492

2a

ABSTRACTS

b

Figure 2. Transformation by mAch of intrinsrc bursts induced by depolarizing current injection (a) into a tonic AP tiring (b). c. Superimposed synaptically induced responses in control condition (burst) and during mAch application (depression of burst). Horizontal bar: pulse of depolarizing current (250 ms. 0.02 nA). Vertical bar: amplitude (50 mV). Triangles: synaptic stimulation.

In epileptic preparations, synaptic bursting wras diminished or blocked by Ach and CCh. This effect wts ascribed to muscarinic mechanisms because it was obtained with mAch (figure 2c) and reduced by atropine. When Cch was applied in combination with atropine, we ohserved a small hyperpolarization of the membrane potential associated with a diminished response to depolarizing current injection and to synaptic stimulation. Contrarily to the other cholinergic agonists, nicotine potentiated synaptic bursts (,figure la. h): under nicotine, the synaptic bursts persisted when the membrane potential was either depolarized by nicotine or hrought back at resting level by current injection (figure 1~).

In bursting pyramidal cells, Ach and Cch increased the number of bursts induced by depolarizing current injection, This effect was also induced by nicotine that favoured the appearance of spontaneous bursting activity. In contrast, mAch, when applied on bursting cells of GWS rats, provoked a transformation of bursts into a tonic AP firing associated with the slow depolarization (fig- ure 2ri,b). When mAch was applied in combination with atropine. the membrane potential was not significantly modified and the epileptic bursts were activated instead of being trasformed in a tonic discharge of APs.

These results suggest that hoth cholinergic receptors are in- volved in the epileptogenic GWS with opposite effects. The ac- tivation of metabotropic muscarinic receptors (by mAch) depressed the ‘epileptic like’ discharges while the activation of ionotropic nicotinic receptors (by nicotine) facilitated them. The simultaneous activation of both types of receptors (by Ach or Cch) had a complex effect favouring intrinsic bursting and depressing synaptic bursts. Interestingly, Cch was more efficient than Ach

and, in some cells, provoked oscillatory variations of the mem- brane potential associated with an important activation of AP fi- ring. This observation might therefore provide a basis for a cholinergic modulation of the epileptic activity through the si- multaneous activation of muscarinic and nicotinic receptors. Our results are also supported by recent findings showing that. in the GABA-injected cortical area, the number of choline acetyltrans- ferase immunopositive neurones is significantly more important in GWS rats than in control mts (Araneda et al., 1094). Given that the cholinergic innervation of the cerebral cortex in normal rats mainly comes from the basal forebrain, the preceding obser- vation suggests the appearance in epileptic rats of neurons neo- synthetizing choline acetyltransferase. This work was supported by a grant from ECOS. U95EOl.

References

Araneda S: Silva-Barrat C: Menini C; Naquet R. High expression of noradrenaline, cholme acetyltransferase and glial ftbrillary acidic protein in the epileptic focus consecutive to GABA withdrawal. An immunocytochemical study. Brain Res. 1994, 655:135-146.

Brailowaky S; Kunimoto M; Menini Ch: Silva-Batrat C; Rrche D; Naquet R. The GABA-withdrawal syndrome: a new model of focal epileptogenesis. Brain Res. 1988,442: 175-I 79.

Silva-Barrat C.. Araneda S.. Menini Ch., Champagnat J., and Naquet R. Burst generation in neocortical neurons after GABA withdrawal in the rat. J. Neurophysiol. 1992,67:715-727.

Silva-Barrat C.. Champagnat J.. Brailowsky S.. Menini Ch. and Naquet R. Relatronship between tolerance to GABAA agonist and hursting properties in neocortical neurons during GABA-withdrawal Syn- drome. Brain Res. lYX9. 498:28Y-298:

S. Simon”, A. Le Gaff”, Y. Frobertb. J. Grassi’, J. MassouliC”

“Laboratoire de Ntwrobiologie Molt%ulaire et Crllulaire, l&vie Normale Supkieure, 46 rue d’Uhn. Paris, Frrrnce ‘CEA, .service de Pharmacologic et d’lmmunologie, Centre d’Etudes de Sacla?; G$-sur-Yvetre. France

In acetylcholinesterase (AChE), acetylcholine is guided along aro- matic residues to the active site, located at the bottom of the

catalytic gorge addin (Sussman et al., 1991). The way of exit of the products is unknown and this led to the hypothesis of a ‘back

door’, opening at the bottom of the gorge (Gilson et al., 1994).

However, this was not demonstrated experimentally and none of the known inhibitors binds to this region. AChE activity is irre- versibly blocked by active site inhibitors or by ligands that bind

to the peripheral site, located at the surface of the protein near

the entrance of the gorge.

We studied Electrophorus elecrricus AChE for several reasons.

