Feb 14, 1991 - Oxford University Press believed that ACh release is ensured by fusion of synaptic vesicles with the presynaptic membrane and exocytosis of.
The EMBO Journal vol. 10 no.7 pp. 1 671 - 1675, 1991
Release of acetylcholine by Xenopus oocytes injected with mRNAs from cholinergic neurons
Antonella Cavalli, Lorenza Eder-Colli, Yves Dunant2, Franpoise Loctin and Nicolas Morel1 Departement de Pharmacologie, CMU, 1211 Geneve 4, Switzerland and 'Neurochimie, Laboratoire de Neurobiologie Cellulaire et Moleculaire, CNRS, 91198 Gif-sur-Yvette, France 2Corresponding author Communicated by J.Mallet
Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain the perikarya of 120 000 cholinergic neurons that innervate the electric organs and are homologous to motor neurons. The injected oocytes accumulated acetyicholine and were able to synthesize [14C]acetylcholine from 1-['4C]acetate. With KCI depolarization and upon treatment with a Ca2' ionophore, they released their endogenous as well as the radiolabelled neurotransmitter in a Ca2+-dependent manner. No synthesis or release were obtained from control oocytes. With respect to their dependency upon Ca2+ concentation, the oocytes injected with Torpedo electric lobe mRNAs released acetylcholine in a manner which closely resembled that found in the native synapses. In contrast to the controls, primed oocytes were also able to release [14C]acetylcholine that was injected a few hours prior to the release trial. inmnunoblot analysis demonstrated that the 15 kd proteolipid antigen of the purified mediatophore, a 200 kd presynaptic protein able to translocate acetylcholine, was expressed in the ACh- releasing oocytes but not in the controls. The present observation may provide a useful approach for investigating the proteins involved in the release of acetylcholine and of other neurotransmitter substances. Key words: acetylcholine release/synaptic transmission/ Torpedo electric organlXenopus oocyte -
Introduction Synaptic transmission of a nerve impulse is an extremely rapid event in which a signal is transferred from one cell to the next by means of a brief chemical impulse. Acetylcholine (ACh) is the neurotransmitter substance that mediates transmission at the neuromuscular junction and in the Torpedo electric organ which is actually a modified neuromuscular system. Electric organs have provided very useful preparations for molecular studies of cholinergic neurotransmission, especially for investigations of the mechanisms by which ACh activates its receptors on the postsynaptic membrane. On the other hand, much less in known on the molecules involved in the release of ACh pulses by the presynaptic motor nerve endings. It is generally Oxford University Press
believed that ACh release is ensured by fusion of synaptic vesicles with the presynaptic membrane and exocytosis of their neurotransmitter content (Ceccarelli and Hurlbut, 1980). However, experiments on the Torpedo electric organ and on other cholinergic systems have also suggested that a mechanism located in the presynaptic plasma membrane might use cytosolic ACh and release it in the synaptic cleft (Muller et al., 1987a). Among the proteins involved in ACh release, the mediatophore protein might play a role (Israel et al., 1984). This protein has been purified from the presynaptic plasma membrane. When incorporated into liposomes prepared with synthetic lipids, it endows them with the ability to release ACh in a Ca2+-dependent manner. The native mediatophore has .an apparent molecular mass of 200 kd but upon dissociation by SDS, the only subunit detected after gel electrophoresis is a 15 kd proteolipid (Israel et al., 1984, 1986; Birman et al., 1986). Using monoclonal antibody mAb 15 kl it was found that the 15 kd proteolipid antigen was present in similar amounts in the presynaptic plasmalemma and in the membrane of synaptic vesicles (Morel et al., 1991). Injection of mRNAs and cDNAs into Xenopus oocytes has provided a powerful tool during the past decade for studying the