Brain Research, 557 (1991) 103-108 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116892K
103
BRES 16892
Inhibition of NMDA-induced protein kinase C translocation by a Zn 2÷ chelator: implication of intracellular Zn 2÷ Akemichi Baba, Susumu Etoh and Heitaroh Iwata Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka (Japan) (Accepted 26 March 1991)
Key words: Protein kinase C; Synaptoneurosome; N-methyl-D-aspartate; Zinc
The role of intracellular Zn 2+ in the translocation of protein kinase C from cytosol to membrane fractions was examined by the [3H]phorbol 12,13-dibutyrate (PDBu) binding method in guinea pig cerebral synaptoneurosomes. N-methyl-o-aspartate (NMDA, 100/~M) and calcium ionophore A23187 (0.3-30 /~M) decreased the binding activity in the cytosol with a concomitant increase in the membrane fractions. Pretreatment of synaptoneurosomes with a heavy metal chelator, N,N,N,N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), inhibited the NMDA- and A23187-induced changes of the distribution of [3H]PDBu binding sites in cytosol and membrane fractions. The inhibitory effect of TPEN was negated by a preincubation of TPEN with equimolar Zn 2+ but not by that with Ca2+. The addition of 500/~M Zn 2+ to the lysate of synaptoneurosomes induced an increase of [3H]PDBu binding activity in the membrane fraction with a concomitant decrease in the cytosol fraction, as did 100 /~M Ca2+. Low concentrations of Zn 2+ (10 /~M), which alone had no effect on the distribution of the binding, significantly enhanced the effect of 10/~M Ca2+ in the lysate. Under those conditions TPEN inhibited the Zn2+-potentiated Ca2+-dependent changes in the binding. These results suggest that intraceUular Zn2+ is essential for the agonist-induced translocation of protein kinase C in guinea pig synaptoneurosomes. INTRODUCTION The Zn 2+ concentration in the mammalian brain, especially in the hippocampus, is relatively high 11. Although the physiological functions of Zn 2+ in the central nervous system have yet to be defined, recent studies suggest the possible involvement of Zn 2÷ in the synaptic transmission in the hippocampus: the turnover (uptake and release) rate of Zn 2+ in mossy fiber terminals of the rat hippocampus is accelerated by nerve stimulation 2' 4,8,16,19. In addition, several lines of evidence implicate a potential interaction of Zn 2÷ with excitatory amino acid neurotransmission 5'9'21'31, especially with N-methyl-i> aspartate ( N M D A ) receptors, one of the subtypes of excitatory amino acid receptors 3°. A n o t h e r recent focus on the biological function of Zn 2+ is its involvement in protein kinase C (PKC) biochemistry. P K C contains a cysteine-repeating sequence, which often appears as a motif of Zn 2+- and DNA-binding domains, 'zinc-fingers '29. Murakami et al. 2s reported that at least one Zn2+-binding site exists which is distinct from Ca2+-hinding sites and that Zn 2÷ may act as an important regulator of PKC. Zn 2+ activates P K C and promotes its binding to plasma membranes in T lymphocytes 1°. By contrast, it is not determined
whether Zn 2+ affects the translocation of P K C in neuronal preparations. This could be an important subject for the evaluation of neurobiological functions of Zn 2÷, because the activation and translocation of P K C seems to be one of the biochemical processes for the maintenance of long-term potentiation, a plastic change in synaptic transmission 1'24, in which the N M D A receptor is involved in certain synapses 26,27. Whereas Zn 2+ inhibits the N M D A receptor, it possibly passes through N M D A receptor-operated channels and thus activates PKC 35. Based on these observations, it is reasonable to assume that Zn 2+ is involved in the translocation of PKC in neuronal cells. We have previously found that guinea pig synaptoneurosomes, which contain both presynaptic and postsynaptic components 18, provide a suitable preparation to examine the effect of N M D A on PKC translocation 13. In the present study, we have examined the role of intracellular Zn 2+ in the translocation of PKC in the guinea pig cerebral synaptoneurosomes. MATERIALS AND METHODS
Preparation of synaptoneurosome-enriched fractions of guinea pig cerebral cortex Synaptoneurosomes were prepared from male Hartley guinea pigs (250-450 g) according to the method described by HoUingsworth et
Correspondence: A. Baba, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565, Japan.
