Evidence for glutamatergic ganglion cells in the avian retina

This dissertation presents structural, immunochemical and neurochemical evidence for glutamatergic retinotectal synaptic transmission, augmenting and extending previous physiological and anatomical studies. The evidence is especially striking when the laminar patterns of ($\sp3$H) L-glutamate receptor binding, ($\sp3$H) L-glutamate high affinity uptake (HAU) and glutamate immunoreactivity (GLIR) of the dorsal tectum are compared. All show high activity in the tectal SGFS, with a peak in the most superficial laminae of SGFS followed by dip in the b-c region, and a second broad peak in deeper SGFS. Uptake and immunoreactivity bear a stronger resemblance to one another than either does to receptor binding, consistent with the fact that HAU and GLIR are localized in the same structures: glutamatergic terminals, intrinsic cell bodies and their processes. Receptor binding, as attested by the lack of enucleation effects, is a marker of postsynaptic receptors. In summary, these results are consistent with the hypothesis that most of the retinal projection to the optic tectum is glutamatergic: (1) A glutamate/aspartate HAU system exists in the superficial laminae, and it is dependent upon an intact retinal input, as shown developmentally and by retinal ablation; (2) Glutamate-like immunoreactivity appears in retinorecipient tectal regions (partially responsive to enucleation), in cell bodies of retinal ganglion cells and displaced ganglion cells, and in a non-tectal ganglion cell projection, the ectomammilary nucleus; (3) Sodium-independent glutamate receptor binding (which remains unchanged by enucleation) is most intense in the retinorecipient regions of the tectum and the ectomammilary nucleus. This binding is pharmacologically typical of a CNS sensory structure, being dominated by the quisqualate/kainate receptor subclass. Thus, as with other sensory systems, a portion of the retinotectal projection has been shown to include glutamatergic afferents with the distribution and properties expected of the primary projection ^

The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons.

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems.

Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia.

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.

Amino acid, peptide and drug transport across monolayers of human intestinal (CAC0-2) cells in vitro

The properties of Caco-2 monolayers were compared on aluminium oxide and nitrocellulose permeable-supports. On nitrocellulose, Caco-2 cells displayed a higher rate of taurocholic acid transport than those cultured on aluminium oxide inserts. In addition, Caco-2 cells grown on these two inserts were not comparable with respect to cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of the aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands, offers a distinct advantage over nitrocellulose inserts for drug transport studies. The carrier-mediated uptake and transport of the imino acid (L-proline) and the acidic amino acids (L-aspartate and L-glutamate) have been studied. At pH7.4, L-proline uptake is mediated via an A-system carrier. Elevated uptake and transport under acidic conditions occurs by activation of a distinct carrier population. Acidic amino acid transport is mediated via a X-AG system. The flux of baclofen, CGP40116 andCGP40117 across Caco-2 monolayers was described by passive transport. The transport of three peptides, thyrotrophin-releasing hormone, SQ29852 and cyclosporin were investigated. Thyrotrophin-releasing hormone transport acrossCaco-2 monolayers was characterised by a minor saturable (carrier-mediated,approximately 25%) pathway, superimposed onto a major non-saturable (diffusional)pathway. SQ29852 uptake into Caco-2 monolayers is described by a major saturable mechanism (Km = 0.91 mM) superimposed onto a minor passive component.However, the initial-rate of SQ29852 transport is consistent with a passive transepithelial transport mechanism. These data highlight the possibility that itsbasolateral efflux is severely retarded such that the passive paracellular transportdictates the overall transepithelial transport characteristics. In addition, modelsuitable for investigating the transepithelial transport of cyclosporin A has been developed. A modification of the conventional Caco-2 model has been developed which has a calcium-free Ap donor-solution and a Bl receiver-solution containing the minimumcalcium concentration required to maintain monolayer integrity (100 μM). The influence of calcium and magnesium on the absorption of [14C]pamidronate was evaluated by comparing its transport across the conventional and minimum calciumCaco-2 models. Ap calcium and magnesium ions retard the Ap-to-Bl flux of pamidronate across Caco-2 monolayers. The effect of self-emulsifying oleic acid-Tween 80 formulations on Caco-2monolayer integrity has been investigated. Oleic acid-Tween 80 (1 0:1) formulations produced a dose-dependent disruption of Caco-2 monolayer integrity. This disruption was related to the oleic acid content of the formulation.

