Glutamate binding in the rat cerebral cortex during ontogeny

Two binding sites for L-glutamate have been identified on adult rat brain cortical membranes. One of these sites is Na+-dependent with Kd of 1.3 micro M and a Bmax of 210 pmol/mg protein. The other is Na+-independent with a Kd of 0.37 micro M and Bmax of 6.2 pmol/mg protein. There is a sharp rise in total number of Na+-independent sites per cortex up to 20 days postnatally followed by a more gradual rise to adult levels at 50 days. Na+-dependent binding is also low at birth rising to a peak at 20 days followed by a drop in total levels of binding to 30 days and then a very sharp rise up to 50 days. The kinetics of binding at 20 days gives a Kd for the Na+-dependent site of 1.77 micro M and a Bmax of 82 pmol/mg protein. The Na+-independent site at 20 days has a Kd = 1.3 micro M and Bmax of 8.47 pmol/mg protein. The ability of several acidic amino acid analogues to displace specifically bound L-glutamate was investigated by estimating IC50 values at 20 and 50 days of age. The Na+-independent site is stereospecific for L-glutamate at both ages, but will also interact with L-aspartate at 20 days. The Na+-dependent site has a similar affinity for L- and D-glutamate and L-aspartate at 50 days. The L-glutamate analogue kainate will not displace any bound L-glutamate.     

Cations differentially affect subpopulations of L-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.     

Effects of maternal protein deficiency on synaptic plasma membranes in offspring

This study examined the influence of maternal protein deficiency during lactation on the accretion and synthesis of synaptic plasma membrane (SPM) proteins in developing offspring. It also examined the activity of Mg2+-dependent, ouabain-sensitive (Na+-K+)-ATPase in SPMs. The results of this study demonstrated that early postnatal undernutrition produces a significant deficit of SPM proteins. Despite this deficit, 10- to 20-day-old undernourished rats incorporated more [3H]-leucine into SPM proteins than age-matched control rats. The specific activity of (Na+-K+)-ATPase was decreased in SPMs from undernourished rats early in development, but was increased at 20 and 34 days of age. Interestingly, 20- and 34-day-old undernourished rats also had an increased proportion of [3H]-leucine-derived radioactivity in the band on SDS gels which corresponded to (Na+-K+)-ATPase. In summary, it appears that maternal deficiency of protein during lactation affects the synaptic plasma membranes in offspring quantitatively and qualitatively.     

Induction of glutamate binding sites in hippocampal membranes by transient exposure to high concentrations of glutamate or glutamate analogs

The number of Na+-independent, Cl--dependent glutamate binding sites in rat hippocampal membranes is increased two- to fourfold after pre-exposing isolated membranes or hippocampal slices to high concentrations (0.1-10 mM) of L-glutamate or of glutamate analogs with high affinity for this binding site, such as quisqualate, homocysteate, or aminoadipate. N-Methylaspartate and kainate are ineffective. A similar binding increase is induced by transient exposure to the dipeptide tyrosylglutamate. The newly induced binding sites appear to be identical with pre-existing Cl--dependent binding sites by several criteria: They have a similar pharmacological profile, they are sensitive to low concentrations of Na+, and the number of sites can be further increased by transient exposure to micromolar calcium concentrations. Moreover, binding of [3H]APB, a ligand selective for the Cl--dependent glutamate binding sites, is also increased after glutamate preincubation. The induction of binding sites by high glutamate concentrations, described herein, is calcium-independent, not inhibited by leupeptin and, therefore, different from the previously described activation of binding sites by a calcium-sensitive protease. The high concentration of ligand needed to induce increased binding suggests the presence in hippocampal membranes of a binding site with low, millimolar affinity that is functionally related to the known high-affinity binding sites. Several interpretations of the observed effects and their implications for the possible relationship between the binding site and the synaptic receptor are discussed.     

Inhibition of synaptosomal [3H]glutamate uptake and [3H]glutamate binding to plasma membranes from brain of young rats by glutaric acid in vitro

Synaptosomes and plasma membrane preparations from brain of 30-day-old rats were incubated with glutaric acid at final concentrations ranging from 10 nM to 1 mM for the determination of glutamate uptake and binding, respectively. [3H]Glutamate uptake into synaptosomes was inhibited by approximately 50% by 1 mM glutaric acid, corresponding to the concentration found in brain of glutaric acidemic children. In addition, in the presence of extracellular Na+ concentrations, the same dose of glutaric acid decreased by about 30% [3H]glutamate binding to brain plasma membranes. The results indicate that the inhibition of both glutamate uptake into synaptosomes and glutamate binding to plasma synaptic membranes by the metabolite could result in elevated concentrations of the excitatory neurotransmitter in the synaptic cleft, potentially causing excitotoxicity to neural cells, a fact that may be related to the brain damage characteristic of glutaric acidemia type I.     