function of postsynaptic receptors and ion channels (Barnard and Bilbe, 1987). The oocyte was used to investigate proteins involved in the presynaptic machinery as well. For example, Gundersen et al. (1985) extracted poly(A)+ mRNAs from the electric lobes of Torpedo and injected this material into oocytes; after a few days choline acetyltransferase activity (ChAT) was expressed and a certain amount of ACh was synthesized from endogenous precursors. The purpose of the present work was to investigate whether such 'cholinergic' oocytes have the capacity to release ACh in a Ca2+-dependent way and to compare the release occurring in this reconstituted system with that observed in the natural synapses. -
Results ACh synthesis by oocytes primed with mRNAs from the electric lobe Poly(A)+ mRNAs were extracted from the electric lobes of Torpedo marmorata and purified using standard techniques. Xenopus laevis oocytes (stage VI) were injected with 50-70 ng mRNAs and maintained at 22°C. Control oocytes were injected either with water or with an equivalent amount of poly(A)+ mRNAs extracted from the Torpedo electric organ (which contain electroplaques as well as axons and nerve terminals of the cholinergic neurons but not their perikarya, that is, not their nuclei). The expression of cholinergic properties in oocytes was monitored by assaying ChAT activity and ACh content. Both increased gradually following injection and reached a maximum value (indicated in Table I a, b) on the fourth day. In control oocytes ChAT activity and ACh content were very
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A.Cavalli et al. Table I. Expression of cholinergic properties in Xenopus oocytes four days after injection of mRNAs extracted from the Torpedo electric lobe, and comparison with controls injected either with water or with mRNAs extracted from the electric organ.
(a) Choline acetyltransferase (nmol/min) (b) ACh content (pmol) (c) ACh released with A23187 (pmol) (d) ACh released with 55 mM KCl (pmol) (e) [14C]ACh synthesized by oocytes incubated with [14C]acetate (Bq) (f) 14C radioactivity released by g) oocytes with A23187 (Bq) (g) 14C radioactivity released by g) oocytes with 55 mM KCI (Bq)
Water (Controls)
Torpedo electric lobe mRNAs
Torpedo electric organ mRNAs
0.15 E 0.18 (3) 0.24 + 0.08 (10) n.d. (3) n.d. (10) n.d. (12) 6.12 i 1.85 (11) 3.15 + 0.55 (14)
1.62 165 75 72 11.06 16.25 8.45
n.d. (2) 0.62 + 0.10 (3)
i 0.11 (4) ± 15 (lO)a + 26 (3) + 17 (10) 4 1.32 (12) + 2.45 (Il)a + 1.23 (14)a
-
0.6 (2) -
1.56
0.72 (3)
Values are expressed as mean S.E. per oocyte; number of experiments in brackets (6- 10 oocytes were tested in each experiment). The sign n.d. means not detected. (a) Choline acetyltransferase activity. (b) ACh content of oocytes. (c) ACh release obtained with oocytes treated for 5 min with A23187 in the presence of 10 mM CaC12 and 1 itM eserine, as in Figure 1. (d) ACh release similarity obtained from oocytes treated for 10 min with the 55 mM KCl medium. In experiments (e-g) the oocytes were incubated overnight with 1 MCi/ml 1-[14C]acetate; the amount of [14C]ACh extracted from the oocytes at the time of the release experiment is indicated in (e); the amount of radioactivity released in the medium after 5 min of A 23187 treatment (10 mM CaCl, no eserine) is shown in (f); the release elicited in the elevated KCl medium in the presence of 10 mM CaC12 for 10 min is shown in (g). ap < 0.01 with paired Student t test.
low or not detectable. This confirmed the observations of Gundersen et al. (1985). Nerve terminals in the Torpedo electric organ use acetate as a preferred external precursor for synthesis of the acetyl moiety of the ACh molecule (Israel and Tucek, 1974). The synthesis of ACh from acetate was also induced in oocytes injected with mRNAs from the electric lobe. When incubated with 1-[14C]acetate, the primed oocytes synthesized [14C]ACh at a rate that was maximal four days after injection of the messenger. No [14C]ACh synthesis was detected in controls oocytes (Table I e).
