E. Edward Mena

Chloride ions enhance L-glutamate binding to rat brain synaptic membranes

Chloride ions increased L-glutamate (L-Glu) binding to synaptic membranes. The binding was saturable and resulted in a 2.5-fold enhancement at concentrations of 20--40 mM chloride. Sodium and potassium ions inhibited only chloride stimulated L-Glu binding. Calcium ions also increased L-Glu binding but this was observed only in the presence of chloride. The anion selectivity of the enhancement of L-Glu binding was similar to that reported for the membrane chloride channel, suggesting that some L-Glu binding sites may be associated with this channel.

Chloride and calcium ions separte L-glutamate receptor populations in synaptic membranes

Cl-/Ca2+-dependent and Cl-/Ca2+-independent L-[3H]glutamate binding sites in rat brain synaptic membranes showed marked differences in their pharmacological properties. One site resembled L-2-amino-4-phosphonobutyrate (L-APB)-sensitive receptors and the other N-methyl-D-aspartate (NMDA) receptors. Inhibition studies demonstrated that L-aspartate was more potent at Cl-/Ca2+-independent than at Cl-/Ca2+-dependent sites although L-glutamate was of similar potency at both sites; the D-isomers of aspartate, glutamate and alpha-aminoadipate exhibited the opposite trend. Quisqualate and ibotenate showed high and low affinity inhibition components in the presence of Cl- and Ca2+, and only low affinity inhibition at Cl-/Ca2+-independent sites. For a series of alpha-amino-omega-phosphono carboxylic acids (propionate-heptanoate), peaks of inhibitory activity in the presence of Cl- and Ca2+ were shifted to l-carbon shorter homologues than in the absence of these ions. These data indicate that the ionic environment is of critical importance for the activity of different physiological receptor populations in vitro.

Pathologic concentrations of ammonium ions block L-glutamate uptake.

Both K+ and NH4+ are potent inhibitors of Na+-dependent L-glutamate binding to synaptic plasma membranes. Thus, the effects of these ions on Na+-dependent L-glutamate uptake were examined in rat forebrain synaptosome preparations. KCl (2 to 4 mM) stimulated L-glutamate uptake 34% over that in K+-free Krebs bicarbonate buffer; NH4+ was without effect. However, in the presence of 4 mM K+, NH4+ blocked the K+-stimulated component of Na+-dependent L-glutamate uptake. These effects were unrelated to ionic strength of Cl- as added Na+ or tris chloride had no effect on L-glutamate uptake. The results suggest that NH4+ could exert some of its toxic effects by blocking a specific L-glutamate uptake site, thereby elevating L-glutamate in the central nervous system.

Freezing eliminates a specific population of l-glutamate receptors in synaptic membranes☆

The binding of L-[3H]glutamate (L-Glu) to freeze-thawed synaptic membranes (SPMs) exhibited saturation kinetics, with Kd 507 nM and Bmax 6.99 pmol/mg protein. The effects of ions, the susceptibility to Triton X-100 and the pharmacological properties of the binding indicated that those sites detected in freeze-thawed SPMs were only of the Cl-/Ca2+-independent type. The Cl-/Ca2+-dependent (2-amino-4-phosphonobutyrate-sensitive) L-Glu binding sites which are additionally present in fresh SPMs are abolished by freezing.

Identification of Synapse Specific Components: Synaptic Glycoproteins, Proteins, and Transmitter Binding Sites

Abstract: Synaptic junctions (SJ) were prepared from synaptic plasma membranes (SPM) by extraction with Triton X‐100 and density gradient centrifugation. These SJs were enriched in certain Concanavalin A (Con A) binding glycoproteins, the 52,000 Mr postsynaptic density (PSD) protein, and receptor sites for l‐glutamate, l‐aspartate, kainic acid (KA) but not quinuclidinyl benzilate (QNB). Various other membrane fractions were extracted by means of the same procedure. Those fractions prepared from light SPMs and crude myelin contained identifiable synaptic junctions and were also highly enriched in the synaptic components. The SJ‐like fraction from mitochondria did not contain any of the characteristic synaptic macromolecules. However, this fraction from microsomes contained levels of the 52,000 Mr PSD protein and binding sites for l‐glutamate (l‐GlU) and l‐aspartate (l‐Asp) similar to true synaptic junctions, although the Con A binding glycoproteins and KA binding sites were nearly absent. On the basis of electron microscopy, the SJ‐like fraction from microsomes did not contain structures recognizable as SJs. Thus, the Con A binding glycoproteins and KA binding sites appear to be excellent markers for the SJ.

