L-glutamic Acid Diethyl Ester Research Articles

Site-specific immobilization of papain on DDI-modified polystyrene beads for the oligo(γ-ethyl-L-glutamate) synthesis

In this paper, a reusable heterogeneous catalyst of papain was prepared and used to catalyze the polymerization for oligo-glutamate. In silico, the 9,9-bis(2,3-epoxypropyl) fluorenyl group (DDI) carried on polystyrene beads (PS-PEG-DDI) showed site-specific binding for sites which are far away the active site of papain. The immobilization of papain on PS-PEG-DDI reached the optimal value of 126 ± 2.5 mg/g beads and 1726.2 ± 34 U/g beads at the papain/beads ratio of 1:5 in phosphate buffer (pH 8.0, 200 mM) at 45 °C for 4 h. The storage stability, pH stability, thermal stability, solvent tolerance and resistance to metal ions of papain were enhanced by the immobilization. The oligomerization of L-glutamic acid diethyl ester hydrochloride via PS-PEG-DDI immobilized papain in phosphate buffer (pH 8, 200 mM) at 45 °C demonstrated the yield of oligo(γ-ethyl-L-glutamate) reached 78.6 ± 1.2% under the conditions of 2 U/ml of papain, 60 mg/ml L-glutamic acid diethyl ester hydrochloride for 4 h. The immobilization increased the yield by about 40%. The analysis by mass spectrometry (MS) and 1H NMR showed an average degree of polymerization (DPavg) of product was approximately 7–8, identical to that produced using free papain. The biocatalytic activity of the PS-PEG-DDI immobilized papain remained stable after 5 repeated uses, all giving a yield of over 70%. The reduced activation energy of the reaction catalyzed by the PS-PEG-DDI immobilized papain suggests a more favorable active conformation was probably achieved following immobilization.

Protease-Catalyzed l-Aspartate Oligomerization: Substrate Selectivity and Computational Modeling.

Poly(aspartic acid) (PAA) is a biodegradable water-soluble anionic polymer that can potentially replace poly(acrylic acid) for industrial applications and has shown promise for regenerative medicine and drug delivery. This paper describes an efficient and sustainable route that uses protease catalysis to convert l-aspartate diethyl ester (Et2-Asp) to oligo(β-ethyl-α-aspartate), oligo(β-Et-α-Asp). Comparative studies of protease activity for oligo(β-Et-α-Asp) synthesis revealed α-chymotrypsin to be the most efficient. Papain, which is highly active for l-glutamic acid diethyl ester (Et2-Glu) oligomerization, is inactive for Et2-Asp oligomerization. The assignment of α-linkages between aspartate repeat units formed by α-chymotrypsin catalysis is based on nuclear magnetic resonance (NMR) trifluoacetic acid titration, circular dichroism, and NMR structural analysis. The influence of reaction conditions (pH, temperature, reaction time, and buffer/monomer/α-chymotrypsin concentrations) on oligopeptide yield and average degree of polymerization (DPavg) was determined. Under preferred reaction conditions (pH 8.5, 40 °C, 0.5 M Et2-Asp, 3 mg/mL α-chymotrypsin), Et2-Asp oligomerizations reached maximum oligo(β-Et-α-Asp) yields of ∼60% with a DPavg of ∼12 (Mn 1762) in just 5 min. Computational modeling using Rosetta software gave relative energies of substrate docking to papain and α-chymotrypsin active sites. The substrate preference calculated by Rosetta modeling of α-chymotrypsin and papain for Et2-Asp and Et2-Glu oligomerizations, respectively, is consistent with experimental results.

