- Email: [email protected]
The effect of scorpion venom tityustoxin on the uptake of acetylcholine in rat brain cortical slices
THE EFFECT OF SCORPION VENOM TITYUSTOXIN ON THE UPTAKE OF ACETYLCHOLINE IN RAT BRAIN CORTICAL SLICES* M. V. GOMEZ. M. E. M. DAI and C. R. DINIZ Departamento de Bioquimica do Instituto de Ciencias Biologicas UFMG. Caixa Postal 2486, Belo Horizonte, Minas Gerais. Brasil (Accrprrd
15 Norrrtther
1978)
Summary--The
effect of tityustoxin on acetylcholine (ACh) uptake in slices of rat brain studied. The toxin reduced the uptake of ACh as a function of its concentration in the incubation time. Tetrodotoxin prevented the inhibition effect of tityustoxin on ACh uptake. tion caused by tityustoxin was noncompetitive with a Ki of 2 x 10m4 M. The mechanism tityustoxin affects the uptake is discussed and it is concluded that, probably, it might be increased release of radioactive ACh.
In the presence of cholinesterase inhibitors, the uptake of acetylcholine (ACh) in brain cortex proceeds against a concentration gradient (Polak and Meeuws, 1966; Schuberth and Sundwall, 1967; Heilbronn. 1969: Kuhar and Simon, 1974). The uptake of ACh was shown to be inhibited by drugs that increase ACh release (Mitchell, 1963; Liang and Quastel, 1969; Polak, 1969). In denervated sympathic ganglion, no reduction of ACh uptake has been found and, in neuronal tissue, ACh appears to accumulate equally well whether cholinergic neurons were present or not (Kuhar and Simon, 1974). These results suggest that there was no association between cholinergic neurons and ACh uptake. Tityustoxin (TsTX), a basic protein purified from the venom of the Brazilian scorpion Tityus serrulatus (Gomez and Diniz, 1966) increased the release of ACh in rat brain cortical slices (Gomez, Dai and Diniz, 1973; Gomez, Diniz and Barbosa, 1975). This effect of TsTX is calcium and sodium-dependent. Calcium is necessary for the release of ACh (Katz and Miledi, 1967) and omission of Ca2 + was found to cause an increased rate of ACh uptake (Heilbronn and Cedergren. 1970) that could be explained as a decreased release of the neurotransmitter. The present paper deals with the influence of TsTX on the accumulation of ACh in slices of rat cerebral cortex. METHODS
was purified by the method of Gomez and Diniz (1966). This method allowed the isolation Tityustoxin
cortex was media and The inhibiby which due to an
of a toxic material which is homogeneous to polyacrylamide gel electrophoresis. Slices were prepared from brain cortex of 5- 7 week-old Holtzman rats as previously described (Gomez et al., 1973). The cortical slices were incubated at 37°C. pH 7.4 in a medium containing (mM): NaCl 120, KCl, 4.6, CaCl, 2.4. KH2P04 1.2, NaHCO, 25. glucose 10, diisopropylfluorophosphate (DFP) 0.01 and 1-3 PCi of r4C-ACh (methyl-r4C-Chloride) (Amersham/Searle 54 mCi/ mol). After incubation, the slices were rinsed with cold incubation medium, blotted on filter paper. transferred to scintillation vials, digested with NCS (Nuclear Chicago Solubilizer) and counted in a Beckman Liquid Scintillation Counter model LS-150. in 15 ml of solution containing per liter of toluene: 2,5 diphenyloxazole, 5.0 g and 1,4 bis-(4-methyl-5phenyloxazolyl) 0.1 g. Radioactivity was calculated by subtracting the blank value incubated simultaneously on an ice bath. Counting efficiency was corrected for each sample by means of the external standard method. In several experiments, r4C-ACh was extracted with ethanol-acetic acid (Aprison and Tdkahashi, 1965) and separated by high voltage electrophoresis using pyridine: water: acetic acid (3 : 500: IO) pH 4.9, 4.000 V for 30 min. The spot corresponding to ACh contained most of the radioactivity and contamination with r4C-choline was less than 5%,. All the experiments were carried out, three times, unless otherwise stated, and duplicates in each experiment were run routinely. The values reported below record the averages of all experiments together with standard deviations.