104 al. 18 with a slight modification. Brains were rapidly removed and the cerebral cortices were dissected and homogenized in 7 Vols. of oxygenated (95% 02/5% CO2) ice-cold Krebs-Ringer (KR) buffer (125 mM NaCI, 3.5 mM KCI, 0.4 mM KH2PO4, 5 mM NaHCO3, 5 mM glucose and 20 mM TES-NaOH, pH 7.4) containing 1.2 mM MgSO 4 and 1 mM EGTA in a glass-glass homogenizer (5 strokes). The homogenate was diluted with 30 ml of the same buffer and filtrated gently through 3 layers of a nylon mesh (160 gm) and then a Millipore 10 #m filter. The filtrate was centrifuged at 1000 g for 15 min. The resulting pellets, synaptoneurosomes, were washed with KR buffer and used for the experiments.
Preparations of cytosol, EGTA-extracted and CHAPS-extracted fractions of guinea pig cerebral synaptoneurosomes Synaptoneurosomes were suspended in KR buffer containing 16 gM bovine serum albumin and incubated at 37 °C for 5 min and therein 1.3 mM CaCI 2 and drugs were added. After another 10 min of incubation, the reaction was terminated by centrifugation at 9000 g for 4 min and then the resulting pellets were washed with a washing buffer (25 mM Tris-HCl, pH 7.6, 10 mM EGTA, 2 mM EDTA and 0.25 M sucrose). The pellet was further washed with 0.25 M sucrose, 25 mM Tris-HCl buffer (pH 7.6) to remove EGTA, resuspended in 500 gl of buffer A (25 mM Tris-HCl, pH 7.6, 100/~M leupeptin) containing 815.8 #M CaCI2 and 1 mM EGTA (corresponding to a 1 #M free Ca 2+ concentration) 6 and was then lysed with Sonifire (Bronson Co.). In the experiment using calcium ionophore A23187, EGTA and EDTA were omitted from the washing buffer. The lysate was centrifuged at 270,000 g for 15 min to yield supernatant (cytosol) and pellet fractions. The pellet was washed with 220/~1 of the buffer A containing 1 pM free Ca 2÷ and resuspended in 220/~1 of buffer A containing 10 mM EGTA and 2 mM EDTA. After standing for 5 min at 4 °C, the suspension was centrifuged (270,000 g, 15 min) to yield the supernatant (EGTAExt.) and the pellet fractions. The pellet, suspended in 1.6 ml of buffer A containing 10 mM EGTA, 2 mM EDTA and 5 mM CHAPS (about 2 mg CHAPS/mg protein), was incubated for 30 min at 4 °C and then centrifuged at 270,000 g for 15 min to yield the supernatant (CHAPS-Ext.). Two hundred microliter of each fraction was filtered through a Sephadex G-25 column (0.75 x 5 cm, equilibrated with 20 mM Tris-HC! buffer containing 5 mM EGTA and 2 mM EDTA, pH 7.5) to eliminate ions and chemicals. The eluate was used for the phorbol ester binding assay.
Treatment of lysate of synaptoneurosomes with divalent cations In the experiment of the lysate of synaptoneurosomes, the prepared synaptoneurosomes were suspended in buffer A and lysed with Sonifire. The lysate (3-5 mg protein) was incubated with divalent cations and drugs at 37 °C for 10 min. Cytosol, EGTA-Ext. and CHAPS-Ext. of incubated preparations were prepared as described above.
