Allosteric modulators of metabotropic glutamate receptors: lessons learnt from mGlu1, mGlu2 and mGlu5 potentiators and antagonists.

Although relatively few G-protein-coupled receptors are Class C, in recent years, this small family of receptors has become a focal point for the discovery of new and exciting allosteric modulators. The mGlu (metabotropic glutamate) receptors are illustrative in the discovery of both positive and/or negative allosteric modulators with unique pharmacological properties. For instance, allosteric modulators of the mGlu2 receptor act as potentiators of glutamate responses in clonal expression systems and in native tissue assays. These potentiators act to increase the affinity of orthosteric agonists for the mGlu2 receptor and shift potency curves for the agonist to the left. In electrophysiological experiments, the potentiators show a unique activation-state-dependent presynaptic inhibition of glutamate release and significantly enhance the receptor-mediated increase in G-protein binding, as seen with autoradiography. Similarly, potentiators of mGlu5 have been described, as well as allosteric antagonists or inverse agonists of mGlu1 and mGlu5. Binding and activity of the modulators have recently indicated that positive and negative allosteric sites can be, but are not necessarily, overlapping. Compared with orthosteric ligands, these modulators display a unique degree of subtype selectivity within the highly conserved mGlu family of receptors and can have very distinct pharmacological properties, such as neuronal frequency-dependent activity. This short review describes some of the unique features of these mGlu1, mGlu2 and mGlu5 allosteric modulators.

Systemic pre-treatment with a group II mGlu agonist, LY379268, reduces hyperalgesia in vivo.

1. Previous studies investigating the role of metabotropic glutamate (mGlu) receptors in nociceptive processing have been hampered by the lack of systemically active, selective, ligands. This study investigates the possible analgesic and/or anti-hyperalgesic properties of the most potent compound to date that has systemic agonist activity at group II mGlu receptors, LY379268. 2. In testing the drug in rats as an analgesic to acute noxious stimuli, LY379268 (in doses up to 3 mg kg(-1) i.p.) did not affect withdrawal latencies to either mechanical or thermal stimulation. 3. However, when a 3 mg kg(-1) dose was given prior to an intraplantar injection of carrageenan, the inflammatory hyperalgesia that developed was significantly delayed compared to saline pre-treated controls, without affecting the inflammation of the paw. A similar dose of the mGlu-inactive enantiomer, LY379267, was not anti-hyperalgesic. 4. In a model of mouse tail withdrawal to warm water, LY379268 (12 mg kg(-1) i.p.), given before a subcutaneous tail injection of capsaicin, reduced the subsequent neurogenic hyperalgesia. 5. Rota-rod testing showed that the drug did not produce a motor impairment in rats at antihyperalgesic doses. 6. The results indicate that systemic activation of this group of mGlu receptors reduces both inflammatory and neurogenic thermal hyperalgesia.

Neuroprotection mediated by glial group-II metabotropic glutamate receptors requires the activation of the MAP kinase and the phosphatidylinositol-3-kinase pathways.

The mGlu2/3 receptor agonists 4-carboxy-3-hydroxyphenylglycine (4C3HPG) and LY379268 attenuated NMDA toxicity in primary cultures containing both neurons and astrocytes. Neuroprotection was abrogated by PD98059 and LY294002, which inhibit the mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI-3-K) pathways, respectively. Cultured astrocytes lost the ability to produce transforming growth factor-beta1 (TGF-beta1) in response to mGlu2/3 receptor agonists when co-incubated with PD98059 or LY294002. As a result, the glial medium was no longer protective against NMDA toxicity. Activation of the MAPK and PI-3-K pathways in cultured astrocytes treated with 4C3HPG or LY379268 was directly demonstrated by an increase in the phosphorylated forms of ERK-1/2 and Akt. Similarly to that observed in the culture, intracerebral or systemic injections of mGlu2/3 receptor agonists enhanced TGF-beta1 formation in the rat or mouse caudate nucleus, and this effect was reduced by PD98059. PD98059 also reduced the ability of LY379268 to protect striatal neurons against NMDA toxicity. These results suggest that activation of glial mGlu2/3 receptors induces neuroprotection through the activation of the MAPK and PI-3-K pathways leading to the induction of TGF-beta.

