Synthesis of diastereoisomeric peptides incorporating cycloglutamic acids. Substrate specificity of vitamin K-dependent carboxylation.

Tripeptides Boc-X-Glu-Val where X is alpha-methyl glutamic acid or various cyclic analogues of glutamic acid, such as 1-amino-1,3-dicarboxycyclohexane (cis or trans-CHGA) or -cyclopentane (cis or trans-CPGA) have been synthesized. Methods for the selective protection, activation, and coupling of such unnatural amino acids are described. The peptides, which are potential competitive inhibitors of the vitamin K-dependent carboxylation, have been preliminarily tested with the rat liver microsomal carboxylase and found to be effective substrates of the carboxylation reaction.     

4-Bromohomoibotenic Acid Selectively Activates a 1-Aminocyclopentane-1S,3R-Dicarboxylic Acid-Insensitive Metabotropic Glutamate Receptor Coupled to Phosphoinositide Hydrolysis in Rat Cortical Slices

Abstract: Glutamate activates a family of receptors, known as metabotropic glutamate receptors (mGluRs), that are coupled to various second messenger systems through G proteins. All mGluR subtypes characterized to date in rat brain slices are activated by the glutamate analogue 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (1 S ,3 R -ACPD). However, few agonists are available that selectively activate specific mGluR subtypes. We report that the glutamate analogue ( R,S )-4-bromohomoibotenate (BrHI) stimulates phosphoinositide hydrolysis in rat cerebral cortical slices in a concentration-dependent manner (EC₅₀ = 190 µ M ). The response to BrHI is stereoselective and is not blocked by ionotropic glutamate receptor antagonists. It is interesting that the responses to BrHI and 1 S ,3 R -ACPD are completely additive, suggesting that these responses are mediated by different receptor subtypes. Consistent with this, the response to BrHI is insensitive to l -2-amino-3-phosphonopropionic acid ( l -AP3), whereas the response to 1 S ,3 R -ACPD is partially blocked by l -AP3. BrHI does not activate metabotropic receptors coupled to changes in cyclic AMP accumulation or activation of phospholipase D. Thus, BrHI seems to activate specifically a phosphoinositide hydrolysis-linked mGluR that is insensitive to 1 S ,3 R -ACPD. This compound may prove useful as a tool for elucidating the roles of different mGluR subtypes in mammalian brain.     

Cellular Mechanisms of Protection by Metabotropic Glutamate Receptors During Anoxia and Nitric Oxide Toxicity

Abstract: Metabotropic glutamate receptors, nitric oxide (NO), and the signal transduction pathways of protein kinase C (PKC) and protein kinase A (PKA) can independently alter ischemic-induced neuronal cell death. We therefore examined whether the protective effects of metabotropic glutamate receptors during anoxia and NO toxicity were mediated through the cellular pathways of PKC or PKA in primary hippocampal neurons. Pretreatment with the metabotropic glutamate receptor agonists (±)-1-aminocyclopentane- trans -1,3-dicarboxylic acid, (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD), and l (+)-2-amino-4-phosphonobutyric acid ( l -AP4) 1 h before anoxia or NO exposure increased hippocampal neuronal cell survival from ∼30 to 70%. In addition, posttreatment with 1 S ,3 R -ACPD or l -AP4 up to 6 h following an insult attenuated anoxic- or NO-induced neurodegeneration. In contrast, treatment with l -(+)-2-amino-3-phosphonopropionic acid, an antagonist of the metabotropic glutamate receptor, did not significantly alter neuronal survival during anoxia or NO exposure. Protection by the ACPD-sensitive metabotropic receptors, such as the subtypes mGluR1α, mGluR2, and mGluR5, appears to be dependent on the modulation of PKC activity. In contrast, l -AP4-sensitive metabotropic glutamate receptors, such as the subtype mGluR4, may increase neuronal survival through PKA rather than PKC. Thus, activation of specific metabotropic glutamate receptors is protective during anoxia and NO toxicity, but the signal transduction pathways mediating protection differ among the metabotropic glutamate receptor subtypes.     