[3H]Phorbol 12,13-dibutyrate binding assay [3H]Phorbol 12,13-dibutyrate ([3H]PDBu) binding was carried out according to the method of Tanaka et al. 32. Fifty microliter of the Sephadex G-25 eluate was added to 200 ~1 of reaction mixture containing 20 mM Tris-maleate (pH 6.8), 100 mM KCI, 2.5 mM CaCI2, 100 #g/ml phosphatidylserine, 30 nM [3H]PDBu and 0.5% dimethylsuifoxide (DMSO) and incubated for 20 min at 30 °C. The incubation was terminated by the addition of 4 ml of ice-cold 0.5% DMSO and a subsequent filtration through 0.3% polyethyleneimine presoaked glass-fiber filters (Whatman GF/B). The filters were washed 4 times with 4 ml of ice-cold 0.5% DMSO. The radioactivity of the filters was counted with a liquid scintillation counter. Non-specific binding was determined in the presence of 15 pM non-radioactive phorbol 12-myristate 13-acetate. The difference between total and non-specific bindings represents specific binding.
Protein kinase C assay Protein kinase C assay was carried out according to the method of Kikkawa et al. 22 with a slight modification. Guinea pig cerebral
cortex was homogenized in 7 Vols. of 25 mM Tris-HCl (pH 7.5) containing 10 mM EGTA, 2 mM EDTA and 100 pM leupeptin, and centrifuged at 270,000 g for 15 min to yield supernatant. The reaction mixture (0.25 ml) contained 20 mM Tris-HCl (pH 7.5), 5 mM magnesium acetate, 50/tg H 1 histone, 1.25 mM CaCIz, 1 pg phosphatidylserine, 0.2 /~g diolein, 10 ~M [y-32p]ATP and the cytosol fraction (25 pg protein). After 3 rain of incubation at 30 °C, the reaction was terminated by the addition of 25% trichloroacetic acid. The acid-precipitable materials were collected by filtration on membrane filters (0.45 pm). The filters were washed 4 times with 4 ml of ice-cold 5% trichloroacetic acid. The radioactivity of the filters was counted with a liquid scintillation counter. The difference of the incorporation of 32p from [~'-32p]ATP into H~ histone between the presence and the absence of CaCI2, phosphatidylserine and diolein presents protein kinase C activity.
45Ca2+ uptake into the synaptoneurosomes Synaptoneurosomes were suspended in oxygenated KR buffer (5-8 mg protein/ml) and each 300 ~1 of suspension was divided into test tubes. After 5 min of incubation at 37 °C, 200 /~1 of drug solutions and 100 pl of 7.8 mM 45CaCIz (final concentration 1.3 mM) were added into the tubes. After 10 min of incubation, the reaction was stopped by the addition of ice-cold KR buffer containing 10 mM EGTA and a subsequent filtration through glass-fiber filters (Whatman GF/B). The filters were washed 4 times with 4 ml of ice-cold KR buffer containing 1 mM EGTA and the radioactivity of the filters was determined.
Materials [3H]Phorboi 12,13-dibutyrate (PDBu) (19.1-20.0 Ci/mmoi) was obtained from New England Nuclear. [y-32p]adenosine 5"-triphosphate (ATP) (37.0 Ci/mmol) and 45CAC!2 (36.3 mCi/mg calcium) were purchased from Amersham. Tris buffer, L-a-phosphatidyl-
L-serine, ethyleneglycol-bis-(13-aminoethylether)N,N,N,N'-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), N,N,N, N'-tetrakis-(2-pyfidylmethyl)-ethylenediamine (TPEN), 1,2-dioleylrac-glycerol (1,2-diolein), leupeptin, N-methyl-o-aspartate, 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propanesulfonate (CHAPS), calcium ionophore A23187 and N-tris[hydroxymethyl]methyl-2aminoethanesulfonic acid (TES) were purchased from Sigma Chemical Co. RESULTS W e s t u d i e d the b i n d i n g of [ 3 H ] P D B u to the t h r e e fractions,
cytosol,
EGTA-Ext.
and
CHAPS-Ext.
