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The G protein-coupled receptor kinase type 4 mediates the homologous desensitisation of type-1 metabotropic glutamate (mGlu1) receptors and is predominantly expressed in the testis. Hence, we searched for the expression of mGlu1 or other mGlu receptor subtypes in rat and human testes. RT-PCR analysis showed the presence of mGlu1, -4 and -5 (but not -2 or -3) receptor mRNA in the rat testis. The presence of mGlu1 and -5 (but not mGlu2/3) receptor proteins was also demonstrated by Western blot analysis. In the rat testis, both mGlu1a and -5 receptors were highly expressed in cells of the germinal line. It is likely that these receptors are functional, because the agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, was able to stimulate inositol phospholipid hydrolysis in slices prepared from rat testes. Immunocytochemical analysis of bioptic samples from human testes showed a high expression of mGlu5 receptors inside the seminiferous tubuli, whereas mGlu1a immunoreactivity was restricted to intertubular spaces. mGlu5 receptors were also present in mature spermatozoa, where they were localised in the mid-piece and tail. This localisation coincided with that of beta-arrestin, a protein that is critically involved in the homologous desensitisation and internalisation of G protein-coupled receptors. Taken collectively, these results offer the first evidence for the expression of any glutamate receptor in testes, and suggest that at least mGlu5 receptors are present and functionally active in mature human sperm.

Cinnabarinic acid is an endogenous metabolite of the kynurenine pathway that meets the structural requirements to interact with glutamate receptors. We found that cinnabarinic acid acts as a partial agonist of type 4 metabotropic glutamate (mGlu4) receptors, with no activity at other mGlu receptor subtypes. We also tested the activity of cinnabarinic acid on native mGlu4 receptors by examining 1) the inhibition of cAMP formation in cultured cerebellar granule cells; 2) protection against excitotoxic neuronal death in mixed cultures of cortical cells; and 3) protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice after local infusion into the external globus pallidus. In all these models, cinnabarinic acid behaved similarly to conventional mGlu4 receptor agonists, and, at least in cultured neurons, the action of low concentrations of cinnabarinic acid was largely attenuated by genetic deletion of mGlu4 receptors. However, high concentrations of cinnabarinic acid were still active in the absence of mGlu4 receptors, suggesting that the compound may have off-target effects. Mutagenesis and molecular modeling experiments showed that cinnabarinic acid acts as an orthosteric agonist interacting with residues of the glutamate binding pocket of mGlu4. Accordingly, cinnabarinic acid did not activate truncated mGlu4 receptors lacking the N-terminal Venus-flytrap domain, as opposed to the mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). Finally, we could detect endogenous cinnabarinic acid in brain tissue and peripheral organs by high-performance liquid chromatography-tandem mass spectrometry analysis. Levels increased substantially during inflammation induced by lipopolysaccharide. We conclude that cinnabarinic acid is a novel endogenous orthosteric agonist of mGlu4 receptors endowed with neuroprotective activity.

The interaction between 5-hydroxytryptamine2A(5-HT2A) serotonin receptors and metabotropic glutamate (mGlu) 2/3 receptors underlies the antipsychotic activity of mGlu2/3 receptor agonists in experimental animals and humans. The molecular nature of this interaction is only partially known. We here report for the first time that pharmacological activation of mGlu2/3 receptors attenuates the stimulation of polyphosphoinositide (PI) hydrolysis mediated by 5-HT2Areceptors in the frontal cortex of living mice. Mice were injected intracerebroventricularly with [myo-3H]inositol and treated with drugs 1 h after a pretreatment with lithium, which blocks the conversion of inositol monophosphate into free inositol. Systemic injection of the mGlu2/3 receptor agonist (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) inhibited the stimulation of PI hydrolysis induced by the hallucinogenic 5-HT2Areceptor agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) without affecting the stimulation by mGlu1/5 or muscarinic receptors. The action of LY379268 was prevented by the preferential mGlu2/3 receptor antagonist (2S,1′S,2′S)-2-(9-xanthylmethyl)-2-(2′-carboxycyclopropyl)glycine (LY341495). N-(4′-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), a selective mGlu2 receptor enhancer, also reduced DOI-stimulated PI hydrolysis when combined with subthreshold doses of LY379268. Systemic LY379268 inhibited DOI-stimulated PI hydrolysis in mice lacking either mGlu2 or mGlu3 receptors but was inactive in double mGlu2/mGlu3 receptor knockout mice, suggesting that both mGlu2 and mGlu3 receptors interact with 5-HT2Areceptors. Surprisingly, contrasting results were obtained in cortical slice preparations, where LY379268 amplified both DOI- and 3,5-dihydroxyphenylglycine-stimulated PI hydrolysis. Amplification was abrogated by the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine, suggesting that experiments in brain slices are biased by an additional component of receptor-stimulated PI hydrolysis. This highlights the importance of in vivo models for the study of the interaction between 5-HT2Aand mGlu2/3 receptors.

