Activation of metabotropic glutamate receptors increases endogenous protein kinase C substrate phosphorylation in adult hippocampal slices

Brain Research 745 Ž1997. 46–54

Research report

Activation of metabotropic glutamate receptors increases endogenous protein kinase C substrate phosphorylation in adult hippocampal slices Frank Angenstein ) , Monika Hirschfelder, Sabine Staak Federal Institute for Neurobiology, Laboratory for Cellular Signalling, P.O. Box 1860, 39008 Magdeburg, Germany Accepted 10 September 1996

Abstract We previously reported ŽStaak, S., Behnisch, T. and Angenstein, F., Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C ŽPKC. isoenzymes a and b, Brain Res., 682 Ž1995. 55–62. that Ca2q-dependent PKC isoenzymes arb and g are not translocated between subcellular compartments after stimulation of glutamate receptor subtypes in hippocampal slices. Extending our previous work in this study in situ phosphorylation of endogenous PKC substrates and the translocation of novel PKC isoenzymes d and e was analysed to detect PKC activation. Two proteins of approximately 94 kDa and 18 kDa were first characterised to be specific PKC substrates. As control of the technique carbachol was shown to increase in situ phosphorylation of the two substrates without any measurable translocation of PKC protein. Activation of metabotropic glutamate receptors by 50 mM DHPG also increased the in situ-phosphorylation by 43.9% Ž94 kDa. and 32.8% Ž18 kDa. compared to controls but did not induce a measurable subcellular redistribution of conventional and novel PKC isoenzymes. Stimulation by 50 mM trans-ACPD or 0.1 mM quisqualate enhanced the in situ phosphorylation in the same range, whereas 0.1 mM NMDA was ineffective. To our knowledge this is the first report showing a direct link between metabotropic glutamate receptor activation and increased endogenous PKC substrate phosphorylation in adult hippocampal slices. This PKC activation was not detectable by a redistribution of enzyme protein between subcellular compartments. We, therefore, conclude, that the failure to detect PKC translocation in physiological experiments is not an indicator for unchanged enzyme activity. Keywords: In situ phosphorylation; Protein kinase C translocation; MARCKS; Neurogranin; Two-dimensional gel electrophoresis; Phorbol ester; Hippocampal slice

1. Introduction The activation of protein kinase C ŽPKC. is long known to be involved in many different cellular processes including cell proliferation and differentiation, gene expression, exocytosis, regulation of receptors and ion channels. Even more complex phenomena, i.e., hippocampal long-term potentiation ŽLTP., long-term depression ŽLTD. and learning and memory formation are thought to be dependent on PKC activation w18,20x. Signal transductiory events that are involved in the activation of PKC are increases in cytoplasmic calcium and the production of the second messenger diacylglycerol w17x. Furthermore, the activity of the enzyme may be regulated by Ca2q-induced membrane association and the interaction of the kinase with phospho-

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Corresponding author. Fax: Ž49. Ž391. 616160.

lipids, namely phosphatidylserine w16x. This PKC translocation between subcellular compartments is very often used and accepted as a measure of enzyme activation. Since the pioneering work of Bazzi and Nelsestuen PKC has been shown to exist in cells in a cytosolic, membrane-associated and membrane-inserted state w2,10x. Cytosolic PKC is thought to be inactive although there is some evidence that the soluble enzyme may retain catalytic activity w19,22,28x. The physiological significance of membrane-associated PKC is largely unknown because it is removed from membranes during preparation by EDTA and EGTA used in conventional PKC assays w11,14x. It was suggested that PKC associates with cell membranes and remains there in an inactive but primed state, sensitised for activation which probably occurs when the enzyme binds diacylglycerol generated in response to receptor-mediated hydrolysis of phospholipids w3,4,7x. Membrane-inserted PKC is no longer regulated by Ca2q and the

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 1 2 9 - 8

F. Angenstein et al.r Brain Research 745 (1997) 46–54

second messenger and, therefore, might represent a persistently activated kinase w2x. Electrophysiological studies revealed that the activation of phospholipase C-coupled receptors is necessary for mechanisms enabling the late phase of LTP w21x. Exploring biochemically the cellular mechanisms of LTP in hippocampal slices we recently reported that redistribution of PKC isoenzymes a and b was not detectable after treatment of slices with glutamate receptor agonists or induction of LTP in the Schaffer collateral-CA1 pathway w27x. One of the possible reasons of the failure to detect PKC translocation in adult hippocampal slices by translocation assays could be the inhomogenity of the tissue. PKC activating phorbol esters should induce translocation of PKC in all cells of the hippocampus whereas activation of metabotropic receptor subtypes reflects the physiology of a slice in a more specific way. We previously reported w27x that Ca2q-dependent PKC isoenzymes are not translocated after stimulation of glutamate receptor subtypes using trans-ACPD, NMDA or a mixture of both as agonists. In this study we used an in situ-phosphorylation assay to detect activation of protein kinase C under identical conditions. The degree of phosphorylation of endogenous PKC substrates was estimated after in situ-labelling of hippocampal slices followed by a quantifiable extraction of these proteins by 2.5% perchloric acid w1x, gelelectrophoretic separation and phosphoimaging. The subcellular distribution of the PKC isoenzymes was analysed by quantitative immunoblotting. We report here, that in adult rat hippocampal slices the activation of metabotropic glutamate receptors and muscarinic cholinergic receptors induces an activation of protein kinase C detectable by in situ-phosphorylation of endogenous specific substrate proteins without any measurable redistribution of enzyme protein between subcellular compartments.