of
g u i n e a pig c e r e b r a l p r e p a r a t i o n s , i n d e p e n d e n t l y , b e c a u s e P K C in t h e s e t h r e e fractions m a y h a v e d i f f e r e n t physiological roles 7. R e s u l t s are e x p r e s s e d as p e r c e n t a g e of [ 3 H ] P D B u - b i n d i n g activity of e a c h f r a c t i o n to t h e total binding activity. A s d e p i c t e d in Fig. 1, t h e p r e t r e a t m e n t of s y n a p t o n e u r o s o m e s with 100 p M N M D A
induced a
d e c r e a s e of the b i n d i n g of [ 3 H ] P D B u in cytosoi with c o n c o m i t a n t increases in E G T A - E x t . and C H A P S - E x t . fractions. W h e n 100 p M T P E N , a p e r m e a b l e h e a v y m e t a l c h e l a t o r 3, was a d d e d s i m u l t a n e o u s l y w i t h N M D A , t h e chelator inhibited the NMDA-induced
c h a n g e s of [3n]-
P D B u b i n d i n g in e a c h fraction. T h e b i n d i n g affinity of Z n 2÷ to T P E N is m u c h h i g h e r t h a n that of C a 2÷ (ref. 3). To clarify w h e t h e r the i n h i b i t o r y action of T P E N is a t t r i b u t e d to its c h e l a t i o n of Z n 2÷ o r C a 2÷, an a q u e o u s solution
of T P E N
was
preincubated
with e q u i m o l a r
a m o u n t s of Z n C I e or CaCI2, a n d t h e n t h e s o l u t i o n was
105
TABLE I
TABLE II
Effect of TPEN on ~5Ca2+ uptake in guinea pig cerebral synaptoneurosomes
Effect of TPEN on PKC activity in the guinea pig cerebral cortex cytosoifraction
Synaptoneurosomes were incubated with 1.3 mM 45CAC12for 10 min at 37 °C and incorporated 45Ca2+ was determined by the filtration method. Values are means + S.E.M. of 4 experiments.
The reaction mixture was incubated for 3 min at 30 °C and contained 20 mM Tris-HC1 (pH 7.5), 5 mM magnesium acetate, 50 #g H 1 histone, 1.25 mM CaCI2, 1 #g phosphatidylserine, 0.2 #g diolein, 10 #M [7-32p]ATP and 25 Mg cytosol fraction. The incorporation of 32p from [7-32p]ATP into H1 histone was determined by the filtration method. The difference of the incorporation of 32p between the presence and the absence of CaC12, phosphatidylserine and diolein presents protein kinase C activity. Values are means + S.E.M. of three experiments.
45CaZ+uptake (nmol/mg protein/lO min)
None 100gM NMDA 40mM KCI
Control
I OOMM TPEN
5.63 + 0.15 6.62 + 0.16" 8.79 + 0.22***
5.77 + 0.17 6.47 + 0.03* 8.93 + 0.11"**
PKC activity (nmol Pi/mg protein/3 min)
*P < 0.05, ***P < 0.001 vs none. None 100MM ZnC12
Control
100#M TPEN
3.51 + 0.11 2.11 + 0.05***
3.59 + 0.40 3.31 + 0.24 tt
***P < 0.001 vs none, **P < 0.01 vs control.
Cytosol
50
Cytosol --ql
4O
.
.
.
.
.
0
.a
EGTA-Ext. 8
25
1 A
o
4
~ ._~ "o ._~
2
"o .Q
0
CHAPS-Ext. E3 o. -1-
II
'
EGTA-Ext. Ill
2 o
15
•,-,
10
"o ._~
5
'
.
'
J
.
.
i
.
7-
,
~, 2 / ' ?
,/?d I _ /
$
-~m 0 m a a. CHAPS-Ext.
50 45
l" e~
0
0
II
,
8O
i
Control TPEN Z n - T P E N Ca-TPEN
***//
60
Fig. 1. The effect of TPEN and its metal complexes on NMDAinduced translocation of [3H]PDBu binding sites in guinea pig synaptoneurosomes. Synaptoncurosomes were preincubated with 100 #M TPEN, Zn-TPEN or Ca-TPEN for 5 min and then incubated in the presence (hatched blocks) or absence (open blocks) of 100/zM NMDA for 10 min. Zn-TPEN and Ca-TPEN show mixtures of 100 #M TPEN and 100 gM ZnCI 2 or 100/zM CaC12, respectively, and these mixtures were prepared 1 h before the addition to synaptoneurosomes. Cytosol, EGTA-Ext. and CHAPSExt. fractions were prepared and then [3H]PDBu binding to each fraction was determined independently as described previously. Results are shown as the percentage of [3H]PDBu binding in each fraction of the total binding. Values are means + S.E.M. of 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs none; t p < 0.05, **P < 0.01 vs control.