Preparation and adsorption capacities evaluation of supramolecular two-component gels nanostructures via fluorine-containing diacid and glutamic acid amino derivative

ABSTRACTIn this paper, new two-component supramolecular gels via 2,2-bis(4-carboxyphenyl)hexafluoropropane and N-(4-Aminobenzoyl)-L-glutamic acid diethyl ester have been prepared and tested in ethanol/water mixed solvents. Scanning electron microscope (SEM), Transmission electron microscope (TEM), powder X-ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FT-IR) were measured to characterize the morphology and nanostructure of the prepared supramolecular gels. The obtained experimental data reveal that different mixed solvents ratios play an important role in regulating formation of nanostructures and the gelation behaviors. Favorable volume ratios in ethanol/water mixed solvents seemed suitable for the formation of supramolecular gels due to cooperation of multi-intermolecular weak forces. SEM and TEM observations indicate that the gelator molecules self-assemble into different aggregate nanostructures including sheets, rods, bar shape, and belt in different solvents ratios. The adsorption capacities of the as-obtained supramolecular gels on model dyes demonstrate good removal rates with the adsorption process according to rather pseudo-first-order model than pseudo-second-order model. The present research work show it potential to prepare new gel soft materials and promising candidates for wastewater treatment.

Preparation and Absorption Capacities of Two-Component Supramolecular Gels

Background: Two-component gels are a class of gel system which have different functionalized chemical groups and nanoparticles. The aim of this paper is to introduce readers to the new two-component gels system composed of N-(4-aminobenzoyl)-L-glutamic acid diethyl ester with polyacrylic acid by the self-assembly in different solvents. The obtained morphological and spectral data demonstrate that the single or mixed solvents make a great difference on the gelation abilities of these studied supramolecular gels. Keywords: Two-component gels, nanostructure, self-assembly, adsorption capacity, xerogels, Supramolecular gels.

The negative effects of alcohol hangover on high-anxiety phenotype rats are influenced by the glutamate receptors of the dorsal midbrain

Alcoholism is a chronic disorder characterized by the appearance of a withdrawal syndrome following the abrupt cessation of alcohol intake that includes symptoms of physical and emotional disturbances, anxiety being the most prevalent symptom. In humans, it was shown that anxiety may increase the probability of relapse. In laboratory animals, however, the use of anxiety to predict alcohol preference has remained difficult. Excitatory amino acids as glutamate have been implicated in alcohol hangover and may be responsible for the seizures and anxiety observed during withdrawal. The dorsal periaqueductal gray (DPAG) is a midbrain region critical for the modulation/expression of anxiety- and fear-related behaviors and the propagation of seizures induced by alcohol withdrawal, the glutamate neurotransmission being one of the most affected. The present study was designed to evaluate whether low- (LA) and high-anxiety rats (HA), tested during the alcohol hangover phase, in which anxiety is the most prevalent symptom, are more sensitive to the reinforcing effects of alcohol when tested in a voluntary alcohol drinking procedure. Additionally, we were interested in investigating the main effects of reducing the excitatory tonus of the dorsal midbrain, after the blockade of the ionotropic glutamate receptors into the DPAG, on the voluntary alcohol intake of HA and LA motivated rats that were made previously experienced with the free operant response of alcohol drinking. For this purpose, we used local infusions of the N-metil D-Aspartato (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-kainate receptors antagonist DL-2-Amino-7-phosphonoheptanoic acid – DL-AP7 (10nmol/0.2μl) and l-glutamic acid diethyl ester – GDEE (160nmol/0.2μl), respectively. Alcohol intoxication was produced by 10 daily bolus intraperitonial (IP) injections of alcohol (2.0g/kg). Peak-blood alcohol levels were determined by gas-chromatography analysis in order to assess blood-alcohol content. Unconditioned and conditioned anxiety-like behavior was assessed by the use of the fear-potentiated startle procedure (FPS). Data collected showed that anxiety and alcohol drinking in HA animals are positively correlated in animals that were made previously familiarized with the anxiolytic effects of alcohol. In addition, anxiety-like behavior induced during alcohol hangover seems to be an effect of changes in glutamatergic neurotransmission into DPAG possibly involving AMPA/kainate and NMDA receptors, among others.