2. Materials and Methods 2.1. Animals Eight-week-old male Wistar rats weighing 220–240 g from our own breeding stock were used for the experiments. 2.2. Materials All chemicals bought from SIGMA ŽDeisenhofen, Germany. or SERVA ŽHeidelberg, Germany. were of analytical grade. The muscarinic agonist carbachol, the glutamatergic agonists N-methyl-D-aspartate ŽNMDA., transŽ 1 S,3 R . -1-amino-1,3-cyclopentanedicarboxylic acid Ž trans-ACPD. and quisqualate as well as 12-O-tetradecanoylphorbol-13-acetate ŽTPA or PMA., staurosporine,

47

chelerythrine, calmidazolium and Rp-adenosine-3X ,5X-cyclic monophosphothioate triethylamine ŽRp-cAMP. were obtained from Research Biochemicals International ŽU.S.A.. and ŽRS.-3,5-dihydroxyphenylglycine ŽDHPG. from Tocris Cookson ŽU.K... The antibodies used were a monoclonal mouse anti-PKC Žclone MC 5. specific for a and b subspecies of the enzyme obtained from Amersham ŽU.K.. or a polyclonal antiserum against PKC g ŽBoehringer, Mannheim., PKC d ŽSIGMA. and PKC e ŽSIGMA.. 32 P and 33 P Žeach 10 mCirml. were also from Amersham ŽU.K... 2.3. Methods 2.3.1. Preparation and incubation of hippocampal slices After decapitation of the animals the brain was rapidly removed, the hippocampus dissected out on ice and cut into 400 mm thick transverse hippocampal slices ŽMcIlwain tissue shopper.. Tissue slices used for the examination of PKC distribution were maintained in a medium containing Žin mM.: NaCl 134.0, KCl 5.0, KH 2 PO4 1.24, MgSO4 1.3, CaCl 2 2.0, NaHCO 3 16.0, glucose 10.0 ŽpH 7.4, 378C. unless otherwise stated. The medium was aerated with carbogen Ž95% O 2 , 5% CO 2 . throughout the experiment. In translocation experiments after a preincubation time of 60 min test substances were added to the experimental slices for the times indicated. Control slices received the appropriate solution. Hippocampal slices used for in situ-phosphorylation were incubated in the same medium as described above except that KH 2 PO4 was substituted by KCl. After 60 min of preincubation 100 mCi 32 P or 33 P Žfinal concentration in the medium 50 mCirml. was added and the slices were prelabelled for 60 min. After this prelabelling time experimental slices were treated with the test substances whereas control slices got appropriate solutions. 2.3.2. Measurement of protein kinase C translocation For each determination of PKC translocation 3 slices were washed shortly in ice-cold buffer containing 25 mM Tris-HCl pH 7.6, 250 mM sucrose, 5 mM dithiothreitol and 1 mM EGTA-Ca2q buffer Žcorresponding to 1 mM free Ca2q . and homogenised in 100 ml of the same buffer including 0.3 mM 4-Ž2-aminoethyl.benzenesulfonyl fluoride ŽAEBSF. and 20 mgrml leupeptin Žbuffer A.. The homogenate was centrifuged at 270,000 g for 15 min to yield cytosol ŽC. and pellet fraction. The pellet was resuspended in buffer A containing 10 mM EGTA and 2 mM EDTA Žbuffer B. and again centrifuged at 270 000 g for 15 min to get a supernatant of EGTA-extractable membrane-associated proteins ŽAP.. The resulting pellet was resuspended in buffer B containing 1% Triton X100 and incubated for 45 min at 48C followed by centrifugation at 270,000 g for 15 min to yield detergent-extractable membrane-bound proteins ŽMP.. The protein concentration in each fraction was determined by the method of Bradford w6x using BSA as a standard.