-
-
...
103
BRES 16892
Inhibition of NMDA-induced protein kinase C translocation by a Zn 2÷ chelator: implication of intracellular Zn 2÷ Akemichi Baba, Susumu Etoh and Heitaroh Iwata Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka (Japan) (Accepted 26 March 1991)
Key words: Protein kinase C; Synaptoneurosome; N-methyl-D-aspartate; Zinc
The role of intracellular Zn 2+ in the translocation of protein kinase C from cytosol to membrane fractions was examined by the [3H]phorbol 12,13-dibutyrate (PDBu) binding method in guinea pig cerebral synaptoneurosomes. N-methyl-o-aspartate (NMDA, 100/~M) and calcium ionophore A23187 (0.3-30 /~M) decreased the binding activity in the cytosol with a concomitant increase in the membrane fractions. Pretreatment of synaptoneurosomes with a heavy metal chelator, N,N,N,N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), inhibited the NMDA- and A23187-induced changes of the distribution of [3H]PDBu binding sites in cytosol and membrane fractions. The inhibitory effect of TPEN was negated by a preincubation of TPEN with equimolar Zn 2+ but not by that with Ca2+. The addition of 500/~M Zn 2+ to the lysate of synaptoneurosomes induced an increase of [3H]PDBu binding activity in the membrane fraction with a concomitant decrease in the cytosol fraction, as did 100 /~M Ca2+. Low concentrations of Zn 2+ (10 /~M), which alone had no effect on the distribution of the binding, significantly enhanced the effect of 10/~M Ca2+ in the lysate. Under those conditions TPEN inhibited the Zn2+-potentiated Ca2+-dependent changes in the binding. These results suggest that intraceUular Zn2+ is essential for the agonist-induced translocation of protein kinase C in guinea pig synaptoneurosomes. INTRODUCTION The Zn 2+ concentration in the mammalian brain, especially in the hippocampus, is relatively high 11. Although the physiological functions of Zn 2+ in the central nervous system have yet to be defined, recent studies suggest the possible involvement of Zn 2÷ in the synaptic transmission in the hippocampus: the turnover (uptake and release) rate of Zn 2+ in mossy fiber terminals of the rat hippocampus is accelerated by nerve stimulation 2' 4,8,16,19. In addition, several lines of evidence implicate a potential interaction of Zn 2÷ with excitatory amino acid neurotransmission 5'9'21'31, especially with N-methyl-i> aspartate ( N M D A ) receptors, one of the subtypes of excitatory amino acid receptors 3°. A n o t h e r recent focus on the biological function of Zn 2+ is its involvement in protein kinase C (PKC) biochemistry. P K C contains a cysteine-repeating sequence, which often appears as a motif of Zn 2+- and DNA-binding domains, 'zinc-fingers '29. Murakami et al. 2s reported that at least one Zn2+-binding site exists which is distinct from Ca2+-hinding sites and that Zn 2÷ may act as an important regulator of PKC. Zn 2+ activates P K C and promotes its binding to plasma membranes in T lymphocytes 1°. By contrast, it is not determined
whether Zn 2+ affects the translocation of P K C in neuronal preparations. This could be an important subject for the evaluation of neurobiological functions of Zn 2÷, because the activation and translocation of P K C seems to be one of the biochemical processes for the maintenance of long-term potentiation, a plastic change in synaptic transmission 1'24, in which the N M D A receptor is involved in certain synapses 26,27. Whereas Zn 2+ inhibits the N M D A receptor, it possibly passes through N M D A receptor-operated channels and thus activates PKC 35. Based on these observations, it is reasonable to assume that Zn 2+ is involved in the translocation of PKC in neuronal cells. We have previously found that guinea pig synaptoneurosomes, which contain both presynaptic and postsynaptic components 18, provide a suitable preparation to examine the effect of N M D A on PKC translocation 13. In the present study, we have examined the role of intracellular Zn 2+ in the translocation of PKC in the guinea pig cerebral synaptoneurosomes. MATERIALS AND METHODS
Preparation of synaptoneurosome-enriched fractions of guinea pig cerebral cortex Synaptoneurosomes were prepared from male Hartley guinea pigs (250-450 g) according to the method described by HoUingsworth et
Correspondence: A. Baba, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565, Japan.
