile. Neocortex, cerebellum, and hippocampus were frozen in liquid nitrogen and stored at -70°C until assayed. PKC assay. Tissues were homogenized by two 5-s strokes of a Polytron homogenizer (Brinkman) in 10 volumes of buffer composed of 50 mM Tris-HCl, pH 7.4, 2 n&f dithiothreitol (DTT), 2 mM EGTA, 1 mA4 phenylmethyl sulfonyl fluoride (PMSF, Sigma), 0.1 mg/ ml leupeptin (Sigma) and centrifuged at 100,OOOg for 1 h at 4°C. The supernatant (cytosolic fraction) was collected. The 100,OOOg pellet (membrane fraction) was reconstituted to the original volume with the homogenizing buffer containing 0.25% Triton X-100 (Sigma). Protein concentrations were determined using the method of Lowry et al. (21). Phosphorylation of lysine-rich histone (Sigma) was assayed in triplicate (24) after having determined the correct Michaelis-Menten conditions for substrate and cofactors. The final assay conditions were: 100 pM free calcium, 30 mM Tris-HCl, pH 7.4, 1.19 mM DTT, 500 pg/ml L-cY-phosphatidyl+serine (PS, Sigma), 25 pg/ml 1,2-dioctanoyl-SN-glycerol (DAG, Sigma), 12 pM [32P]ATP (0.5-1.0 X lo6 cpm, NEN, Montreal, Quebec) 10 mA4 MgCl,, 250 pg/ml ly-
AND
SHERWIN
sine-rich histone and cytosolic or particulate fraction (2-7 pg protein) in a final volume of 40 ~1. The reaction components minus ATP and MgCl, were preincubated at 30°C and phosphorylation was initiated by adding ATP and MgCl,. After 1 min, a 25-~1 aliquot was spotted on a phosphocellulose paper strip (Whatman P81) (44) which was transferred to a beaker containing 75 mh4phosphoric acid. The strips were rinsed three times with 75 mM phosphoric acid, once with methanol, and air-dried. The incorporation of [32P] into histone was measured by liquid scintillation spectrometry. Blanks (complete reaction mixture minus enzyme fraction) were used to correct for nonspecific binding. PKC activity was calculated as the difference in activity in the presence or absence of DAG, PS, and calcium. Results were analyzed using a one-way analysis of variance with Duncan’s multiple comparisons. Differences were considered significant if P < 0.05 was achieved. RESULTS We found that between 77 to 84% of the total PKC activity was present in the cytosolic fraction. This percentage did not change significantly at any time during the postictal period varying from 79 to 81% in the neocortex, from 81 to 84% in the hippocampus, and from 77 to 80% in the cerebellum. As shown in Table 1 a comparison of the three brain regions assayed in the sham ECS control groups showed that total PKC activity was significantly higher in cerebellum than in neocortex (15%,
P < 0.05). Protein kinase C activity was not significantly altered in either the membrane or cytosolic fraction of neocortex, cerebellum, or hippocampus at any time following the last repeated ECS when compared with the sham ECS controls (Table 1). In particular there was no evidence of a reduction in the enzymic activity of PKC in membranes prepared from neocortex or hippocampus 24 h following the last repeated ECS. There was also no evidence of transduction of PKC activity from cytosol to membranes at 30 s, 15 min, 60 min, 240 min, or 24 h postictally. The relative distribution of PKC enzymic activity in the membrane versus the cytosolic fraction was remarkably consistent during the 24-h postictal period. DISCUSSION Our study shows that repeated ECS does not alter membrane or cytosolic PKC activity in neocortex, hippocampus, or cerebellum within 24 h of the last seizure. Repeated ECS has been found to decrease the number of [3H]PDBu binding sites in rat neocortex and cerebellum when sampled 24 h postictally (11). This decrease in receptor density (B,) was not associated with a change in the apparent dissociation constant (Kd)
PROTEIN
KINASE
C AND
ELECTROCONVULSIVE
TABLE Effect
Time
interval
Neocortex 30 s 15 min 60 min 240 min 24 h Sham ECS Hippocampus 30 s 15 min 60 min 240 min 24 h Sham ECS Cerebellum 30s 15 min 60 min 240 min 24 h Sham ECS
Note. Values * Significantly
are mean different
1
of Repeated ECS on PKC Activity PKC
activity
299
SEIZURES
and Intracellular
(nmol/min/mg
Distribution
protein) Cytosol
Total PKC (nmol/min/mg
activity protein)
n
Membrane
5 6 5 5 6 14
1.52 1.41 1.70 1.65 1.63 1.50
+: + + t t +
0.12 0.09 0.08 0.15 0.22 0.09
5.87 6.02 6.63 6.96 6.07 5.98
t + + f f +
0.65 0.28 0.25 0.33 0.57 0.26
7.39 7.43 8.33 8.61 7.70 7.48
+ 0.55 * 0.31 ?z 0.26 -t 0.43 + 0.71 -t 0.33
5 6 5 5 6 14
1.47 1.40 1.59 1.42 1.43 1.50
+ +-+ i t r
0.05 0.05 0.17 0.09 0.10 0.09
6.42 7.16 6.27 6.05 6.24 6.77
f 0.16 + 0.40 -c 0.33 -1- 0.30 2 0.25 + 0.18
7.89 8.56 7.86 7.47 7.67 8.27
f 2 t 2 f i
0.21 0.40 0.44 0.33 0.21 0.23
5 5 5 5 6 13
1.70 1.77 1.87 1.63 1.82 1.73
* + t tr -t
0.11 0.12 0.18 0.07 0.19 0.12
6.80 6.06 6.34 6.51 6.23 6.88
t f f ? A k
8.50 7.83 8.21 8.14 8.05 8.61
+ + + + + +
0.21 0.37 0.56 0.24 0.56 0.45*
t SEM. PKC activity is expressed in nmol s2P transferred from the neocortex sham ECS group (P < 0.05, one-way