The interaction between 5-hydroxytryptamine2A(5-HT2A) serotonin receptors and metabotropic glutamate (mGlu) 2/3 receptors underlies the antipsychotic activity of mGlu2/3 receptor agonists in experimental animals and humans. The molecular nature of this interaction is only partially known. We here report for the first time that pharmacological activation of mGlu2/3 receptors attenuates the stimulation of polyphosphoinositide (PI) hydrolysis mediated by 5-HT2Areceptors in the frontal cortex of living mice. Mice were injected intracerebroventricularly with [myo-3H]inositol and treated with drugs 1 h after a pretreatment with lithium, which blocks the conversion of inositol monophosphate into free inositol. Systemic injection of the mGlu2/3 receptor agonist (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) inhibited the stimulation of PI hydrolysis induced by the hallucinogenic 5-HT2Areceptor agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) without affecting the stimulation by mGlu1/5 or muscarinic receptors. The action of LY379268 was prevented by the preferential mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). N-(4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), a selective mGlu2 receptor enhancer, also reduced DOI-stimulated PI hydrolysis when combined with subthreshold doses of LY379268. Systemic LY379268 inhibited DOI-stimulated PI hydrolysis in mice lacking either mGlu2 or mGlu3 receptors but was inactive in double mGlu2/mGlu3 receptor knockout mice, suggesting that both mGlu2 and mGlu3 receptors interact with 5-HT2Areceptors. Surprisingly, contrasting results were obtained in cortical slice preparations, where LY379268 amplified both DOI- and 3,5-dihydroxyphenylglycine-stimulated PI hydrolysis. Amplification was abrogated by the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine, suggesting that experiments in brain slices are biased by an additional component of receptor-stimulated PI hydrolysis. This highlights the importance of in vivo models for the study of the interaction between 5-HT2Aand mGlu2/3 receptors.

Knowing that expression of metabotropic glutamate 2 (mGlu2) receptors in the dorsal root ganglia is regulated by acetylation mechanisms, we examined the effect of two selective and chemically unrelated histone deacetylase (HDAC) inhibitors, N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide (MS-275) and suberoylanilide hydroamic acid (SAHA), in a mouse model of persistent inflammatory pain. Although a single subcutaneous injection of MS-275 (3 mg/kg) or SAHA (5-50 mg/kg) was ineffective, a 5-day treatment with either of the two HDAC inhibitors substantially reduced the nociceptive response in the second phase of the formalin test, which reflects the development of central sensitization in the dorsal horn of the spinal cord. Analgesia was abrogated by a single injection of the mGlu2/3 receptor antagonist (αS)-α-amino-α-[(1S,2S)-2-carboxycyclopropyl]-9H-xantine-9-propanoic acid (LY341495; 1 mg/kg, i.p.), which was inactive per se. Both MS-275 and SAHA up-regulated the expression of mGlu2 receptors in the dorsal root ganglion (DRG) and spinal cord under conditions in which they caused analgesia, without changing the expression of mGlu1a, mGlu4, or mGlu5 receptors. Induction of DRG mGlu2 receptors in response to SAHA was associated with increased acetylation of p65/RelA on lysine 310, a process that enhances the transcriptional activity of p65/RelA at nuclear factor-κB-regulated genes. Transcription of the mGlu2 receptor gene is known to be activated by p65/RelA in DRG neurons. We conclude that HDAC inhibition produces analgesia by up-regulating mGlu2 receptor expression in the DRG, an effect that results from the amplification of NF-κB transcriptional activity. These data provide the first evidence that HDAC inhibitors cause analgesia and suggest that HDACs are potential targets for the epigenetic treatment of pain.

