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1. 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. 2. Specific binding sites for [(3)H]-guanosine were detected on membrane preparations from rat brain. The kinetics of [(3)H]-guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122 x 10(7) M(-1) min(-1) and 1.69 min(-1), respectively. A single high affinity binding site for [(3)H]-guanosine with a K(D) of 95.4 +/- 11.9 nM and B(max) 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 [(3)H]-guanosine was: guanosine=6-thio-guanosineinosine6-thio-guanineguanine. 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. 5. The addition of GTP produced a small concentration-dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G-protein. 6. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.

Several studies have shown that guanine-based purines exert biological effects on the central nervous system, possibly through membrane receptor. In a parallel work, we have identified the first guanosine G protein-coupled receptor GPR23, known as LPA4 receptor, involved in the modulation of guanosine-mediated antiproliferative effects in human glioma cell lines. Here, we performed in different brain areas the following studies: by PCR, the expression levels of GPR23; by [3H]-Guanosine radioligand binding assay, the binding properties of GPR23; by [35S] GTPγS binding assay, the receptor activation properties of guanosine. Among the examined areas, the cerebral cortex showed the highest GPR23 expression levels and affinity binding site for [3H]-Guanosine (KD 143.8 nM and Bmax 3713 nM) as compared to other brain regions with the following rank order: cerebral cortex>hippocampus>striatum>spinal cord. T! he activation of a G protein-coupled receptor in response to guanosine showed in the cerebral cortex an EC50 92 nM. The binding site for [3H]-guanosine was highly specific and both lysophosphatidic acid and guanine agonists were 10 times less effective than guanosine in displacing 50 nM [3H]-guanosine binding. Overall these data demonstrate the existence of different levels of GPR23 mRNAs and of guanosine binding in the brain areas examined. Nevertheless at this stage of study the guanosine binding observed could include, in addition to GPR23, other unidentified receptors.

Several studies have shown that guanine-based purines exert biological effects on the central nervous system (CNS), possibly through membrane receptors, but at the present there are not reports related to the identification of such specific receptor(s). We have identified the first guanosine G protein-coupled receptor GPR23, also known as LPA4 receptor, involved in the modulation of guanosine-mediated antiproliferative effects in human glioma cell line (U87). We report that the silencing of GPR23 reduces significantly the antiproliferative effects of guanosine, while stably transfected cell clones over-expressing GPR23 increase sensitivity to guanosine. [3H] Guanosine radioligand binding assay reveals that [3H]-Guanosine binding to membrane fractions is greatly enhanced by GPR23 overexpression, and inhibited by GPR23 silencing. Furthermore, in [35S] GTPγS binding assay experiments, Guanosine causes a functional G protein coupled receptor activation in U87GPR23 overexp! ressing cells with an EC50= 8,067 nM. The binding site for [3H]-guanosine is highly specific and both lysophosphatidic acid (LPA) and guanine agonists are 10 times less effective than guanosine in displacing 50 nM [3H]-guanosine binding. In order to correlate the effects of guanosine in the CNS to a putative activation of GPR23, we performed, in different brain areas, the following investigations: by PCR, the expression levels of GPR23; by [3H]-Guanosine radioligand binding assay, the binding properties of Guanosine; by [35S] GTPγS binding assay, the receptor activation properties in response to Guanosine. Among the examined brain areas, the cerebral cortex shows the highest GPR23 expression levels which correlate with the highest Bmax values for [3H]-Guanosine as compared to other brain regions, with the following rank order: cerebral cortex>hippocampus>striatum>spinal cord. [35S] GTPγS binding assay experiments confirm an activation of a G protein-cou! pled receptor in response to guanosine in the cerebral cortex (EC50 10 0 nM). Although these observations do not exclude a possible involvement of other unidentified receptors, they can suggest an involvement of GPR23 in the functional response of cerebral cortex to Guanosine. Overall, together these data suggest that GPR23 may act as a functional membrane receptor for Guanosine.