(S)-3,4-DCPG, a potent and selective mGlu8a receptor agonist, activates metabotropic glutamate receptors on primary afferent terminals in the neonatal rat spinal cord.

(S)-3,4-Dicarboxyphenylglycine (DCPG) has been tested on cloned human mGlu1-8 receptors individually expressed in AV12-664 cells co-expressing a rat glutamate/aspartate transporter and shown to be a potent and selective mGlu8a receptor agonist (EC(50) value 31+/-2 nM, n=3) with weaker effects on the other cloned mGlu receptors (EC(50) or IC(50) values >3.5 microM on mGlu1-7). Electrophysiological characterisation on the neonatal rat spinal cord preparation revealed that (S)-3,4-DCPG depressed the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) giving a biphasic concentration-response curve showing EC(50) values of 1.3+/-0.2 microM (n=17) and 391+/-81 microM (n=17) for the higher and lower affinity components, respectively. The receptor mediating the high-affinity component was antagonised by 200 microM (S)-alpha-methyl-2-amino-4-phosphonobutyrate (MAP4, K(D) value 5.4+/-1.5 microM (n=3)), a group III metabotropic glutamate (mGlu) receptor antagonist. The alpha-methyl substituted analogue of (S)-3,4-DCPG, (RS)-3,4-MDCPG (100 microM), antagonised the effects of (S)-3,4-DCPG (K(D) value 5.0+/-0.4 microM, n=3) in a similar manner to MAP4. (S)-3,4-DCPG-induced depressions of the fDR-VRP in the low-affinity range of the concentration-response curve were potentiated by 200 microM (S)-alpha-ethylglutamate (EGLU), a group II mGlu receptor antagonist, and were relatively unaffected by MAP4 (200 microM). However, depressions of the fDR-VRP mediated by the AMPA selective antagonist (R)-3,4-DCPG were not potentiated by EGLU, suggesting that the low-affinity component of the concentration-response curve for (S)-3,4-DCPG is not due to antagonism of postsynaptic AMPA receptors. It is suggested that the receptor responsible for mediating the high-affinity component is mGlu8. The receptor responsible for mediating the low-affinity effect of (S)-3,4-DCPG has yet to be identified but it is unlikely to be one of the known mGlu receptors present on primary afferent terminals or an ionotropic glutamate receptor of the AMPA or NMDA subtype.

A novel, competitive mGlu(5) receptor antagonist (LY344545) blocks DHPG-induced potentiation of NMDA responses but not the induction of LTP in rat hippocampal slices.

1. We have investigated the pharmacological properties of LY344545, a structurally related epimer of the broad spectrum competitive metabotropic glutamate receptor antagonist, LY341495. We have found that LY344545 also antagonizes competitively nearly all mGlu receptor subtypes, but with a wide spectrum of activity. The order of potency for the human receptor isoforms was mGlu(5a) (IC(50) of 5. 5+/-0.6 microM)>mGlu(2)=mGlu(3)>mGlu(1alpha)=mG lu(7)>mGlu(6)=mGlu(8). No significant mGlu(4) receptor antagonist activity was detected at the highest concentration used (100 microM). 100 microM LY344545 displaced 50+/-5% of [(3)H]-CGP39653 binding, but less than 30% of [(3)H]-kainate or [(3)H]-AMPA in radioligand binding assays. 2. LY344545 antagonized L-glutamate stimulated Ca(2+) release in CHO cells transfected with mGlu receptors in a concentration dependent manner with a 10 fold higher affinity for the rat mGlu(5a) receptor (K:(i)=2.1+/-0.6 microM) compared to the rat mGlu(1alpha) receptor (K:(i)=20.5+/-2.1 microM). 50 microM (1S, 3R)-ACPD-induced Ca(2+) rises in hippocampal CA1 neurones were also antagonized (IC(50)=6. 8+/-0.7 microM). 3. LY344545 antagonized 10 microM (S)-3,5-DHPG-induced potentiation of NMDA depolarizations in CA1 neurones (EC(50)=10. 6+/-1.0 microM). At higher concentrations (> or =100 microM), LY344545 was an NMDA receptor antagonist. 4. LY344545 also blocked the induction, but not the expression, of LTP at CA3 to CA1 synapses with an IC(50)>300 microM. This effect is consistent with its weak activity at NMDA receptors. 5. These results demonstrate that the binding of ligands to mGlu receptor subtypes is critically dependent on the spatial orientation of the same molecular substituents within a given chemical pharmacophore. The identification of LY344545 as the first competitive antagonist to show selectivity towards mGlu(5) receptors supports the potential to design more selective and potent competitive antagonists of this receptor. 6. These results further indicate that mGlu receptor-mediated potentiation of NMDA responses is not essential for the induction of LTP.