Knowing that expression of metabotropic glutamate 2 (mGlu2) receptors in the dorsal root ganglia is regulated by acetylation mechanisms, we examined the effect of two selective and chemically unrelated histone deacetylase (HDAC) inhibitors, N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide (MS-275) and suberoylanilide hydroamic acid (SAHA), in a mouse model of persistent inflammatory pain. Although a single subcutaneous injection of MS-275 (3 mg/kg) or SAHA (5-50 mg/kg) was ineffective, a 5-day treatment with either of the two HDAC inhibitors substantially reduced the nociceptive response in the second phase of the formalin test, which reflects the development of central sensitization in the dorsal horn of the spinal cord. Analgesia was abrogated by a single injection of the mGlu2/3 receptor antagonist (αS)-α-amino-α-[(1S,2S)-2-carboxycyclopropyl]-9H-xantine-9-propanoic acid (LY341495; 1 mg/kg, i.p.), which was inactive per se. Both MS-275 and SAHA up-regulated the expression of mGlu2 receptors in the dorsal root ganglion (DRG) and spinal cord under conditions in which they caused analgesia, without changing the expression of mGlu1a, mGlu4, or mGlu5 receptors. Induction of DRG mGlu2 receptors in response to SAHA was associated with increased acetylation of p65/RelA on lysine 310, a process that enhances the transcriptional activity of p65/RelA at nuclear factor-κB-regulated genes. Transcription of the mGlu2 receptor gene is known to be activated by p65/RelA in DRG neurons. We conclude that HDAC inhibition produces analgesia by up-regulating mGlu2 receptor expression in the DRG, an effect that results from the amplification of NF-κB transcriptional activity. These data provide the first evidence that HDAC inhibitors cause analgesia and suggest that HDACs are potential targets for the epigenetic treatment of pain.

Alzheimer's disease (AD), the leading cause of senile dementia, has become a considerable social and economical problem. Current AD therapeutics provide mainly symptomatic short-term benefit, rather than targeting disease mechanisms. The hallmarks for AD are ß-amyloid plaques, neurofibrillary tangles, and regionalized neuronal loss. Additional neuropathological features have been described that may provide some clues to the mechanism by which neurons die in AD. Specifically, the aberrant expression of cell cycle proteins and the presence of de novo-replicated DNA in neurons have been described both in AD brain and in culture models of the disease. The unscheduled cell cycle events are deleterious to neurons, which undergo death rather than complete the cell cycle. Although our understanding of the neuronal cell cycle is not complete, experimental evidence suggests that compounds able of arresting the aberrant cell cycle will yield neuroprotection. This review focuses on drug development centered on the cell cycle hypothesis of AD.

Alzheimers disease (AD), the leading cause of senile dementia, has become a considerable social and economical problem. Current AD therapeutics provide mainly symptomatic short-term benefit, rather than targeting disease mechanisms. The hallmarks for AD are ß-amyloid plaques, neurofibrillary tangles, and regionalized neuronal loss. Additional neuropathological features have been described that may provide some clues to the mechanism by which neurons die in AD. Specifically, the aberrant expression of cell cycle proteins and the presence of de novo-replicated DNA in neurons have been described both in AD brain and in culture models of the disease. The unscheduled cell cycle events are deleterious to neurons, which undergo death rather than complete the cell cycle. Although our understanding of the neuronal cell cycle is not complete, experimental evidence suggests that compounds able of arresting the aberrant cell cycle will yield neuroprotection. This review focuses on drug development centered on the cell cycle hypothesis of AD.

