Antagonist activity of alpha-substituted 4-carboxyphenylglycine analogues at group I metabotropic glutamate receptors expressed in CHO cells.
1. We have investigated the antagonist properties of 6 alpha-substituted phenylglycine analogues based on the structure of 4-carboxyphenylglycine (4-CPG) for group I metabotropic glutamate receptors (mGlu1alpha and mGlu5a) permanently expressed in CHO cells. 2. (S)-4-CPG and (S)-MCPG were the most selective mGlu1alpha receptor antagonists. Longer chain alpha-carbon substitutions resulted in a progressive loss of antagonist affinity at mGlu1alpha receptors but not at mGlu5a receptors. Thus mGlu1alpha receptor antagonists require small aliphatic groups at the alpha-position. Alpha-cyclopropyl-4-CPG showed a tendency towards mGlu5a selectivity, suggesting that bulky groups at this position may favour mGlu5a receptor antagonism. 3. We demonstrate that the mGlu5a receptor displays agonist-dependent antagonism. L-glutamate-induced Ca2+ release in mGlu5a receptor expressing cells was more susceptible to antagonism by cyclic alpha-carbon derivatives than (S)-3,5-dihydroxyphenylglycine (DHPG)-induced Ca2+ release in the same cell line. 4. The data presented suggests that mGlu1alpha and mGlu5a receptors have different steric and/or conformational requirements for the binding of antagonists and different amino acids which could interact with agonists. 5. These phenylglycine analogues could provide leads for the development of subtype selective antagonists. (+info)
Mechanisms involved in the metabotropic glutamate receptor-enhancement of NMDA-mediated motoneurone responses in frog spinal cord.
1. The metabotropic glutamate receptor (mGluR) agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) (10-100 microM) depolarized isolated frog spinal cord motoneurones, a process sensitive to kynurenate (1.0 mM) and tetrodotoxin (TTX) (0.783 microM). 2. In the presence of NMDA open channel blockers [Mg2+; (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801); 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] and TTX, trans-ACPD significantly potentiated NMDA-induced motoneurone depolarizations, but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA)- or kainate-induced depolarizations. 3. NMDA potentiation was blocked by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) (240 microM), but not by alpha-methyl-(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (MCCG) (290 microM) or by alpha-methyl-(S)-2-amino-4-phosphonobutyrate (L-MAP4) (250 microM), and was mimicked by 3,5-dihydroxyphenylglycine (DHPG) (30 microM), but not by L(+)-2-amino-4-phosphonobutyrate (L-AP4) (100 microM). Therefore, trans-ACPD's facilitatory effects appear to involve group I mGluRs. 4. Potentiation was prevented by the G-protein decoupling agent pertussis toxin (3-6 ng ml(-1), 36 h preincubation). The protein kinase C inhibitors staurosporine (2.0 microM) and N-(2-aminoethyl)-5-isoquinolinesulphonamide HCI (H9) (77 microM) did not significantly reduce enhanced NMDA responses. Protein kinase C activation with phorbol-12-myristate 13-acetate (5.0 microM) had no effect. 5. Intracellular Ca2+ depletion with thapsigargin (0.1 microM) (which inhibits Ca2+/ATPase), 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetyl methyl ester (BAPTA-AM) (50 microM) (which buffers elevations of [Ca2+]i), and bathing spinal cords in nominally Ca2+-free medium all reduced trans-ACPD's effects. 6. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) (100 microM) and chlorpromazine (100 microM) diminished the potentiation. 7. In summary, group I mGluRs selectively facilitate NMDA-depolarization of frog motoneurones via a G-protein, a rise in [Ca2+]i from the presumed generation of phosphoinositides, binding of Ca2+ to calmodulin, and lessening of the Mg2+-produced channel block of the NMDA receptor. (+info)
Selenium redox biochemistry of zinc-sulfur coordination sites in proteins and enzymes.