Neuroprotective effects of LY379268, a selective mGlu2/3 receptor agonist: investigations into possible mechanism of action in vivo.

The mechanisms underlying the neuroprotective effects of the group II metabotropic glutamate receptor (mGluR) agonist LY379268 were investigated in a gerbil model of global ischemia. LY379268 (10 mg/kg i.p.) 30 or 60 min after 5-min bilateral carotid artery occlusion (BCAO) attenuated the ischemia-induced hyperactivity and provided protection in the CA1 hippocampal cells. This neuroprotective effect was maintained (P <.001) when histological analysis was performed 14 and 28 days after BCAO. Furthermore, 24- or 48-h pretreatment with LY379268, 10 mg/kg i.p., before 5-min BCAO markedly reduced (P <.001 and P <.05, respectively) the damage to CA1 hippocampal neurons. This result is consistent with the induction of neuroprotective factors or a very long brain half-life. To study the possible induction of neuroprotective factors as contributing to this action of LY379268, brains were examined for expression of neurotrophic factors. Results indicated that LY379268 (10 mg/kg i.p.) failed to alter the expression of transforming growth factor-beta, brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor in the hippocampal regions of brains taken from gerbils sacrificed at 6, 24, 72, and 120 h postinjection. The new group II mGlu antagonist, LY341495, administered 1 h before 5-min BCAO, attenuated the neuroprotective effect of LY379268 administered 24 h before 5-min BCAO. Complementary pharmacokinetic studies showed that a significant receptor-active concentration persisted in the brain 24 h after LY379268 10 mg/kg i.p. We conclude that group II mGluR occupancy, rather than induction of neuroprotective factors, explains the long-lasting neuroprotective effect of LY379268 in the gerbil model of global ischemia.

Evaluation of the activity of a novel metabotropic glutamate receptor antagonist (+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) in the in vitro neonatal spinal cord and in an in vivo pain model.

The cyclobutylglycine (+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) has been identified as a functionally potent metabotropic glutamate receptor antagonist. It is most potent on the two group I metabotropic glutamate receptors, 1alpha and 5alpha, with IC50 values of 1.0+/-0.4 microM and 1.6+/-1.4 microM, respectively. In this study, LY393053 has also been evaluated electrophysiologically on native group I metabotropic glutamate receptors in an in vitro spinal cord preparation as well as behaviourally, in a mouse model of visceral pain. LY393053 dose-dependently antagonised group I agonist, (RS)-3, 5-dihydroxyphenylglycine, or a broad-spectrum agonist (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation of spinal motoneurons. The apparent Kd values were estimated to be 0.3 microM against (RS)-3, 5-dihydroxyphenylglycine-induced depolarisation and 0.5 microM against (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation, respectively. On the other hand, the dorsal root-ventral root potential elicited at 8 x threshold was depressed by LY393053 with IC50 values of 9.0+/-0.7 microM and 12.7+/-1.7 microM on monosynaptic and polysynaptic responses, respectively. When investigated using the mouse acetic acid writhing test, LY393053 showed significant analgesic effects at doses of 1-10 mg/kg intraperitoneally. An ED50 value of 6.0 mg/kg was obtained in this test. By revealing a potent effect of LY393053 in antagonising the native group I metabotropic receptor-mediated responses in the spinal cord in rodents, and an antinociceptive efficacy in a mouse visceral pain model, these results, therefore, provide additional evidence in support of the analgesic potential of metabotropic glutamate receptor antagonists.

Evaluation of agonists and antagonists acting at Group I metabotropic glutamate receptors in the thalamus in vivo.