* Supported by Conselho National de Desenvolvimento Cientifico e Tecnologico and FINEP. One of the authors (M.V,G) is a Research Fellow of CNPq.
RESULTS
Abbreviations-TsTX: Tityustoxin obtained from the venom of the scorpion Titgus serrularus. ACh: Acetylcholine, DFP: Diisopropylfluorophosphate, TTX: Tetrodotoxin.
The effect of TsTX on ACh uptake as a function of ACh concentration is shown in Figure 1. Tityustoxin reduced the uptake in all the studied concen-
\.I?
IS 6
A
515
M. V. GOMEZ. M. E. M. DAI and c‘. R. DIVU
510
I”
2u
IUU
ACETYLCHOLI~E
( PM 1
Fig. 1. ER’ect of TsTX on the uptake of ACh as a function of ACh concentration; o control: l with TsTX 2 x 10. ’ M. Cerebral cortex slices were preincubated for 10 min in Krebs medium containing (mM): NaCl 120; KCI 4.6; CaCI, 2.4; KH2P304 1.2; NaHCO, 25, glucose 10 and DFP 0.01: pH 7.4. Each slice was then transferred to flasks with the above medium containing the indicated concentrations of ACh and 1-3 ,uCi of ?-ACh. After incubation for 60min at 37°C’. the radioactivity in the slices was counted as described in the text. The values represent the means _+S.D. for three experiments. For other detail:, see the text.
60
O
75
65
30 TIME
60
(min)
Fig. 7. E&t of TsTX on ACh uptake as function of incubation time: 0 control: 0 TsTX 2 x IO-’ M. The cortical slices were incubated at the indicated times as described in the legend of’ Figure 1 and in the presence of 100 /IM of ACh (3 &i of ‘%‘Y-ACh). Values represent the means &S.D. for three experiments. For other details see the legend to Figure I and the text.
Tityustoxin
517
effect on ACh uptake
60
I
L
I
0
I
5x167
I
1 x166
I
I
2 x 10-6
6x166 TITYUSTOXIN
1M 1
Fig. 3. Effect of TsTX concentration on the uptake of ACh in cortical slices of rat brain: 0 control; l with TsTX. The slices were incubated in the presence of lOO/rM of ACh containing 1-3 ntci of 14C-ACh for 60 min at the indicated concentrations TsTX. Values are means kS.D. for three experiments. For other details see legend to Figure 1 and the text.
trations of ACh. The inhibition was maximum, about 40::, at a concentration of 1OOpM of ACh in the incubation medium. Figure 2 shows the inhibition of ACh uptake by the toxin as a function of incubation time. At 15, 30 and 60min, TsTX reduced the uptake by 15. 31 and 40% respectively. The inhibitory effect of
100
TsTX was dependent on the toxin dose (Fig. 3). At concentrations of 0.25, 0.5, 2.0 and 4.0pM, TsTX reduced the uptake of ACh by 21, 30. 34 and 36%. respectively. The inhibition reached a plateau at a concentration of 2pM of TsTX. Figure 4 shows a typical Lineweaver-Burk plot obtained for the uptake
’
ln g Y 5 -0”I 50 .
Fig. 4. Effect of TsTX on the variation of the reciprocal of uptake of ACh into rat brain cortical slices: 0 control; l with TsTX 2 x 10e6 M. The cortical slices were incubated for 6Omin at the indicated concentrations of ACh (IL3 &i of 14C-ACh). Values express the means &-SD. for three experiments. For other details see the legend to Figure 1 and the text.