a) mRNA
b) mRNA
Control
(1 OmM CaCI2)
(O CaCI,)
(10mM CaCI,)
15 pmol 2 min
ACh standards
a,
Transmitter release by primed oocytes An ACh release experiment is illustrated in Figure 1. Oocytes were incubated for 5 min in a medium containing a Ca2+ ionophore (A23187), and also eserine (10-6 M) to prevent ACh hydrolysis. Subsequently, the oocytes were removed and ACh was assayed in the medium. No ACh was detected in the medium of control oocytes whereas those injected with electric lobe mRNAs liberated a substantial amount of transmitter, provided that calcium was present in the solution. On average, the oocytes treated for 5 min with ionophore A23187 in the presence of 10 mM Ca2+ released 75 pmol ACh, that is, 45% of their initial ACh content (165 pmol/oocyte, see Table I b,c). Another way to trigger ACh release was to depolarize the oocytes in a medium containing a high KCI concentration (55 mM) with an equivalent reduction of NaCl. Again, the oocytes primed with electric lobe mRNAs released ACh in the medium provided that Ca2+ was present. The amount of transmitter released with KCI was similar to that released with the A23187 trial (Table I c,d).
Release of [14C]ACh injected into oocytes before stimulation Control oocytes did not accumulate any endogenous ACh. It was therefore not surprising that they were unable to release the neurotransmitter. To investigate more precisely if the release mechanism is really absent in control oocytes, we designed the following experiments. Oocytes at the fourth day after mRNAs injection and control oocytes were reinjected with 83 Bq of [14C]acetylcholine prior to the
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10
| 10
10
a, 0. E
(a
(a
C,)
CO
w
.m
5
0
Fig. 1. (a) ACh release by oocytes injected with mRNA from cholinergic neurons. Four days after injection of 1 ng poly(A)+ mRNAs extracted from the Torpedo electric lobes, oocytes were incubated for 5 min in a medium containing the calcium ionophore and eserine. Panel a shows the assay of the ACh recovered in the medium. The light emission at the beginning of the trace resulted from the addition of 10 ,u of the incubating medium (sample) and was due to oxidation of choline and possibly other compounds present in the fluid. Then 4.5 U of acetylcholinesterase (AChE) were added which was sufficient to overcome the inhibition by eserine and to hydrolyse the ACh specifically; this produced the second peak of light emission (arrow). Subsequently, standard doses of an ACh solution were applied to calibrate the response. (b) Other mRNA-primed oocytes from the same pool were incubated in the same medium except that CaCl2 was omitted and 10 mM EDTA present. No ACh release took place. Control: the same experiment was made with control oocytes (injected with water). CaCl2 was present but no ACh could be detected in the medium at the end of incubation.
release experiment. Three hours later a few oocytes of each group were treated with TCA for extraction and measurement of their ACh content, the remaining was used for testing ACh release with the ionophore A23187 method. Extraction showed that 80 % of the injected labelled transmitter was still present at the time of the release experiment (65.8 Bq in controls and 69.5 Bq in the primed oocytes). Upon stimulation with ionophore A23187, only those oocytes injected with mRNAs from the electric lobe released signifi-
Neurotransmitter release expressed in oocyte 10-
a) QWy
10-
(KCI)
O
b) Oocyaes (A 23187) 0
1 3 1
& g
3
0 0
a
i
L
V
5-
5O
0
0
5
I10
n-X
0
....
.. 5
lO
*4,4F Dal
E .E 9
& Fig. 3. Detection of the Torpedo presynaptic 15 kd proteolipid antigen in injected oocytes. The first four lanes show the reaction obtained with the indicated amounts of mediatophore purified from plasma membranes of Torpedo synaptosomes. The other lanes refer to the material extracted from 1 and 3 oocytes injected either with water (C, controls), or with electric organ mRNAs (0), or with electric lobe mRNAs (L). The reaction indicating the presence of the 15 kd antigen is present only in extracts from the electric lobe primed oocytes, corresponding in this experiment to - 1 ng mediatophore per oocyte.