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F. Angenstein et al.r Brain Research 745 (1997) 46–54

The proteins of these fractions were dissolved in a sample buffer Ž50 mM dithiothreitol, 3 mM EDTA, 4% SDS, 20% glycerol, 250 mM Tris-HCl, 0.01% bromophenol blue, pH 8.0. and loaded onto the gel Ž5–20%.. Samples from control and experimental slices were applied to one gel. After separation the proteins were transferred onto nitrocellulose Ž0.45 mm pore size. in a transfer buffer Ž25 mM Tris, 192 mM glycine, 0.02% SDS, 20% methanol. for 90 min at a constant current Ž200 mA. as described by Tobwin et al. w30x using the tank blotting system ŽMighty small II, Hoefer scientific instruments.. Additional protein binding sites on the nitrocellulose were saturated by incubation in 10 mM Tris-buffered saline ŽTBS; pH 7.4. containing 5% dry milk powder for 2 h at 378C. After a short wash with TBS an antibody specific for the arb PKC Žclone MC 5, Amersham at a dilution of 1:1000., the polyclonal antiserum against PKC g Ž1:1000., PKC d Ž1:2000. or PKC e Ž1:2000. was applied overnight at 48C. The nitrocellulose was washed with TBS and TBS containing 0.1% Tween 20 and the immunoreactivity was revealed using a peroxidase conjugated anti-mouse IgG ŽBoehringer, 1:5000. or anti rabbit antibody ŽSIGMA, 1:10000. and the ECL system ŽAmersham.. PKC immunoreactivity was quantified by scanning densitometry ŽBioImager, Millipore. and linear with respect to the protein concentration between 10–35 mgrgel lane. An equal amount of proteins Žabout 15 mg. from control and experimental slices was used on the same immunoblot. The amount of PKC in each fraction was calculated by measuring the peak area of the immunostained 80 kDa protein and expressed as per cent of the whole PKC ŽC q AP q MP s 100%.. 2.3.3. Determination of endogenous substrate phosphorylation Three control or experimental slices were removed and washed quickly in an ice-cold solution containing in mM: HEPES ŽpH 7.4. 20, NaF 100, Na 4 P2 O 7 10, EDTA 4 and 2.5% perchloric acid ŽPCA.. They were then homogenised in 200 ml of the same buffer including 1 mM AEBSF, 20 mgrml leupeptin and 250 mgrml p-nitrophenylphosphate. Then a 200 ml aliquot of the homogenate was centrifuged for 15 min at 14,000 g to get a supernatant of PCA-soluble proteins. The PCA-soluble proteins were then precipitated by 80 ml 30% trichloroacetic acid for 45 min at 48C and centrifuged again. The resulting pellet was washed with 350 ml ethanol and dissolved in 50 ml sample buffer Ž250 mM Tris-HCl, 50 mM dithiothreitol, 3 mM EDTA, 4% SDS, 20% glycerol and 0.01% bromophenol blue, pH 8.0.. Each 25 ml were used for electrophoretic separation Ž5–20% SDS gel.. The gel was dried and used for phosphoimaging on a Bio-Imaging Analyzer BAS 1000 ŽFUJIX.. For two-dimensional separation of the proteins 14 slices were used. The TCA precipitated 2.5% PCA soluble pro-

teins were rehomogenised in 40 ml TrisrHCl solution Ž10 mM, pH 7.4.. SDS, DTT and bromphenol blue were added to the homogenate to reach final concentrations of 1.7%, 0.4% and 0.002%, respectively. Isoelectric focusing has been carried out in tube gels 1.5 mm i.d., 64 mm in length prepared as outlined by Hochstrasser w12x. Samples adjusted to equal amounts of protein Žabout 2 mg. were loaded on the basic side of the tube gels and focused with a constant voltage of 100 V, 200 V, 400 V, 600 V each for 1 h 15 min and finally with 800 V for 10 min and 1000 V for 5 min. Tube gels were transferred to a stacking gel containing 0.125 mM TrisrHCl, pH 6.8, 5% acrylamide, 0.14% bisacrylamide, 0.1% SDS, 2 mM EDTA, 0.02% Žvrv. glycerol with an extra slot for a molecular weight standard using a transfer buffer as in w12x. The separation gel Ž70 mm = 100 mm = 1.5 mm. contained 0.375 mM TrisrHCl pH 8.8, 13% acrylamide, 0.3% bisacrylamide, 0.1% SDS and bromphenol blue. 2.3.4. Statistical analysis Data are expressed as mean " S.E.M. and the statistical significance of differences between control and experimental slices was evaluated using Student’s t-test. Difference in P-value of less than 0.05 were considered to be significant.

3. Results It was the aim of the present study to visualise activation of PKC in adult hippocampal slices either by measuring translocation of PKC isoenzymes between subcellular compartments or in situ-phosphorylation of specific substrates after stimulation of metabotropic receptors. In situ phosphorylation of hippocampal slices in the presence of 50 mCirml 33 Pi for 60 min led to phosphorylation of a large number of proteins in the cytosolic and membranebound fractions. Since several PKC substrate proteins are known to share the unusual property to be soluble in 2.5% perchloric acid ŽPCA. w1x we examined whether proteins extracted and enriched in this way from hippocampal slices after in situ-phosphorylation could be utilised as an index of intracellular PKC activation. In this extracts a basal phosphorylation of at least six different proteins Žapproximately 94, 85, 70, 46, 20 and 18 kDa. was detectable by phosphoimaging. The degree of protein phosphorylation was linear with respect to protein concentration for the 94 kDa and 18 KDa phosphoproteins whereas the phosphorylation profile of the other proteins was somewhat inconsistent under the conditions used ŽFig. 1.. The phosphorylation of both proteins depends to a different degree on the presence of extracellular calcium. Calcium chelation by 1 mM EGTA in the incubation medium reduced the phosphorylation of the 94 kDa protein