SIX Table
M. V. GOMI.I. M. E. M. DAI and f’. R. Drur~ I. Effects of TsTX and TTS on the upiakc m rat brain cortical slices
Tetrodotoxin 5 x 10~ i M ‘Tityustoxin 2 x IO ’ M Tityustoxin 2 x IO- ’ M
of h(‘h
.l(,~rto~~ii,t/il~,~~~[,~~f.\ --We are grateful to Dr David L. Nelson for reading thi< manuscript. We thank Terezinhn Stockier Barhosa and Anthnio Soares Usual dos Santos for expert technical assistance. We we grateful to Miss Marly Fonseca Silvn for secretarial assistance.
94.7 5 4.95s 63.7 i 3.45+ 93.4 + 6.2*
PIUS
Tctrodotoxin
of thi: toxin on AC’h uptake t.Table 1t. Thus. the observed i~lhibitory effect on ACh uptake. caused h> TSTX might possibly be cxpiained as the result of an increased release of the ACh that is taken up.
5 x IO-’ M
Brain cortical slices were rncuhated for hOrnin as indicated in the legend to Figure 2. Values represent the means *SD. for 3 experiments. For other details see the text. Tests for significance * P < 0. I ; i P < 0.005.
Aprison. M. H. and Takahashi. R. (1965). Biochemistry of the acian central nervous system. J. .~‘~,~r~~~~~~~~~. 12:
?‘I 230. of ACh in the presence or absence of 7 /tM of TsTX. The toxin caused a noncompetitive inhibition of ACh uptake. K, = 2 x 10e4M. Table 1 shows the effect of tetrodotoxin (TTX) on the uptake of ACh in the presence of TsTX: TTX had no cffcct on ACh uptake. but. incubated in the presence of TsTX. it prevented the inhibitory effect caused by the scorpion toxin.
In the presence of the cholinestcrasc jnhib~tor. DFP. to prevent ACh hydrolysis. TsTX reduced the uptake of ACh in rat brain cortical slices. This effect was dependent on toxin concentration. incubation time and ACh concentration in the incubation medium. The ability to alter the ACh uptake seems to he a common process for drugs that increase (Mitchel, 1963; Liang and Quastcl, 1969: Polak, 1969) or decrease (Carson. Jenden and Noble. 1975) ACh release. Tityustoxin increased the release of free ACh thus reducing tissue ACh of cortical slices of rat brain (Gomez er ui.. 1973. 1975), while. in the present paper. the toxin reduced the ACh uptake. Being a basic protein, TsTX carries a positive charge at physjoiogical PH. It has been shown that drugs charged with a cationic group could inhibit ACh
uptake
by a mechanism
of competition
for the
(Polak. 1969). In spite of its charge. the effect of TsTX on the inhibition of ACh uptake could not be explained by this mechanism. since the inhibition caused by the TsTX was noncompetitive (Fig. 4). The effect could not be atanionic
tributable
sites in the membrane
to
a cholinesterase
or
phospholipase
ac-
of the toxin since the venom from the scorpion 7ir 1’14s.s~,r~~{~~~~f~.~ is completely devoid of these activities (Dini and Goncalvcs, 1960). ietrodotoxin, a drug that had no effect on ACh
tivity
uptake
(Liang
and
Quastel.
1969).