(D E al
Ca2W concentration (mM)
Fig. 2. Ca2 +-dependency of transmitter release in oocytes injected with electric lobe mRNAs. (a) Release triggered by high KCI as a function of Ca2+ concentration. The different symbols refer to experiments carried out on different oocyte preparations. (b) Release triggered by A23187 as a function of Ca2+ concentration. (c) Ca2+ dependency of synaptic transmission tested on intact Torpedo electrogenic tissue. Release was measured either electrophysiologically by recording the amplitude of the electroplaque response in response to a single stimulus (open symbols), or by assaying the labelled neurotransmitter overflowing from the tissue in response to a brief train of impulses (filled symbols; from Dunant et al., 1980; Muller et al., 1987b). (d) Release measured by luminescence and triggered by A23187 from nerve terminals (synaptosomes) isolated from the Torpedo electric organ (filled symbols) and from proteoliposomes containing the mediatophore protein extract from the Torpedo electric organ presynaptic membrane (open symbols; from Birman et al., 1987).
cant amounts of the radiolabelled transmitter (6.9 Bq per oocyte) whereas control oocytes only released negligible amounts of radioactivity (0.3 Bq per oocytes; 2 experiments with 9 oocytes in each). Therefore, the mechanism of release was apparently not present in control oocytes whereas it was
expressed in those primed with electric lobe mRNAs.
Ca2, -dependency of ACh release [14C]ACh accumulation using [14C]acetate proved to give an easy
test for ACh release. The oocytes were incubated
overnight with [14C]acetate; the labelled precursor was then removed and the oocytes, either 'cholinergic' or controls, were treated with ionophore A23187 in the presence or absence of Ca2+. In other experiments the oocytes were depolarized in the high KCl medium. Control oocytes lost in the medium a small amount of radioactivity (1-10 Bq per oocyte) that was equivalent with and without Ca2+. Thus control oocytes were used in all experiments to provide a base-line value for the 'cholinergic' oocytes that were tested in parallel. With KCl depolarization, the oocytes primed with electric
lobe mRNAs released radioactivity in addition to the baseline value provided that Ca2+ was present (Figure 2a). The dependency of ACh release with different Ca2+ concentrations had a steep slope and compared well with the relationship observed for synaptic transmission in situ, either by recording the amplitude of the electroplaque potential in response to a single nerve stimulus, or by measuring the amount of ACh overflowing after a brief train of impulses (Figure 2c; Hill coefficient 3-4, see Dunant et al., 1980; Muller et al., 1987b). With the ionophore A23187, transmitter release was also dependent on the Ca2+ concentration but the relationship was somewhat different; the slope was less steep than with KCl (Figure 2b). This relationship resembled that observed when ACh release was triggered by A23187 in nerve terminals (synaptosomes) isolated from the electric organ and in proteoliposomes equipped with the mediatophore protein (Figure 2d; see Birman et al., 1986). With a CaCl2 concentration (10 mM) ensuring a maximum rate of release, the amounts of 14C-radioactivity liberated by primed oocytes over the blank values were 10. 13 Bq and 5.30 Bq with A 23187 and KCl, respectively (Table I f,g). This represents an important proportion of the [14C]ACh present in such oocytes prior to the release trial (Table I e). This will be analysed in the Discussion. Magnesium ions are known to antagonize the effects of Ca2+ on transmitter release. In an experiment, ACh release was triggered by KCI depolarization in the presence of 1 mM CaCl2 with and without 0.5 mM MgCl2. Release was reduced to 53% by Mg2+ (1 experiment, mean of 9 oocytes for each condition). We also tested the effect of cetiedil on the amount of ACh released by oocytes. Cetiedil is a drug that blocks ACh release from intact synapses, from synaptosomes and from proteoliposomes containing the mediatophore (Morot1673
A.Cavalli et al.