Neutral Aminopeptidase and Dipeptidyl Peptidase IV Activities in Plasma of Monosodium Glutamate Obese and Food-deprived Rats

Biometric parameters, glycemia and activity levels of plasma neutral aminopeptidase (APN) and dipeptidyl peptidase IV (DPPIV) were measured in monosodium glutamate obese and food-deprived rats (MSG-FD), to analyze the involvement of these enzymes in such situations. Plasma APN was distinguished as sensitive (PSA) (K(m) = 7.8 x 10(-5) mol/l) and predominantly insensitive (APM) (K(m) = 21.6 x 10(-5) mol/l) to puromycin, whereas DPPIV was sensitive (DPPIV-DS) (K(m) = 0.24 x 10(-5) mol/l) and predominantly insensitive (DPPIV-DI) (K(m) = 7.04 x 10(-5) mol/l) to diprotin A. Although unchanged in the MSG and food-deprived animals, APM activity levels were closely correlated with body mass, Lee index, and mass of retroperitoneal fat pad in the food deprived, but not in the MSG animals. DPPIV-DI activity levels decreased by 33% and were correlated with body mass, Lee index, and mass of periepididymal fat pad in the food-deprived MSG rats. These data suggest that APM and DPPIV-DI are respectively related to the downregulation of somatostatin in food-deprived rats, and to the recovery of energy balance in MSG obese rats during food deprivation.

Obesity induced by neonatal treatment with monosodium glutamate impairs microvascular reactivity in adult rats: Role of NO and prostanoids

Background and aim: given that obesity is an independent risk factor for the development of cardiovascular diseases we decided to investigate the mechanisms involved in microvascular dysfunction using a monosodium glutamate (MSG)-induced model of obesity, which allows us to work on both normotensive and normoglycemic conditions. Methods and results: Male offspring of Wistar rats received MSG from the second to the sixth day after birth. Sixteen-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia and insulin resistance, with no alteration in glycemia and blood pressure. The effect of norepinephrine (NE), which was increased in MSG rats, was potentiated by L-nitro arginine methyl ester (L-NAME) or tetraethylammonium (TEA) and was reversed by indomethacin and NS-398. Sensitivity to acetylcholine (ACh), which was reduced in MSG rats, was further impaired by L-NAME or TEA, and was corrected by indomethacin, NS-398 and tetrahydrobiopterin (BH4). MSG rats displayed increased endothelium-independent relaxation to sodium nitroprusside. A reduced prostacyclin/tromboxane ratio was found in the mesenteric beds of MSG rats. Mesenteric arterioles of MSG rats also displayed reduced nitric oxide (NO) production along with increased reactive oxygen species (ROS) generation; these were corrected by BH4 and either L-NAME or superoxide dismutase, respectively. The protein expression of eNOS and cyclooxygenase (COX)-2 was increased in mesenteric arterioles from MSG rats. Conclusion: Obesity/insulin resistance has a detrimental impact on vascular function. Reduced NO bioavailability and increased ROS generation from uncoupled eNOS and imbalanced release of COX products from COX-2 play a critical role in the development of these vascular alterations (C) 2010 Elsevier B.V. All rights reserved.

Obesity: effects of physical exercise and metformin on metabolic aspects of monosodium glutamate treated rats

The present study investigated the effects of swimming training and metformin on metabolic aspects of obese rats. Wistar rats were divided into control (C), obese (O), Trained Obese (TO) and metformin obese (MO) groups. Obesity was induced by subcutaneous monosodium glutamate injection (4 mg/g body weight). Exercise program consisted in swimming 1 h/day, 5 days/week, for 8 weeks, supporting a load corresponding to 5% of body weight. Metformin was dissolved in the drinking water (1.4 mg/ml) for 8 weeks. At the end of the experimental period, rats were sacrificed and blood was collected for determinations of serum glucose, insulin and triglycerides and hematocrit. Samples of gastrocnemius muscle and liver were removed to evaluate triglycerides content MSG-induced obesity, increased serum glucose, insulin and triglycerides, while physical training was able to recover serum glucose and insulin and metformin treatment recovered serum insulin and slightly reduced the serum glucose. MSG-induced obesity also increased liver triglycerides content and physical training and metformin administration recovered these parameters. It was concluded that in MSG obese rats, physical exercise and metformin induced important metabolic alterations associated with an improvement in glucose homeostasis and in liver fat content. Obesity and Metabolism 2009; 5: 129-133.

Toxicity of hypercaloric diet and monosodium glutamate: oxidative stress and metabolic shifting in hepatic tissue.

