1. Specific [3H]-guanosine binding sites in rat brain membranes
- Author
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Michel P. Rathbone, Eva S. Werstiuk, Renata Ciccarelli, Giulia Bombi, Ugo Traversa, and Patrizia Di Iorio
- Subjects
Pharmacology ,G protein ,Guanine ,Guanosine ,Biology ,Adenosine ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Guanosine binding ,medicine ,Extracellular ,Purine metabolism ,Nucleoside ,medicine.drug - Abstract
Extracellular guanosine has diverse effects on many cellular components of the central nervous system, some of which may be related to its uptake into cells and others to its ability to release adenine-based purines from cells. Yet other effects of extracellular guanosine are compatible with an action on G-protein linked cell membrane receptors. Specific binding sites for [3H]-guanosine were detected on membrane preparations from rat brain. The kinetics of [3H]-guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122×107 M−1 min−1 and 1.69 min−1, respectively. A single high affinity binding site for [3H]-guanosine with a KD of 95.4±11.9 nM and Bmax of 0.57±0.03 pmol mg−1 protein was shown. This site was specific for guanosine, and the order of potency in displacing 50 nM [3H]-guanosine was: guanosine=6-thio-guanosine>inosine>6-thio-guanine>guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine caffeine, theophylline, GDP, GMP and ATP were unable to significantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well-characterized receptors for adenosine and purines. The addition of GTP produced a small concentration-dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G-protein. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine. Keywords: Guanosine, rat brain membranes, purine analogues, guanosine binding sites Introduction Guanosine is a naturally occurring compound that, like its adenine-based counterpart adenosine, shows a spectrum of biological activities. Adenine-based purines are released extracellularly from many cell types, including neurons and astrocytes, and function as important intracellular signalling molecules that mediate diverse biological effects (Rathbone et al., 1998a, 1998b; 1999). Recently it has become recognized that both neurons and glia also release guanine-based purines. GTP is stored in synaptic vesicles (Wagner et al., 1978) and indirect evidence indicates that guanosine can be released from neurons following depolarization (Fredholm & Vernet, 1979). Moreover the amount of guanine-based purines released from astrocytes under both basal conditions and after various types of stimulation, including hypoxia/hypoglycemia, is much greater than that of their adenine-based counterparts (Ciccarelli et al., 1999). In cultured astrocytes inhibition of ecto-5′-nucleotidase activity by α,β-MeADP significantly reduces accumulation of extracellular guanosine indicating that it, like extracellular adenosine, is largely derived from the extracellular metabolism of guanine-derived nucleotides (Caciagli et al., 2000). Both extracellular guanosine and GTP can exert trophic effects in the nervous system (Rathbone et al., 1998b; 1999), including stimulation of astrocyte proliferation (Kim et al., 1991; Ciccarelli et al., 2000) synthesis and release of trophic factors from astrocyte cultures (Middlemiss et al., 1995; Caciagli et al., 2000) and the enhancement of differentiation of PC12 cells and hippocampal neurons in vitro (Gysbers & Rathbone, 1996a; Rathbone & Juurlink, 1993). Guanosine also exerts neuroprotective effects in vivo (Caciagli, personal communication) and in vitro (Rathbone et al., 1998b; Caciagli et al., 2000). Some of the actions of the guanosine may be mediated intracellularly after its uptake. Yet many trophic effects of guanine-based purines are not substantially affected by the nucleoside uptake inhibitors, such as NBTI or dipyridamole (Gysbers & Rathbone, 1992), indicating that they are independent of intracellular mechanisms. Guanosine also stimulates the release of adenine-based purines from astrocytes, which may, in turn, be responsible for some other effects of guanosine (Ciccarelli et al., 2000). But this explanation is also incomplete, since many of the effects of guanine-based purines persist in the presence of adenosine-receptor and/or P2 purine receptor antagonists (Gysbers & Rathbone, 1992). An alternative possibility is that there are distinct receptors for guanine-based purines. Gysbers et al. (2000) found that PC12 cells expressed binding sites for GTP with features consistent with a cell membranes receptor. Moreover, several of the effects of guanosine may be mediated through G-protein dependent signalling pathways involving cyclic nucleotides or MAP kinase pathway (Rathbone et al., 1991; Gysbers & Rathbone, 1996b; Caciagli et al., 2000) raising the possibility that some of the effects of guanosine involve activation of cell-surface receptors. If the brain does indeed contain specific receptors for guanosine, it should be possible to identify a high-affinity binding site for guanosine in brain membranes. This study reports the characterization of such a guanosine binding site.
- Published
- 2002