Gaudry-Talarmain et al., 1989). In two experiments, cetiedil (250 ,4m) inhibited [14C]ACh release from our injected oocytes by 86% and 100%, respectively Expression of the mediatophore subunit in primed oocytes After the release experiments, the oocytes were frozen, a proteolipid fraction were prepared by extraction in organic solvents followed by analysis for the presence of the 15 kd proteolipid subunit of the mediatophore by immunoblotting using the monoclonal antibody mAb 15 kl. The antigen could be detected in organic extracts derived from a single mRNA-primed oocyte but not from control oocytes (Figure 3). By comparison with known amounts of mediatophore purified from Torpedo synaptosomes, it was possible to estimate that injected oocytes had synthesized 1-10 ng antigen after a 4 day period. Controls injected with mRNAs extracted from the Torpedo electric organ As mentioned above, the electric organ does not contain the nuclei of the cholinergic neurons and oocytes injected with mRNAs from this tissue did not express choline acetyltransferase and did not accumulate ACh (Gundersen et al., 1985; Table I a, b). Similarly, these oocytes did not synthesize [14C]ACh from [14C]acetate and did not release the radiolabelled transmitter (Table I e, g). The 15 kd proteolipid antigen was not detected in organic extracts of oocytes injected with mRNAs from the electric organ
(Figure 3).
Discussion The present results indicated that the process of ACh release can be induced in frog oocytes injected with mRNAs from cholinergic neurons. The electric lobe of Torpedo was used as an abundant source of relevant mRNAs since it is composed of a homogeneous population of cholinergic
perikarya. With respect to pharmacology and to the dependency upon Ca2+ concentration, ACh release induced in these oocytes was similar to that observed with the native synapses, either by using electrophysiology in situ (Dunant et al., 1980; Muller et al., 1987b), or by using isolated nerve terminals, or synaptosomes (Birman et al., 1986). Primed oocytes released a large part ( > 40%) of the ACh
they had accumulated (Table I b -d). This proportion was still greater for the radiolabelled transmitter (Table I e -g). It should be recalled in this connection that the radioactivity released by controls is not [14C]ACh and that the release of the labelled neurotransmitter can only be evaluated as the difference between primed oocytes and controls in these experiments (see Materials and methods). Nevertheless, the amount released is nearly as high as the content of [14C]ACh that was present in oocytes at the time of experiment. This suggests that the rate of synthesis was accelerated during stimulation. Experiments in progress support this view (unpublished data). On the other hand, the primed oocytes only released 10% of the [14C]ACh that was artificially injected 3 h before stimulation. It can be concluded that a good part of this injected transmitter was not available at the sites of release whereas the ACh synthesized by the oocytes was in a more appropriate
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situation, suggesting that the synthesizing machinery was expressed at the vicinity of the releasing mechanism. It was expected that a number of presynaptic proteins had been expressed in 'cholinergic' oocytes. Choline acetyltransferase activity was assessed here, confirming previous results (Gundersen et al., 1985). High affinity choline uptake was recently demonstrated using the same system by O'Regan et al. (1991). It has been proposed that a presynaptic membrane protein, the mediatophore, plays some role in the process of ACh release (Israel et al., 1984 and 1986). We therefore looked for this protein using mAb 15 kl and a very sensitive detection assay. The 15 kd proteolipid antigen was indeed expressed in the primed oocytes in measurable amounts. Obviously, the oocyte system described here should provide a good approach to assess the role of presynaptic proteins such as the mediatophore in the mechanism of ACh release. A further issue could be to use this technique to investigate the release of other neurotransmitters such as amino acids which are difficult to study in situ in the central nervous system.