Firstly, tt has been used extensively for studies of acetylcholine hydrolysis. Secondly, its catalytic turn-over is the highest among studied AChEs. Thirdly, three monoclonal antibodies that inhibit

specifically its catalytic activity have been isolated (Remy et al., 1995). Two of them recognized overlapping but different epitopes

Xth International Symposium on Cholinergic Mechanisms 493

(Elec-403 and Elec-410); their binding is competitive with that

of peripheral site ligands. The third antibody (Elec-408) binds to another site and its inhibitory effect is additive with the other

two antibodies, thus defining a new regulatory site on the enzyme. In order to determine the binding sites of these antibodies, we constructed chimeric molecules composed of parts of Elecrropho-

rus AChE and rat AChE (unrecognized by the antibodies). The sensitivity of these chimeras to the antibodies allows us to define

regions implicated in their recognition. By site directed mutage- nesis, we have been able to abolish the inhibitory effects of each of the three antibodies. These experiments confirm that two an- tibodies bind to the peripheral site and recognize distinct epitopes with at least one common residue.The third antibody binds to another part of the enzyme, corresponding to the theoretical ‘back door’. We hope that an analysis of the inhibition mechanism of this antibody will allow us to confirm the ‘back door’ hypothesis and to analyze its possible function during the catalytic cycle.

A ribbon three-dimensional model of Electrophorus AChE. The entrance of the catalytic gorge is indicated by an arrow; the active serine of the catalytic site and residues involved in the binding of the three antibodies are shown in space filling view.

References

Suasman, J. L., Hare], M.. Frolow. F.. Oefner, C., Goldman, A., Toker. L. and Silman, I. (1991) Atomic structure of acetylcholinesterase from Torpedo californiccr: a prototypic acetylchohne-binding pro- tein Science 253, 872-879.

Gilson, M. K., Straatsma, T. P., .Mc Gammon, J. A.. Rtpoll, D. R.. Faerman. C. H., Axelsen, P. H., Silman. 1. and Sussman, I. L ( 1994) Open “back door” in a molecular dynamtcs simulation (11 acetylcholinesterase. Science 263, 3276-1278.

R&y, M.H., Frobert, Y. and Grassi, J. (1995) Characterization 01 me noclonal antibodies that strongly inhibit E/ecrr@~,horu.r elerfricn\ acetylcholinesterase. J. Biochem. 231, 651-658.

induced by activation of muscarinic m3 receptors

B.E. Slack

Department of Pathology und Laborutor~ Medicine. Boctorr University School of Medicine. 85 Eust Newton Street, Rm MI007, Boston, MA 02118, USA

Muscarinic receptors are characterized by seven transmembrane domains, and signal by coupling to heterotrimeric GTP binding

(G) proteins. Of the five muscarinic receptor isoforms that have been cloned (Peralta et al., 1987; Bonner et al., 1988); three (ml. m3, and m5) stimulate phosphatidylinositol 4,5 bisphosphate (PIP2) breakdown, and two (m2 and m4) inhibit adenylyl cyclase (Peralta et al., 1988; Sandmann et al., 1991). Activation of PIPZ- coupled receptors results in hydrolysis of PIP2 to form diacylgly- cerol and inositol-1,4.5-trisphosphate, with subsequent activation of protein kinase C (PKC). Muscarinic receptors activate other ki-

nases as well, including mitogen-activated protein kinase (Crespo et al., 1994). and tyrosine kinases of the Src family (Wan et al., 1996; Taai et al.. 1997). A variety of proteins are tyrosine phos- phorylated following muscarinic receptor activation, including the

focal adhesion-associated proteins paxillin and focal adhesion ki- nase (FAK) (Gutkind and Robbins, 1992; Petryniak et al., 1996).

Focal adhesions are attachment sites found in cultured cells; at these sites the extracellular domains of cell-surface integrins bind to immobilized extracellular matrix (ECM) proteins such as fibronectin (Burridge et al., 1988). Focal adhesion formation is initiated experimentally by the plating of cell suspensions onto immobilized ECM proteins (Burridge et al., 1997). and results in clustering of the mtegrins, and the association of their intracellular domains with cyt.oskeletal proteins that anchor polymerized actin

filaments (stress fibers) to these sites. A number of signaling proteins are recruited to focal adhesions, including the adapter protein paxil-

lin, and the tyrosine kinase FAK (Clark and Brugge, 1995). Thus focal adhesions have both structural and signaling functions. Focal adhesions are also formed following the addition of growth factors or G protein-coupled receptor ligands to quiescent cells (Ridley and Hall, 1992; Seutferlein and Rozengurt, 1994). Exposure either

to immobilized ECM proteins, or to soluble receptor ligands. results in transient tyroaine phosphorylation of similar sets of pro- teins. including tensin, pl30’“‘. paxillin and FAK (Burridge et al. 1997). In tibroblasts. G protein-coupled receptor-mediated tyrosine phosphorylation and focal adhesion formation are dependent on

cytoskeletal integrity, on activation of the small molecular G-protein Rho. and on actomyosin contmctility (Rankin et al., 1994; Chrza- nowska-Wodnicka and Butridge, 1996; Sinnett-Smith et al., 1993)

The experiments described in this report were carried out in human embryonic kidney cells stably expressing muscarinic m3 receptors (HEK-m3). Treatment of serum-deprived monolayera of HEK-m3 cells with the muscarinic receptor ligand carbachol rc- sulted in time- and concentration-dependent increases in tyrosine phosphorylation of the proteins paxillin and FAK (assessed by Western blot). The response approached maximal values within

10 min, and at a carbachol concentration of I pM. Although tyrosine phosphorylation of paxillin and FAK elicited by a variety of receptor ligands in Swiss 1T3 cells was reported to be tndc pendent of PKC (Sinnett-Smith et al., 1993). the response to carp bachol of HEK-m3 cells appeared to be partially dependent on this enzyme, since it was reduced in the presence of 2.5 pM 01