which is a measure of the affinity of the receptors for the test ligand. The density (B,,,) of phorbol ester binding sites reflects the amount of PKC protein bound to membranes and this does not strictly correlate with PKC enzymic activity (12, 28). Repeated ECS may merely downregulate membrane recognition sites for phorbol esters without affecting protein kinase C histone phosphorylase activity (42). Moreover, certain analytical techniques may preferentially detect one or more of the isoforms of PKC or the access of [3H]PDBu to PKC protein in various membrane preparations might be a limiting factor (12, 28). Diacylglycerol activates PKC primarily through a reversible PKC/membrane complex while phorbol esters induce a sustained activation of PKC by forming an irreversible PKC/membrane complex (9). In rat neocortex, (~-~a subtype adrenoceptors which are coupled to membrane PI metabolism are localized by autoradiography to lamina 1 and lamina Va and Vc sparing layer Vb pyramidal cells (4). Noradrenergic fibers originating in the locus coeruleus (LC) are widely distributed in cerebral cortex including the outermost lamina and many cortical neurons have adrenoceptors. Lamina 1 a-Is adrenoceptors are preferentially increased 24 h following repeated ECS, but this rise is not accompanied by a change in PI turnover or PKC activity. Perhaps these seizure-induced binding sites are comprised of precursor or degraded forms of the a(-ia adrenoceptor in which the ligand recognition sites have
0.20 0.27 0.39 0.18 0.49 0.38'
into histone/min/mg ANOVA with Duncan’s
protein. multiple
comparisons).
been preserved but no longer functionally connected to the PI signal transduction mechanism (4,8,31). We found average levels of PKC activity to be of the same order of magnitude as reported for rat striatal neuronal cultures by a similar assay (43), but higher than those reported for whole rat brain by other techniques of measurement (14, 18, 20). We found si~ificantly more total PKC activity in cerebellum compared to neocortex and an intermediate value in hippocampus. Only a few studies have simultaneously quantitated PKC activity in different brain regions. An earlier report showed almost identical levels of PKC between rat neocortex and cerebellum (18). Comparisons with previous studies are hindered, however, by differences in methods of tissue preparation or techniques such as autoradiography (45) and immunocytochemistry (26). From 77 to 84% of the total PKC activity was recovered from the cytosolic fraction. These percentages are in accordance with some previous studies (36, 43) but differ notably from other observations where about one-third of the activity was found to be present in the cytosolic fraction (6, 10, 14, 24). We recently reported that from 67 to 70% of total PKC activity was present in cytosolic fractions prepared from specimens of histologically normal human brain excised to permit the removal of deep-seated tumors (40). Protein kinase C activity and intracellular distribution was not altered in focally epileptic neocortex.
300
VERNET,
ROSTWOROWSK~,
Absolute comparisons of PKC activity are difficult, especially because extraction conditions which aim to prevent irreversible proteolytic activation of PKC by the Ca’+-dependent protease calpain (16) influence its subcellular distribution (1, 14, 26). We prevented PKC activation by means of high concentrations of a Ca2+ chelator and by adding the protease inhibitors PMSF and leupeptin during the homogenization step. Moreover, the detergent Triton X-100 was employed in order to unmask all PKC activity in the cortical particulate fractions (14). Comparisons between studies are also complicated by the findings that PKC is known to be a family of subspecies which exhibit subtle individual enzymatic properties and different activation characteristics (27). Slightly different techniques can thuspreferentially detect one or the other of these isozymes. Moreover, the relative distribution of protein kinase C subspecies varies in different regions of the CNS (36). The data presented in this study show that repeated ECS treatment does not cause a prolonged change in PKC activity or subcellular distribution. These results do not, however, rule out the possibility that PKC could be activated and translocated from cytosol to membrane for only a very brief period (22). As recently demonstrated in monoblastoid U937 cells PKC activity may be downregulated for exogenous histone substrates but preserved when tested with certain peptide substrates (42). Both a single ECS seizure or bicuculline-induced status epilepticus (2,34,46) produce an accumulation of DAG in brain, which by 60 s has already returned to basal levels (33). Chronic ECS treatment might thus induce a recurrent short-lived activation of PKC which would not alter the activity or subcellular distribution of the enzyme. More protracted membrane changes including a redudtion in phorbol ester binding sites may result from PKC-mediated phosphorylation reactions (19, 29, 35).
4.
5.
6. 7.
8.
9.