inhibited
the
of ACh stimulated by TsTX (Gomer tit tri.. 1973; lY75) and is also capable of inhibiting the effect
release
Carson. V. G.. Jcnden. D. J. and Noble. E. P. (1975). Acctylcholinc (A&h) uptake in cortical slabs from C5-BI mice after acute and/or chronic ethanol treatment. P,oL,. U’rsr. Pkornlclc,. 19: 341 345. Dimz. C. R. and Con&es. J. M. (1960). Separation 01 biolo~lcally actice components from scorpion venoms by zone electrophoresis. Biochim. hioph!,s. Ac,rcc 41 : 470-477. Gomez, M. V. and Diniz. C. R. (1966). Separation of toxic components from Brazilian scorpion T. serrdntus. Mrrm irw. BzcrfInrrrt~ 33: 889-902. Gctmer.. M. V.. Dai, M. E. M. and Diniz. C. R. (197.1). Effect of scorpion venom, tityustoxin on the release of ~lc~tylcho)ine from incubated slices of rat brain. J. Nrwrxh<‘nr. 20: 1051~~l061. Gomez, M. V.. Diniz. C. R. and Barbosd, T. S. (1975). A comparison of the effects of scorpion tityustoxin and ouabain on the release of acetylcholine from incubated slices of rat brain. J. Ncwochew 24: 331-336. Heilbronn. E. (1969). The effect of phospholipases on the uptake of atropinc and acetylcholine by slices of mouse and brain cortex. J. Nrurochm. 16: 627-635. Heilhronn. E. and Ccdergren. E. (1970). Chemically induced changes in the acetylcholine uptake and storage capacity of brain tissue. In: Druys und Cholinwyic Mwlrur~isnts11~rhe C’NS (Heilbronn, E. and Winter. A.. Eds). pp. 245 269. Forsvarets Forskningsanstalt, Research Institute of National Defence, Stockholm. Kat/. B. and Miledi. R. (1967). Ionic requirements ol synaptic transmitw release. ,X‘urwc 215: 6.51. Kuhar. M. J. and Simon, J. R. (1974). Acctylcholinc uptake: lack of association with cholinergic neurons. .I. h’cwoc4wt~~. 22: I I3S- 1136. Liang. C‘. C. and Quastel, J. H. (1969). ElTects of drugs on the uptake of acetylcholine in rat brain cortex slices. Biochor~. Phimmrc. 18: I I X7 I 194. Mitchel. J. F. (1963). The spontaneous and evoked release of acetylcholine from the cerebral cortex. J. Physiol. 165: 116. Polak. R. I.. (1969). The influence of drugs on the uptake of acetylcholine by slices of rat cerebrat cortex. Br. .I. Phiirrtltr<. 3h: I44 152. Polak, R. L. and Mceuws. M. M. (1966). The influcncc of atropine on the release and uptake of acetylchohne
by the is&ted
cerebral cortex of the rat.
Biochrru.
f’frc~r-
IPlilC. IS: 989 -992.
Schubcrth. J. and Sundwall, A. (1967). Effects of some drugs on the uptake of acetylcholinc in cortex slices of mouse brain. J. Nwrochwl. 14: 807 8 12.
15 Norrrtther
1978)
Summary--The
effect of tityustoxin on acetylcholine (ACh) uptake in slices of rat brain studied. The toxin reduced the uptake of ACh as a function of its concentration in the incubation time. Tetrodotoxin prevented the inhibition effect of tityustoxin on ACh uptake. tion caused by tityustoxin was noncompetitive with a Ki of 2 x 10m4 M. The mechanism tityustoxin affects the uptake is discussed and it is concluded that, probably, it might be increased release of radioactive ACh.
In the presence of cholinesterase inhibitors, the uptake of acetylcholine (ACh) in brain cortex proceeds against a concentration gradient (Polak and Meeuws, 1966; Schuberth and Sundwall, 1967; Heilbronn. 1969: Kuhar and Simon, 1974). The uptake of ACh was shown to be inhibited by drugs that increase ACh release (Mitchell, 1963; Liang and Quastel, 1969; Polak, 1969). In denervated sympathic ganglion, no reduction of ACh uptake has been found and, in neuronal tissue, ACh appears to accumulate equally well whether cholinergic neurons were present or not (Kuhar and Simon, 1974). These results suggest that there was no association between cholinergic neurons and ACh uptake. Tityustoxin (TsTX), a basic protein purified from the venom of the Brazilian scorpion Tityus serrulatus (Gomez and Diniz, 1966) increased the release of ACh in rat brain cortical slices (Gomez, Dai and Diniz, 1973; Gomez, Diniz and Barbosa, 1975). This effect of TsTX is calcium and sodium-dependent. Calcium is necessary for the release of ACh (Katz and Miledi, 1967) and omission of Ca2 + was found to cause an increased rate of ACh uptake (Heilbronn and Cedergren. 1970) that could be explained as a decreased release of the neurotransmitter. The present paper deals with the influence of TsTX on the accumulation of ACh in slices of rat cerebral cortex. METHODS