104 al. 18 with a slight modification. Brains were rapidly removed and the cerebral cortices were dissected and homogenized in 7 Vols. of oxygenated (95% 02/5% CO2) ice-cold Krebs-Ringer (KR) buffer (125 mM NaCI, 3.5 mM KCI, 0.4 mM KH2PO4, 5 mM NaHCO3, 5 mM glucose and 20 mM TES-NaOH, pH 7.4) containing 1.2 mM MgSO 4 and 1 mM EGTA in a glass-glass homogenizer (5 strokes). The homogenate was diluted with 30 ml of the same buffer and filtrated gently through 3 layers of a nylon mesh (160 gm) and then a Millipore 10 #m filter. The filtrate was centrifuged at 1000 g for 15 min. The resulting pellets, synaptoneurosomes, were washed with KR buffer and used for the experiments.
Preparations of cytosol, EGTA-extracted and CHAPS-extracted fractions of guinea pig cerebral synaptoneurosomes Synaptoneurosomes were suspended in KR buffer containing 16 gM bovine serum albumin and incubated at 37 °C for 5 min and therein 1.3 mM CaCI 2 and drugs were added. After another 10 min of incubation, the reaction was terminated by centrifugation at 9000 g for 4 min and then the resulting pellets were washed with a washing buffer (25 mM Tris-HCl, pH 7.6, 10 mM EGTA, 2 mM EDTA and 0.25 M sucrose). The pellet was further washed with 0.25 M sucrose, 25 mM Tris-HCl buffer (pH 7.6) to remove EGTA, resuspended in 500 gl of buffer A (25 mM Tris-HCl, pH 7.6, 100/~M leupeptin) containing 815.8 #M CaCI2 and 1 mM EGTA (corresponding to a 1 #M free Ca 2+ concentration) 6 and was then lysed with Sonifire (Bronson Co.). In the experiment using calcium ionophore A23187, EGTA and EDTA were omitted from the washing buffer. The lysate was centrifuged at 270,000 g for 15 min to yield supernatant (cytosol) and pellet fractions. The pellet was washed with 220/~1 of the buffer A containing 1 pM free Ca 2÷ and resuspended in 220/~1 of buffer A containing 10 mM EGTA and 2 mM EDTA. After standing for 5 min at 4 °C, the suspension was centrifuged (270,000 g, 15 min) to yield the supernatant (EGTAExt.) and the pellet fractions. The pellet, suspended in 1.6 ml of buffer A containing 10 mM EGTA, 2 mM EDTA and 5 mM CHAPS (about 2 mg CHAPS/mg protein), was incubated for 30 min at 4 °C and then centrifuged at 270,000 g for 15 min to yield the supernatant (CHAPS-Ext.). Two hundred microliter of each fraction was filtered through a Sephadex G-25 column (0.75 x 5 cm, equilibrated with 20 mM Tris-HC! buffer containing 5 mM EGTA and 2 mM EDTA, pH 7.5) to eliminate ions and chemicals. The eluate was used for the phorbol ester binding assay.
Treatment of lysate of synaptoneurosomes with divalent cations In the experiment of the lysate of synaptoneurosomes, the prepared synaptoneurosomes were suspended in buffer A and lysed with Sonifire. The lysate (3-5 mg protein) was incubated with divalent cations and drugs at 37 °C for 10 min. Cytosol, EGTA-Ext. and CHAPS-Ext. of incubated preparations were prepared as described above.