AND SHERWIN troconvulsive shock and reserpine increase Lu,-adrenoceptor binding sites but not norepinephrine stimulated phosphoinositide hydrolysis in rat brain. Eur. J. Pharmacol. 156: 267-270. BLENDY, J. A., L. J. GRIMM, D. C. PERRY, L. WEST-J• HNSRUD, AND K. J. KELLER. 1990. Electroconvulsive shock differentially increases binding to (Y,adrenergic receptor subtypes in discrete regions of rat brain. J. Neurosci. 10: 2580-2586. CASTAGNA, M., Y. TAKAI, K. KAIBUCHI, K. SANO, U. KIKKAWA, ANDY. NISHIZUKA. 1982. Direct activation of calcium-activated, phosphoiipid-dependent protein kinase by tumor-promoting phorbol esters. J. Bid. G’hem. 257: 7847-7851. CUMFUNE, R. C., AND J. C. LA MANNA. 1990. Protein kinase C activity in rat brain cortex. J. Neu~~m. 55: 826-831. DIAZ-GUERRA, M. J. M., AND L. BOXA. 1990. Lack of translocation of protein kinase C from the cytosol to the membranes in vasopressin-stimulated hepatocytes. Biochem. J. 269: 163-168. DUBEAU, F., AND A. L. SHERWIN. 1989. Effect of repeated versus single electroconvulsive seizures on adrenergic-mediated phosphatidylinositol hydrolysis in rat neocortex. Exp. Neurol. 105: 206-210. EDERVEEN, A. G. H., S. J. VAN EMST-DE VFUES, J. H. H. M. DE PONT, AND P. H. G. M. WILLEMS. 1991. Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in rabbit pancreatic acini. Eur. J. Biochem. 195: 679683.
10. GIRARD, P. R., G. J. MAZZEI, AND J. F. KUO. 1986. immunolo~cal quanti~tion of phospholipid/~a’+-dependent protein kinase and its fragments: Tissue levels, subcellular distribution, and ontogenic changes in brain and heart. J. Bid. Chem. 261: 370375.
11. GLEITER, C. H., J. DECKERT, D. J. NUTT, AND P. J. MARANGOS. 1988. The effect of acute and chronic electroconvulsive shock on [3Hlphorbol-dibutyrate binding to rat brain membranes. Neurothem. Res. 13: 1023-1026. 12. HOUSEY, G. M., M. D. JOHNSON, W. L. W. HSIAO, C. A. O’BRIAN, J. P. MURPHY, P. KIRSCHMEIER, AND I. B. WEINSTEIN. 1988. Overproduction of protein kinase C causes disordered growth control in rat fibroblasts. Cell 52: 343-354. 13. KENDALL, D. A., E. BROWN, AND S. R. NAHORSKI. 1985. culadrenoceptor-me~ated inositol phospholipid hydrolysis in rat cerebral cortex: Relationship between receptor occupancy and response and effects of denervation. Eur. J. Pharmacol. 114: 41-52.
ACKNOWLEDGMENTS This work was supported by the Medical Research Council of Canada. Dr. Vernet is a postdoctoral fellow of the Fonds Decker of Surgery, CHUV, Lausanne and Janggen-Poehn Stiftung, St. Gallen, Switzerland. We greatly appreciated the cooperation of Ms. J. Green and Mr. R. Jones. We thank Drs. J. Ante1 and V. W. Yong for helpful advice and Mrs. M. Nicholls-Spence for preparing the manuscript. REFERENCES 1. ALOYO, V. J., H. ZWIERS, P. N. E. DE GFZAAN,AND W. H. GISPEN. 1988. Phosphorylation of the neuronal protein kinase C substrate B-50: In vitro assay conditions alter sensitivity to ACTH. Neurochem. Res. 13: 343-348. 2, AVELDANO DE CALDIRONI, M. I., AND N. G. BAZAN. 1979. (Ymethyl-p-tyrosine inhibits the production of free arachidonic acid and diacylglycerols in brain after a single electroconvulsive shock. Neurochem. Res. 4: 213-221. 3. BLENDY, J. A., C. A. STOCKMEIER, AND K. J. KERR. 1988. Elec-
14. KIKKAWA, U., Y. TAKAI, R. MINAKUCHI, S. INOHARA, AND Y. NISHIZUKA. 1982. Calcium-activated, phospholipid-dependent protein kinase from rat brain: Subcellular distribution, purification and properties. J. Biol. Chem. 257: 13,341-13,348. 15. KISHIMOTO, A., Y. TAKAI, T. MORI, U. KIKKAWA, AND Y. NISHIZUKA. 1980. Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J. Bid. Chem. 255: 2273-2276. 16. KISHIMOTO, A., K. MIKAWA, K. HASHIMO~, I. YAS~.X)A, S. TANAKA, M. TOMINAGA, T. KURODA, AND Y. NISHIZUKA. 1989. Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain). J. Bid. Chem. 264: 40884092. 17. KRAFT, A. S., AND W. B. ANDERSON. 1983. Phorbol esters increase the amount of Cazc, phospolipid-dependent protein kinase associated with plasma membrane. Nature 301: 621-623. 18. Kuo, J. F., R. G. G. ANDERSSON, B. C. WISE, L. MACKERLOVA, I. SALOMONSSON, N. L. BRACKETT, N. KATOH, M. SHC~JI,AND R. W. WRENN. 1980. Calcium-dependent protein kinase C: Widesnread occurrence in various tissues and uhvla of the animal
PROTEIN
19.
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22.
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24.
25.