Recordings were made from single neurones in the ventrobasal thalamus of anaesthetised rats in order to evaluate the properties of several agonists and antagonists of Group I mGlu receptors. The selective mGlu1 receptor antagonist LY367385 was found to reduce excitatory responses to iontophoretically applied ACPD and DHPG whereas the mGlu5 agonist CHPG was resistant to antagonism. The antagonists LY367366 and LY393053 reduced responses to all three agonists, but without reducing responses to NMDA or AMPA. Although AIDA was also found to reduce mGlu agonist-evoked responses, this antagonist also produced significant reductions in responses to NMDA and AMPA. These data suggest that there are functional mGlu1 and mGlu5 receptors in the thalamus. Furthermore, LY367385 is a useful tool for investigating mGlu1 functions whereas LY367366 and LY393053 have a broader spectrum of action. The usefulness of AIDA as an antagonist in physiological experiments would appear to be limited by its effects against NMDA and AMPA.

DHPG-induced LTD in area CA1 of juvenile rat hippocampus; characterisation and sensitivity to novel mGlu receptor antagonists.

We have used extracellular microelectrode recording to characterise a form of long-term depression (LTD) of synaptic transmission that can be induced by metabotropic glutamate (mGlu) receptor activation in the CA1 region of the young (12-18 day old) rat hippocampus. Activation of group I mGlu receptors by the specific agonist 3,5-dihydroxyphenylglyine (DHPG) induced LTD of field excitatory postsynaptic potentials (fEPSPs). The mGlu5 selective agonist 2-chloro-5-hydroxyphenylglycine was also capable of inducing LTD. In contrast, the group II specific agonist DCG-IV had no effect on synaptic transmission, whilst the group III receptor agonist (S)-2-amino-4-phosphonobutyrate elicited a depression that reversed fully upon agonist washout. DHPG-induced LTD could still be generated after prior saturation of electrically-induced NMDA receptor-dependent LTD. DHPG-induced LTD was reversed by tetanic stimulation comprising 100 shocks delivered at 100 Hz. A novel mGlu receptor antagonist, (RS)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) that potently inhibits mGlu1 and mGlu5 receptors, prevented the induction of DHPG-induced LTD. Like other mGlu receptor antagonists, LY393053 also reversed pre-established DHPG-induced LTD. In contrast, a potent mGlu1 selective antagonist (S)-2-methyl-4-carboxyphenylglycine (LY367385) did not prevent the induction of DHPG-induced LTD. In conclusion, DHPG, probably via activation of mGlu5 receptors, is able to induce a robust form of LTD in the CA1 region of the young rat hippocampus that is mechanistically distinct from NMDA receptor-dependent homosynaptic LTD.

Neuroprotection by metabotropic glutamate receptor glutamate receptor agonists: LY354740, LY379268 and LY389795.

In rat cortical neuronal cultures, metabotropic glutamate (mGlu) receptor agonists: LY354740 (+)-2-aminobicyclo[3.1.0]hexane-2,6dicarboxylate); LY379268 (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate, and LY389795 (-)-2-thia-4-aminobicyclo[3.1.0]-hexane-4,6-dicarboxylate, were neuroprotective against toxicity induced by N-methyl-D-aspartic acid (NMDA), kainic acid and staurosporine as measured by release of lactate dehydrogenase (LDH) activity into culture supernatants and DNA fragmentation by oligonucleosome formation. The potencies of the agonists were at least 100 times greater in reducing nucleosome formation than LDH release indicating a differential effect on neurons dying by apoptosis than by necrosis. In vivo studies showed that LY354740 was able to mediate a partial protection against apoptosis in CA1 hippocampal cells under ischaemic conditions where substantial CA1 cell loss occurred. The effects of the agonists in vitro were: (a) reversed by mGlu receptor antagonist LY341495, (b) enhanced by the presence of glial cells, (c) abrogated by RNA and protein synthesis inhibitors, and (d) unaltered by inhibition of endogenous adenosine activity. These results suggest that group II mGlu receptor agonists may represent a novel therapeutic strategy for the treatment of neurodegenerative diseases.

Neuroprotective actions of novel and potent ligands of group I and group II metabotropic glutamate receptors.