Surprising metal binding properties of phytochelatin-like peptides prepared by protease-catalysis

Phytochelatins (PCs) consist of alternating glutamic acid and cysteine residues ([γGlu–Cys]–Xaa) and are responsible for binding to heavy metals for cellular metal homeostasis and detoxification. This paper describes the papain-catalyzed synthesis of cysteine-rich peptides as potential PC mimics. By adjusting the feed ratio of L-cysteine ethyl ester (L-Et-Cys) and L-glutamic acid diethyl ester (L-(Et)2-Glu) followed by de-esterification, α-linked oligo(L-Glu-co-47%L-Cys) was prepared that closely matches the 1 : 1 L-Glu-to-L-Cys molar ratio of PC peptides. Plots of absorbance difference as a function of total metal-to-peptide ([M]total–[P]total) molar ratio were constructed for titrations with Zn(II), Cd(II), Co(II) and Ni(II). A series of equations were generated to evaluate dissociation constants and the number of metal ions per peptide molecule in metal–peptide complexes. All of the four complexes have on average two oligo(L-Glu-co-47%L-Cys) molecules per divalent cation. The binding of metals to oligo(L-Glu-co-47%L-Cys) weakens in the order of Cd(II) > Zn(II) > Ni(II) > Co(II), the same trend observed for [(γGlu–Cys)4]–Gly. The peptide quantity required to sequester a given amount of Zn(II) and Cd(II) when using oligo(L-Glu-co-47%L-Cys) is only about twice as much as the quantity for perfect sequence peptide (γGlu–Cys)2–Gly. Furthermore, when [M]total is low, random oligo(L-Glu-co-47%L-Cys) sequesters much higher fractions of Co(II) and Ni(II) than does (γGlu–Cys)2–Gly. Results from this work provide the first evidence that, in some cases, uniform peptides synthesized by tedious solid or liquid phase peptide synthetic methods, can be replaced with peptide mixtures prepared by facile protease-catalyzed peptide synthetic methods without substantial loss in product performance.

Design, Synthesis, and X-ray Crystal Structure of Classical and Nonclassical 2-Amino-4-oxo-5-substituted-6-ethylthieno[2,3-d]pyrimidines as Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors and as Potential Antitumor Agents

N-{4-[(2-Amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)thio]benzoyl}-L-glutamic acid 2 and 13 nonclassical analogues 2a-2m were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in the synthesis was 2-amino-6-ethyl-5-iodothieno[2,3-d]pyrimidin-4(3H)-one, 7, to which various arylthiols were attached at the 5-position. Coupling 8 with L-glutamic acid diethyl ester and saponification afforded 2. X-ray crystal structures of 2 and 1 (the 6-methyl analogue of 2), DHFR, and NADPH showed for the first time that the thieno[2,3-d]pyrimidine ring binds in a "folate" mode. Compound 2 was an excellent dual inhibitor of human TS (IC50 = 54 nM) and human DHFR (IC50 = 19 nM) and afforded nanomolar GI50 values against tumor cells in culture. The 6-ethyl substitution in 2 increases both the potency (by 2-3 orders of magnitude) as well as the spectrum of tumor inhibition in vitro compared to the 6-methyl analogue 1. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from Toxoplasma gondii.

Mass spectrometric and kinetic studies on slow progression of papain-catalyzed polymerization of l-glutamic acid diethyl ester

Papain polymerizes l-glutamic acid diethyl ester (Glu-di-OEt) regioselectively, resulting in the formation of poly (γ-ethyl α- l-glutamic acid) with various degrees of polymerization of less than 13. Reaction temperatures below 20 °C were appropriate for the reaction in terms of suppression of non-enzymatic degradation of Glu-di-OEt and an increase in the peptide yield, while the reaction was preceded by a pronounced induction period. Mass spectrometric analyses of the reaction conducted at 0 °C revealed that the accumulation of the initial dimerization product, l-glutamyl- l-glutamic acid triethyl ester (Glu-Glu-tri-OEt), was limited during the induction period, and that a sequential polymer derived from a further elongation of the dimer was the tetramer, but not the trimer. Kinetic analyses of acyl transfer reactions with Glu-di-OEt and Glu-Glu-tri-OEt as acyl acceptors and Nα-benzoyl- l-arginine ethyl ester as an acyl donor affirmed that Glu-Glu-tri-OEt bound more strongly than Glu-di-OEt both to the S- and S′-subsites of papain. Therefore, what occurred during the initial stage of the polymerization was interpreted as follows: the rate of the papain-catalyzed dimerization of Glu-di-OEt was extremely slow, once Glu-Glu-tri-OEt was initially synthesized it exclusively bound to the active site of papain, and then papain utilized the dimer in polymerization effectively rather than the monomer.