[3H]Phorbol 12,13-dibutyrate binding assay [3H]Phorbol 12,13-dibutyrate ([3H]PDBu) binding was carried out according to the method of Tanaka et al. 32. Fifty microliter of the Sephadex G-25 eluate was added to 200 ~1 of reaction mixture containing 20 mM Tris-maleate (pH 6.8), 100 mM KCI, 2.5 mM CaCI2, 100 #g/ml phosphatidylserine, 30 nM [3H]PDBu and 0.5% dimethylsuifoxide (DMSO) and incubated for 20 min at 30 °C. The incubation was terminated by the addition of 4 ml of ice-cold 0.5% DMSO and a subsequent filtration through 0.3% polyethyleneimine presoaked glass-fiber filters (Whatman GF/B). The filters were washed 4 times with 4 ml of ice-cold 0.5% DMSO. The radioactivity of the filters was counted with a liquid scintillation counter. Non-specific binding was determined in the presence of 15 pM non-radioactive phorbol 12-myristate 13-acetate. The difference between total and non-specific bindings represents specific binding.
Protein kinase C assay Protein kinase C assay was carried out according to the method of Kikkawa et al. 22 with a slight modification. Guinea pig cerebral
cortex was homogenized in 7 Vols. of 25 mM Tris-HCl (pH 7.5) containing 10 mM EGTA, 2 mM EDTA and 100 pM leupeptin, and centrifuged at 270,000 g for 15 min to yield supernatant. The reaction mixture (0.25 ml) contained 20 mM Tris-HCl (pH 7.5), 5 mM magnesium acetate, 50/tg H 1 histone, 1.25 mM CaCIz, 1 pg phosphatidylserine, 0.2 /~g diolein, 10 ~M [y-32p]ATP and the cytosol fraction (25 pg protein). After 3 rain of incubation at 30 °C, the reaction was terminated by the addition of 25% trichloroacetic acid. The acid-precipitable materials were collected by filtration on membrane filters (0.45 pm). The filters were washed 4 times with 4 ml of ice-cold 5% trichloroacetic acid. The radioactivity of the filters was counted with a liquid scintillation counter. The difference of the incorporation of 32p from [~'-32p]ATP into H~ histone between the presence and the absence of CaCI2, phosphatidylserine and diolein presents protein kinase C activity.
45Ca2+ uptake into the synaptoneurosomes Synaptoneurosomes were suspended in oxygenated KR buffer (5-8 mg protein/ml) and each 300 ~1 of suspension was divided into test tubes. After 5 min of incubation at 37 °C, 200 /~1 of drug solutions and 100 pl of 7.8 mM 45CaCIz (final concentration 1.3 mM) were added into the tubes. After 10 min of incubation, the reaction was stopped by the addition of ice-cold KR buffer containing 10 mM EGTA and a subsequent filtration through glass-fiber filters (Whatman GF/B). The filters were washed 4 times with 4 ml of ice-cold KR buffer containing 1 mM EGTA and the radioactivity of the filters was determined.
Materials [3H]Phorboi 12,13-dibutyrate (PDBu) (19.1-20.0 Ci/mmoi) was obtained from New England Nuclear. [y-32p]adenosine 5"-triphosphate (ATP) (37.0 Ci/mmol) and 45CAC!2 (36.3 mCi/mg calcium) were purchased from Amersham. Tris buffer, L-a-phosphatidyl-
L-serine, ethyleneglycol-bis-(13-aminoethylether)N,N,N,N'-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), N,N,N, N'-tetrakis-(2-pyfidylmethyl)-ethylenediamine (TPEN), 1,2-dioleylrac-glycerol (1,2-diolein), leupeptin, N-methyl-o-aspartate, 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propanesulfonate (CHAPS), calcium ionophore A23187 and N-tris[hydroxymethyl]methyl-2aminoethanesulfonic acid (TES) were purchased from Sigma Chemical Co. RESULTS W e s t u d i e d the b i n d i n g of [ 3 H ] P D B u to the t h r e e fractions,
cytosol,
EGTA-Ext.
and
CHAPS-Ext.
