Validation of high-affinity binding sites for succinic acid through distinguishable binding of gamma-hydroxybutyric acid receptor-specific NCS 382 antipodes.

Gamma-hydroxybutyric acid (GHB) binding to multiple sites for the tricarboxylic acid cycle intermediate succinic acid (SUC) has been disclosed recently. In order to better characterize these targets, distinguishable binding of GHB receptor-specific NCS 382 antipodes to [(3)H]-SUC or [(3)H]-GHB labelled sites in rat brain synaptic membranes was explored. Eutomer binding parameters suggest identity of the high-affinity target for SUC with a synaptic GHB receptor subtype.

gamma-Hydroxybutyrate binds to the synaptic site recognizing succinate monocarboxylate: a new hypothesis on astrocyte-neuron interaction via the protonation of succinate.

Succinate (SUC), a citrate (CIT) cycle intermediate, and carbenoxolone (CBX), a gap junction inhibitor, were shown to displace [3H]gamma-hydroxybutyrate ([3H]GHB), which is specifically bound to sites present in synaptic membrane subcellular fractions of the rat forebrain and the human nucleus accumbens. Elaboration on previous work revealed that acidic pH-induced specific binding of [3H]SUC occurs, and it has been shown to have a biphasic displacement profile distinguishing high-affinity (K(i,SUC) = 9.1 +/- 1.7 microM) and low-affinity (K(i,SUC) = 15 +/- 7 mM) binding. Both high- and low- affinity sites were characterized by the binding of GHB (K(i,GHB) = 3.9 +/- 0.5 microM and K(i,GHB) = 5.0 +/- 2.0 mM) and lactate (LAC; K(i,LAC) = 3.9 +/- 0.5 microM and K(i,LAC) = 7.7 +/- 0.9 mM). Ligands, including the hemiester ethyl-hemi-SUC, and the gap junction inhibitors flufenamate, CBX, and the GHB binding site-selective NCS-382 interacted with the high-affinity site (in microM: K(i,EHS) = 17 +/- 5, K(i,FFA) = 24 +/- 13, K(i,CBX) = 28 +/- 9, K(i,NCS-382) = 0.8 +/- 0.1 microM). Binding of the Na+,K+-ATPase inhibitor ouabain, the proton-coupled monocarboxylate transporter (MCT)-specific alpha-cyano-hydroxycinnamic acid (CHC), and CIT characterized the low-affinity SUC binding site (in mM: K(i,ouabain) = 0.13 +/- 0.05, K(i,CHC) = 0.32 +/- 0.07, K(i,CIT) = 0.79 +/- 0.20). All tested compounds inhibited [3H]SUC binding in the human nucleus accumbens and had K(i) values similar to those observed in the rat forebrain. The binding process can clearly be recognized as different from synaptic and mitochondrial uptake or astrocytic release of SUC, GHB, and/or CIT by its unique GHB selectivity. The transient decrease of extracellular SUC observed during epileptiform activity suggested that the function of the synaptic target recognizing protonated succinate monocarboxylate may vary under different (patho)physiological conditions. Furthermore, we put forward a hypothesis on the synaptic activity-regulated signaling between astrocytes and neurons via SUC protonation.

Characterization of specific succinate binding site in brain synaptic membranes.

A synaptic receptor for gamma-hydroxybutyric acid (GHB) --a naturally occurring metabolite of succinic acid--interacting succinate has been disclosed in rat and human nucleus accumbens (NA) subcellular fractions, but the molecular properties of this recognition site were not characterised. To address the presumed recognition site for succinate, the pharmacological profile of [3H]succinate binding to synaptic membranes prepared from rat forebrain and human NA samples has been investigated. Specific [3H]succinate binding sites in the human NA synaptic membrane fraction showed a strong pH-dependence and were characterized by binding of succinate (IC50 succinate=2.9+/-0.6 microM), GHB (IC50 GHB=2.1 +/-1.3 microM) and gap junction blocker carbenoxolone (IC50 = 7.1 +/-5.8 microM). A similar [3H]succinate binding profile was found in rat forebrain synaptic membrane fractions. We conclude the existence of a pHo-dependent synaptic membrane binding site for the intermediary metabolite succinate. The pharmacological properties of this recognition site may possibly suggest the existence of a hemichannel-like target protein for succinate.

Metabolic GHB precursor succinate binds to gamma-hydroxybutyrate receptors: characterization of human basal ganglia areas nucleus accumbens and globus pallidus.

Binding of the metabolic gamma-hydroxybutyrate (GHB) precursor succinate to NCS-382-sensitive [3H]GHB-labeled sites in crude synaptosomal or purified synaptic membrane fractions prepared from the human nucleus accumbens (NA), globus pallidus (GP) and rat forebrain has been shown. This site can be characterized by binding of ethyl hemisuccinate and gap-junction blockers, including carbenoxolone hemisuccinate and beta-GRA. There was no significant binding interaction between GABAB receptor ligands (CGP 55845, (R)-baclofen) and these [3H]GHB-labeled sites. GHB, NCS-382 and succinate binding profile of [3H]GHB-labeled sites in rat forebrain, human NA or GP synaptic membranes were similar. The synaptic fraction isolated from the rat forebrain was characterized by GHB binding inhibition constants: Ki,NCS-382 = 1.2 +/- 0.2 microM, Ki,GHB = 1.6 +/- 0.3 microM and Ki,SUCCINATE = 212 +/- 66 microM. In crude membranes containing mainly extrasynaptic membranes, distinct GHB and GABAB receptor sites were found in the NA. By contrast, extrasynaptic GABAB receptor sites of rat forebrain and GP were GHB- and succinate-sensitive, respectively. The heterogeneity of GABAB sites found in native membranes indicates GABAB receptor-dependent differences in GHB action. Based on these findings, we suggest that succinate (and possibly drugs available as succinate salt derivatives) can mimic some of the actions of GHB.