KINASE
C AND ELECTROCONVULSIVE
kingdom and comparison of effects of phospholipid, calmodulin and trifluoperazine. Proc. N&l. Acad. Ski. USA 77: 7039-7043. LEEB-LUNDBERG, L. M. F., S. COTECCHIA, J. W. LOMASNEY, J. F. DE BERNARDIS, R. J. LEFKOWITZ, AND M. G. CARON. 1985. Phorbol esters promote a,-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism. Proc. Natl. Acad. Sci. USA 82: 5651-5655. LOUIS, J-C., E. MAGAL, AND E. YAVIN. 1988. Protein kinase C alterations in the fetal rat brain after global ischemia. J. Biol. Chem. 263: 19,282-19,285. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-275. MARTIN, T. F. J., K. P. HSIEH, AND B. W. PORTER. 1990. The sustained second phase of hormone stimulated diacylglycerol accumulation does not activate protein kinase C in GH, cells. J. Biol. Chem. 265: 7623-7631. MICHEL, M. C., G. HANFT, AND G. GROSS. 1990. a,,-but not (Y,~adrenoceptors mediate inositol phosphate generation. NuuynSchmiedebergs Arch. Pharmacol. 341: 385-387. NEARY, J. T., L. 0. B. NORENBERG, AND M. D. NORENBERG. 1988. Protein kinase C in primary astrocyte cultures: Cytoplasmic localization and translocation by a phorbol ester. J. Neurothem. 50: 117991184. NEUMAN, M. E., I. MISKIN, AND B. LERER. 1987. Effects of single and repeated electroconvulsive shock administration on inositol phosphate accumulation in rat brain slices. J. Neurochem. 49: 19-23.
26. NISHIZUKA, Y. 1986. Studies and perspectives of protein kinase C. Science 233: 305-312. 27. NISHIZUKA, Y. 1988. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334: 661-665. 28. ONO, Y., T. FUJII, K. OGITA, U. KIKKAWA, K. IGARASHI, AND Y. NISHIZUKA. 1989. Protein kinase C zeta subspecies from rat brain: its structure, expression and properties. Proc. N&l. Acad. Sci. USA
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29. ORELLANA, S., P. A. SOLSKI, AND J. H. BROWN. 1987. Guanosine 5’-O-(thiotriphosphate)-dependent inositol triphosphate formation in membranes is inhibited by phorbol ester and protein kinase C. J. Biol. Chem. 262: 1638-1643. 30. PELECH, S. L., D. L. CHAREST, S. L. HOWARD, H. B. PADDON, AND H. SALARI. 1990. Protein kinase C activation by platelet activating factor is independent of enzyme translocation. Biochim. Biophys. Acta 1051: 100-107. 31. PILC, A., E. CHALECKA-FRANASZEK, I. NALEPA, J. VETULANI, AND S. J. ENNA. 1988. The influence of electroshock on adrenoceptor function in brain cerebral cortex: Selectivity for the (Y adrenoceptor site. Eur. J. Pharmacol. 156: 143-147. 32. POST, R. M., F. PUTNAM, T. W. UHDE, AND S. R. WEISS. 1986. Electroconvulsive therapy as an anticonvulsant. Implications for its mechanisms of action in affective illness. Ann. N. Y. Acad. Sci. 462:
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33. REDDY, T. S., AND N. G. BAZAN. 1987. Arachidonic acid, stearic acid, and diacylglycerol accumulation correlates with the loss of
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phosphatidylinositol 4,5-biphosphate in cerebrum 2 seconds after electroconvulsive shock: Complete revision of changes 5 minutes after stimulation. J. Neurosci. Res. 18: 449-455. 34. RODRIGUEZ DE TURCO, E. B., S. MORELLI DE LIBERTI, AND N. G. BAZAN. 1983. Stimulation of free fatty acid and diacylglycerol accumulation in cerebrum and cerebellum during bicuculline-induced status epilepticus. Effect of pretreatment with or-methylp-tyrosine and p-chlorophenylalanine. J. Neurochem. 40: 252259.
35. SCHOEPP, D. D., AND B. G. JOHNSON. 1988. Selective inhibition of excitatory amino acid stimulated phosphoinositide hydrolysis in the rat hippocampus by activation of protein kinase C. Biothem.
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36. SHEARMAN, M. S., Z. NAOR, U. KIKKAWA, AND Y. NISHIZUKA. 1987. Differential expression of multiple protein kinase C subspecies in rat central nervous tissue. Biochem. Biophys. Res. Commun. 147: 911-919. 37. SHERWIN, A. L., M. DE ROODE, F. DUBEAU, D. GUEVREMONT, AND N. MILLS. 1989. Transient changes in cortical (pi adrenoceptors and seizure threshold following electroconvulsive seizures in rats. Epilepsy Res. 3: 49-54. 38. VADNAL, R. E., AND N. G. BAZAN. 1987. Electroconvulsive shock stimulates polyphosphoinositide degredation and inositol trisphosphate accumulation in rat cerebrum: Lithium pretreatment does not potentiate the changes. Neurosci. Lett. 80: 75-79. 39. VADNAL, R. E., AND N. G. BAZAN. 1988. Carbamazepine inhibits electroconvulsive shock-induced inositol trisphosphate (IP,) accumulation in rat cerebral cortex and hippocampus. Biochem. Biophys. Res. Commun. 153: 128-134. 40. VERNET, O., V. W. YONG, K. ROST~OROWSKI, M. FADICH, A. OLIVIER, R., AND A. L. SHERWIN. 1991. Protein kinase C activity and intracellular distribution in surgically excised human epileptic neocortex. Brain Res. 547: 319-322. 41. VETULANI, J. L., A. ANTKIEWICZ-MICHALUK, A. ROKOSZ-PELC, AND A. PILC. 1983. Chronic electroconvulsive treatment enhances the density of [3H]prazosin binding sites in the central nervous system of the rat. Brain Res. 275: 392-395. 42. WAYS, K., R. RIDDLE, M. WAYS, AND P. COOK. 1990. Effect of phorbol esters on cytosolic protein kinase C content and activity in the human monoblastoid U937 cell. J. Biol. Chem. 266: 12581264. 43. WEISS, S., J. ELLIS, D. D. HENDLEY, AND R. H. LENOX. 1989. Translocation and activation of protein kinase C in striatal neurons in primary culture: Relationship to phorbol dibutyrate actions on the inositol phosphate generating system and neurotransmitter release. J. Neurochem. 52: 530-536. 44. WITT, J. J., AND R. ROSKOSKI, JR. 1975. Rapid protein kinase assay using phosphocellulose-paper absorption. Anal. Biochem. 66: 253-258. 45. WORLEY, P. F., J. M. BARABAN, E. B. DE Souz~, AND S. H. SNYDER. 1986. Mapping second messenger systems in the brain: Differential localizations of adenylate cyclase and protein kinase C. Proc. Natl. Acad. Sci. USA 83: 4053-4057. 46. YOSHIDA, S., M. IKEDA, R. BUSTO, M. SANTISO, E. MARTINEZ, AND M. D. GINSBERG. 1987. Cerebral phosphoinositide, triacylglycerol and energy metabolism during sustained seizures induced by bicuculline. Brain. Res. 412: 114-124.
