Received: April 28, 2003.  Accepted: September 29, 2003.  Published online: September 10, 2004.

Inhibition of L-glutamate and GABA synaptosome uptake by crotoxin, the major neurotoxin from Crotalus durissus  terrificus venom

Cecchini A.L.^1,4; Soares A.M.^{2; Giglio J.R.3; Amara S.4; Arantes E.C.1}

1 Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, São Paulo, Brasil; 2 Unidade de Biotecnologia, Universidade de Ribeirão Preto (UNAERP), Ribeirão Preto, São Paulo, Brasil; 3 Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, São Paulo, Brasil; 4 Vollum Institute, Oregon Health Sciences University, Portland, Oregon, USA.

ABSTRACT. This paper describes a brief study on the crotoxin mechanism of action, regarding the transport of GABA and L-glutamate in rats cortico-cerebral synaptosomes and in heterologous systems, such as COS-7 cells expressing gabaergic transporters, and C6 glioma cells and Xenopus oocytes expressing glutamatergic transporters. Crotoxin concentrations over 1 mM caused an inhibitory effect of ³H-L-glutamate and ³H-GABA, and reversibly inhibited L-glutamate uptake by C6 glioma cells. When COS-7 cells were assayed, no inhibition of the ³H-GABA transport could be evidenced. Crotoxin kept its inhibitory effect on neurotransmitters uptake even when Ca²⁺ ions were removed from the medium, therefore, independently of its PLA2 activity. In addition, high concentrations (2 mM) of BPB did not avoid the action of crotoxin on the neurotransmitters uptake. Crotoxin also inhibited ³H-L-glutamate, independently on Na⁺ channel blockade by TTX. In addition, an evaluation of the lactic dehydrogenase activity indicated that uptake inhibition does not involve a hydrolytic action of crotoxin upon the membrane. We may also suggest that crotoxin acts, at least partially, altering the electrogenic equilibrium, as evidenced by confocal microscopy, when a fluorescent probe was used to verify cell permeability on C6 glioma cells in presence of crotoxin.

Key words: crotoxin, L-glutamate, GABA, Crotalus durissus terrificus.

Correspondence to:

E. C. Arantes - Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP; Avenida do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil. Phone: 55 16 6024275, fax: 55 16 6332960 E-mail: ecabraga@fcfrp.usp.br