Excitation of rat hippocampal neurones by the stereoisomers of cis- and trans-1-amino-1,3-cyclopentane dicarboxylate

Intracellular recordings were obtained from rat hippocampal neurons during the microiontophoretic ejection of the stereoisomers of cis- and trans-1-amino-1,3-cyclopentane dicarboxylate into the dendritic region (stratum radiatum) of the impaled cells. L-(+)-cis-1-Amino-1,3-cyclopentane dicarboxylate, D(+)-trans-1-amino-1,3-cyclopentane dicarboxylate, and L-(-)-trans-1-amino-1,3-cyclopentane dicarboxylate all evoked patterns of excitation resembling that elicited by kainate. All of these responses were unaffected by D-(-)-2-amino-5-phosphonovalerate but were antagonized at comparable currents by kynurenate. The excitation produced by D-(-)-cis-1-amino-1,3-cyclopentane dicarboxylate was similar to that evoked by N-methyl-D-aspartate. At low ejection currents a slow depolarization triggered rhythmic burst firing, each burst consisting of a depolarizing shift in membrane potential upon which were superimposed four to five action potentials. These responses were antagonized both by D-(-)-2-amino-5-phosphonovalerate and by kynurenate. The results are discussed with respect to the conformational requirements considered to be necessary for interaction at the kainate and N-methyl-D-aspartate receptors on CA1 pyramidal neurones. It is important to note that the isopropylene side chain of kainate is absent from the 1-amino-1-3-cyclopentane dicarboxylate molecule.     

Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology

Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.     

Modulation by Ionotropic Excitatory Amino Acids and Potassium of (±)-1-Aminocyclopentane-trans-1,3-Dicarboxylic Acid-Stimulated Phosphoinositide Hydrolysis in Mouse Cerebellar Granule Cells

Abstract: The effect of ionotropic excitatory amino acids and potassium on the formation of inositol phosphates elicited by the metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was studied in mouse cerebellar granule cells. In Mg²⁺-containing buffers, NMDA (50–100 µM), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 10–1,000 µM), and high potassium (10–30 mM) enhanced synergistically the response to a maximally effective concentration of 500 µMtrans-ACPD. Potentiation of the trans-ACPD response was blocked by higher concentrations of NMDA (>500 µM) and potassium (>35 mM) but not by AMPA (up to 1 mM). The potentiation by NMDA of the trans-ACPD-stimulated phosphoinositide hydrolysis was blocked by d,l -2-amino-5-phosphonopentanoic acid (APV), a competitive NMDA-receptor antagonist. Under Mg²⁺-free conditions, the accumulation of inositol phosphates in the presence of trans-ACPD alone was equal to that attained by trans-ACPD in Mg²⁺-containing buffers when costimulated with maximally enhancing concentrations of NMDA (50 µM). trans-ACPD potentiated synergistically the NMDA-evoked increases in cytosolic free-Ca²⁺ levels in Mg²⁺-containing but not in Mg²⁺-free solutions, and moreover did not enhance the AMPA-evoked increases in cytosolic free-Ca²⁺ levels. The calcium ionophore A23187 caused a dose-dependent increase in inositol phosphate accumulation but did not enhance the response stimulated by trans-ACPD alone. These results demonstrate the existence of cross talk between metabotropic and ionotropic glutamate receptors in cerebellar granule cells. The exact mechanism remains unclear but appears to involve interplay of G protein-coupled phospholipase C activation and regulated elevation of cytosolic free-Ca²⁺ levels. This study may provide a framework for future investigations at the cellular and molecular level that clarify the functional relevance and molecular mechanisms that are described.     

Effect of various analogues of D-glutamic acid on the D-glutamate-adding enzyme from Escherichis coli

Abstract Twenty-four analogues of D-glutamic acid were tested as substrates or inhibitors of the D-glutamate-adding enzyme from Escherichia coli . The best substrates were, in decreasing order of specific activity, D- erythro -4-methylglutamic acid, D- erythro - methylglutamic acid, DL-homocysteic acid, (±)- trans -1-amino-3-carboxy-cyclopentanecarboxylic acid and (±)- trans -1-amino-3-carboxy-cyclohexanecarboxylic acid. Among the different stereoisomers, only the D- erythro isomers for methylglutamic acids, and the trans isomers for the cyclic analogs, were substrates. Apart from the D- erythro -3 and 4-methylglutamic acids and DL-homocysteic acid, none of the examined compounds significantly inhibited the addition of radioactive D-glutamic acid to UDP-N-acetylmuramyl-L-alanine.     