115,
297-303.
(1992)
Protein Kinase C Activity and Subcellular Distribution in Rat Brain following Repeated Electroconvulsive Seizures OLIVIER VERNET, KLARA ROSTWOROWSKI, Montreal
neurological
Institute,
McGill
University,
Protein kinase C (PKC) activity was measured in samples of neocortex, cerebellum, and hippoeampus from adult rats receiving a series of 10 electroeonvulsive seizures (ECS). Rats were sacrificed immediately and at various intervals from 16 min to 24 h after the last seizure. From 77 to 84% of total PKC activity was found in the cytosol versus the membrane fraction. PKC activity in cerebellum was significantly higher than in neocortex (IS%, P < 0.05). Repeated ECS treatment-did not affect total PKC activity nor its distribution between membrane and cytosolic fractions when compared with sham ECS controls. This finding is in keeping with reports that adrenergic-stimulated phosphoinositol turnover is not altered 24 h following repeated ECS. o issz Academic
Press,
Inc.
INTRODUCTION Among the many kinases, the calcium/phospholipiddependent kinase or protein kinase C (PKC) plays a fundamental role in the transmembrane signaling of many extracellular agonists (26). This enzyme is especially abundant in brain (18) and has been implicated in neurotransmitter release, ion channel regulation, and neuronal plasticity (26). In the presence of phospholipid and physiological concentrations of calcium, the second messenger diacylglycerol (DAG), produced by receptormediated phosphoinositide (PI) hydrolysis, activates PKC at the plasma membrane level (15). Tumor promoting phorbol esters can substitute for DAG and directly activate PKC (5) causing the translocation of enzyme from cytosol to cellular membrane (9, 17). Activating of PKC by platelet-activating factor, however, does not require the association of cytosolic PKC with membrane (30), while in GH, cells thyrotropin-releasing hormone stimulation merely elicits a brief translocation (22). In hepatocytes activation of PKC in response to Ca” mobilizing hormones involves preexistent membrane-bound enzyme only without affecting the soluble enzyme (7). Gleiter et al. (11) employed [‘Hlphorbol-12,13butyrate ([3H]PDBu) binding as a marker of mem-
AND ALLAN L. SHERWIN
Montreal,
Q&bee,
Cmuda
H3A
2B4
brane-bound PKC in neural tissues from rats subjected to 10 once-daily electroconvulsive seizures (ECS). They observed a 30% diminution in the density (B,,) of 13H]PDBu binding sites in neocortex and cerebellum but not hippocampus 24 h after the last treatment. No change was noted following a single seizure. They suggested that repeated ECS downregulates PI breakdown leading to a reduction in DAG-activated membranebound PKC. Phorbol ester binding, however, does not permit measurements of the subcellular distribution of PKC since it determines only the amount of membranebound PKC protein. Moreover, phorbol ester binding sites may not strictly correlate with enzyme activity (12, 28). ECS alters the biochemical parameters of a variety of neurotransmitter systems (3, 32). Repeated daily ECS significantly increases the density of a-l adrenoceptors (37, 41) which are coupled to PI metabolism (13). This increase which is comprised of the (Y--1a subtype is confined to a band of binding in the outermost lamina of the neocortex (4). Michel et al. (23) reported that a-1B sites mediate PI hydrolysis in rat neocortex. In an early report Neuman et al. (25) observed that single or repeated ECS treatment increased PI hydrolysis in rat neocortex but not in caudate nucleus or hippocampus. Blendy et ~1. (3) and Dubeau et al. (8), however, were not able to demonstrate a change in either basal or norepinephrine-induced PI hydrolysis in rat cortex sampled 24 h after repeated ECS. Pile et al. (31) noted no change in a-l receptor-coupled PI hydrolysis but observed a marked reduction in the CAMP response which involves an interaction between ar and @receptors. Vadnal and Bazan (38) used an in uiuo model to measure the acute effects of ECS on radio-labeled polyphosphoinositides and inositol phosphates. ECS increased the level of inositol trisphosphate (IP,) but diminished the endogenous mass of phosphatidyl4,5-biphosphate (PIP,). They concluded that ECS stimulates the inosito1 lipid cycle in brain possibly due to neurotransmitter release. Pretreatment of animals with lithium did not affect inositol monophosphate levels but did appear to attenuate the ECS-induced changes in PIP, and IP, suggesting that lithium has other sites of action in addi-
297 All
Copyright 0 1992 rights of reproduction
0014.4886192 $3.00 by Academic Press, Inc. in any form reserved.