Synthesis of N-(5,7-diamino-3-phenyl-quinoxalin-2-yl)-3,4,5-substituted anilines and N-[4[(5,7-diamino-3-phenylquinoxalin-2-yl)amino]benzoyl]- l-glutamic acid diethyl ester: Evaluation of in vitro anti-cancer and anti-folate activities

Several diamino quinoxalines were designed, synthesized and evaluated as anti-tumor agents. Two compounds showed the most potent cytotoxic activities against the leukemia CCRF-CEM cell line (GI 50 < 0.01 μM) and the ovarian cancer cell line OVCAR-4 (GI 50 = 0.03 μM), respectively, with comparable/better activities than Methotrexate (MTX). Docking calculations of the complexes of hDHFR with the most active compounds identified the binding mode of the described molecules with respect to MTX.

Ultrasound induced formation of organogel from a glutamic dendron

New l-glutamic acid based dendritic compounds: N-(2-naphthacarbonyl)- l-glutamic acid diethyl ester (NGE) and N-(2-naphthacarbonyl)-1,5-bis( l-glutamic acid diethyl ester)- l-glutamic diamide (NBGE) were designed. Although NGE could not form any gels in common solvents, NBGE could form stable gels in hexane, toluene, and water under ultrasound. Three dimensional network structures composed of fibers with various diameters were observed in the gel by SEM and TEM. FTIR spectral measurement revealed that ultrasound during cooling of the solution could destroy some of the hydrogen bond interactions and caused the gel formation. In solution, no CD signal was detected because the naphthyl chromophore is far from the chiral center. In the gel, however, CD signals assigned to the naphthyl group were observed, which indicated that the chirality of the chiral center could be transferred to the chromophore in the supramolecular organogel system.

Postsynaptic α4β2 and α7 type nicotinic acetylcholine receptors contribute to the local and endogenous acetylcholine-mediated synaptic transmissions in nigral dopaminergic neurons

The local and endogenous nicotinic neuronal transmissions of dopaminergic neurons in the substantia nigra were confirmed electrophysiologically using a slice-patch technique. After identifying dopaminergic neurons based on their electrophysiological characteristics, miniature postsynaptic inward currents were recorded in the presence of atropine (a muscarinic acetylcholine receptor antagonist), bicuculline (a GABA receptor antagonist) and L-glutamic acid diethyl ester (GDEE) (a non-selective glutamate receptor antagonist). Under conditions that eliminated muscarinic, GABAergic and glutamatergic synaptic transmissions, we found miniature currents that were inhibited by the specific neuronal nicotinic receptor antagonists, dihydro-β-erythroidine (DHβE) and/or methyllycaconitine (MLA) (selective α4β2 and/or α7 nicotinic acetylcholine receptor antagonists, respectively). Under the same extracellular conditions, local stimulations in the vicinity of a target neuron evoked excitatory postsynaptic inward currents (EPSCs). These EPSCs were elicited in an extracellular Ca 2+ dependent manner and were also blocked by DHβE and/or MLA. These results suggest that dopaminergic neurons in the substantia nigra receive excitatory cholinergic inputs that are mediated via at least two types of postsynaptic nicotinic receptors, namely α7 and α4β2 subtypes.

Mouse Cerebellar Adenosine-Glutamate Interactions and Modulation of Ethanol-Induced Motor Incoordination