Methylmalonate administration decreases Na+,K+-ATPase activity in cerebral cortex of rats

Buffered methylmalonate (MMA) was injected s.c. into rats twice a day at 8 h intervals from 5 to 25 days of age (chronic treatment), or into 10-day-old rats three times a day at 1 h intervals (acute treatment). Control rats received saline in the same volumes. Na+,K+-ATPase and Mg2+-ATPase activities were determined in the synaptic plasma membranes from cerebral cortex of rats. Na+,K+-ATPase activity was reduced by 30-40% in MMA-treated rats, whereas Mg2+-ATPase activity was not. In contrast, MMA at final concentrations ranging from 0.1 to 2.0 mM had no in vitro effect on these enzyme activities. However, when brain homogenates were incubated with 2 mM MMA before membrane preparation, Na+,K+-ATPase activity was decreased by 44%. Furthermore, this reduction was totally prevented by the simultaneous addition of glutathione and MMA, suggesting that oxidation of thiol groups or other oxidative damage to the enzyme could be responsible for this effect.     

Kinetic and pharmacologic characterization of gamma-aminobutyric acid receptive sites from mammalian brain

Sodium-dependent (+Na) and sodium-independent (-Na) receptive sites for gamma-aminobutyric acid (GABA) have been characterized using synaptic plasma membranes from bovine and rat brain. Synaptic plasma membranes were prepared from either rat cerebellar cortex or calf cerebral cortex by discontinuous sucrose gradient flotation centrifugation of crude mitochondrial pellets, and assayed using equilibrium ligand binding assays to obtain the maximum binding capacity (Bmax) and the thermodynamic constant (KD). Values for KD from equilibrium studies were subsequently confirmed by kinetic analyses of association and dissociation reactions. The KD for +Na GABA binding (5.0 +/- 0.2 micron) corresponds to the apparent Michaelis constant for neuronal GABA transport (3.8 +/- 0.1 micron)22, while the KD for -Na binding (0.17 +/- 0.04 micron) agrees with that determined by Enna and Snyder for the putative postsynaptic receptor. Maximal binding activities of about 5 and 55 pmole/mg protein were obtained for -Na and +Na binding respectively. The pharmacologic specificities of the two sites were determined using competition binding studies. Nipecotic acid and diaminobutyric acid inhibit both synaptosomal GABA uptake (Ki approximately 25 micron and 120 micron respectively) and +Na binding of GABA to synaptic plasma membrane (IC50 approximately 40 micron and 350 micron respectively) but do not inhibit -Na binding. Bicuculline inhibits -Na [3H]GABA binding at low concentrations (IC50 approximately 15 micron), while affecting the uptake and +Na binding of [3H]GABA only at high concentrations (IC50 approximately 520 micron and 300 micron respectively). beta-Alanine inhibits the -Na binding site (IC50 approximately 100 micron), but is ineffective at the +Na binding site and does not interfere with synaptosomal uptake of GABA. Finally, chlorpromazine and N-ethylmaleimide inhibit the +Na binding, albeit at high concentrations (IC50 approximately 600 micron and 5 mM respectively) but are ineffective at the -Na binding site. From these results the -Na binding site is tentatively identified as a postsynaptic receptor and the +Na binding site is identified as the neuronal uptake receptive site.     

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.     

Thyroxine regulates the activity and the concentration of synaptic plasma membrane Na,K-ATPase in the developing rat brain cortex

The administration of thyroxine to neonatal rats stimulates the activity of synaptic membrane Na,K-ATPase in the brain cortex of euthyroid and hypothyroid animals. Thyroxine also increases NA,K-ATPase activity of isolated neuronal perikarya in neonatal rats. Binding studies employing [3H]ouabain indicate that the hormone increases the amount of Na,K-ATPase in the synaptic membranes of two-week-old rats. Thyroxine treatment however did not affect synaptic membrane Na,K-ATPase in rats aged 30 days. The results suggest that thyroid hormones are important for the maturation of the synaptic plasma membrane during the critical period of rat brain development.     