298
VERNET,
ROSTWOROWSKI,
tion to an inhibition of inositol-1-phosphatase. In contrast, carbamazepine-ECS-treated rats showed enhanced labeling of PIP,, decreased IP, levels, andinhibition of IP, accumulation (39). Both these drugs may elicit differential effects on cell signal transduction perhaps at the G protein phospholipase C level (39). The aim of the present study was to determine if daily repeated ECS treatment reduces membrane-bound PKC activity. The data show that membrane and cytosolic PKC activities remain remarkably consistent when examined at various intervals following the last seizure. MATERIALS
AND
METHODS
Animals. Male Sprague-Dawley rats (Charles River, St. Constant, Quebec) weighing between 250 and 300 g were used in all experiments. Rats were housed in constant temperature animal quarters with a light/dark cycle and allowed access to food and water ad libitum. Electroconuulsiue seizures. Maximal ECS were induced by delivering current (100 mA, 60 Hz, 200 ms) through saline-moistened soft earclip electrodes (37). Each stimulus induced a generalized tonic-clonic seizure lasting 20 to 30 s. Test animals received a series of 10 daily treatments. Matched control rats were handled in the same manner daily but no current was delivered (sham ECS). Animals were sacrificed by decapitation at fixed intervals just at the end of the seizure (30 s after the shock) or 15 min, 60 min, 240 min, and 24 h after the last ECS. Sham ECS animals were sacrificed at 30 s, 240 min, or 24 h. Brains were rapidly removed and dissected free from blood vessels on an ice-chilled ceramic tile. Neocortex, cerebellum, and hippocampus were frozen in liquid nitrogen and stored at -70°C until assayed. PKC assay. Tissues were homogenized by two 5-s strokes of a Polytron homogenizer (Brinkman) in 10 volumes of buffer composed of 50 mM Tris-HCl, pH 7.4, 2 n&f dithiothreitol (DTT), 2 mM EGTA, 1 mA4 phenylmethyl sulfonyl fluoride (PMSF, Sigma), 0.1 mg/ ml leupeptin (Sigma) and centrifuged at 100,OOOg for 1 h at 4°C. The supernatant (cytosolic fraction) was collected. The 100,OOOg pellet (membrane fraction) was reconstituted to the original volume with the homogenizing buffer containing 0.25% Triton X-100 (Sigma). Protein concentrations were determined using the method of Lowry et al. (21). Phosphorylation of lysine-rich histone (Sigma) was assayed in triplicate (24) after having determined the correct Michaelis-Menten conditions for substrate and cofactors. The final assay conditions were: 100 pM free calcium, 30 mM Tris-HCl, pH 7.4, 1.19 mM DTT, 500 pg/ml L-cY-phosphatidyl+serine (PS, Sigma), 25 pg/ml 1,2-dioctanoyl-SN-glycerol (DAG, Sigma), 12 pM [32P]ATP (0.5-1.0 X lo6 cpm, NEN, Montreal, Quebec) 10 mA4 MgCl,, 250 pg/ml ly-
AND
SHERWIN
sine-rich histone and cytosolic or particulate fraction (2-7 pg protein) in a final volume of 40 ~1. The reaction components minus ATP and MgCl, were preincubated at 30°C and phosphorylation was initiated by adding ATP and MgCl,. After 1 min, a 25-~1 aliquot was spotted on a phosphocellulose paper strip (Whatman P81) (44) which was transferred to a beaker containing 75 mh4phosphoric acid. The strips were rinsed three times with 75 mM phosphoric acid, once with methanol, and air-dried. The incorporation of [32P] into histone was measured by liquid scintillation spectrometry. Blanks (complete reaction mixture minus enzyme fraction) were used to correct for nonspecific binding. PKC activity was calculated as the difference in activity in the presence or absence of DAG, PS, and calcium. Results were analyzed using a one-way analysis of variance with Duncan’s multiple comparisons. Differences were considered significant if P < 0.05 was achieved. RESULTS We found that between 77 to 84% of the total PKC activity was present in the cytosolic fraction. This percentage did not change significantly at any time during the postictal period varying from 79 to 81% in the neocortex, from 81 to 84% in the hippocampus, and from 77 to 80% in the cerebellum. As shown in Table 1 a comparison of the three brain regions assayed in the sham ECS control groups showed that total PKC activity was significantly higher in cerebellum than in neocortex (15%,
P < 0.05). Protein kinase C activity was not significantly altered in either the membrane or cytosolic fraction of neocortex, cerebellum, or hippocampus at any time following the last repeated ECS when compared with the sham ECS controls (Table 1). In particular there was no evidence of a reduction in the enzymic activity of PKC in membranes prepared from neocortex or hippocampus 24 h following the last repeated ECS. There was also no evidence of transduction of PKC activity from cytosol to membranes at 30 s, 15 min, 60 min, 240 min, or 24 h postictally. The relative distribution of PKC enzymic activity in the membrane versus the cytosolic fraction was remarkably consistent during the 24-h postictal period. DISCUSSION Our study shows that repeated ECS does not alter membrane or cytosolic PKC activity in neocortex, hippocampus, or cerebellum within 24 h of the last seizure. Repeated ECS has been found to decrease the number of [3H]PDBu binding sites in rat neocortex and cerebellum when sampled 24 h postictally (11). This decrease in receptor density (B,) was not associated with a change in the apparent dissociation constant (Kd)