During the past two decades, many pharmacological strategies have been investigated for the management of painful neuropathies. However, neuropathic pain still remains a clinical challenge. A combination of therapies is often required, but unfortunately in most cases adequate pain relief is not achieved. Recently, attention has been focused on the physiological and pharmacological effects of L-acetylcarnitine in neurological disorders. There are a number of reports indicating that L-acetylcarnitine can be considered as a therapeutic agent in neuropathic disorders including painful peripheral neuropathies. In this review article, we will examine the antinociceptive and the neuroprotective effects of Lacetylcarnitine as tested in clinical studies and in animal models of nerve injury.

Wnts function through the activation of at least three intracellular signal transduction pathways, of which the canonical β-catenin mediated pathway is the best understood. Aberrant canonical Wnt signaling has been involved in both neurodegeneration and cancer. An impairment of Wnt signals appears to be associated with aspects of neurodegenerative pathologies while overactivation of Wnt signaling is a common theme in several types of human tumors. Therefore, although therapeutic approaches aimed at modulating Wnt signaling in neurodegenerative and hyperproliferative diseases might impinge on the same molecular mechanisms, different pharmacological outcomes are required. Here we review recent developments on the understanding of the role of Wnt signaling in Alzheimer's disease and CNS tumors, and identify possible avenues for therapeutic intervention within a complex and multi-faceted signaling pathway.

Wnts function through the activation of at least three intracellular signal transduction pathways, of which the canonical β-catenin mediated pathway is the best understood. Aberrant canonical Wnt signaling has been involved in both neurodegeneration and cancer. An impairment of Wnt signals appears to be associated with aspects of neurodegenerative pathologies while overactivation of Wnt signaling is a common theme in several types of human tumors. Therefore, although therapeutic approaches aimed at modulating Wnt signaling in neurodegenerative and hyperproliferative diseases might impinge on the same molecular mechanisms, different pharmacological outcomes are required. Here we review recent developments on the understanding of the role of Wnt signaling in Alzheimer’s disease and CNS tumors, and identify possible avenues for therapeutic intervention within a complex and multi-faceted signaling pathway.

We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by l-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the Gβγ subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with Gβγ subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a “switch molecule” by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.

A new series of compounds were designed as structural analogues of the α1-AR ligand RN5 (4), characterized by a tricyclic 5H-pyrimido[5,4-b]indole-(1H,3H)2,4-dione system connected through an alkyl chain to a phenylpiperazine (PP) moiety. These compounds were synthesized and tested in binding assays on human α1A-AR, α1B-AR, and α1D-AR subtypes expressed in HEK293 cells. Several structural modifications were performed on the PP moiety, the tricyclic system, and the connecting alkyl chain. Many of the new molecules showed a preferential affinity for the α1D-AR subtype. Some compounds, including 39 and 40, displayed substantial α1D-AR selectivity with respect to α1A-AR, α1B-AR, serotonergic 5-HT1A, 5-HT1B, 5-HT2A, and dopaminergic D1 and D2 receptors. Two conformationally rigid analogues of 4, useful for studying the architecture of the receptor/ligand complex, were also prepared and tested. A subset of the new compounds was then used to evolve a preliminary pharmacophore model for α1D-AR antagonists, based on a more generalized model we had developed for α1-AR antagonists. This new model rationalized the relationships between structural properties and biological data of the pyrimido[5,4-b]indole compounds, as well as other compounds.