F. Angenstein et al.r Brain Research 745 (1997) 46–54

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Fig. 1. Autoradiography of 2.5% PCA soluble proteins isolated and enriched from different numbers of hippocampal slices. The degree of the 94 kDa and 18 kDa protein phosphorylation was linear with respect to protein concentration.

only by 22.4% " 3.7% whereas the phosphorylation of the 18 kDa protein was completely abolished ŽTable 1.. The PKC inhibitors staurosporine Ž0.1 mM. or chelerythrine Ž10 mM. when present in the incubation medium during the labelling period prevented the basal phosphory-

lation mainly of the 94 and 18 kDa proteins whereas inhibitors of the Ca2qrcalmodulin-dependent protein kinase, calmidazolium Ž10 mM., or of protein kinase A, Rp-cAMP Ž0.1 mM. were nearly ineffective ŽTable 1.. To further characterise PCA-soluble proteins different

Fig. 2. A: effect of 1 mM TPA on phosphorylation degree of 2.5% PCA-soluble proteins. The inhibitors staurosporine Ž0.1 mM. or chelerythrine Ž10 mM. were added to the incubation medium after the prelabelling period and 5 min before application of the phorbol ester. The strongly increased phosphorylation of the 94 and 18 kDa proteins was abolished by staurosporine and partially inhibited by chelerythrine. B: effect of 1 mM TPA on subcellular distribution of PKC immunoreactivity in cytosolic ŽC., membrane-associated ŽAP. and membrane-inserted protein fraction ŽMP.. PKC levels were quantified by scanning densitometry, see Table 3.

F. Angenstein et al.r Brain Research 745 (1997) 46–54

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Table 1 Effect of different kinase inhibitors on the basal phosphorylation of the 94 kDa and 18 kDa protein Final concentration Control EGTA Staurosporine Chelerythrine Calmidazolium Rp-cAMP

1 mM 0.1 mM 10 mM 10 mM 0.1 mM

94 kDa 100.0 77.6"3.7 19.5"3.3 55.1"9.3 81.6"1.9 86.5"5.4

18 kDa

a a a

100.0 n.d. 35.4"4.8 51.2"9.7 99.4"5.3 92.2"5.4

n

a a

6 6 6 3 6

Hippocampal slices were preincubated for 60 min in a phosphate free medium. Immediately before the addition of 32 Pi to the incubation medium Žfinal concentration 50 mCirml. the kinase inhibitors were applied at the final concentrations indicated. The labelling time was 60 min. Values are given as per cent of control, n.d. not detectable, a P - 0.05..

kinase activators and inhibitors were used. In situ phosphorylation of the PCA-soluble proteins in the presence of PKC activating phorbol ester TPA Ž1 mM. for 15 min induced a dramatic increase in phosphorylation state of the 94 and 18 kDa proteins ŽFig. 2A.. This effect of TPA on the phosphorylation state was accompanied by a translocation of PKC isoenzymes arbrgrre into the membraneinserted form, PKCd immunoreactivity in the cytosol is lost completely ŽFig. 2B.. The TPA-induced enhanced phosphorylation was prevented by simultaneous application of 0.1 mM staurosporine and was partially inhibited by addition of 10 mM chelerythrine ŽFig. 2A.. Two D-gel electrophoretic separation of the phosphoproteins revealed the TPA-induced increased phosphorylation of an acidic protein Žp I s 4.5. in the 94 kDa range and of a basic protein Ž8.0 - p I - 8.5. in the 18 kDa range ŽFig. 3.. In contrast, activation of adenylate cyclase by 10 mM forskolin or the addition of the calcium ionophor A23187 at a concentration of 10 mM which has been shown to increase the intracellular calcium concentration did not affect the phosphorylation of the two proteins. The effects of different kinase activators and inhibitors on the phosphorylation state of the 94 kDa and 18 kDa proteins were quantified and summarised in Table 2. We concluded, that these proteins are good candidates to be specific PKC substrates and useful indicators of PKC activation after in situ-labelling of hippocampal slices. In a third series of experiments we finally addressed the question about the relationship of PKC redistribution between subcellular compartments and the phosphorylation states of the two identified substrates after stimulation of metabotropic receptors by comparing translocation and in situ-phosphorylation data under identical conditions. Muscarinic acetylcholine and metabotropic glutamate receptors are known to be coupled to phospholipase C and, subsequently, phosphoinositide hydrolysis and formation of diacylglycerol w5,23x. However, an increased in situ-phosphorylation of PKC substrates after stimulation of these

Table 2 In situ phosphorylation of the 94 kDa and 18 kDa proteins after stimulation of different second messengers pathways ŽTPA, Forskolin, A23187. or muscarinic acetylcholine Žcarbachol. and glutamate ŽDHPG, transACPD, quisqualate, NMDA. receptors Final 94 kDa concentration Control TPA TPArStaurosporine TPArChelerythrine Forskolin A23187 Carbachol CarbrStaurosporine CarbrChelerythrine DHPG DHPGrStaurosporine DHPGrChelerythrine trans-ACPD quisqualate NMDA