PROTEIN
KINASE
C AND
ELECTROCONVULSIVE
TABLE Effect
Time
interval
Neocortex 30 s 15 min 60 min 240 min 24 h Sham ECS Hippocampus 30 s 15 min 60 min 240 min 24 h Sham ECS Cerebellum 30s 15 min 60 min 240 min 24 h Sham ECS
Note. Values * Significantly
are mean different
1
of Repeated ECS on PKC Activity PKC
activity
299
SEIZURES
and Intracellular
(nmol/min/mg
Distribution
protein) Cytosol
Total PKC (nmol/min/mg
activity protein)
n
Membrane
5 6 5 5 6 14
1.52 1.41 1.70 1.65 1.63 1.50
+: + + t t +
0.12 0.09 0.08 0.15 0.22 0.09
5.87 6.02 6.63 6.96 6.07 5.98
t + + f f +
0.65 0.28 0.25 0.33 0.57 0.26
7.39 7.43 8.33 8.61 7.70 7.48
+ 0.55 * 0.31 ?z 0.26 -t 0.43 + 0.71 -t 0.33
5 6 5 5 6 14
1.47 1.40 1.59 1.42 1.43 1.50
+ +-+ i t r
0.05 0.05 0.17 0.09 0.10 0.09
6.42 7.16 6.27 6.05 6.24 6.77
f 0.16 + 0.40 -c 0.33 -1- 0.30 2 0.25 + 0.18
7.89 8.56 7.86 7.47 7.67 8.27
f 2 t 2 f i
0.21 0.40 0.44 0.33 0.21 0.23
5 5 5 5 6 13
1.70 1.77 1.87 1.63 1.82 1.73
* + t tr -t
0.11 0.12 0.18 0.07 0.19 0.12
6.80 6.06 6.34 6.51 6.23 6.88
t f f ? A k
8.50 7.83 8.21 8.14 8.05 8.61
+ + + + + +
0.21 0.37 0.56 0.24 0.56 0.45*
t SEM. PKC activity is expressed in nmol s2P transferred from the neocortex sham ECS group (P < 0.05, one-way
which is a measure of the affinity of the receptors for the test ligand. The density (B,,,) of phorbol ester binding sites reflects the amount of PKC protein bound to membranes and this does not strictly correlate with PKC enzymic activity (12, 28). Repeated ECS may merely downregulate membrane recognition sites for phorbol esters without affecting protein kinase C histone phosphorylase activity (42). Moreover, certain analytical techniques may preferentially detect one or more of the isoforms of PKC or the access of [3H]PDBu to PKC protein in various membrane preparations might be a limiting factor (12, 28). Diacylglycerol activates PKC primarily through a reversible PKC/membrane complex while phorbol esters induce a sustained activation of PKC by forming an irreversible PKC/membrane complex (9). In rat neocortex, (~-~a subtype adrenoceptors which are coupled to membrane PI metabolism are localized by autoradiography to lamina 1 and lamina Va and Vc sparing layer Vb pyramidal cells (4). Noradrenergic fibers originating in the locus coeruleus (LC) are widely distributed in cerebral cortex including the outermost lamina and many cortical neurons have adrenoceptors. Lamina 1 a-Is adrenoceptors are preferentially increased 24 h following repeated ECS, but this rise is not accompanied by a change in PI turnover or PKC activity. Perhaps these seizure-induced binding sites are comprised of precursor or degraded forms of the a(-ia adrenoceptor in which the ligand recognition sites have
0.20 0.27 0.39 0.18 0.49 0.38'
into histone/min/mg ANOVA with Duncan’s
protein. multiple
comparisons).
been preserved but no longer functionally connected to the PI signal transduction mechanism (4,8,31). We found average levels of PKC activity to be of the same order of magnitude as reported for rat striatal neuronal cultures by a similar assay (43), but higher than those reported for whole rat brain by other techniques of measurement (14, 18, 20). We found si~ificantly more total PKC activity in cerebellum compared to neocortex and an intermediate value in hippocampus. Only a few studies have simultaneously quantitated PKC activity in different brain regions. An earlier report showed almost identical levels of PKC between rat neocortex and cerebellum (18). Comparisons with previous studies are hindered, however, by differences in methods of tissue preparation or techniques such as autoradiography (45) and immunocytochemistry (26). From 77 to 84% of the total PKC activity was recovered from the cytosolic fraction. These percentages are in accordance with some previous studies (36, 43) but differ notably from other observations where about one-third of the activity was found to be present in the cytosolic fraction (6, 10, 14, 24). We recently reported that from 67 to 70% of total PKC activity was present in cytosolic fractions prepared from specimens of histologically normal human brain excised to permit the removal of deep-seated tumors (40). Protein kinase C activity and intracellular distribution was not altered in focally epileptic neocortex.