Apomorphine, given by a single injection, repeated injections, or by continuous infusion, was tested for neuroprotective effects in mice administered methamphetamine or N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in order to induce striatal dopamine (DA) depletion. In the first part of the study, the DA agonist (R)-apomorphine was administered at various doses (1, 5, and 10 mgkg), 15 min before methamphetamine (5 mgkg × 3, 2 h apart). Mice were sacrificed 5 days later. In the second part, apomorphine was administered either continuously by subcutaneous minipump (cumulative daily dose of 0.5, 1, and 3.15 mgkg), or as single, repeated daily injections (up to 5 mgkg) starting 40 h afteran acute administration of MPTP (30 mgkg). Mice were sacrificed at different time intervals (up to 1 month) following MPTP injection. In all the animals, the integrity of striatal DA terminals was evaluated by measuring striatal DA levels and TH immunohistochemistry. Apomorphine dose-dependently prevented methamphetamine toxicity. These effects were neither due to a decrease in the amount of striatal methamphetamine nor to the hypothermia, and they were not reversed by the DA antagonist haloperidol. Moreover, chronic, continuous (but not pulsatile) administration of apomorphine rescued damaged striatal dopaminergic terminals. These findings confirm a protective effect of apomorphine that also consists of a neurorescue of damaged striatal DA terminals. This suggests a new hypothesis about the long-term benefits observed during continuous apomorphine administration in Parkinson's disease patients.

Recent evidence suggests a link between the aberrant re-expression of cell cycle proteins in adult neurons of the Alzheimer's disease brain and the process of apoptotic degeneration. Here we will discuss this unexpected phenomenon as related to the mechanisms of β-amyloid toxicity, and its significance for therapeutic possibilities.

The effects of combined treatment with cyclophosphamide (CTX) and interferon-β (IFN-β) are described in selected patients with "rapidly transitional" multiple sclerosis. This form of multiple sclerosis is extremely active with very frequent and severe attacks which produce a dramatic increase on the expanded disability status scale (EDSS). Ten patients with rapidly transitional multiple sclerosis were previously treated with interferon-β, but none benefited by this treatment. Monthly treatment with intravenous CTX, from 500 mg/m² to 1500 mg/m² to obtain a chronic lymphocytopenia (600/mm³ to 900/mm³) produced a marked and significant reduction in the number of relapses (p<0.0001), disability previously accumulated (p<0.0001), and a reduction of T2 MRI burden of lesion. This particular group of patients benefited by combining cyclophosphamide and IFN-β. The possibility is considered of carrying out further studies to test the efficacy of the association between the two drugs for patients who are not responsive to IFN-β or other active disease modifying therapies.

Aggregates of β‐amyloid peptide (βAP), the main constituent of amyloid plaques in Alzheimer's brain, kill neurons by a not yet defined mechanism, leading to apoptotic death. Here, we report that both full‐length βAP(j_40) or (1–42) and its active fragment βAP(25_35) act as proliferative signals for differentiated cortical neurons, driving them into the cell cycle. The cycle followed some of the steps observed in proliferating cells, including induction of cyclin Di, phosphorylation of retinoblastoma, and induction of cyclin E and A, but did not progress beyond S phase. Inactivation of cyclin‐dependent protein kinase‐4 or ‐2 prevented both the entry into S phase and the development of apoptosis in βAP(25_35)‐treated neurons. We conclude that neurons must cross the G1/S transition before succumbing to βAP signaling, and therefore multiple steps within this pathway may be targets for neuroprotective agents.—Copani, A., Condorelli, F., Caruso, A., Vancheri, C., Sala, A., Giuffrida Stella, A. M., Canonico, P. L., Nicoletti, F., Sortino, M. A. Mitotic signaling by β ‐amyloid causes neuronal death. FASEB J.13, 2225–2234 (1999)

The role of group I metabotropic glutamate (mGlu) receptors in neurodegeneration is controversial because of the contradictory effects of mGlu15 agonists in in vitromodels 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 vitroand in vivo. Furthermore, intraventricular administration of LY367385 reduced hippocampal cell death in gerbils subjected to transient global ischemia.

Reactive magnetron sputtering deposition processes based on rotating or double cathodes allow the deposition of multiple TiO2//SiO2layers on glass for obtaining antireflective coatings for large area applications with a good homogeneity and high resistance against environmental attacks. By increasing the number of TiO2//SiO2layers, the luminous reflectance of the coated glass decreases but the calculation of angular daylight and energy parameters requires the use of an increasing number of complementary analytical techniques due to the complexity of the coating. In this paper we show how Rutherford back-scattering, neutron reflectivity and spectrophotometry can be successfully utilised to perform a quite complete characterisation of commercial antireflective coatings based on three TiO2//SiO2layers.