Background It was demonstrated previously that cerebellar adenosine modulates ethanol-induced motor incoordination via A1 subtype of adenosine receptors. Several reports suggest the involvement of brain glutamate mechanisms, in particular N-methyl-d-aspartate (NMDA) receptor sites, in the central nervous system (CNS) actions of ethanol. Mutually antagonistic functional responses as a result of glutamate and adenosine within the brain regions have also been well documented. Methods With the use of rotorod performance as the test response, this study was conducted to evaluate possible functional interactions between cerebellar adenosine and glutamate and its consequence on ethanol-induced motor incoordination. Except for ethanol, which was injected intraperitoneally, all drugs used were microinfused directly into the cerebellum. Results Direct intracerebellar microinfusion of glutamate (125, 250, and 500 ng) and the antagonist l-glutamic acid diethyl-ester (125, 250, and 500 ng) markedly and dose-dependently attenuated and accentuated, respectively, ethanol-induced motor incoordination, suggesting an involvement of glutamate. Subsequently, intracerebellar microinfusions of NMDA (125, 250, and 500 ng) and its antagonists AP-5 [(+)-2-amino-5-phosphoropentanoic acid; 125, 250, and 500 ng] and (+)-MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a. d]-cyclo-hepten-5,10-imine hydrogen maleate; 25, 50, and 100 ng] significantly attenuated and accentuated, respectively, ethanol-induced motor incoordination in a dose-related manner, indicating participation of NMDA receptor. The attenuation of ethanol-induced motor incoordination by glutamate and NMDA was receptor mediated as it was antagonized by their receptor antagonists. Adenosine A1-selective agonist N6-cyclohexyladenosine and antagonist 8-cyclopentyl-1,3-dipropylxanthine functionally opposed the attenuation by glutamate and NMDA and the accentuation by L-glutamic acid diethyl-ester, AP-5, and (+)-MK-801, respectively, of ethanol-induced motor incoordination. Conclusions These results suggest a functional antagonism between glutamate NMDA and adenosine A1 receptors exhibiting a co-modulation of ethanol-induced motor incoordination within the cerebellum.

Neurotransmitters in the thalamus relaying visceral input to the insular cortex in the rat.

Neurotransmitters relaying ascending visceral information were examined by comparing the response of neurons in the insular cortex to vagal stimulation (0.8 Hz, 2 mA) before and after neurotransmitter antagonist injections (200 nl) in the ventroposterior parvocellular nucleus of the thalamus (VPpc). Cobalt (10 mM; presynaptic blocker) and kynurenate (100 microM; nonspecific excitatory amino acid antagonist) injections in the VPpc resulted in an attenuation (73-100 and 38-98%, respectively) of the evoked cortical response. Injections of the specific N-methyl-D-aspartate (NMDA) antagonist DL-2-amino-5-phosphonopentanoic acid (200 microM and 2 mM) did not affect the vagally evoked response, whereas the nonspecific non-NMDA antagonist L-glutamic acid diethylester (200 microM) attenuated the vagally evoked response by 66-100%. Three concentrations of the DL-alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA)-specific antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 and 200 microM and 2 mM) attenuated the vagally evoked cortical response by 29 +/- 9, 31 +/- 10, and 59 +/- 8%, respectively. The more selective AMPA antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (200 microM and 2 mM) inhibited the vagally evoked cortical response by 53 +/- 8 and 52 +/- 3%, respectively. Phentolamine (0.1 and 1.0 microM), a general alpha-adrenergic antagonist, and picrotoxin (0.1 and 1.0 microM), a GABA(A) antagonist, did not affect the vagally evoked response. Atropine, a muscarinic cholinergic antagonist, decreased the vagally evoked response by 40 +/- 2% at a concentration of 0.1 microM, but a higher concentration of 1.0 microM had no effect. These results indicate that the non-NMDA excitatory amino acid receptor is necessary for the relay of visceral information in the VPpc. Muscarinic receptors may modulate visceral neuronal excitability in the VPpc, although the exact interaction between the inhibitory (m2) and excitatory (m3 or m5) muscarinic receptor types found in the thalamus is not known.

Excitation of Rat Striatal Large Neurons by Dopamine and / or Glutamate Released From Nerve Terminals via Presynaptic Nicotinic Receptor (α4β2 Type) Stimulation

Previous in vivo experiments using rats anesthetized with chloral hydrate have revealed that nicotine applied iontophoretically increased firing of striatal neurons receiving excitatory dopaminergic input from the substantia nigra, and nicotine-induced firing was inhibited by domperidone, a dopamine D2 antagonist. The results suggest that nicotine increases release of dopamine from the terminals of dopaminergic neurons. Therefore, we performed the present patch clamp study using slice and acutely dissociated preparations of the rat striatum to elucidate the mechanisms underlying the nicotine-induced excitation of striatal neurons. Application of nicotine (100 microM) to large striatal neurons in slice preparations did not produce any effect on the resting membrane potential, but did increase the frequency of miniature postsynaptic potentials (mpps) and action potentials in all 15 neurons tested. The nicotine-induced increase in mpps and action potentials were inhibited during simultaneous application of domperidone; L-glutamic acid diethyl ester hydrochloride, a non-selective glutamate receptor antagonist; and/or dihydro-beta-erythroidine, a central nicotinic acetylcholine receptor (alpha4beta2 type) antagonist. Postsynaptic current was not induced by nicotine applied by U-tube in 96% of acutely dissociated striatal neurons. The present findings suggest that nicotine mainly acts on the presynaptic nicotinic receptors in the nerve terminals to release neurotransmitters such as dopamine and/or glutamate, thereby activating the striatal large neurons.