The role of group I metabotropic glutamate (mGlu) receptors in neurodegeneration is controversial because of the contradictory effects of mGlu1/5 agonists in in vitro models of neuronal cell death. In this study, novel and selective antagonists of mGlu1 and mGlu5: LY367385 and LY367366 were found to show consistent neuroprotective effects against N-methyl-D-aspartate (NMDA)-induced excitotoxicity in vitro and in vivo. Furthermore, intraventricular administration of LY367385 reduced hippocampal cell death in gerbils subjected to transient global ischemia. Previous studies have also shown that activation of group II mGlu receptors may contribute to neuroprotective mechanisms in vitro and in vivo. Three potent group II mGlu agonists--LY354740, LY379268 and LY389795--were found to attenuate both NMDA excitotoxicity and staurosporine-induced neuronal cell death. LY354740 and LY379268 were protective against transient global ischemia in gerbils when dosed intraperitoneally. These results support the view that antagonists of mGlu1 and mGlu5 and agonists of group II mGlu receptors may be useful agents in the therapeutic treatment of neurodegenerative disease.

Sulphur-containing amino acids are agonists for group 1 metabotropic receptors expressed in clonal RGT cell lines.

Comparison of the pharmacological effects of a range of sulphur-containing amino acids on human mGluR1alpha and mGluR5a has been undertaken. cDNAs of each mGluR were transfected into a Syrian hamster tumour cell line AV12-664 that was previously transfected with the rat glutamate-aspartate transporter protein (GLAST). The L-isomers of cysteine sulphinic acid (CSA), homocysteine sulphinic acid (HCSA), cysteic acid (CA) and serine-O-sulphate (SOS) stimulated PI hydrolysis in human mGluR1alpha and mGluR5a cells with full agonist effects. D-CSA, the only active D-isomer, was a partial agonist for mGluR5a whereas L-sulphocysteine (S-CYS) showed weak agonist-like effects at high concentrations on both mGluR1alpha and mGluR5a. L-Homocysteic acid was inactive on both mGluR1alpha and mGluR5a cells. Treatment of mGluR cultures with glutamate pyruvate transaminase did not alter the potencies of the S-amino acids on PI hydrolysis responses. Inhibitor constants (Ki) obtained for L-HCSA, L-CSA, L-CA and L-SOS in [3H]glutamate receptor binding studies with mGluR1alpha cells indicated that L-HCSA, L-CSA, L-CA and L-SOS can bind specifically to mGluR1 with L-HCSA showing the highest affinity. These results confirm that certain endogenously produced S-amino acids may interact directly with group 1 mGluRs.

LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors.

The in vitro pharmacology of a structurally novel compound, LY341495, was investigated at human recombinant metabotropic glutamate (mGlu) receptor subtypes expressed in non-neuronal (RGT, rat glutamate transporter) cells. LY341495 was a nanomolar potent antagonist of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-induced inhibition of forskolin-stimulated cAMP formation at mGlu2 and mGlu3 receptors (respective IC50S of 0.021 and 0.014 microM). At group I mGlu receptor expressing cells, LY341495 was micromolar potent in antagonizing quisqualate-induced phosphoinositide (PI) hydrolysis, with IC50 values of 7.8 and 8.2 microM for mGlu1a and mGlu5a receptors, respectively. Among the human group III mGlu receptors, the most potent inhibition of L-2-amino-4-phosphonobutyric acid (L-AP4) responses was seen for LY341495 at mGlu8, with an IC50 of 0.17 microM. LY341495 was less potent at mGlu7 (IC50 = 0.99 microM) and least potent at mGlu4 (IC50 = 22 microM). Binding studies in rat brain membranes also demonstrated nanomolar potent group II mGlu receptor affinity for LY341495, with no appreciable displacement of ionotropic glutamate receptor ligand binding. Thus, LY341495 has a unique range of selectivity across the mGlu receptor subtypes with a potency order of mGlu3 > or = mGlu2 > mGlu8 > mGlu7 >> mGlu1a = mGlu5a > mGlu4. In particular, LY341495 is the most potent antagonist yet reported at mGlu2, 3 and 8 receptors. Thus, it represents a novel pharmacological agent for elucidating the function of mGlu receptors in experimental systems.