Characterization of Na+-dependent binding sites of [3H]glutamate in synaptic membranes from rat brain

Some biochemical characteristics of Na+-dependent binding of [3H]L-glutamic acid (Glu) were studied using crude synaptic membrane preparations from the rat brain as compared with Na+-independent binding. In vitro addition of sodium chloride (1-100 mM) exhibited a significant enhancement of [3H]Glu binding to synaptic membranes in a concentration-dependent manner independent of the incubation temperature employed (2 or 30 degrees C). In contrast, sodium acetate elicited a concentration-dependent augmentation of the binding at 2 degrees C to a significantly greater extent than that found at 30 degrees C. It was found that the binding found in the presence of 100 mM sodium acetate reached its maximal value within 10 min of incubation followed by a rapid decline up to 60 min at 30 degrees C, while gradually increasing up to 60 min at 2 degrees C. The Na+-independent basal binding was significantly activated by the alteration of incubation temperature from 2 to 30 degrees C and reached equilibrium within 10 min of incubation at both incubation temperatures. The Na+-dependent binding was more promptly attenuated by the addition of excess of non-radioactive Glu (1 mM) at 30 degrees C than that at 2 degrees C, whereas the Na+-independent binding was greatly suppressed by the addition at 2 degrees C in comparison with that at 30 degrees C. Quisqualic acid induced a considerably less-potent inhibition of the Na+-dependent binding than that of the Na+-independent binding. Neither N-methyl-D-aspartic acid nor kainic acid had such a significant effect on each binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurophysiological and Neurochemical Studies on the Action of the Anticonvulsant γ-Hydroxy, γ-Ethyl, γ-Phenyl-Butyramide

The effect of gamma-hydroxy, gamma-ethyl, gamma-phenyl-butyramide (HEPB) on afterdischarges produced by hippocampal stimulation in cats was studied. HEPB notably diminished the duration of afterdischarges and in some cats blocked their propagation into the substantia nigra and the amygdala. HEPB treatment also antagonized the enhancement of afterdischarge duration produced by subconvulsive doses of bicuculline, whereas treatment with diphenylhydantoin strongly potentiated this effect of bicuculline. The intracisternal injection of HEPB or gamma-aminobutyric acid (GABA) in mice resulted in a potentiation of strychnine-induced convulsions. On the other hand, neurochemical experiments in mouse brain cortex slices and in synaptosomes demonstrated that HEPB did not affect the high affinity uptake of [3H] GABA, its spontaneous or Ca2+ dependent release stimulated by depolarizing K+ concentrations, and its Na+ independent binding to synaptic plasma membranes.     

In vitro effects of D-2-hydroxyglutaric acid on glutamate binding, uptake and release in cerebral cortex of rats

Neurological dysfunction is common in patients with D-2-hydroxyglutaric aciduria (DHGA). However, the mechanisms underlying the neuropathology of this disorder are far from understood. In the present study, we investigated the in vitro effects of D-2-hydroxyglutaric acid (DGA) at various concentrations (0.1-1.0 mM) on various parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomal preparations, Na(+)-dependent L-[3H]glutamate uptake by synaptosomal preparations and Na(+)-independent L-[3H]glutamate uptake by synaptic vesicles, as well as of Na(+)-independent and dependent L-[3H]glutamate binding to synaptic plasma membranes from cerebral cortex of male adult Wistar rats. We observed that DGA significantly increased synaptosomal L-[3H]glutamate uptake, without altering the other parameters. Although these findings do not support a direct excitotoxic action for DGA since the metabolite did not affect important parameters of the main neurotransmission system, they do not exclude a direct action of DGA on NMDA or other glutamate receptors. More comprehensive studies are therefore necessary to evaluate the exact role of DGA on neurotransmission.     