Selenium has been increasingly recognized as an essential element in biology and medicine. Its biochemistry resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidation states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a dozen selenoproteins. We have chosen to examine zinc-sulfur centers as possible targets of selenium redox biochemistry. Selenium compounds release zinc from zinc/thiolate-coordination environments, thereby affecting the cellular thiol redox state and the distribution of zinc and likely of other metal ions. Aromatic selenium compounds are excellent spectroscopic probes of the otherwise relatively unstable functional selenium groups. Zinc-coordinated thiolates, e.g., metallothionein (MT), and uncoordinated thiolates, e.g., glutathione, react with benzeneseleninic acid (oxidation state +2), benzeneselenenyl chloride (oxidation state 0) and selenocystamine (oxidation state -1). Benzeneseleninic acid and benzeneselenenyl chloride react very rapidly with MT and titrate substoichiometrically and with a 1:1 stoichiometry, respectively. Selenium compounds also catalyze the release of zinc from MT in peroxidation and thiol/disulfide-interchange reactions. The selenoenzyme glutathione peroxidase catalytically oxidizes MT and releases zinc in the presence of t-butyl hydroperoxide, suggesting that this type of redox chemistry may be employed in biology for the control of metal metabolism. Moreover, selenium compounds are likely targets for zinc/thiolate coordination centers in vivo, because the reactions are only partially suppressed by excess glutathione. This specificity and the potential to undergo catalytic reactions at low concentrations suggests that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compounds in antiinflammatory and anticarcinogenic agents. (+info)
Antagonist pharmacology of metabotropic glutamate receptors coupled to phospholipase D activation in adult rat hippocampus: focus on (2R,1'S,2'R,3'S)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine versus 3, 5-dihydroxyphenylglycine.
Metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) appear to be distinct from any known mGlu receptor subtype linked to phospholipase C or adenylyl cyclase. The availability of antagonists is necessary for understanding the role of these receptors in the central nervous system, but selective ligands have not yet been identified. In a previous report, we observed that 3, 5-dihydroxyphenylglycine (3,5-DHPG) inhibits the PLD response induced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate in adult rat hippocampal slices. We now show that the antagonist action of 3, 5-DHPG (IC50 = 70 microM) was noncompetitive in nature and nonselective, because the drug was also able to reduce PLD activation elicited by 100 microM norepinephrine and 1 mM histamine. In the search for a selective and more potent antagonist, we examined the effects of sixteen stereoisomers of 2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG) on the PLD-specific transphosphatidylation reaction resulting in the formation of [3H]phosphatidylethanol. The (2R,1'S,2'R,3'S)-PCCG stereoisomer (PCCG-13) antagonized the formation of [3H]phosphatidylethanol induced by 100 microM (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylate in a dose-dependent manner and with a much lower IC50 value (25 nM) compared with 3,5-DHPG. In addition, increasing concentrations of PCCG-13 were able to shift to the right the agonist dose-response curve but had no effect when tested on other receptors coupled to PLD. The potent, selective, and competitive antagonist PCCG-13 may represent an important tool for elucidating the role of PLD-coupled mGlu receptors in adult hippocampus. (+info)
Dual effect of alkylresorcinols, natural amphiphilic compounds, upon liposomal permeability.
The effect of 5-n-alkylresorcinols, natural amphiphilic compounds, upon properties of phospholipid vesicles depends on their localization asymmetry. A significant increase of the bilayer permeability is observed when the title compounds are present only in the external medium. When these amphiphiles are preincorporated into the bilayer during its formation, the resulting liposomes effectively encapsulate water-soluble solutes which still remain in liposomes after 25 h. Additionally, the size of liposomes made of alkylresorcinol-phosphatidylcholine mixtures after eight cycles of freezing and thawing only (180-200 nm) is severalfold smaller than the size of vesicles prepared in a similar way from phospholipids only and the resulting liposomes are more homogeneous. These liposomes modified with alkylresorcinols are also stable during 40 day storage at both 4 degrees C and 20 degrees C, in contrast to control liposomes that already strongly aggregate after 10 days. (+info)
Differential effects of metabotropic glutamate receptor antagonists on bursting activity in the amygdala.