Novel 5-substituted 1-pyrazolol analogues of ibotenic acid: synthesis and pharmacology at glutamate receptors

5-Substituted 1-pyrazolol analogues of ibotenic acid have been synthesized and pharmacologically characterized on ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). The syntheses involved introduction of bromide, alkyls, phenyl and arylalkyls in the 5-position of 1-benzyloxypyrazole leading to 5-substituted (RS)-2-amino-(1-hydroxy-4-pyrazolyl)acetic acids (5a-l). The pharmacological activities of the synthesized analogues ranged from the 5-cyclopropylmethyl analogue (5f) with weak but selective affinity for NMDA receptors (IC(50)=35 microM), over the 5-n-propyl analogue (5c), which was a selective mGluR2 agonist (EC(50)=72 microM), to the 5-cyclohexylmethyl analogue (5g), which was a selective mGluR2 antagonist (K(i)=32 microM), and the 5-phenylethyl analogue (5j), which was a weak but apparently selective mGluR1 antagonist (K(i)=230 microM). This series of compounds afforded GluR ligands with a broad spectrum of pharmacological profiles, and showing potential for development of new compounds with subtype-selective activities at various GluRs.     

Stimulation of Postsynaptic and Inhibition of Presynaptic Adenylyl Cyclase Activity by Metabotropic Glutamate: Receptor Activation

Abstract: To determine the subcellular distribution of cyclic AMP-coupled metabotropic glutamate receptors (mGluRs), the effects of glutamate agonists on adenylyl cyclase activity were examined using two hippocampal membrane preparations. These were synaptosomes (SY), which are composed of presynaptic terminals, and synaptoneurosomes (SN), which are composed of both pre-and postsynaptic elements. In SY, a water-soluble analogue of forskolin (7β-forskolin) increased enzyme activity ˜ 10-fold at the highest concentration tested. The selective metabotropic receptor agonist (1S,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3 R -ACPD) inhibited enzyme activity as did glutamate and quisqualate. l -Amino-4-phosphobutanoate ( l -AP4) had no effect on enzyme activity at any concentration tested. The metabotropic receptor antagonist l -2-amino-3-phosphopropionic acid ( l -AP3) was not effective in the SY in antagonizing the agonist-induced decreases in adenylyl cyclase activity by glutamate or 1S,3 R -ACPD. It was, however, effective at antagonizing quisqualate-induced decreases in enzyme activity. In SN, at the highest concentration tested, 7β-forskolin produced a 60-fold increase in adenylyl cyclase activity. As was observed in SY, glutamate decreased adenylyl cyclase activity in SN. In contrast, 1S,3 R -ACPD, quisqualate, and l -AP4 increased adenylyl cyclase activity. In the SN, l -AP3 was ineffective in antagonizing any agonist-induced increases (1S,3 R -ACPD, l -AP4, and quisqualate) or decreases (glutamate) in adenylyl cyclase activity. The data suggest that postsynaptic metabotropic glutamate receptor activation results in stimulation of adenylyl cyclase activity, whereas inhibition of this enzyme appears to be mediated at least partly through presynaptic mechanisms.     

Functional role of astrocyte glutamate receptors and carbon monoxide in cerebral vasodilation response to glutamate

In newborn pigs, vasodilation of pial arterioles in response to glutamate is mediated via carbon monoxide (CO), a gaseous messenger endogenously produced from heme degradation by a heme oxygenase (HO)-catalyzed reaction. We addressed the hypothesis that ionotropic glutamate receptors (iGluRs), including N-methyl-D-aspartic acid (NMDA)- and 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid (AMPA)/kainate-type receptors, expressed in cortical astrocytes mediate glutamate-induced astrocyte HO activation that leads to cerebral vasodilation. Acute vasoactive effects of topical iGluR agonists were determined by intravital microscopy using closed cranial windows in anesthetized newborn pigs. iGluR agonists, including NMDA, (±)1-aminocyclopentane-cis-1,3-dicarboxylic acid (cis-ACPD), AMPA, and kainate, produced pial arteriolar dilation. Topical L-2-aminoadipic acid, a gliotoxin that selectively disrupts glia limitans, reduced vasodilation caused by iGluR agonists, but not by hypercapnia, bradykinin, or sodium nitroprusside. In freshly isolated and cultured cortical astrocytes constitutively expressing HO-2, iGluR agonists NMDA, cis-ACPD, AMPA, and kainate rapidly increased CO production two- to threefold. Astrocytes overexpressing inducible HO-1 had high baseline CO but were less sensitive to glutamate stimulation of CO production when compared with HO-2-expressing astrocytes. Glutamate-induced astrocyte HO-2-mediated CO production was inhibited by either the NMDA receptor antagonist (R)-3C4HPG or the AMPA/kainate receptor antagonist DNQX. Accordingly, either antagonist abolished pial arteriolar dilation in response to glutamate, NMDA, and AMPA, indicating functional interaction among various subtypes of astrocytic iGluRs in response to glutamate stimulation. Overall, these data indicate that the astrocyte component of the neurovascular unit is responsible for the vasodilation response of pial arterioles to topically applied glutamate via iGluRs that are functionally linked to activation of constitutive HO in newborn piglets.     

