γ‐Hydroxybutyrate binds to the synaptic site recognizing succinate monocarboxylate: A new hypothesis on astrocyte–neuron interaction via the protonation of succinate

Abstract

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.

Calcium signals in the nucleus accumbens: Activation of astrocytes by ATP and succinate

“…Binding of CBX to SUC receptor [4-6], however, could be accounted for some blockade of ATP-driven Ca 2+ signal propagation as detailed below.…”

“…SUC binding sites have previously been disclosed in rat forebrain and human NAc membrane homogenates [4,6], however, their functions have not been assigned yet. Identity of SUC-sensitive GHB [5] and GHB-sensitive SUC [4] binding sites raised the possibility to relate SUC and different GHB actions [47-50].…”

“…Identity of SUC-sensitive GHB [5] and GHB-sensitive SUC [4] binding sites raised the possibility to relate SUC and different GHB actions [47-50]. Importantly, the binding site of GHB/SUC interacts with CBX [4-6], a blocker of gap-junctions that are major players of astroglial Ca 2+ signalling [51]. We have also shown previously that GHB activates intracellular store-dependent Ca 2+ transients in NAc astrocytes [3].…”

“…Previously, we also showed that binding sites for GHB are shared with citric acid cycle intermediate succinic acid (SUC) and the gap-junction blocker carbenoxolone hemisuccinate (CBX), as disclosed in NAc membrane homogenates isolated from rat and human brain tissues [4-6]. These findings raised the possibility that SUC, similarly to GHB may also evoke Ca 2+ transients in NAc astrocytes.…”

Polyamidoamine dendrimer impairs mitochondrial oxidation in brain tissue

“…Astroglial Ca 2+ signals were monitored in the astroglia-rich stratum radiatum area in the hippocampal CA3 region after bulk loading of the rat hippocampal slice with the Ca 2+ sensitive fluorescent dye Fluo-4 [10,24] (Figure 2A-C, n = 7 slices). Astroglial localization of the dye was confirmed by colocalization with the astroglia-specific SR101 marker (Figure 2A, Left ).…”