100 242.0"22.3 50.7"9.7 182.6"17.8 89.1"6.0 112.7"8.3 138.1"11.5 39.3"9.3 90.3"3.7 143.9"9.0 61.1"3.4 97.6"4.8 131.1"10.3 124.4"6.4 91.0"6.5

1 mM

10 mM 10 mM 0.1 mM

50 mM

50 mM 0.1 mM 0.1 mM

a

a

a

a

18 kDa

n

100 182.2"17.4 a 78.4"15 188.9"16.0 86.4"8.2 109.6"7.9 133.1"7.1 a 43.1"3.8 94.7"11.5 132.4"5.8 a 55.4"7.2 104.1"12.2 137.4"11.6 a 126.4"9.7 105.8"10.5

6 4 4 3 3 6 4 4 7 2 2 6 4 3

PKC activators alone or in combination with PKC inhibitors were added for 15 min except NMDA which was applied for only 5 min. Basal phosphorylation remained unaffected under these conditions Ž a P - 0.05..

receptors has never been shown in mature hippocampal slices. Carbachol Ž0.1 mM. applied for 2 min as well as 15 min into the incubation medium did not alter the subcellular distribution of PKC a r b r g r d r e in hippocampal slices ŽFig. 4, Table 3.. The phosphorylation of the 94 and 18 kDa proteins, however, was increased Ž138.1 " 11.5% and

Table 3 Percentage distribution of different PKC isoenzymes in cytosolic ŽC., membrane-associated ŽAP. and membrane-inserted fraction ŽMP. isolated from hippocampal slices after application of 0.1 mM carbachol, 50 mM DHPG or 1 mM TPA for 15 min

PKC a r b control carbachol DHPG TPA PKC g control carbachol DHPG TPA PKC d control carbachol DHPG TPA PKC e control carbachol DHPG TPA

C

AP

MP

Recovery

n

n.d. n.d. n.d. n.d. 7.5"3.6 8.8"3.8 9.4"3.3 4.9"2.9 46.1"1.0 46.8"3.9 45.9"2.7 n.d. 42.8"2.1 42.3"3.2 45.2"2.6 14.0"1.0

52.0"3.7 51.1"4.7 54.3"3.9 19.9"4.9 36.3"2.7 33.5"2.2 34.7"2.3 5.2"1.9 8.4"3.3 5.8"1.5 6.1"1.8 n.d. 15.7"2.1 13.4"2.5 13.3"1.8 1.8"0.8

48.0"3.7 48.9"4.7 45.7"3.9 80.1"4.9 56.2"2.1 57.7"1.8 55.9"2.0 89.9"2.0 45.5"2.8 47.4"2.8 48.0"2.4 100 41.5"1.3 44.3"3.6 41.5"2.6 84.2"1.7

100 97.8"6.2 98.2"7.4 94.2"11.2 100 102.1"5.8 105.3"7.2 76.4"5.8 100 101.1"6.1 94.3"6.9 49.7"4.3 100 93.3"12.2 92.8"9.5 64.8"8.7

5 4 3 4 3 3 3 3 3 3 3 3 3 3 3 3

In TPA treated slices significant amounts of PKCd re immunoreactivity are lost Žsee recovery., therefore it remains unclear whether these isoenzymes are translocated. Žn.d. not detectable..

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Fig. 3. Two-dimensional separation of 2.5% PCA-soluble phosphoproteins extracted from hippocampal slices. The slices were labelled in situ with 32 Pi for 60 min and then treated with 1 mM TPA or with 0.1 mM staurosporine in order to activate or to inhibit the PKC. The TPA-stimulatable 94 kDa phosphoprotein is an acidic protein ŽpI-4.5. whereas the 18 kDa protein is basic Ž8.0 - p I - 8.5.. The obvious TPA-induced increased phosphorylation of a 46 kDa protein Žp I s 5.0. cannot detected consistently in 1D-gels, perhaps because it comigrates with a group of basic phosphoproteins in the same molecular weight range which do not respond to TPA.

Fig. 4. A: in situ-phosphorylation of 2.5% perchloric acid soluble protein of hippocampal slices after muscarinic receptor stimulation. After 60 min labelling with 32 Pi the slices were stimulated with 0.1 mM carbachol for 15 min in presence or without the PKC inhibitors 0.1 mM staurosporine or 10 mM chelerythrine. The PKC inhibitors were added immediately before stimulation of muscarinic receptor. Carbachol induces an increased phosphorylation of the 94 and 18 kDa proteins although to a lower extent compared to phorbol ester treatment. This agonist-induced phosphorylation can be completely abolished by staurosporine and by chelerythrine. B: effect of 0.1 mM carbachol on subcellular distribution of PKC immunoreactivity in cytosolic ŽC., membrane-associated ŽAP. and membrane-inserted protein fraction ŽMP.. PKC levels were quantified, see Table 3.