Synthesis and metabotropic glutamate receptor antagonist activity of N1-substituted analogs of 2R,4R-4-aminopyrrolidine-2,4-dicarboxylic acid.

A series of N1-substituted derivatives of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) has been prepared as constrained analogs of gamma-substituted glutamic acids and examined for their effects at recombinant metabotropic glutamate receptor (mGluR) subtypes in vitro. Appropriate substitution of the N1 position of 2R,4R-APDC resulted in the identification of a number of selective group II mGluR antagonists.

2,3'-disubstituted-2-(2'-carboxycyclopropyl)glycines as potent and selective antagonists of metabotropic glutamate receptors.

2-(9-Xanthylmethyl)-2-(2'-carboxycyclopropyl) glycine 6e is a novel metabotropic glutamate receptor antagonist. A series of alpha, C-3' disubstituted (carboxycyclopropyl)glycines 6f-n were prepared. Antagonist activity was observed for all these compounds at group 2 and group 3 mGluRs. Although they were slightly less active on group 2 mGluRs than non C-3' substituted 6e, the compounds 6f-n were more selective with lesser or no activity on group 1 mGluR subtypes (IC50 values greater than 100 microns).

The potent mGlu receptor antagonist LY341495 identifies roles for both cloned and novel mGlu receptors in hippocampal synaptic plasticity.

Understanding the roles of metabotropic glutamate (mGlu) receptors has been severely hampered by the lack of potent antagonists. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-y l)propanoic acid) has been shown to block group II mGlu receptors in low nanomolar concentrations (Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist at group II metabotropic glutamate receptors. Neuropharmacology 37, 1-12) but can be used in higher concentrations to block all hippocampal mGlu receptors, identified so far by molecular cloning (mGlu1-5,7,8). Here we have further characterised the mGlu receptor antagonist activity of LY341495 and have used this compound to investigate roles of mGlu receptors in hippocampal long-term potentiation (LTP) and long-term depression (LTD). LY341495 competitively antagonised DHPG-stimulated PI hydrolysis in AV12-664 cells expressing either human mGlu1 or mGlu5 receptors with Ki-values of 7.0 and 7.6 microM, respectively. When tested against 10 microM L-glutamate-stimulated Ca2+ mobilisation in rat mGlu5 expressing CHO cells, it produced substantial or complete block at a concentration of 100 microM. In rat hippocampal slices, LY341495 eliminated 30 microM DHPG-stimulated PI hydrolysis and 100 microM (1S,3R)-ACPD-inhibition of forskolin-stimulated cAMP formation at concentrations of 100 and 0.03 microM, respectively. In area CA1, it antagonised DHPG-mediated potentiation of NMDA-induced depolarisations and DHPG-induced long-lasting depression of AMPA receptor-mediated synaptic transmission. LY341495 also blocked NMDA receptor-independent depotentiation and setting of a molecular switch involved in the induction of LTP; effects which have previously been shown to be blocked by the mGlu receptor antagonist (S)-MCPG. These effects may therefore be due to activation of cloned mGlu receptors. In contrast, LY341495 did not affect NMDA receptor-dependent homosynaptic LTD; an effect which may therefore be independent of cloned mGlu receptors. Finally, LY341495 failed to antagonise NMDA receptor-dependent LTP and, in area CA3, NMDA receptor-independent, mossy fibre LTP. Since in the same inputs these forms of LTP were blocked by (S)-MCPG, a novel type of mGlu receptor may be involved in their induction.

A 71-kD heat shock protein (hsp) from Mycobacterium tuberculosis has modulatory effects on experimental rat arthritis.

The effects of a mycobacterial 71-kD hsp antigen have been investigated for its ability to modulate arthritis in rats. Subcutaneous injection (base of tail) of increasing amounts of hsp71 from Mycobacterium tuberculosis (MTB) produced dose-dependent differential inhibitory effects on induction of arthritis by MTB and CP20961 in rats. As little as 1 microgram of the hsp71 produced a reduction in MTB arthritis, whereas complete protection was observed when 50 micrograms were administered. When 71-kD-treated rats were challenged with CP20961, all developed reduced symptoms of arthritis compared with control rats, but in this model no complete protection was observed over the dose range studied. The effects of 71-kD pretreatment on collagen II arthritis were not significant, but in general symptoms of arthritis were milder than in the control group. The same pattern of results was observed previously when hsp65 was used in the different models. These results show that the modulatory effects of hsp on adjuvant arthritis are not restricted to the hsp65 series, but are also mediated by a member of the hsp70 family.