Cultured cerebellar granule cells underwent apoptotic degeneration when grown in medium containing 10 instead of 25 mM K+. Knowing that apoptosis is associated with changes in the expression of primary response genes, we have measured c-fos, zif/268, and c-jun mRNA levels during maturation of cultured granule cells grown in 10 or 25 mM K+. The constitutive expression of c-fos and zif/268 was differentially regulated by extracellular K+ concentration at 5 days of maturation in vitro (DIV), when cells grown under suboptimal conditions (i.e. in 10 mM K+) are committed to degenerate. At this stage, c-fos mRNA levels were detectable only in cultures grown in 25 mM K+, whereas zif/268 mRNA levels were dramatically elevated in cultures grown in 10 mM K+. This provides one of the few conditions in which c-fos and zif/268 are differentially regulated in nerve cells. Substantial changes in c-jun, or ß-actin mRNA levels were detectable only at 7 DIV, when the percentage of apoptotic cells had already reached a plateau in ultures grown in 10 mM K+. We speculate that changes in the expression of zif/268 are important in the gene program associated with the induction of apoptosis by trophic deprivation in cultured neurons.

The pharmacological profile of (2S,1'S,2'S,3'R)-2-(2'-Carboxy-3'-phenylcyclopropyl)glycine (PCCG-IV) at metabotropic glutamate receptor (mGluR) subtypes mGluR1a, mGluR2, mGluR4a, and mGluR5 was examined. PCCG-IV potently antagonized glutamate-induced inhibition of forskolin-stimulated cAMP formation in baby hamster kidney cells expressing mGluR2 in a competitive manner (KB = 8.2 +/- 0.4 microM). PCCG-IV was a weak agonist at mGluR4a but inactive at the cloned phosphoinositide-coupled mGluRs (mGluR1a and mGluR5a). PCCG-IV was significantly more potent and selective as an antagonist at mGluR2 compared with previously described mGluR2 antagonists, including alpha-methyl-4-carboxyphenylglycine. In mice cortical neurons, PCCG-IV antagonized the neuroprotective effects of a selective mGluR2 agonist, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine, at low doses (0.2-20 microM), whereas a higher dose of PCCG-IV (80 microM) was similarly neuroprotective to L-2-amino-4-phosphonobutanoate. The neuroprotective effect of PCCG-IV was blocked by an antagonist of mGluR4a, alpha-methyl-4-phosphonophenylglycine. Thus, PCCG-IV is a novel and useful tool for delineating the physiological roles of group II mGluRs in the central nervous system.

Prolonged exposure of cultured cortical cells or cultured cerebellar granule cells to the residue 25-35 fragment of beta-amyloid peptide (beta AP), beta AP(25-35), induced neuronal apoptosis, as revealed by morphological analysis, fluorescent chromatin staining, and immunodetection of oligonucleosomes released from the nucleus into the cytoplasm. beta AP(25-35)-induced apoptosis was insensitive to ionotropic glutamate receptor antagonists but was substantially attenuated by the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid. The neuroprotective action of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid was antagonized by (RS)-alpha-methyl-4-carboxyphenylglycine and was mimicked by (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (a selective agonist of mGluR2 and -3 subtypes) and by L-2-amino-4-phosphobutanoate and L-serine-O-phosphate (selective agonists of mGluR4, -6, and -7 subtypes). However, whereas all of these drugs behaved as neuroprotectants in cultured cortical cells, only L-2-amino-4-phosphobutanoate and L-serine-O-phosphate [and not (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine] reduced beta AP(25-35)-induced apoptosis in cultured cerebellar granule cells. The neuroprotective activity of mGluR agonists may be related to their ability to inhibit membrane Ca2+ conductance, because drugs that block voltage-sensitive Ca2+ channels, such as nimodipine or Co2+, could also attenuate beta AP(25-35)-induced apoptosis.