Materials and methods Torpedo marmorata were anaesthesized with tricaine at a concentration of 0.33 g/l of seawater. The two electric lobes were carefully dissected and promptly frozen in liquid nitrogen. The mRNAs were isolated from 6-7 lobes by two extractions with guanidinium thiocyanate-phenol-chloroform, a modification of the method of Chomczynski and Sacchi (1987), that included a precipitation in 5 M LiCl. Diethylpyrocarbonate (0.05%) was added to all solutions to inhibit RNAses and all glass vessels and injection pipettes were treated with silicone to avoid adsorption. Then the RNA was washed in 75% and then 95% ethanol. It was dissolved in 0.5% SDS and isolated by chromatography on Type III oligo(dT) cellulose (from Collaborative Research Inc., Lexington, MA, USA) with the usual binding and elution protocol. Isolated poly(A)+ mRNAs were dried with speed vacuum centrifugation. The same method was used to obtain mRNAs from the Torpedo electric organ. Xenopus laevis oocytes at stage VI were injected with 50-70 ng mRNA freshly diluted in H20 (1 ng/nl). The oocytes were kept at 22°C in an oocyte medium containing 82.5 mM NaCl, 2.5 mM KCI, 1 mM CaC12, 1 mM MgCI2, 5 mM HEPES, 5 g/l Polyvinylpyrrolidon, NaOH to pH 7.5, until the day of experiment. Activity of choline acetyltransferase and measurement of ACh and [14C]ACh content ChAT activity was measured as described by Fonnum (1968) modified by Rossier et al. (1973). For the measurement of transmitter content, the oocytes were treated with trichloroacetic acid, homogenized and centrifuged. The acid was extracted with water-saturated ether from the supernatant. Unlabelled ACh was measured by luminescence (Israel and Lesbats, 1981), and [14C]ACh by scintillation spectroscopy after separation on high-voltage electrophoresis (Dunant et al., 1980).
ACh and [14]ACh release The oocytes were incubated for various times (usually 5 min) in a medium containing 82.5 mM NaCl, 2.5 mM KCI, 10 mM CaCI2, 10 $AM A23187 (a calcium ionophore) and 1 AM eserine (50 yI medium per oocyte). The oocytes were removed at the end of incubation and ACh was analysed in the medium by a luminescence reaction (Israel and Lesbats, 1981). Alternatively, ACh release was triggered for 10 min in a medium in which KCI was raised to 55 mM and NaCI reduced to 30 mM. CaCI2 was either absent or present at various concentrations to study the function describing the Ca2+ dependency of transmitter release. To study [ 4C]ACh release, primed oocytes and controls were incubated overnight in the presence of l-[14C]acetate (1 ACi/ml). Before the release experiment, the labelled precursor was washed out for 60 min in the standard oocyte medium. For the release run, the oocytes were individually transferred either to the high-KCImedium or to that containing 10 /LM ionophore A23187 as described above. In both cases the background leakage of radioactivity
Neurotransmitter release expressed in oocyte remained low with control oocytes (50-600 c.p.m./oocyte) at the end of the 5 min incubation for all the Ca2 + concentrations tested; this control value was taken as a blank and release by the mRNA injected oocytes can be measured by comparison with the blank. One experiment was designed to verify that the difference in the radioactivity released between primed and control oocytes really corresponded to [14C]ACh. Release was elicited in the high-KCl medium in the presence of a cholinesterase inhibitor (ecothiopate iodide, 1 PM). Controls released 3.75 Bq of 14C radioactivity per oocytes; primed oocytes released 11.0 Bq. The difference was therefore 7.3 Bq. Samples of the medium were run on high-voltage electrophoresis and, from the medium of primed oocytes, 8.0 Bq could be identified as [14C]ACh. No [14C]ACh was detected in the medium of control oocytes.