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F. Angenstein et al.r Brain Research 745 (1997) 46–54

Fig. 5. A: in situ-phosphorylation of 2.5% perchloric acid soluble protein of hippocampal slices after metabotropic glutamate receptor stimulation. After the labelling time the slices were stimulated with 50 mM DHPG for 15 min in presence or without the PKC inhibitor 10 mM chelerythrine. B: effect of 50 mM DHPG on subcellular distribution of PKC immunoreactivity in cytosolic ŽC., membrane-associated ŽAP. and membrane-inserted protein fraction ŽMP. of hippocampal slices. PKC levels were quantified, see Table 3.

133.1 " 7.1%, respectively.. This enhanced 32 Pi incorporation was blocked by 0.1 mM staurosporine and by 10 mM chelerythrine ŽFig. 4, Table 2.. Activation of the metabotropic glutamate receptors by DHPG Ž50 mM. also induced an increased in situ-phosphorylation of both proteins which was abolished by PKC inhibitors ŽFig. 5A.. The subcellular distribution of the isoenzymes remained unchanged ŽFig. 5, Table 3.. The same holds true for 50 mM trans-ACPD and tendentiously 0.1 mM quisqualate. The stimulation of ionotropic glutamate receptors by a relatively high concentration of NMDA Ž0.1 mM. was ineffective ŽTable 2..

4. Discussion The objective of the present study was to better understand the relationship between the intracellular compartmentalisation of conventional and novel PKC isoforms arbrgrdre and the activation state of the enzyme in hippocampal slices after stimulation of metabotropic receptors. As the physiological phenomena of interest, i.e., LTP and LTD are calcium-dependent a monoclonal antibody

recognising the Ca2q-regulated arb isoforms and a polyclonal antiserum against PKC g were used for measuring PKC redistribution by quantitative immunoblotting. Furthermore diacylglycerol activated isoenzymes PKCdre could be involved in synaptic plasticity and were, therefore, included in this study. To compare the subcellular distribution of the enzyme with the phosphorylation state of endogenous substrates of this kinase we first had to characterise the PCA-soluble proteins as PKC substrates. It turns out that two of them, namely the 94 kDa and 18 kDa proteins seem to be specific PKC substrates for the following reasons: Ž1. The basal phosphorylation of both proteins is reduced by the PKC inhibitors staurosporine and chelerythrine although to a different degree. Possibly, the concentration of chelerythrine used in our experiments does not influence membrane-inserted PKC. Ž2. The phosphorylation of both proteins was clearly stimulated by the PKC activator TPA whereas the activation of protein kinase A or Ca2qrcalmodulin-dependent protein kinases was ineffective. On the basis of the following biochemical properties: Ža. molecular weight 94 kDa, isoelectric point 4.5 ŽFig. 3.;

F. Angenstein et al.r Brain Research 745 (1997) 46–54

Žb. solubility in 2.5% perchloric acid; and Žc. phosphorylation under low calcium conditions we assume that the 94 kDa protein could be the MARCKS protein Žmyristoylated alanine-rich C kinase substrate.. It has been shown that MARCKS is preferentially phosphorylated by calcium-dependent PKC ŽPKCa . as well as novel PKC isoforms ŽPKCdre . but not by atypical PKCz w9x. Therefore, MARCKS phosphorylation is a good indicator for the activation of the PKC isoenzymes examined. The second protein of interest seems also to be a specific PKC substrate: Ža. it has a molecular weight of about 18 kDa and an isoelectric point of 8–8.5; Žb. is soluble in 2.5% perchloric acid; and Žc. is phosphorylated in a calcium-dependent manner. We, therefore, assume that this protein might be neurogranin Žalso known as BICKs or RC3.. The expression of neurogranin in developing rat brain parallels that of PKC g and both proteins have been shown to be located postsynaptically w13x. Taken all data together, the in situ-phosphorylation of the 94 kDa protein Žpossibly MARCKS. and of the 18 kDa protein Žpossibly neurogranin. seems to be a reasonable indicator of PKC activation state in adult hippocampal slices as already shown before in other systems w15,31x. Actually, this technique is also useful to proteins isolated from adult hippocampal slices. The question about PKC subcellular distribution and in situ-phosphorylation of the two substrates was addressed after stimulation of muscarinic cholinergic or different glutamate receptor subtypes. As control for the capability of the technique the muscarinic acetylcholine receptor agonist carbachol Ž0.1 mM. which has been shown to activate phospholipase C at this concentration and subsequently to cause formation of diacylglycerol and inositoltrisphosphate w23x was used. Carbachol enhanced the in situ-PKC substrate phosphorylation without a measurable redistribution Žincreased membrane insertion. of the enzyme. Activation of metabotropic glutamate receptor subtypes by DHPG, trans-ACPD or quisqualate caused a comparable increase in the in situ-phosphorylation of PKC substrates in hippocampal slices. DHPG did not alter the subcellular distribution of the conventional or novel PKC isoenzymes under these conditions. As reported before a translocation of the Ca2q-dependent isoenzymes was also not demonstrable using trans-ACPD or quisqualate as agonists w26,27,32x. To our knowledge, this is the first report showing a direct link between metabotropic glutamate receptor activation and increased PKC substrate phosphorylation in adult hippocampal slices. In cultured hippocampal neurons or organotypic hippocampal slices glutamate or ibotenate have been shown to phosphorylate MARCKS, whereas trans-ACPD was ineffective w24–26,29x. However, under the experimental conditions described in this report transACPD or quisqualate did also not affect phospholipid hydrolysis. In a synaptosomal preparation of young rats trans-ACPD provoked an increased MARCKS phosphory-