of
g u i n e a pig c e r e b r a l p r e p a r a t i o n s , i n d e p e n d e n t l y , b e c a u s e P K C in t h e s e t h r e e fractions m a y h a v e d i f f e r e n t physiological roles 7. R e s u l t s are e x p r e s s e d as p e r c e n t a g e of [ 3 H ] P D B u - b i n d i n g activity of e a c h f r a c t i o n to t h e total binding activity. A s d e p i c t e d in Fig. 1, t h e p r e t r e a t m e n t of s y n a p t o n e u r o s o m e s with 100 p M N M D A
induced a
d e c r e a s e of the b i n d i n g of [ 3 H ] P D B u in cytosoi with c o n c o m i t a n t increases in E G T A - E x t . and C H A P S - E x t . fractions. W h e n 100 p M T P E N , a p e r m e a b l e h e a v y m e t a l c h e l a t o r 3, was a d d e d s i m u l t a n e o u s l y w i t h N M D A , t h e chelator inhibited the NMDA-induced
c h a n g e s of [3n]-
P D B u b i n d i n g in e a c h fraction. T h e b i n d i n g affinity of Z n 2÷ to T P E N is m u c h h i g h e r t h a n that of C a 2÷ (ref. 3). To clarify w h e t h e r the i n h i b i t o r y action of T P E N is a t t r i b u t e d to its c h e l a t i o n of Z n 2÷ o r C a 2÷, an a q u e o u s solution
of T P E N
was
preincubated
with e q u i m o l a r
a m o u n t s of Z n C I e or CaCI2, a n d t h e n t h e s o l u t i o n was
105
TABLE I
TABLE II
Effect of TPEN on ~5Ca2+ uptake in guinea pig cerebral synaptoneurosomes
Effect of TPEN on PKC activity in the guinea pig cerebral cortex cytosoifraction
Synaptoneurosomes were incubated with 1.3 mM 45CAC12for 10 min at 37 °C and incorporated 45Ca2+ was determined by the filtration method. Values are means + S.E.M. of 4 experiments.
The reaction mixture was incubated for 3 min at 30 °C and contained 20 mM Tris-HC1 (pH 7.5), 5 mM magnesium acetate, 50 #g H 1 histone, 1.25 mM CaCI2, 1 #g phosphatidylserine, 0.2 #g diolein, 10 #M [7-32p]ATP and 25 Mg cytosol fraction. The incorporation of 32p from [7-32p]ATP into H1 histone was determined by the filtration method. The difference of the incorporation of 32p between the presence and the absence of CaC12, phosphatidylserine and diolein presents protein kinase C activity. Values are means + S.E.M. of three experiments.
45CaZ+uptake (nmol/mg protein/lO min)
None 100gM NMDA 40mM KCI
Control
I OOMM TPEN
5.63 + 0.15 6.62 + 0.16" 8.79 + 0.22***
5.77 + 0.17 6.47 + 0.03* 8.93 + 0.11"**
PKC activity (nmol Pi/mg protein/3 min)
*P < 0.05, ***P < 0.001 vs none. None 100MM ZnC12
Control
100#M TPEN
3.51 + 0.11 2.11 + 0.05***
3.59 + 0.40 3.31 + 0.24 tt
***P < 0.001 vs none, **P < 0.01 vs control.
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Fig. 1. The effect of TPEN and its metal complexes on NMDAinduced translocation of [3H]PDBu binding sites in guinea pig synaptoneurosomes. Synaptoncurosomes were preincubated with 100 #M TPEN, Zn-TPEN or Ca-TPEN for 5 min and then incubated in the presence (hatched blocks) or absence (open blocks) of 100/zM NMDA for 10 min. Zn-TPEN and Ca-TPEN show mixtures of 100 #M TPEN and 100 gM ZnCI 2 or 100/zM CaC12, respectively, and these mixtures were prepared 1 h before the addition to synaptoneurosomes. Cytosol, EGTA-Ext. and CHAPSExt. fractions were prepared and then [3H]PDBu binding to each fraction was determined independently as described previously. Results are shown as the percentage of [3H]PDBu binding in each fraction of the total binding. Values are means + S.E.M. of 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs none; t p < 0.05, **P < 0.01 vs control.
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