Detection of the mediatophore protein Pooled frozen oocytes were homogenized in a glass-glass Potter device with 0.1 mM EDTA, 10 mM Tris pH 8 buffer (10 oocytes/ml) followed by the addition of an equal volume of chloroform/methanol (v/v) mixture. After centrifugation (25 000 g, 30 min), the material at the interface between the aqueous and organic phases was collected and resuspended in 0.2 ml H20 and 5 ml chloroform/methanol (v/v) for 30 min on ice. Precipitated proteins were removed by centrifugation (25 000 g, 30 min). After evaporation of the organic solvents, extracted proteolipids were resuspended in H20 and delipidated by successive ether washings. SDS-gel electrophoresis was carried out on a 8-18% linear acrylamide gradient gel and protein bands were transferred onto nitrocellulose filters as previously described (Morel et al., 1989). A monoclonal antibody directed to the 15 kd proteolipid found in purified mediatophore fractions (mAb 15 kl) was allowed to bind overnight to the blot. Its binding was indirectly visualized using biotinylated anti-mouse Ig antibodies and streptavidin-conjugated alkaline phosphatase (Amersham, RPN 1001 and 1234). Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were used as substrates of the phosphatase.
Acknowledgements We are grateful to Dr D.Runger for help with the oocyte technology, to Drs M.Israel and A.C.Kato for useful criticism, and to N.Collet, S.Bonnet and F.Pillonel for assistance with the manuscript. The work was supported by a grant from the Fonds National Suisse pour la Recherche Scientifique.
References Barnard,E.A. and Bilbe,G. (1987) In Turner,A.J. and Bachelard,H.S. (eds) Neurochemistry: A Practical Approach. IRL Press, Oxford, pp. 243 -269. Birman,S., Israel,M., Lesbats,B. and Morel,N. (1986) J. Neurochem., 47, 433-444. Ceccarelli,B. and Hurlbut,W.P. (1980) Physiol. Rev., 60, 396-441. Chomczynski,P. and Sacchi,N. (1987) Anal. Biochem., 162, 156-159. Dunant,Y., Eder,L. and Servetiadis-Hirt,L. (1980) J. Physiol. Lond., 298, 185 -203. Fonnum,F. (1968) Biochem. J., 109, 389-398. Gundersen,C.B., Jenden,D.J. and Miledi,R. (1985) Proc. Natl. Acad. Sci. USA, 82, 608-611. Israel,M. and Tucek,S. (1974) J. Neurochem., 22, 487-491. Israel,M. and Lesbats,B. (1981) J. Neurochem., 37, 1475-1483. Israel,M., Lesbats,B., Morel,N., Manaranche,R., Gulik-Krzywicki,T. and Dedieu,J.-C. (1984) Proc. Natl. Acad. Sci. USA, 81, 277-281. Israel,M., Morel,N., Lesbats,B., Birman,S. and Manaranche,R. (1986) Proc. Natl. Acad. Sci. USA, 83, 9226-9230. Morel,N., Israel,M., Synguelakis,M. and Le Gal la Salle,G. (1989) Neurochem. Int., 15, 169-177. Morel,N., Synguelakis,M. and Le Gal La Salle,G. (1991) J. Neurochem., 56, 1401-1408. Morot-Gaudry-Talarmain,Y., Diebler,M.-F., Robba,M., Lancelot,J.-C., Lesbats,B. and Israel,M. (1989) Eur. J. Pharmacol., 166, 427-433. Muller,D., Garcia-Segura,L.M., Parducz,A. and Dunant,Y. (1987a) Proc. Natl. Acad. Sci. USA, 84, 590-594. Muller,D., Loctin,F. and Dunant,Y. (1987b) Eur. J. Pharmacol., 133, 225-234. O'Regan,S., Birman,S. and Meunier,F.M. (1991) Neurochem. Int., 19, 87-92. Rossier,J., Baumann,A. and Benda,P. (1973) FEBS Lett., 32, 321-324. Received on August 2, 1990; revised
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