Chloride-dependent binding sites for L-[3H]glutamate on dendrodendritic synaptosomal membranes of rat olfactory bulb

Dendrodendritic synapses occur between granule cell dendrites and secondary dendrites of mitral cells within the olfactory bulb and are attainable in a subcellular fraction (DDS). Since the mitral cells are thought to utilize an excitatory amino acid as a neurotransmitter, we determined the pharmacologic specificity of Na+-independent L-[3H]glutamate binding to fresh membranes of DDS in 50 mM Tris-HCl, pH 7.1. Binding of L-glutamate to membranes of DDS was specific, Cl(-)-dependent, and saturable. Scatchard plots were analyzed by nonlinear regression analyses using the computer program LIGAND, and the data was best-fitted to a one-site model with KD of 0.56 +/- 0.04 microM and an apparent Bmax of 48 +/- 5 pmol/mg protein. Hill plots also indicated the presence of one site and no cooperativity (nH = 0.99 +/- 0.03). However, the relative effectiveness of several compounds in inhibiting L-glutamate binding to membranes of DDS clearly demonstrated the presence of more than one site. Electrophysiological studies suggest that 2-amino-4-phosphonobutyrate (APB) is a potent antagonist of evoked responses elicited by stimulation of mitral cell axons and that quisqualate is a potent agonist; both of these compounds were highly effective inhibitors of L-glutamate binding to DDS membranes. APB displaced about 70% of the sites labeled with 200 nM L-glutamate with a KI of 1.6 microM, whereas quisqualate inhibition of L-glutamate binding yielded a line that was curvilinear in the Scatchard plot and was resolved into two sites of relatively high affinity (KI values of 0.02 and 0.65 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+, K+ ATPase activity is markedly reduced by cis-4-decenoic acid in synaptic plasma membranes from cerebral cortex of rats

We have previously demonstrated that octanoic (OA) and decanoic acids (DA) inhibit Na+, K+ ATPase activity in synaptic plasma membranes from rat brain. The objective of the present study was to investigate the in vitro effects of the other metabolites that accumulate in tissues of medium-chain acyl-CoA dehydrogenase (MCAD)-deficient patients, namely cis-4-decenoic acid (cDA), octanoylcarnitine (OC), hexanoylcarnitine (HC), hexanoylglycine (HG), phenylpropionylglycine (PPG) and suberoylglycine (SG), on Na+, K+ ATPase activity in synaptic plasma membrane from cerebral cortex of 30-day-old rats. cDA, the pathognomonic compound found in this disorder, provoked the strongest inhibition on this enzyme activity at concentrations as low as 0.25 mM, whereas OC inhibited this activity at 1.0 mM and higher concentrations in a dose-dependent manner. In contrast, HC, HG, PPG and SG did not affect Na+, K+ ATPase activity. Furthermore, pre-treatment of cortical homogenates with the antioxidant enzymes catalase plus superoxide dismutase totally prevented cDA-induced Na+, K+ ATPase inhibition. We also provided evidence that cDA, as well as OA and DA, caused lipid peroxidation, which may explain, at least in part, the inhibitory properties of these compounds towards Na+, K+ ATPase. Considering that Na+, K+ ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings, especially those regarding to the marked inhibitory effect of cDA, may be involved in the pathophysiology of the neurological dysfunction of MCAD-deficient patients.     

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.     

L-[35S]cysteic acid selectively detects chloride-dependent L-glutamate transporters in synaptic membrane

Na+-independent L-[35S]cysteic acid (CA) accumulation in rat cortical synaptic membrane was examined. In the absence of Cl-, the accumulation was not observed. Addition of Cl- revealed the accumulation in a dose-dependent manner. Br- and NO3- also did. Ca2+-enhanced the Cl- -dependent accumulation, whereas low concentrations of Na+ reduced it. L-[35S]CA accumulation was inhibited by quisqualate, L-glutamate (L-Glu), L-cysteine sulfinate, D,L-homocysteic acid and D,L-2-amino-4-phosphonobutyrate (D,L-APB) potently. L-CA inhibited L-[3H]Glu accumulation in synaptic membrane in the presence of Cl-. The maximal inhibition of L-CA was equal to that of D,L-APB, but L-CA did not inhibit L-[3H]Glu accumulation in the absence of Cl-. These results show that L-[35S]CA selectively detects the Cl- -dependent L-Glu transporters in synaptic membrane.     

Influence of age on N-methyl-D-aspartate antagonist binding sites in the rat brain studied by in vitro autoradiography

The N-methyl-D-aspartate (NMDA) receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In this study, an effect of age on NMDA antagonist binding sites was investigated through quantitative in vitro autoradiography with 3H-3-((+)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). 3H-CPP binding sites were most concentrated in the hippocampus and cerebral cortex where NMDA receptors have been demonstrated to be involved in synaptic transmission. In aged rats, 3H-CPP binding sites in the hippocampus and cerebral cortex were not significantly changed. As for other brain regions, there was an age-dependent decline of binding sites only in the caudate-putamen and nucleus accumbens. Our previous study revealed that strychnine-insensitive glycine receptors were markedly reduced in telencephalic regions in the aged rat brain. Taking these findings into consideration, it is concluded that glycine receptors but not NMDA antagonist binding sites are severely altered in telencephalic regions of aged animals. It is considered that within the NMDA receptor complex, glycine receptors may be selectively affected in the aging process.     