Differential effects of metabotropic glutamate receptor antagonists on bursting activity in the amygdala. Metabotropic glutamate receptors (mGluRs) are implicated in both the activation and inhibition of epileptiform bursting activity in seizure models. We examined the role of mGluR agonists and antagonists on bursting in vitro with whole cell recordings from neurons in the basolateral amygdala (BLA) of amygdala-kindled rats. The broad-spectrum mGluR agonist 1S,3R-1-aminocyclopentane dicarboxylate (1S,3R-ACPD, 100 microM) and the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 20 microM) evoked bursting in BLA neurons from amygdala-kindled rats but not in control neurons. Neither the group II agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I, 10 microM) nor the group III agonist L-2-amino-4-phosphonobutyrate (L-AP4, 100 microM) evoked bursting. The agonist-induced bursting was inhibited by the mGluR1 antagonists (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG, 500 microM] and (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG, 300 microM]. Kindling enhanced synaptic strength from the lateral amygdala (LA) to the BLA, resulting in synaptically driven bursts at low stimulus intensity. Bursting was abolished by (S)-4C3HPG. Further increasing stimulus intensity in the presence of (S)-4C3HPG (300 microM) evoked action potential firing similar to control neurons but did not induce epileptiform bursting. In kindled rats, the same threshold stimulation that evoked epileptiform bursting in the absence of drugs elicited excitatory postsynaptic potentials in (S)-4C3HPG. In contrast (+)-MCPG had no effect on afferent-evoked bursting in kindled neurons. Because (+)-MCPG is a mGluR2 antagonist, whereas (S)-4C3HPG is a mGluR2 agonist, the different effects of these compounds suggest that mGluR2 activation decreases excitability. Together these data suggest that group I mGluRs may facilitate and group II mGluRs may attenuate epileptiform bursting observed in kindled rats. The mixed agonist-antagonist (S)-4C3HPG restored synaptic transmission to control levels at the LA-BLA synapse in kindled animals. The different actions of (S)-4C3HPG and (+)-MCPG on LA-evoked bursting suggests that the mGluR1 antagonist-mGluR2 agonist properties may be the distinctive pharmacology necessary for future anticonvulsant compounds. (+info)
Arg-gingipain inhibition and anti-bacterial activity selective for Porphyromonas gingivalis by malabaricone C.
Effects of malabaricon C, isolated from nutmeg (Myristica fragrans), on Arg-gingipain activity and growth of several kinds of anaerobic and aerobic microorganisms were investigated. Malabaricone C irreversibly inhibited Arg-gingipain by 50% at a concentration of 0.7 microgram/ml and selectively suppressed Porphyromomas gingivalis growth. (+info)
Group I metabotropic glutamate receptors mediate an inward current in rat substantia nigra dopamine neurons that is independent from calcium mobilization.
Metabotropic glutamate receptors modulate neuronal excitability via a multitude of mechanisms, and they have been implicated in the pathogenesis of neurodegenerative processes. Here we investigated the responses mediated by group I metabotropic glutamate receptors (mGluRs) in dopamine neurons of the rat substantia nigra pars compacta, using whole cell patch-clamp recordings in combination with microfluorometric measurements of [Ca(2+)](i) and [Na(+)](i). The selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) was bath-applied (20 microM, 30 s to 2 min) or applied locally by means of short-lasting (2-4 s) pressure pulses, delivered through an agonist-containing pipette positioned close to the cell body of the neuron. 3,5-DHPG evoked an inward current characterized by a transient and a sustained component, the latter of which was uncovered only with long-lasting agonist applications. The fast component coincided with a transient elevation of [Ca(2+)](i), whereas the total current was associated with a rise in [Na(+)](i). These responses were not affected either by the superfusion of ionotropic excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphono-pentanoic acid (D-APV), nor by the sodium channel blocker tetrodotoxin (TTX). (S)-alpha-methyl-4-carboxyphenylglycine (S-MCPG) and the more selective mGluR1 antagonist 7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (CPCCOEt) depressed both 3,5-DHPG-induced inward current components and, although less effectively, the associated [Ca(2+)](i) elevations. On repeated agonist applications the inward current and the calcium transients both desensitized. The time constant of recovery from desensitization differed significantly between these two responses, being 67.4+/-4.4 s for the inward current and 28.6+/-2.7 s for the calcium response. Bathing the tissue in a calcium-free/EGTA medium or adding thapsigargin (1 microM) to the extracellular medium prevented the generation of the [Ca(2+)](i) transient, but did not prevent the activation of the inward current. These electrophysiological and fluorometric results show that the 3, 5-DHPG-induced inward current and the [Ca(2+)](i) elevations are mediated by independent pathways downstream the activation of mGluR1. (+info)