300
VERNET,
ROSTWOROWSK~,
Absolute comparisons of PKC activity are difficult, especially because extraction conditions which aim to prevent irreversible proteolytic activation of PKC by the Ca’+-dependent protease calpain (16) influence its subcellular distribution (1, 14, 26). We prevented PKC activation by means of high concentrations of a Ca2+ chelator and by adding the protease inhibitors PMSF and leupeptin during the homogenization step. Moreover, the detergent Triton X-100 was employed in order to unmask all PKC activity in the cortical particulate fractions (14). Comparisons between studies are also complicated by the findings that PKC is known to be a family of subspecies which exhibit subtle individual enzymatic properties and different activation characteristics (27). Slightly different techniques can thuspreferentially detect one or the other of these isozymes. Moreover, the relative distribution of protein kinase C subspecies varies in different regions of the CNS (36). The data presented in this study show that repeated ECS treatment does not cause a prolonged change in PKC activity or subcellular distribution. These results do not, however, rule out the possibility that PKC could be activated and translocated from cytosol to membrane for only a very brief period (22). As recently demonstrated in monoblastoid U937 cells PKC activity may be downregulated for exogenous histone substrates but preserved when tested with certain peptide substrates (42). Both a single ECS seizure or bicuculline-induced status epilepticus (2,34,46) produce an accumulation of DAG in brain, which by 60 s has already returned to basal levels (33). Chronic ECS treatment might thus induce a recurrent short-lived activation of PKC which would not alter the activity or subcellular distribution of the enzyme. More protracted membrane changes including a redudtion in phorbol ester binding sites may result from PKC-mediated phosphorylation reactions (19, 29, 35).
4.
5.
6. 7.
8.
9.
AND SHERWIN troconvulsive shock and reserpine increase Lu,-adrenoceptor binding sites but not norepinephrine stimulated phosphoinositide hydrolysis in rat brain. Eur. J. Pharmacol. 156: 267-270. BLENDY, J. A., L. J. GRIMM, D. C. PERRY, L. WEST-J• HNSRUD, AND K. J. KELLER. 1990. Electroconvulsive shock differentially increases binding to (Y,adrenergic receptor subtypes in discrete regions of rat brain. J. Neurosci. 10: 2580-2586. CASTAGNA, M., Y. TAKAI, K. KAIBUCHI, K. SANO, U. KIKKAWA, ANDY. NISHIZUKA. 1982. Direct activation of calcium-activated, phosphoiipid-dependent protein kinase by tumor-promoting phorbol esters. J. Bid. G’hem. 257: 7847-7851. CUMFUNE, R. C., AND J. C. LA MANNA. 1990. Protein kinase C activity in rat brain cortex. J. Neu~~m. 55: 826-831. DIAZ-GUERRA, M. J. M., AND L. BOXA. 1990. Lack of translocation of protein kinase C from the cytosol to the membranes in vasopressin-stimulated hepatocytes. Biochem. J. 269: 163-168. DUBEAU, F., AND A. L. SHERWIN. 1989. Effect of repeated versus single electroconvulsive seizures on adrenergic-mediated phosphatidylinositol hydrolysis in rat neocortex. Exp. Neurol. 105: 206-210. EDERVEEN, A. G. H., S. J. VAN EMST-DE VFUES, J. H. H. M. DE PONT, AND P. H. G. M. WILLEMS. 1991. Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in rabbit pancreatic acini. Eur. J. Biochem. 195: 679683.
10. GIRARD, P. R., G. J. MAZZEI, AND J. F. KUO. 1986. immunolo~cal quanti~tion of phospholipid/~a’+-dependent protein kinase and its fragments: Tissue levels, subcellular distribution, and ontogenic changes in brain and heart. J. Bid. Chem. 261: 370375.
11. GLEITER, C. H., J. DECKERT, D. J. NUTT, AND P. J. MARANGOS. 1988. The effect of acute and chronic electroconvulsive shock on [3Hlphorbol-dibutyrate binding to rat brain membranes. Neurothem. Res. 13: 1023-1026. 12. HOUSEY, G. M., M. D. JOHNSON, W. L. W. HSIAO, C. A. O’BRIAN, J. P. MURPHY, P. KIRSCHMEIER, AND I. B. WEINSTEIN. 1988. Overproduction of protein kinase C causes disordered growth control in rat fibroblasts. Cell 52: 343-354. 13. KENDALL, D. A., E. BROWN, AND S. R. NAHORSKI. 1985. culadrenoceptor-me~ated inositol phospholipid hydrolysis in rat cerebral cortex: Relationship between receptor occupancy and response and effects of denervation. Eur. J. Pharmacol. 114: 41-52.
ACKNOWLEDGMENTS This work was supported by the Medical Research Council of Canada. Dr. Vernet is a postdoctoral fellow of the Fonds Decker of Surgery, CHUV, Lausanne and Janggen-Poehn Stiftung, St. Gallen, Switzerland. We greatly appreciated the cooperation of Ms. J. Green and Mr. R. Jones. We thank Drs. J. Ante1 and V. W. Yong for helpful advice and Mrs. M. Nicholls-Spence for preparing the manuscript. REFERENCES 1. ALOYO, V. J., H. ZWIERS, P. N. E. DE GFZAAN,AND W. H. GISPEN. 1988. Phosphorylation of the neuronal protein kinase C substrate B-50: In vitro assay conditions alter sensitivity to ACTH. Neurochem. Res. 13: 343-348. 2, AVELDANO DE CALDIRONI, M. I., AND N. G. BAZAN. 1979. (Ymethyl-p-tyrosine inhibits the production of free arachidonic acid and diacylglycerols in brain after a single electroconvulsive shock. Neurochem. Res. 4: 213-221. 3. BLENDY, J. A., C. A. STOCKMEIER, AND K. J. KERR. 1988. Elec-
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KINASE
C AND ELECTROCONVULSIVE
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