was purified by the method of Gomez and Diniz (1966). This method allowed the isolation Tityustoxin
cortex was media and The inhibiby which due to an
of a toxic material which is homogeneous to polyacrylamide gel electrophoresis. Slices were prepared from brain cortex of 5- 7 week-old Holtzman rats as previously described (Gomez et al., 1973). The cortical slices were incubated at 37°C. pH 7.4 in a medium containing (mM): NaCl 120, KCl, 4.6, CaCl, 2.4. KH2P04 1.2, NaHCO, 25. glucose 10, diisopropylfluorophosphate (DFP) 0.01 and 1-3 PCi of r4C-ACh (methyl-r4C-Chloride) (Amersham/Searle 54 mCi/ mol). After incubation, the slices were rinsed with cold incubation medium, blotted on filter paper. transferred to scintillation vials, digested with NCS (Nuclear Chicago Solubilizer) and counted in a Beckman Liquid Scintillation Counter model LS-150. in 15 ml of solution containing per liter of toluene: 2,5 diphenyloxazole, 5.0 g and 1,4 bis-(4-methyl-5phenyloxazolyl) 0.1 g. Radioactivity was calculated by subtracting the blank value incubated simultaneously on an ice bath. Counting efficiency was corrected for each sample by means of the external standard method. In several experiments, r4C-ACh was extracted with ethanol-acetic acid (Aprison and Tdkahashi, 1965) and separated by high voltage electrophoresis using pyridine: water: acetic acid (3 : 500: IO) pH 4.9, 4.000 V for 30 min. The spot corresponding to ACh contained most of the radioactivity and contamination with r4C-choline was less than 5%,. All the experiments were carried out, three times, unless otherwise stated, and duplicates in each experiment were run routinely. The values reported below record the averages of all experiments together with standard deviations.
* Supported by Conselho National de Desenvolvimento Cientifico e Tecnologico and FINEP. One of the authors (M.V,G) is a Research Fellow of CNPq.
RESULTS
Abbreviations-TsTX: Tityustoxin obtained from the venom of the scorpion Titgus serrularus. ACh: Acetylcholine, DFP: Diisopropylfluorophosphate, TTX: Tetrodotoxin.
The effect of TsTX on ACh uptake as a function of ACh concentration is shown in Figure 1. Tityustoxin reduced the uptake in all the studied concen-
\.I?
IS 6
A
515
M. V. GOMEZ. M. E. M. DAI and c‘. R. DIVU
510
I”
2u
IUU
ACETYLCHOLI~E
( PM 1
Fig. 1. ER’ect of TsTX on the uptake of ACh as a function of ACh concentration; o control: l with TsTX 2 x 10. ’ M. Cerebral cortex slices were preincubated for 10 min in Krebs medium containing (mM): NaCl 120; KCI 4.6; CaCI, 2.4; KH2P304 1.2; NaHCO, 25, glucose 10 and DFP 0.01: pH 7.4. Each slice was then transferred to flasks with the above medium containing the indicated concentrations of ACh and 1-3 ,uCi of ?-ACh. After incubation for 60min at 37°C’. the radioactivity in the slices was counted as described in the text. The values represent the means _+S.D. for three experiments. For other detail:, see the text.
60
O
75
65
30 TIME
60
(min)
Fig. 7. E&t of TsTX on ACh uptake as function of incubation time: 0 control: 0 TsTX 2 x IO-’ M. The cortical slices were incubated at the indicated times as described in the legend of’ Figure 1 and in the presence of 100 /IM of ACh (3 &i of ‘%‘Y-ACh). Values represent the means &S.D. for three experiments. For other details see the legend to Figure I and the text.