[3H]Inositolhexakisphosphate (InsP6) binds to a single population of specific and saturable recognition sites in membranes prepared from cerebral hemispheres, anterior pituitaries, or cultured cerebellar neurons. Binding is temperature and pH dependent, exhibits slow association and dissociation kinetics, and differentiates among various inositolpolyphosphates (InsP6 is much more potent than inositol-1,3,4,5,6-pentakis- and inositol-1,3,4,5-tetrakisphosphate, whereas inositol-1,4,5-trisphosphate is inactive as a displacer). In membranes from cerebral hemispheres, saturation analysis reveals a KD value of 33 +/- 4 nM and a maximal density (Bmax) of 152 +/- 23 pmol/mg of protein. Both affinity and density of [3H]InsP6 binding are lower in membranes from anterior pituitaries. In cultured cerebellar neurons, micromolar concentrations of divalent cations enhance both [3H]InsP6 binding and InsP6-stimulated 45Ca2+ uptake, suggesting that activation of specific receptors may be involved in the stimulation of 45Ca2+ uptake produced by InsP6. These data support the recent view that InsP6, as other inositolpolyphosphates, may act as a signal molecule in excitable 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)>(1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>ß-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as a-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 1S,3R-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.

The serum levels of the heterodimeric cytokine IL-12 were measured by solid-phase ELISA in a group of healthy subjects, multiple sclerosis (MS) patients with secondary chronic progressive course of the disease and patients suffering from other neurological diseases (OND) Serum levels of IL-12 higher than 5 pg/ml (limit of sensitivity of the assay) were only found in 2/30 (6.7%) of the healthy subjects and none of the 8 subjects with OND. In contrast, IL-12 was found in the majority of CPMS patients’ sera (10/15, 66.7%) with values ranging between 5.5 and 18.6 pg/ml. These results are suggestive for an up-regulated production of IL-12 in CPMS.

In rat hippocampal slices, the novel metabotropic glutamate receptor (mGluR) ligand, (1 S,1′ R,2′ R,3′ R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) enhanced the stimulation of polyphosphoinositide (PPI) hydrolysis elicited by quisqualate or by submaximal concentrations of ibotenate or (1 S,3 R)-1aminocyclopentane-1,3-dicarboxylic acid (1 S,3 R-ACPD). The enhancing effect of DCG-IV was (i) specific for mGluR agonists, (ii) restricted to hippocampal slice preparation, (iii) reversible, and (iv) not subject to homologous desensitization. In addition, DGC-IV did not interact with l-2-amino-4-phosphonobutanoate (AP4), a noncompetitive antagonist of mGluRs coupled to PPI hydrolysis in brain slices [32]. The action of DCG-IV on quisqualate-stimulated PPI hydrolysis was insensitive to antagonists of ionotropic glutamate receptors and did not appear to be a consequence of a reduction in the intracellular levels of cAMP [14]. When the stimulation of PPI hydrolysis was measured as a function of the incubation time, DCG-IV potentiated quisqualate-stimulated PPI hydrolysis after 60 min of incubation, when quisqualate had already reached its maximal effect. Knowing that activation of protein kinase C (PKC) limits the extent of mGluR agonist-stimulated PPI hydrolysis over time, we have studied the enhancing effect of DCG-IV in the presence of the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected [9], TPA reduced quisqualate-stimulated PPI hydrolysis in control slices, but was inactive in slices incubated in the presence of DCG-IV. Taken collectively, these results suggest that DCG-IV positively modulates the activity of mGluRs coupled to PPI hydrolysis through a mechanism, which involves PKC-mediated phosphorylation processes.

The findings of an epidemiological survey of multiple sclerosis (MS) in the urban area of Catania reveal a new and in some respects original pattern of MS cases recorded on 31 December 1989 (prevalence day).