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

Synthesis of oxytocin antagonists containing conformationally constrained amino acids in position 2

Analogues of oxytocin containing D-Trp, 2-amino-1,2,3,4-tetrahydronaphthalene-1-carboxylic acid (Atc) or 1,2,3,4-tetrahydro-beta-carboline-1-carboxylic acid (Car) with R or S configurations in position 2 were synthetized, and their receptor bindings were tested on isolated guinea-pig uterus, rat liver and rat kidney inner medulla plasma membranes. The peptides were synthetized in the solid phase by using racemates of Car and Atc. The resulting diastereomeric mixtures were separated by means of RP-HPLC. The binding to the oxytocin receptor was somewhat decreased for the Atc isomers and dramatically decreased for both R- and S-Car, while the D-Trp-containing analogue displayed a relatively high receptor affinity. However, the V1 receptor affinities were almost the same as those of the parent peptide for the Car-containing analogues and dramatically decreased for the S-Atc substituted analogue, which has a relatively high OT/V1 receptor selectivity of 44.5.     

The Effects of l-Glutamate and trans-(±)-1-Amino-1,3-Cyclopentanedicarboxylate on Phosphoinositide Hydrolysis Can Be Pharmacologically Differentiated

Abstract: The excitatory amino acid analogues l -glutamate ( l -Glu), l -aspartate ( l -Asp), d -Asp, and trans -(±)-1-amino-1,3-cyclopentanedicarboxylate ( trans -ACPD) stimulate the hydrolysis of phosphoinositides (PI). In the present studies, the effects of noncompetitive and competitive inhibitors on PI hydrolysis stimulated by excitatory amino acid analogues were examined. When agonist and inhibitor were added simultaneously to hippocampal tissue, the noncompetitive inhibitor l -2-amino-3-phosphonopropionate ( l -AP3) did not block the effects of l -Glu, l -Asp, or d -Asp at concentrations that block the effects of trans -ACPD by more than 80%. When tissue was pre-incubated with l -AP3, the effects of l -Glu, l -Asp, or d -Asp were blocked (IC₅₀ values between 65 and 210 µ M ). Unlike l -AP3, l -aspartate-β-hydroxamate ( l -AβHA) inhibited PI hydrolysis stimulated by trans -ACPD, l -Glu, l -Asp, or d -Asp when agonist and inhibitor were added simultaneously in hippocampus; its effects were not time-dependent. In cerebellum, both l -AP3 and l -AβHA had agonist activity. Inhibition by the recently identified competitive inhibitor (+)-α-methyl-4-carboxyphenylglycine [(+)-MCPG] of PI hydrolysis was also examined. (+)-MCPG blocked PI hydrolysis stimulated by trans -ACPD, l -Asp, or d -Asp in both hippocampus and cerebellum (IC₅₀ values between 220 and 1,700 µ M ). The effects of (+)-MCPG were consistent with a competitive mechanism of action. (+)-MCPG (up to 3 m M ) blocked PI hydrolysis stimulated by l -Glu by less than 25% in both hippocampus and cerebellum.     

Excitatory Amino Acid-Induced Formation of Inositol Phosphates in Guinea-Pig Cerebral Cortical Slices: Involvement of Ionotropic or Metabotropic Receptors?

In cerebral cortical slices from the guinea-pig, quinoxalinedione derivatives antagonised the generation of 3H-inositol phosphates evoked by the excitatory amino acids quisqualate and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid but were without effect on the trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid and L-glutamate responses. Omission of calcium from the medium reduced the accumulation of 3H-inositol phosphates induced by incubation with trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid (incubation for 45 min) by greater than 50%, whereas the responses to L-glutamate and the two other amino acid analogues were reduced by approximately 20%. Generation of inositol 1,4,5-trisphosphate over a 30-s period by treatment with quisqualate, trans-DL-1-amino-1,3-cyclopentane-dicarboxylic acid, KCl, and carbachol was abolished in the presence of nominally calcium-free medium. L-Glutamate induced a large, rapid increase in inositol 1,4,5-trisphosphate mass (more than three-fold), which was, however, unaffected by omission of calcium from the medium. These results indicate that of the excitatory amino acids tested, only L-glutamate may be classed as a metabotropic receptor agonist in guinea-pig cerebral cortical slices with respect to generation of inositol phosphates. The other agents appear to stimulate accumulation of inositol phosphates, at least in part through some mechanism requiring the presence of extracellular Ca2+, presumably Ca2+ entry.     