Tityustoxin
517
effect on ACh uptake
60
I
L
I
0
I
5x167
I
1 x166
I
I
2 x 10-6
6x166 TITYUSTOXIN
1M 1
Fig. 3. Effect of TsTX concentration on the uptake of ACh in cortical slices of rat brain: 0 control; l with TsTX. The slices were incubated in the presence of lOO/rM of ACh containing 1-3 ntci of 14C-ACh for 60 min at the indicated concentrations TsTX. Values are means kS.D. for three experiments. For other details see legend to Figure 1 and the text.
trations of ACh. The inhibition was maximum, about 40::, at a concentration of 1OOpM of ACh in the incubation medium. Figure 2 shows the inhibition of ACh uptake by the toxin as a function of incubation time. At 15, 30 and 60min, TsTX reduced the uptake by 15. 31 and 40% respectively. The inhibitory effect of
100
TsTX was dependent on the toxin dose (Fig. 3). At concentrations of 0.25, 0.5, 2.0 and 4.0pM, TsTX reduced the uptake of ACh by 21, 30. 34 and 36%. respectively. The inhibition reached a plateau at a concentration of 2pM of TsTX. Figure 4 shows a typical Lineweaver-Burk plot obtained for the uptake
’
ln g Y 5 -0”I 50 .
Fig. 4. Effect of TsTX on the variation of the reciprocal of uptake of ACh into rat brain cortical slices: 0 control; l with TsTX 2 x 10e6 M. The cortical slices were incubated for 6Omin at the indicated concentrations of ACh (IL3 &i of 14C-ACh). Values express the means &-SD. for three experiments. For other details see the legend to Figure 1 and the text.
SIX Table
M. V. GOMI.I. M. E. M. DAI and f’. R. Drur~ I. Effects of TsTX and TTS on the upiakc m rat brain cortical slices
Tetrodotoxin 5 x 10~ i M ‘Tityustoxin 2 x IO ’ M Tityustoxin 2 x IO- ’ M
of h(‘h
.l(,~rto~~ii,t/il~,~~~[,~~f.\ --We are grateful to Dr David L. Nelson for reading thi< manuscript. We thank Terezinhn Stockier Barhosa and Anthnio Soares Usual dos Santos for expert technical assistance. We we grateful to Miss Marly Fonseca Silvn for secretarial assistance.
94.7 5 4.95s 63.7 i 3.45+ 93.4 + 6.2*
PIUS
Tctrodotoxin
of thi: toxin on AC’h uptake t.Table 1t. Thus. the observed i~lhibitory effect on ACh uptake. caused h> TSTX might possibly be cxpiained as the result of an increased release of the ACh that is taken up.
5 x IO-’ M
Brain cortical slices were rncuhated for hOrnin as indicated in the legend to Figure 2. Values represent the means *SD. for 3 experiments. For other details see the text. Tests for significance * P < 0. I ; i P < 0.005.
Aprison. M. H. and Takahashi. R. (1965). Biochemistry of the acian central nervous system. J. .~‘~,~r~~~~~~~~~. 12:
?‘I 230. of ACh in the presence or absence of 7 /tM of TsTX. The toxin caused a noncompetitive inhibition of ACh uptake. K, = 2 x 10e4M. Table 1 shows the effect of tetrodotoxin (TTX) on the uptake of ACh in the presence of TsTX: TTX had no cffcct on ACh uptake. but. incubated in the presence of TsTX. it prevented the inhibitory effect caused by the scorpion toxin.