Cholinergic and glutamatergic transmission in medial vestibular nucleus neurons responding to lateral roll tilt in rats

The responses of the medial vestibular nucleus (MVN) neurons to lateral tilt and the neurotransmitters mediating otolith information to MVN neurons were investigated using rats. A computer-operated goniometer was tilted 20° clockwise and counterclockwise at an angular speed of 5°/s and paused in the inclined positions for 10 s to record neuronal responses in the static phase. The 185 MVN neurons recorded were classified into eight types according to their responses to tilt (α, β, γ, δ, ε, ζ, η and θ). A majority showed increased firing in response to ipsilateral tilting and decreased firing in response to contralateral tilting (α type: 31.4%) or exhibited the reverse pattern (β type: 36.8%). Further, other groups of neurons increased (γ type) or decreased (δ type) firing rates to either side tilting and increased (ε and ζ type) or decreased (η and θ type) firing only on one side. Atropine or l-glutamic acid diethyl ester hydrochloride (GDEE) applied microiontophoretically antagonized tilt-induced firing of α type neurons in 58.8% or 60.0%, respectively, and of β type neurons in 66.7% or 58.3%, respectively. When the effects of atropine and GDEE were examined in the same neurons, antagonizing effects of both drugs on tilt-induced firing were obtained in 28.6% and 40.0% of α and β type neurons, respectively. These results suggest that both acetylcholine and glutamate act as neurotransmitters in the transmission of otolith information to most MVN neurons.

Quenching studies of pyrenoyl glutamic acid derivatives

The preparation and full characterization of fluorescent labeled N-pyrenoyl–L-glutamic acid diethyl ester (2) and 4-(N-Boc–L-glutamide(O-benzyl ester))–N,N-dimethylaniline (3) by the DCC–HOBT protocol is described. The benzyl group in 3 was removed by hydrogenation giving the free acid 4-(N-Boc–L-glutamide(OH))–N,N-dimethylaniline (4). The steady-state and time-resolved fluorescence quenching studies were performed in order to distinguish ground state quenching taking place in a hydrogen-bonded complex and dynamic quenching. Weak molecular interactions between the fluorophore 2 and the quencher 4 leading to a 1∶1 complex were detected by 1H-NMR titration experiments. The association constants were determined by fluorescence methods to be in the order of 3.8–4.0 × 103 M–1 for all systems investigated. The bimolecular quenching constants kq are in the order of 1.0–1.28 × 1010 M–1 s–1, indicating a diffusion controlled electron transfer process.

Adenosine and glutamate modulate the cardiovascular responses of angiotensins II and III in the area postrema of rats

The purpose of this study was to determine the interactions of the renin-angiotensin system with adenosine and glutamate in the area postrema (AP) of rats. Male Sprague-Dawley rats were anesthetized with urethane. Adenosine, angiotensins (Ang) II, III and their antagonist 1,3-Dipropyl-8- p-sulfophenylxanthine (DPSPX), [Sar 1Ile 7]Ang III and glutamate antagonist, l-glutamic acid diethyl ester (GDEE) were microinjected into the AP of rats. Our results demonstrated that microinjection of DPSPX significantly attenuated the depressor and bradycardic effects of Ang II and III at low (9.6 pmol) and high dose (480 pmol) of Ang II in normotensive rats. To test the interaction of glutamate and renin-angiotensin system, we found that glutamate antagonist, GDEE, markedly lowered depressor and bradycardic responses of Ang II but did not influence Ang III in rats. On the other hand, microinjection of the Ang antagonist [Sar 1Ile 7]Ang III 10 min prior to the injection of adenosine significantly altered the cardiovascular effects of adenosine in the AP. In conclusion, the endogenous adenosine and glutamate may influence the renin-angiotensin system on cardiovascular responses in the AP of rats.