Autoradiographic analysis in rat brain of the postnatal ontogeny of voltage-dependent Na+ channels, Ca2+-dependent K+ channels and slow Ca2+ channels identified as receptors for tetrodotoxin, apamin and (-)-desmethoxyverapamil

The postnatal development of the distribution of 3 different ionic channel proteins in rat brain was studied using light microscopic autoradiography. [3H]Ethylenediaminetetrodotoxin, [125I]apamin and (-)-[3H]desmethoxyverapamil were used to label one class of voltage-dependent Na+ channel proteins, one class of Ca2+-dependent K+ channel proteins, and the slow Ca2+ channel protein, respectively. Ca2+-dependent K+ channel proteins are detected very early in the germinative zone. They are associated to neuronal somas during their migration and their maturation. In hippocampus and cerebral cortex, apamin binding sites are already present at birth and their density increases to day 20 postnatal when the adult localization is established. Slow Ca2+ channel protein development occurs later in CNS ontogenesis. The development of slow Ca2+ channels seems to follow the development of dendrites. Density of these channel proteins increases regularly until adult age. At the resolution level of this analysis, Na+ channel proteins are absent in diencephalon at birth. Their appearance and their increase in density are strictly correlated to the synaptogenesis in particular in cerebral and cerebellar cortex and hippocampus. Although cerebellum, neocortex and hippocampus have been particularly analyzed, other brain structures have also been examined.     

Differential coupling of opioid binding sites to guanosine triphosphate binding regulatory proteins in subcellular fractions of rat brain.

In this study, we present evidence for the occurrence of mu, delta, and kappa opioid binding sites in synaptic plasma membranes (SPM) and microsomes of rat brain. Binding to all three opioid classes was inhibited by 5'-guanylylimidodiphosphate (Gpp[NH]p) in SPM, while microsomal sites proved to be insensitive to this GTP analog. Sensitivity was restored upon solubilization of microsomes with digitonin, suggesting that opioid receptors are physically separated from G proteins in this fraction. Modulation of microsomal binding by Na+ and Mn++ was greater than that of SPM. Pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation revealed the presence of G proteins with alpha-subunit molecular weights of 40 kDa in both subcellular fractions. Basal low Km GTPase activity in SPM was greater than in microsomes. Etorphine elicited a concentration-dependent stimulation of guanosine triphosphatase (GTPase) activity in SPMs but not in microsomes, indicating functional coupling of opioid receptors to G protein in the former and an uncoupling in the latter. Microsomes from 3-day-old rat brain contained more mu opioid sites and they were more sensitive to Gpp(NH)p inhibition than those in adults. These results are consistent with the hypothesis that opioid binding sites in adult microsomes are internalized and G protein uncoupled, while those in neonates are newly synthesized, coupled receptors.     

In vivo and in vitro effects of homocysteine on Na,K-ATPase activity in parietal, prefrontal and cingulate cortex of young rats

In the present study we determined the effect of chronic administration of homocysteine on Na+,K+-ATPase activity in synaptic membranes from parietal, prefrontal and cingulate cortex of young rats. We also studied the in vitro effect of homocysteine on this enzyme activity and on some oxidative stress parameters, namely thiobarbituric acid-reactive substances (TBA-RS) and total radical-trapping antioxidant potential (TRAP) in the same cerebral structures. For the in vivo studies, we induced elevated levels of homocysteine in blood (500 microM), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 micromol/g of body weight) twice a day at 8 h intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Rats were killed 12 h after the last injection. Chronic administration of homocysteine significantly decreased (50%) Na+,K+-ATPase activity in parietal, increased (36%) in prefrontal and did not alter in cingulate cortex of young rats. In vitro homocysteine decreased Na+,K+-ATPase activity and TRAP and increased TBA-RS in all cerebral structures studied. It is proposed that the alteration of Na+,K+-ATPase and induction of oxidative stress by homocysteine in cerebral cortex may be one of the mechanisms related to the neuronal dysfunction observed in human homocystinuria.