The present study examines the effects of a hypercaloric diet on hepatic glucose metabolism of young rats, with and without monosodium glutamate (MSG) administration, and the association of these treatments with evaluating markers of oxidative stress. Male weaned Wistar rats (21 days old) from mothers fed with a hypercaloric diet or a normal diet, were divided into four groups (n=6): control (C) fed with control diet; (MSG) treated with MSG (4 mg/g) and control diet; (HD) fed with hypercaloric diet and (MSG-HD) treated with MSG and HD. Rats were sacrificed after the oral glucose tolerance test (OGTT), at 45 days of treatments. Serum was used for insulin determination. Glycogen, hexokinase(HK), glucose-6-phosphatase(G6PH), lipid hydroperoxide, superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were determined in liver. HD rats showed hypoglycemia, hyperinsulinemia, and high hepatic glycogen, HK and decreased G6PH. MSG and MSG-HD had hyperinsulinemia, hyperglycemia, decreased HK and increased G6PH in hepatic tissue. These animals had impaired OGTT. HD, MSG and MSG-HD groups had increased lipid hydroperoxide and decreased SOD in hepatic tissue. Hypercaloric diet and monosodium glutamate administration induced alterations in metabolic rate of glucose utilization and decreased antioxidant defenses. Therefore, the hepatic glucose metabolic shifting induced by HD intake and MSG administration were associated with oxidative stress in hepatic tissue.

Monosodium glutamate in standard and high-fiber diets: Metabolic syndrome and oxidative stress in rats

Objective: This study determined the effects of adding monosodium glutamate (MSG) to a standard diet and a fiber-enriched diet on glucose metabolism, lipid profile, and oxidative stress in rats. Methods: Male Wistar rats (65 ± 5 g, n = 8) were fed a standard diet (control), a standard diet supplemented with 100 g of MSG per kilogram of rat body weight, a diet rich in fiber, or a diet rich in fiber supplemented with 100 g of MSG per kilogram of body weight. After 45 d of treatment, sera were analyzed for concentrations of insulin, leptin, glucose, triacylglycerol, lipid hydroperoxide, and total antioxidant substances. A homeostasis model assessment index was estimated to characterize insulin resistance. Results: Voluntary food intake was higher and feed efficiency was lower in animals fed the standard diet supplemented with MSG than in those fed the control, fiber-enriched, or fiber- and MSG-enriched diet. The MSG group had metabolic dysfunction characterized by increased levels of glucose, triacylglycerol, insulin, leptin, and homeostasis model assessment index. The adverse effects of MSG were related to an imbalance between the oxidant and antioxidant systems. The MSG group had increased levels of lipid hydroperoxide and decreased levels of total antioxidant substances. Levels of triacylglycerol and lipid hydroperoxide were decreased in rats fed the fiber-enriched and fiber- and MSG-enriched diets, whereas levels of total antioxidant substances were increased in these animals. Conclusions: MSG added to a standard diet increased food intake. Overfeeding induced metabolic disorders associated with oxidative stress in the absence of obesity. The fiber-enriched diet prevented changes in glucose, insulin, leptin, and triacylglycerol levels that were seen in the MSG group. Because the deleterious effects of MSG, i.e., induced overfeeding, were not seen in the animals fed the fiber-enriched diets, it can be concluded that fiber supplementation is beneficial by discouraging overfeeding and improving oxidative stress that is induced by an MSG diet. © 2005 Elsevier Inc. All rights reserved.

Lipid Metabolism of Monosodium Glutamate Obese Rats after Partial Removal of Adipose Tissue

We analyzed the effects of partial fat pad removal on retroperitoneal and epididymal fat depots and carcass metabolism of control (C) and MSG-obese (M) rats. Three-month-old C and M male Wistar rats were submitted to either partial surgical excision of epididymal and retroperitoneal fat tissue (lipectomy, L) or sham surgery (S) and studied after 7 or 30 days. Retroperitoneal and epididymal tissue re-growth after lipectomy was not observed, as indicated by the low pads weight of the L groups. The lipolysis rate was stimulated in LC7 and LM7, probably due to surgical stress and low insulin levels. In LM7, but not in LC7, in vivo lipogenesis rate increased in retroperitoneal and epididymal fat tissue, as did the diet-derived lipid accumulation in epididymal fat tissue. Although these local increases were no longer present in LM30, this group showed a large increase in the percentage of small area adipocytes in both pads as well as increased carcass lipogenesis rate. The present data showed that the partial removal of fat depots affected the metabolism of control and MSG-obese rats differently. In the obese animals only, it stimulated both local and carcass lipogenesis rate as well as adipocyte differentiation, i.e. responses likely to favor excised tissue re-growth and/or compensatory growth of non-excised depots.