NMDA and Not Non-NMDA Receptor Antagonists Are Protective against Seizures Induced by Homocysteine in Neonatal Rats

Homocysteine induces seizures in adult, as well as in immature, experimental animals, but the mechanism of its action is still unknown. The aim of the present study was to examine whether homocysteine in immature animals may act via excitatory amino acids receptors. Seizures were induced in 7-day-old rats by ip administration of homocysteine (16.5 mmol/kg) and the effects of selected antagonists at NMDA and non-NMDA receptor sites were investigated. The anticonvulsant effect was evaluated not only in terms of behavioral changes, but also in terms of some indicators of brain energy metabolism. Rat pups were sacrificed during generalized clonic-tonic seizures, corresponding approximately to 15-30 min after homocysteine administration. Comparable time intervals were used for sacrificing pups in the groups with protective drugs. Non-NMDA antagonists, l-glutamic acid diethylester (GDEE) (4 mmol/kg, ip) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (NBQX) (two doses, 30 mg/kg each, ip), failed to protect neonatal rats against homocysteine-induced seizures. Although NBQX prevented the tonic phase, the severity of clonic movements was even more pronounced. Metabolic changes accompanying the seizures (decreases of glucose and glycogen and a rise of lactate) were also not influenced by GDEE or NBQX pretreatment. On the contrary, NMDA antagonists, both competitive (AP7, 0.33 mmol/kg, ip) and noncompetitive (MK-801, 0.5 mg/kg, ip), had a clear-cut anticonvulsant effect. They not only suppressed the behavioral signs of seizures, but also prevented most of the metabolic changes accompanying seizures. Whether incomplete normalization of lactate levels reflects subclinical seizure activity or whether it is due to the effect of anatagonists (AP7, MK-801) themselves is not clear. All drugs, besides GDEE, when given alone caused a rise in brain glucose. This is the first demonstration that seizures induced by homocysteine can be blocked by NMDA antagonists. The findings thus suggest that, at least in neonatal rats, enhanced activation of NMDA receptors is implicated in homocysteine-induced seizures.

Effects of ketamine and L-glutamic acid diethyl ester on spatial and nonspatial learning tasks in rats

An NMDA antagonist, ketamine, at the highest dose tested (15 mg/kg), impaired the acquisition of a hole-board spatial learning task but not the acquisition of a left-right alternation task. A non-NMDA (quisqualate) antagonist, L-glutamic acid diethyl ester (LGDE), did not impair the acquisition of either task. Both drugs had effects on different aspects of a go-no go discrimination task and a straight runway task, ketamine tending to activate and LGDE tending to slow rats. These results concur with previous research regarding the sensitivity of some spatial tasks to NMDA antagonism. Non-NMDA antagonists affect behavior without causing spatial deficits.

Striatal N-methyl-D-aspartate receptors in haloperidol-induced catalepsy

Bilateral ablation of the frontal cortex of rats markedly reduced the catalepsy induced by haloperidol (1 mg/kg i.p.). Similarly, the selective antagonist of N-methyl-D-aspartate (NMDA) receptors, D(−)-2-amino-5-phosphonopentanoic acid (10 μg/side), injected bilaterally into the rostral part of the caudate-putamen (CP) reduced haloperidol-induced catalepsy whereas its injection into the intermediate part of the CP was ineffective. The quisqualate receptor antagonist, L-glutamic acid diethyl ester (100 μg/side), did not affect haloperidol-induced catalepsy when injected into the rostral part of the CP. On the other hand, NMDA (1μg/side) injected bilaterally into the rostral part of the CP was able to restore haloperidol-induced catalepsy in frontally decorticated rats without any notable cataleptic effect of its own. These findings suggest that a certain degree of tonic stimulatory effect of corticostriatal glutamatergic pathways on NMDA receptors within the rostral part of the CP is a prerequisite for the expression of the cataleptogenic action of haloperidol.