In the presence of the cholinestcrasc jnhib~tor. DFP. to prevent ACh hydrolysis. TsTX reduced the uptake of ACh in rat brain cortical slices. This effect was dependent on toxin concentration. incubation time and ACh concentration in the incubation medium. The ability to alter the ACh uptake seems to he a common process for drugs that increase (Mitchel, 1963; Liang and Quastcl, 1969: Polak, 1969) or decrease (Carson. Jenden and Noble. 1975) ACh release. Tityustoxin increased the release of free ACh thus reducing tissue ACh of cortical slices of rat brain (Gomez er ui.. 1973. 1975), while. in the present paper. the toxin reduced the ACh uptake. Being a basic protein, TsTX carries a positive charge at physjoiogical PH. It has been shown that drugs charged with a cationic group could inhibit ACh
uptake
by a mechanism
of competition
for the
(Polak. 1969). In spite of its charge. the effect of TsTX on the inhibition of ACh uptake could not be explained by this mechanism. since the inhibition caused by the TsTX was noncompetitive (Fig. 4). The effect could not be atanionic
tributable
sites in the membrane
to
a cholinesterase
or
phospholipase
ac-
of the toxin since the venom from the scorpion 7ir 1’14s.s~,r~~{~~~~f~.~ is completely devoid of these activities (Dini and Goncalvcs, 1960). ietrodotoxin, a drug that had no effect on ACh
tivity
uptake
(Liang
and
Quastel.
1969).
inhibited
the
of ACh stimulated by TsTX (Gomer tit tri.. 1973; lY75) and is also capable of inhibiting the effect
release
Carson. V. G.. Jcnden. D. J. and Noble. E. P. (1975). Acctylcholinc (A&h) uptake in cortical slabs from C5-BI mice after acute and/or chronic ethanol treatment. P,oL,. U’rsr. Pkornlclc,. 19: 341 345. Dimz. C. R. and Con&es. J. M. (1960). Separation 01 biolo~lcally actice components from scorpion venoms by zone electrophoresis. Biochim. hioph!,s. Ac,rcc 41 : 470-477. Gomez, M. V. and Diniz. C. R. (1966). Separation of toxic components from Brazilian scorpion T. serrdntus. Mrrm irw. BzcrfInrrrt~ 33: 889-902. Gctmer.. M. V.. Dai, M. E. M. and Diniz. C. R. (197.1). Effect of scorpion venom, tityustoxin on the release of ~lc~tylcho)ine from incubated slices of rat brain. J. Nrwrxh<‘nr. 20: 1051~~l061. Gomez, M. V.. Diniz. C. R. and Barbosd, T. S. (1975). A comparison of the effects of scorpion tityustoxin and ouabain on the release of acetylcholine from incubated slices of rat brain. J. Ncwochew 24: 331-336. Heilbronn. E. (1969). The effect of phospholipases on the uptake of atropinc and acetylcholine by slices of mouse and brain cortex. J. Nrurochm. 16: 627-635. Heilhronn. E. and Ccdergren. E. (1970). Chemically induced changes in the acetylcholine uptake and storage capacity of brain tissue. In: Druys und Cholinwyic Mwlrur~isnts11~rhe C’NS (Heilbronn, E. and Winter. A.. Eds). pp. 245 269. Forsvarets Forskningsanstalt, Research Institute of National Defence, Stockholm. Kat/. B. and Miledi. R. (1967). Ionic requirements ol synaptic transmitw release. ,X‘urwc 215: 6.51. Kuhar. M. J. and Simon, J. R. (1974). Acctylcholinc uptake: lack of association with cholinergic neurons. .I. h’cwoc4wt~~. 22: I I3S- 1136. Liang. C‘. C. and Quastel, J. H. (1969). ElTects of drugs on the uptake of acetylcholine in rat brain cortex slices. Biochor~. Phimmrc. 18: I I X7 I 194. Mitchel. J. F. (1963). The spontaneous and evoked release of acetylcholine from the cerebral cortex. J. Physiol. 165: 116. Polak. R. I.. (1969). The influence of drugs on the uptake of acetylcholine by slices of rat cerebrat cortex. Br. .I. Phiirrtltr<. 3h: I44 152. Polak, R. L. and Mceuws. M. M. (1966). The influcncc of atropine on the release and uptake of acetylchohne
by the is&ted
cerebral cortex of the rat.
Biochrru.
f’frc~r-
IPlilC. IS: 989 -992.
Schubcrth. J. and Sundwall, A. (1967). Effects of some drugs on the uptake of acetylcholinc in cortex slices of mouse brain. J. Nwrochwl. 14: 807 8 12.