L-glutamate and l-aspartate bind to separate sites in rat brain synaptic membranes

The specific binding of L-glutamate (L-Glu) and L-aspartate (L-Asp) was measured in rat brain synaptic plasma membranes (SPMs). A distinction between the binding sites for these amino acids was made on the basis of the kinetics, ion effects, pharmacology and chemical susceptibility of the binding. The existence of distinct binding sites for L-Glu and L-Asp is consistent with electrophysiological data that mammalian neurons possess separate receptors for these amino acids.

Regional distribution and ionic requirement of Cl−/Ca2+-activated and Cl−/Ca2+-independent glutamate receptors in rat brain

Recent studies have shown that Cl- and Ca2+ ions increase [3H]glutamate binding to rat forebrain synaptic plasma membranes by expressing a new class of glutamate receptors. We examined the regional distribution of these two classes of glutamate binding sites and further characterized their ionic requirements. Significant differences in both Cl-/Ca2+-independent (basal) and Cl-/Ca2+-activated receptors, as well as the ratios of these two receptor classes were observed among different areas of the CNS. Cl- and Ca2+ appeared to act synergistically, with Cl-ion an absolute requirement for Ca2+ stimulation, in expressing these additional binding sites. Ca2+ alone did not affect glutamate binding.

Cations differentially affect subpopulations ofl-glutamate receptors in rat synaptic plasma membranes

Several cations were examined for their ability to specifically affect one of the 3 L-glutamate (L-Glu) binding sites in rat forebrain synaptic plasma membranes (i.e. Na+-dependent, Cl--dependent and Cl--independent). Na+-dependent binding was potently inhibited by K+ and NH4+ ions. Other monovalent cations tested (Cs+, Li+, triethylammonium) had no effect on this binding site. Polyvalent cations (Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Cr3+) also had little effect on the Na+-dependent L-Glu binding site. Cl--dependent L-Glu binding was potently inhibited by Na+ ions but was not affected by other monovalent ions. All of the divalent cations were potent inhibitors of both Cl--dependent and -independent binding. The results show that these binding sites of L-Glu can be distinguished by their response to cations and suggest possible novel modes of regulation in vivo.

The effect of entorhinal cortical ablation on the distribution of muscarinic cholinergic receptors in the rat hippocampus

Removal of the entorhinal cortical projection to the hippocampus in adult rats decreased the density of muscarinic cholinergic receptors in the denervated dentate gyrus outer molecular layer at two days postlesion. Thirty days following the lesion (in adults and neonates) there is a small receptor density increase in the outer molecular layer (may be due to tissue shrinkage), and a larger increase in the lacunosum-moleculare. The receptor density decrease seen two days postlesion suggests the presence of presynaptic muscarinic receptors on the lost entorhinal cortical fibers. The distribution and extent of the receptor changes seen at 30 days postlesion are inconsistent with the cholinergic fiber reorganization which follows an entorhinal cortical lesion, but are consistent with a proposed model of non-cholinergic afferent mediated control of muscarinic receptor density in the rat hippocampus.

Lesion-induced alterations of lectin binding sites in the rat dentate gyrus

Abstract The pattern of binding of horseradish peroxidase conjugated lectins (Concanavalin A, fucose binding protein, Ricinus communis agglutinin) was examined in the rat hippocampal formation both prior to and following a lesion of the entorhinal cortex. In normal animals, Concanavalin A binding sites were concentrated around the granular and pyramidal cell bodies. Receptors were less concentrated in the stratum radiatum, stratum oriens and the dentate molecular layer. Receptors of fucose binding protein were concentrated in the granular and pyramidal cells, the boundary between the first and second quarters of the molecular layer and at the hippocampal fissure. Ricinus communis agglutinin binding sites were highest in the first 1/4 of the molecular layer and lowest in the stratum lacunosum-moleculare. Three days after an entorhinal lesion, the binding of Concanavalin A and fucose binding protein in the molecular layer was increased and while Ricinus communis agglutinin binding was unchanged. At thirty days post-lesion there was an increase in the binding of Concanavalin A and fucose binding protein in the molecular layer and stratum lacunosum-moleculare, whereas Ricinus communis agglutinin binding sites increased only in the molecular layer. The extensive alterations of lectin receptors that occur simultaneously with reactive synaptogenesis may indicate that membrane-bound glycoconjugates have an important role in this process.