Pharmacological analysis of 4-carboxyphenylglycine derivatives: comparison of effects on mGluR1 alpha and mGluR5a subtypes.

The antagonist effects of the 4-carboxyphenylglycines: (S)-4-carboxy-3hydroxyphenylglycine (4C3HPG), (S)-4-carboxyphenylglycine (4CPG) and (+)-alpha-methyl-4-carboxyphenylglycine (M4CPG) were compared on functional responses of human metabotropic glutamate receptor (mGluR) subtypes mGluR1 alpha and mGluR5a. These receptors both belong to group 1 type mGluRs which couple to the phosphoinositide (PI) hydrolysis/[Ca2+]i mobilization signal transduction pathway and are closely related in both structure and agonist pharmacology. In this study, the IC50 values obtained for quisqualate induced PI hydrolysis responses show that although all the phenylglycines are antagonists for both mGluR1 alpha and mGluR5a, the compounds exhibit differential potencies at these receptor subtypes. The 4C3HPG derivative was the most potent antagonist for both mGluR1 alpha (IC50 range: 19-50 microM) and mGluR5a (IC50 range: 53-280 microM). 4CPG produced an IC50 range of 4r-72 microM for mGluR1 alpha and 150-156 microM for mGluR5a cells. The potency of the M4CPG could not be distinguished from that of 4CPG with IC50 ranges of 29-100 microM and 115-210 microM for mGluR1 alpha and mGluR5a respectively. Further characterization of the dose-response effects of the compounds on quisqualate induced [Ca2+]i mobilization showed that although the magnitude of phenylglycine inhibition was reduced for both mGluR subtypes compared to those observed for stimulation of PI hydrolysis (except for 4C3HPG on mGluR1 alpha), similar differences in the relative potencies of the phenylglycines between mGluR1 alpha (IC50s: 40 +/- 10 microM for 4C3HPG: 300-1000 microM for 4CPG and M4CPG) and mGluR5a (IC50s: > 1000 microM) were evident.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-reactivity to proteoglycans in bacterial arthritis: lack of evidence for in vivo role in induction of disease.

Cross-reactivity between bacterial epitopes and cartilage components has been assumed to play a role in the pathology of bacterial-induced arthritis models. In this study, we report prominent proteoglycan (PG) depletion in Safranin-O stained ankle joint sections from collagen-induced arthritic rats. In adjuvant arthritis and streptococcal cell wall-induced arthritis (SCW-A), however, only limited PG degradation was observed. In vitro, PG fractions were able to stimulate T lymphocytes from these arthritic rats. To investigate the contribution of cross-reactivity, Lewis rats were primed with SCW in Freund's incomplete adjuvant (SCW/FIA). This immunization protocol resulted in in vitro stimulatory responses to the SCW antigens and cartilage PG antigens, but not to joint inflammation per se. Next, papain was injected intraarticularly to create a situation in which a large amount of potential cross-reactive cartilage epitopes are released. Interestingly, no inflammatory reaction could be observed in the papain-injected joints of SCW/FIA-primed rats. These data suggest that cross-reactivity between bacterial epitopes and PG does not seem to be a key element in the onset of joint inflammation in bacterial-induced arthritis. However, it cannot be ruled out that at later time points cross-reactivity will contribute to joint inflammation.

In vitro and in vivo effects of proteoglycan fractions in adjuvant treated rats.

Differential stimulatory effects by proteoglycan fractions: chondroitin (CS)- and keratan-sulphate regions; link protein and binding region were observed in cultures of spleen and lymph node lymphocytes taken from normal and adjuvant treated Lewis rats. In vivo, none of the fractions induced symptoms of arthritis but that pretreatment with the CS rich region produced an inhibition of Mycobacterium tuberculosis induced arthritis.