805 Characterization of Imido [8- 3 H] Guanosine 5' -Triphosphate Binding Sites to Rat Brain Membranes Liane N. Rotta, 1,2,4 Félix A. A. Soares, 1 Cristina W. Nogueira, 3 Lúcia H. Martini, 1 Marcos L. S. Perry, 1 and Diogo O. Souza 1 (Accepted October 27, 2003) Besides their well-defined intracellular roles in transmembrane signals transduction, guanine derivatives play important roles by acting from the outside of neural cell membranes. These roles are mediated by two different pool sites in cell membranes: G proteins, which bind to specific (GDP and GTP) intracellular guanine derivatives, and sites that bind to extracellular guanine derivatives. In this study we investigated some methodological characteristics of both guanine derivatives binding sites (intracellular and extracellular) in rat brain neural membranes. By inves- tigating the binding of a poorly hydrolyzed GTP analogue and the adenylate cyclase activity in neural membranes, we observed some distinctiveness of guanine derivatives binding sites: stability to washing procedures (extracellular) and modulation of adenylate cyclase activity (intra- cellular). These results allow dealing with each site separately, which could be useful for discrim- inating the roles of extracellular and intracellular guanine derivatives in the central nervous system. KEY WORDS: Binding sites; guanine derivatives; G protein; adenylate cyclase. INTRODUCTION Most excitatory synapses in the central nervous sys- tem (CNS) have glutamate as a neurotransmitter, which acts via ionotropic and/or metabotropic receptors. Inotropic receptors are cation-specific ion channels, whereas metabotropic receptors are coupled to GTP-binding pro- teins (G proteins), modulating second messengers system (1,2). Besides the physiological roles of glutamate in plas- tic processes in the CNS, such as those involved in devel- opment and ageing, learning and memory modulation, and environmental interaction (1–4), overstimulation of the glutamatergic system is implicated in a variety of neuronal insults, including neurodegenerative disorders and acute brain injuries (5–8). The role of intracellular guanine derivatives (GDs) on transmembrane signals transduction is well known (9). G proteins are intrinsic membrane proteins that couple activated receptors to membrane effectors. When bound to guanosine diphosphate (GDP), G proteins are in the inactive state; when a neurotransmitter binds to its receptors, there is a conformational change in G pro- teins, which release GDP and bind guanosine triphos- phate (GTP). The active complex GTP/G protein modulates the activities of specific effectors, generating cell responses to the neurotransmitter. An intrinsic GTPase activity of G proteins hydrolyses bound GTP 0364-3190/04/0400–0805/0 © 2004 Plenum Publishing Corporation Neurochemical Research, Vol. 29, No. 4, April 2004 (© 2004), pp. 805–809 1 Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, 90035-003, Porto Alegre, RS, Brazil. 2 Curso de Farmácia, Universidade Luterana do Brasil, Canoas, RS, Brazil. 3 Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil. 4 Address reprint requests to: Liane N. Rotta, Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Rua Ramiro Barcelos, 2600-Anexo, CEP 900 35-003, Porto Alegre, RS, Brazil, Tel: +55 51 3316 5558; Fax: +55 51 3316-5540 or 5535; E-mail: lianerotta@hotmail.com