X-ray Studies on Crystalline Complexes Involving Amino Acids and Peptides. XIII. Effect of Chirality on Molecular Aggregation: The Crystal Structures of L-Arginine D-Aspartate and L-Arginine D-Glutamate Trihydrate

The crystal structures of (1) L-arginine D-asparate, C6HIsN40~.C4H6NO4 [triclinic, P1, a=5.239(1), b=9.544(1), c=14.064(2)A, a=85"58(1), /3=88.73 (1), ~/=84.35 (1) °, Z=2] and (2) L-arginine D-glutamate trihydrate, C6H15N40~-.CsHsNO4.3H20 [monoclinic, P2~, a=9.968(2), b=4.652(1), c=19.930 (2) A, fl = 101.20 (1) °, Z = 2] have been determined using direct methods. They have been refined to R =0.042 and 0.048 for 2829 and 2035 unique reflections respectively [I>2cr(I)]. The conformations of the two arginine molecules in the aspartate complex are different from those observed so far in the crystal structures of arginine, its salts and complexes. In both complexes, the molecules are organized into double layers stacked along the longest axis. The core of each double layer consists of two parallel sheets made up of main-chain atoms, each involving both types of molecules. The hydrogen bonds within each sheet and those that interconnect the two sheets give rise to EL-, DD- and DE-type head-to-tail sequences. Adjacent double layers in (1) are held together by side-chain-side-chain interactions whereas those in (2) are interconnected through an extensive network of water molecules which interact with sidechain guanidyl and carboxylate groups. The aggregation pattern observed in the two LD complexes is fundamentally different from that found in the corresponding EL complexes.

Effect of beta-n-oxalyl-L-alpha, beta-diaminopropionic acid on glutamate uptake by synaptosomes

THE unusual amino acid beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (ODAP), isolated from the seeds of Lathyrus sativus is a potent neurotoxin1−3. It produces biochemical changes in the brain typical of an excitant amino acid and is implicated in the aetiology of human neurolathyrism caused by eating the seeds of L. sativus 4−6. It may act as a glutamate antagonist: ODAP inhibits glutamate oxidation7 possibly by inhibiting glutamate uptake in bovine brain mitochondria; it also acts as a competitive inhibitor of glutamate uptake in certain strains of yeast8, and a similar process might occur at the synaptic level. Any effect of ODAP on glutamate uptake at synapses is significant in view of the neurotransmitter function of glutamate, which seems to be neuroexcitory as well as neurotoxic9−12. But Balcar and Johnston13 have shown with rat brain slices that ODAP does not inhibit the glutamate uptake by the high affinity system.

X-ray studies on crystalline complexes involving amino acids and peptides. XV. Crystal structures of L-lysine D-glutamate and L-lysine D-aspartate monohydrate and the effect of chirality on molecular aggregation

L-Lysine D-glutamate crystallizes in the monoclinic space group P2(1) with a = 4.902, b = 30.719, c = 9.679 A, beta = 90 degrees and Z = 4. The crystals of L-lysine D-aspartate monohydrate belong to the orthorhombic space group P2(1)2(1)2(1) with a = 5.458, b = 7.152, c = 36.022 A and Z = 4. The structures were solved by the direct methods and refined to R values of 0.125 and 0.040 respectively for 1412 and 1503 observed reflections. The glutamate complex is highly pseudosymmetric. The lysine molecules in it assume a conformation with the side chain staggered between the alpha-amino and the alpha-carboxylate groups. The interactions of the side chain amino groups of lysine in the two complexes are such that they form infinite sequences containing alternating amino and carboxylate groups. The molecular aggregation in the glutamate complex is very similar to that observed in L-arginine D-aspartate and L-arginine D-glutamate trihydrate, with the formation of double layers consisting of both types of molecules. In contrast to the situation in the other three LD complexes, the unlike molecules in L-lysine D-aspartate monohydrate aggregate into alternating layers as in the case of most LL complexes. The arrangement of molecules in the lysine layer is nearly the same as in L-lysine L-aspartate, with head-to-tail sequences as the central feature. The arrangement of aspartate ions in the layers containing them is, however, somewhat unusual. Thus the comparison between the LL and the LD complexes analyzed so far indicates that the reversal of chirality of one of the components in a complex leads to profound changes in molecular aggregation, but these changes could be of more than one type.