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lation only when applied in combination with arachidonic acid w8x. In contrast to our in situ experiments in such a preparation the effect on presynaptic terminals is studied predominantly. Possible reasons for the discrepancies in the measurement of PKC activation by in situ substrate phosphorylation and enzyme translocation might be. Ž1. Calcium-dependent translocation of the conventional isoenzymes into a membrane-associated state is already induced by submerged incubation of slices w27x. This inactive but primed membrane-associated PKC could become activated upon receptor-mediated formation of diacylglycerol. Our observation that chelerythrine is able to inhibit completely the metabotropic glutamate and muscarinic acetylcholine receptor mediated enhancement of PKC substrate phosphorylation supports this hypothesis. This inhibitor seems not affect membrane-inserted PKC as shown by our TPArchelerythrine blockade experiments. However, as reported by Chakravarthy et al. in hippocampal tissue a membrane-bound and still inactive form of PKC seems not to be detectable w7x. Ž2. Alternatively, the formation of constitutively active PKM by proteolysis of the PKC a r b r g r d r e cannot be absolutely excluded, although, we have never observed the appearance of a 50 kDa protein in the cytosolic fraction or a significant decrease in immunostaining of these PKC isoenzymes after activation of phospholipase C-coupled receptors. Additionally, we have observed some PKCarb immunostaining in a crude cytoskeletal fraction. The biochemical and physiological role of this subfraction is unknown. Ž3. The translocation of the kinase is often described as a very transient event Žwithin 2 min. whereas the measurement of an increased phosphorylation of its substrates 15 min after receptor stimulation reflects the summarily result of phosphorylation and dephosphorylation processes. Ž4. Predictably, in contrast to phorbol ester treatment the stimulation of metabotropic receptors activates PKC only in subset of neurons and the detection of a possible translocation is behind the scope of an assay in which all of the PKC of a slice is sampled. In conclusion, activation of metabotropic glutamate receptors or muscarinic cholinergic receptors induced an activation of PKC measurable by in situ-phosphorylation of different endogenous specific substrate proteins. This enzyme activation is not paralleled by a detectable redistribution of enzyme protein between subcellular compartments. Therefore, the failure to detect PKC translocation in physiological experiments is not a definite indicator for unchanged enzyme activity.

References w1x Baudier, J., Bronner, C., Kligman, D. and Cole, R.D., Protein kinase C substrates from bovine brain, J. Biol. Chem., 264 Ž1989. 1824– 1828.

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w2x Bazzi, M.D. and Nelsestuen, G.L., Properties of membrane inserted protein kinase C, Biochemistry, 27 Ž1988. 7589–7593. w3x Bell, R.M., Protein kinase C activation by diacylglycerol second messengers, Cell, 45 Ž1986. 631–632. w4x Bell, R.M. and Bruns, D.J., Lipid activation of protein kinase C, J. Biol. Chem., 266 Ž1991. 4661–4664. w5x Boss, V., Desai, M.A., Smith, T.S. and Conn, P.J., trans- ACPD-induced phosphoinositide hydrolysis and modulation of hippocampal pyramidal cell excitability do not undergo parallel developmental regulation, Brain Res., 594 Ž1992. 181–188. w6x Bradford, M.M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of proteindye binding, Anal. Biochem., 72 Ž1976. 248–254. w7x Chakravarthy, B.R., Whitfield, J.F. and Durkin, J.P., Inactive membrane protein kinase Cs: a possible target for receptor signalling, Biochem. J., 304 Ž1994. 809–816. w8x Coffey, E.T., Herrero, I., Sihra, T.S., Sanchez-Prieto, J. and Nicholls, ´ D.G., Glutamate exocytosis and MARCKS phosphorylation are enhanced by a metabotropic glutamate receptor coupled to a protein kinase C synergistically activated by diacylglycerol and arachidonic acid, J. Neurochem., 63 Ž1994. 1303–1310. w9x Fujise, A., Mizuno, K., Ueda, Y., Osada, S., Hirai, S., Takayanagi, A., Shimuzi, N., Owada, M.K., Nakajima, H. and Ohno, S., Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristyolated alanine-rich protein kinase C substrate, J. Biol. Chem., 269 Ž1994. 31642–31648. w10x Golapakrishna, R., Barsky, S.H., Thomas, T.P. and Anderson, W.B., Factors influencing chelator-stable, detergent-extractable, phorboldiester induced membrane association of protein kinase C, J. Biol. Chem., 261 Ž1989. 16438–16445. w11x Hannun, Y.A., Loomis, C.R. and Bell, R.M., Activation of protein kinase C by triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine, J. Biol. Chem., 260 Ž1985. 10039–10043. w12x Hochstrasser, D.F., Harrington, M.G., Hochstrasser, A.-C., Miller, M.J. and Merril, C.R., Methods for increasing the resolution of two-dimensional protein electrophoresis, Anal. Biochem., 173 Ž1988. 424–435. w13x Huang, F.L., Yoshida, Y., Nakabayashi, H., Young, W.S. and Huang, K.P., Immunocytochemical localization of protein kinase C isoenzyme in rat brain, J. Neurosci., 8 Ž1988. 4734–4744. w14x Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S.I. and Nishizuka, Y., Calcium-activated, phopholipid-dependent protein kinase from rat brain, J. Biol. Chem., 257 Ž1982. 13341–13341. w15x Klann, E., Chen, S.-J. and Sweatt, J.D., Increased phosphorylation of a 17-kDa protein kinase C substrate ŽP 17. in long-term potentiation., J. Neurochem., 58 Ž1992. 1576–1579. w16x Kraft, A.S. and Anderson, W.B., Phorbol esters increase the amount of calcium, phospholipid dependent protein kinase associated with plasma membrane, Nature, 301 Ž1983. 621–623. w17x Nishizuka, Y., Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C, Science, 258 Ž1992. 607–614. w18x Otani, S. and Ben-Ari, Y., Biochemical correlates of long-term potentiation in hippocampal synapses, Int. ReÕ. Neurobiol., 35 Ž1993. 1–44.