Further probing of the substrate specificities and inhibition of enzymes involved at an early stage of glycosylphosphatidylinositol (GPI) biosynthesis

1-D-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-1-O-hexadecyl-myo-inositol (14), 1-D-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1-(octadecyl phosphate) (18), 1-D-6-O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (24), 1-D-6-O-(2-amino-2-deoxy-alpha-D-mannopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (30) and the corresponding 2-amino-2-deoxy-alpha-D-galactopyranosyl analogue 36 have been prepared and tested in cell-free assays as substrate analogues/inhibitors of alpha-(1 --> 4)-D-mannosyltransferases that are active early on in the glycosylphosphatidylinositol (GPI) biosynthetic pathways of Trypanosoma brucei and HeLa (human) cells. The corresponding N-acetyl derivatives of these compounds were similarly tested as candidate substrate analogues/inhibitors of the N-deacetylases present in both systems. Following on from an early study, 1-L-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-2-O-methyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (44) was prepared and tested as an inhibitor of the trypanosomal alpha-(1 --> 4)-D-mannosyltransferase. A brief summary of the biological evaluation of the various analogues is provided.     

A metabotropic l-Glutamate receptor agonist: Pharmacological difference between rat central neurones and crayfish neuromuscular junctions

1. 2S,3S,4S-2-(carboxycyclopropyl)glycine (l-CCG-I), a conformationally restricted glutamate analogue, is a potent metabotropic l-glutamate receptor agonist in the mammalian central nervous system.2. Depolarizing actions of l-CCG-I and trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) in the newborn rat spinal motoneurone are temperature-sensitive, and are not depressed by 3-[(±)-2-carboxypiperazin-4-yl] propyl-1-phosphonic acid (CPP) and/or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX).3. l-CCG-I and trans-ACPD induced oscillatory responses in Xenopus oocytes injected with rat brain mRNA. Oocytes with oscillatory responses to l-CCG-I and trans-ACPD showed reversal potential of about −20 mV, which was very close to the equilibrium potential of chloride ions.4. In rat hippocampal synaptoneurosomes, l-CCG-I stimulated phosphoinositide hydrolysis in a concentration dependent manner. l-CCG-I was less potent than quisqualate but more potent than trans-ACPD.5. At low concentrations, l-CCG-I did not cause any depolarization of newborn rat spinal motoneurones, but reduced substantially amplitudes of monosynaptic reflexes.6. At the crayfish neuromuscular junction l-CCG-I, acting presynaptically, reduced the amplitude of excitatory junctional potentials. This action was prevented by application of picrotoxin but not pertussis toxin. The actions of trans-ACPD differ from those of either l-CCG-I or ibotenate at the crayfish neuromuscular junction.7. l-CCG-I has a potential to provide further useful information on metabotropic l-glutamate receptor function.     

Exploring the interest of 1,2-dithiolane ring system in peptide chemistry. Synthesis of a chemotactic tripeptide and x-ray crystal structure of a 4-amino-1,2-dithiolane-4-carboxylic acid derivative.

Due to their relevant biological functions and specific chemical reactivity 1,2-dithiolanes (five-membered cyclic disulfides) represent an emerging class of heterocyclic compounds. However, despite the extensive research centered on lipoic acid and its analogues, only very few data are at the present available on peptides containing this ring system. We report here synthesis, conformation and bioactivity of a fMLF-OMe analogue, namely For-Met-Adt-Phe-OMe (7), in which the residue of the 4-amino-1,2-dithiolane-4-carboxylic acid (Adt) (4) replaces the central L-leucine. The crystal conformation of the synthetic intermediate Boc-Adt-OMe (5) is also described and compared to that of lipoic acid (R-1,2-dithiolane-3-pentanoic acid) (3) and asparagusic acid (1,2-dithiolane-4-carboxylic acid) (2).     