The effects of different doses of the dopamine (DA) receptor agonist apomorphine on the activity of the γ-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase (GAD, EC 4.1.1.15) were investigated in rat substantia nigra in comparison with haloperidol and sulpiride, two DA receptor blocking agents. Results obtained show that low doses (10,35 μg/kg, s.c.) of apomorphine induce a decrease in nigral GAD activity whilst an opposite effect is observed with the highest dose (1000 μg/kg, s.c.). No significant change is observed following injection of the intermediate doses (100 and 500 μg/kg, s.c.). Moreover, sulpiride at the dose used (2 mg/kg, i.p.) induces an increase in GAD activity whilst no effect follows systemic injection of the same dose of haloperidol. The results are discussed in light of recent neurochemical and behavioral data.

In order to confirm the existence of metabotropic glutamate receptors in astroglial cultures and to provide information on different receptor subtypes, the expression of different mOIuRs was analysed in cultures highly enriched in rat astroglial cells. mRNA levels for mGluR1, 2, 3, 4, 7 were undetectable by Northern blot analysis in primary type-1 astroglial cultures derived from total cerebral hemispheres, cerebral cortex and striatum. Interestingly, these cultures expressed a low, but detectable, level of mGluR5 mRNA. The more sensitive technique Reverse Transcription-Polymerase Chain Reaction (RT-PCR) confirmed the presence of mGluR5 transcript in cultured astrocytes and, in addition, revealed the presence of mGluR3 mRNA. The lack of expression of mGluR5 in CG-4 cells, a rat cell line able to differentiate in type-2 astrocytes or oligodendrocytes depending on the culture conditions, suggested that the presence of mGluR5 was not a general feature of cells of glial origin. Moreover, all the examined mGluR transcripts were undetectable by RT-PCR in CG4 cells. In order to confirm the possible expression of mGluR5 in cell of glial origin we examined the mRNA levels for this receptor in tissue samples from human gliomas obtained after surgical resection of the tumors: only 1 sample (grade II astrocytoma), out of 8 examined, showed the presence of mGluR5 mRNA. In conclusion our data show that the only cloned metabotropic receptor linked to phosphoinositide hydrolysis, whose expression is detectable in cultured type-1 astrocytes, is mGluR5. It remains to be established if the low level of expression of mGluR3 could be responsible for the group II metabotropic glutamate receptor activity previously observed in cultured astroglial cells.

Objective: to investigate the relationship between disease activity and in vitro cytokine, soluble(s)CD23 and polyclonal and anti-DNA antibody production by PBMC from patients with active systemic lupus erythematosus (SLE).Methods: cytokines, sCD23 and immunoglobulins were estimated by ELISA in unstimulated and polyclonal mitogen-stimulated culture supernatants.Results: PHA-induced IL-2 and IFN-y production were decreased, whereas spontaneous and PHA-induced IL-6 and IL-10 production were increased in cultures of SLE lympho cytes. Conversely, spontaneous and PHA-stimulated IL-4 and sCD23 production was com parable between patients and controls. Finally, we found an increase in in vitro spontaneous polyclonal and anti-DNA IgG secretion.Conclusions: We demonstrated an expanded type-2 cytokine profile with no correlation with parameters of disease activity.

A consensus structure for the active site of terminal oxidases has been recently proposed by Hosler et al. [(1993) J. Bioenerg. Biomem. 25, 121–1351]. We exploit the novel structural information to propose a hypothesis for the large difference in the rate of internal electron transfer found when experiments are started either with the reduced or with the oxidized enzyme. This rationale also allows us to discuss the oxidation state of the prevailing oxygen reacting species with reference to the concentration of the two substrates (oxygen and cytochrome c) and to the structural state of the oxidase.

We have studied the expression of a‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K±(10, 25, or 40 mM) with or without 100 μM N‐methyl‐D‐aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mMK±(K10) and increased dramatically in cultures grown in 25 mMK±(K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 ± NMDA). In cultures grown in 40 mMK±(K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and ‐3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA‐stimulated 4SCa2±influx and 40‐[3H]phorbol 12, 13‐dibutyrate binding in intact cells. Both functional parameters increased along with the K±concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits. These results indicate that functional diversity of AMPA receptors can be generated by the degree of chronic depolarization and/or exposure to NMDA in neurons developing in primary culture.