w19x Otani, S., Ben-Ari, Y. and Roisin-Lallemand, M.P., Metabotropic receptor stimulation coupled to weak tetanus leads to long-term potentiation and a rapid elevation of cytosolic protein kinase C activity, Brain Res., 613 Ž1993. 1–9. w20x Reymann, K.G. and Staak, S. In P.L. Canonico, U. Scapagnini, F. Pamparana and A. Routtenberg ŽEds.., Protein Kinase C in the CNS Focus on Neuronal Plasticity: Proceedings, M asson, MilanorParisrBarcelona, 1994, pp. 31–56. w21x Riedel, G., Casabona, G. and Reymann, K.G., Inhibition of long-term potentiation in the dentate gyrus of freely moving rats by the metabotropic glutamate receptor antagonist MCPG, J. Neurosci., 15 Ž1995. 87–98. w22x Sacktor, T.C., Osten, P., Valsamis, H., Jiang, X. and Sublette, E., Persistent activation of the z isoform of protein kinase C in the maintenance of long-term potentiation, Proc. Natl. Acad. Sci. USA, 90 Ž1993. 8342–8346. w23x Salles, ´ J., A., W. and Fain, J.N., Modulation of the phospholipase C activity in rat brain cortical membranes by simultaneous activation of distinct monoaminergic and cholinergic muscarinic receptors, Mol. Brain Res., 20 Ž1993. 111–117. w24x Scholz, W.K., An ibotenate-selective metabotropic glutamate receptor mediates protein phosphorylation in cultured hippocampal pyramidal neurons, J. Neurochem., 62 Ž1994. 1764–1772. w25x Scholz, W.K. and Palfrey, H.C., Glutamate-stimulated protein-phosphorylation in cultured hippocampal pyramidal neurons, J. Neurosci., 11 Ž1991. 2422–2432. w26x Shaffer, L.M., Han, Y.-F. and Dokas, L.A., Phorbol ester- and glutamate-sensitive phosphorylation of hippocampal membrane proteins from adult and neonatal rats, DeÕ. Brain Res., 73 Ž1993. 133–139. w27x Staak, S., Behnisch, T. and Angenstein, F., Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C isoenzymes a and b, Brain Res., 682 Ž1995. 55–62. w28x Suzuki, T., Okumura-Noji, K., Ogura, A., Tanaka, R., Nakamura, K. and Kudo, Y., Calpain may produce a Ca2q-independent form of kinase C in long-term potentiation, Biochem. Biophys. Res. Commun., 189 Ž1992. 1515–1520. w29x Tan, S.-E., Wenthold, R.J. and Soderling, T.R., Phosphorylation of AMPA-Type glutamate receptors by calciumr calmodulin-dependent protein kinase II and protein kinase C in cultured hippocampal neurons, J. Neurosci., 14 Ž1994. 1123–1129. w30x Tobwin, H., Staehelin, T. and Gorden, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose: procedure and some applications, Proc. Natl. Acad. Sci. USA, 76 Ž1979. 4350– 4354. w31x Trilivas, I., McDonough, P.M. and Brown, J.H., Dissociation of protein kinase C redistribution from the phosphorylation of its substrates, J. Biol. Chem., 266 Ž1991. 8431–8438. w32x Zatz, M., Translocation of protein kinase C in rat hippocampal slices, Brain Res., 385 Ž1986. 174–178.