Glutamate Activates Phospholipase D in Hippocampal Slices of Newborn and Adult Rats

Abstract: Phospholipase D (PLD) is activated by many neuro-transmitters in a novel signal transduction pathway. In the present work, PLD activity was studied comparatively in hippocampal slices of newborn and adult rats. Basal PLD activity in adult rats was almost three times higher than in newborn rats. In newborn rats, L-glutamate and 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD) time- and concentrationdependently enhanced the formation of [³H]phosphatidylpropanol ([³H]PP) and of [³H]phosphatidic acid in the presence of 2% propanol. N -MethylD-aspartate and kainate (both 1 m M ) caused small, but significant increases (∼50%). whereas α-amino-3-hydroxy-5-methylisoxazole-4-propionate (100 μ M ) was ineffective. Maximally effective concentrations of glutamate (1 m M ) and of 1 S ,3 R -ACPD (300 μ M ) increased the PLD activity to almost 300% of basal activity; the EC₅₀ values were 199 and 47 μ M , respectively. Glutamate receptor antagonists, such as DL-2-amino-3-phosphonopropionic acid (AP3). DL-2-aminc-5-phosphonovalenic acid, and kynurenate (all 1 m M ) did not inhibit the glutamate-evoked increase of PP formation. In slices of adult rats, the response to 1 S ,3 R -ACPD was significant, but small, whereas glutamate was effective only in the presence of the glutamate uptake inhibitor L-aspartate-β-hydroxarnate. It is concluded that glutamate activates PLD in rat hippocampus through an AP3-resistant metabotropic receptor. This effect is subject to ontogenetic development, with one important factor being glutamate uptake.     

Synthesis of fluorescent and radiolabeled analogues of phosphatidic acid

Procedures for the synthesis of fluorescent and radiolabeled analogues of phosphatidic acid are described. The fluorophore 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was coupled to 6-amino-caproic acid and 12-aminododecanoic acid by reaction of NBD-chloride with the amino acids under mild alkaline conditions at room temperature. 1,2-Dioleoyl-sn-[U-14C]glycerol 3-phosphate was prepared by acylation of sn-[U-14C]glycerol 3-phosphate with oleic acid anhydride using dimethylaminopyridine as the catalyst. This compound was converted to 1-oleoyl-sn-[U-14C]glycerol 3-phosphate by hydrolysis with phospholipase A2. The lysophosphatidic acid was reacylated with NBD-aminocaproyl imidazole or NBD-aminododecanoyl imidazole to form the fluorescent, radiolabeled analogue of phosphatidic acid. Fluorescent, non-radiolabeled analogues of phosphatidic acid were prepared by phospholipase D hydrolysis of fluorescent phosphatidylcholine.     

In Vitro and In Vivo Pharmacology of trans- and cis-(±)-1-Amino-1,3-Cyclopentanedicarboxylic Acid: Dissociation of Metabotropic and Ionotropic Excitatory Amino Acid Receptor Effects

This study explored further the function of the metabotropic excitatory amino acid receptor in the rat brain. The trans and cis isomers of (+-)-1-amino-1,3-cyclopentane-dicarboxylic acid (ACPD) were characterized for relative affinities at ionotropic and metabotropic excitatory amino acid receptors in vitro, as well as ability to produce in vivo excitatory or excitotoxic effects in rats. trans-ACPD was about 12 times more potent in vitro as an agonist for metabotropic excitatory amino acid receptors when compared to its ability to displace N-methyl-D-aspartate (NMDA) ([3H]CGS-19755) receptor binding, cis-ACPD was about 30 times more potent as a displacer of [3H]CGS-19755 binding than as a stimulant of phosphoinositide hydrolysis. When administered intraperitoneally to neonatal rats, both cis- and trans-ACPD produced convulsions that were prevented by the competitive NMDA receptor antagonists, LY233053 and LY274614. cis-ACPD was six times more potent as a convulsant when compared to trans-ACPD. Both compounds were examined for excitotoxic effects in vivo following stereotaxic injection into the mature or neonatal rat striatum. Doses of trans-ACPD of up to 5,000 or 1,200 nmol produced few signs of striatal neuronal degeneration in the mature or neonatal brain, respectively. However, cis-ACPD produced extensive dose-related neuronal degeneration at doses of 100-1,000 nmol in the mature brain and 50-200 nmol in the neonatal brain. These studies suggest that, unlike the ionotropic excitatory amino acid receptors, activation of the metabotropic excitatory amino acid receptor does not result directly in excitatory effects, such as excitotoxicity.