RESUMEN
Glutamate carboxypeptidase II (GCPII) inactivates the peptide neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Inhibitors of GCPII increase extracellular NAAG levels and are efficacious in animal models of clinical disorders via NAAG activation of a group II metabotropic glutamate receptor. mGluR2 and mGluR3 knock-out (ko) mice were used to test the hypothesis that mGluR3 mediates the activity of GCPII inhibitors ZJ43 and 2-PMPA in animal models of memory and memory loss. Short- (1.5 h) and long- (24 h) term novel object recognition tests were used to assess memory. Treatment with ZJ43 or 2-PMPA prior to acquisition trials increased long-term memory in mGluR2, but not mGluR3, ko mice. Nine month-old triple transgenic Alzheimer's disease model mice exhibited impaired short-term novel object recognition memory that was rescued by treatment with a NAAG peptidase inhibitor. NAAG peptidase inhibitors and the group II mGluR agonist, LY354740, reversed the short-term memory deficit induced by acute ethanol administration in wild type mice. 2-PMPA also moderated the effect of ethanol on short-term memory in mGluR2 ko mice but failed to do so in mGluR3 ko mice. LY354740 and ZJ43 blocked ethanol-induced motor activation. Both GCPII inhibitors and LY354740 also significantly moderated the loss of motor coordination induced by 2.1 g/kg ethanol treatment. These data support the conclusion that inhibitors of glutamate carboxypeptidase II are efficacious in object recognition models of normal memory and memory deficits via an mGluR3 mediated process, actions that could have widespread clinical applications.
Asunto(s)
Intoxicación Alcohólica/metabolismo , Enfermedad de Alzheimer/metabolismo , Glutamato Carboxipeptidasa II/metabolismo , Memoria/fisiología , Receptores de Glutamato Metabotrópico/deficiencia , Urea/análogos & derivados , Intoxicación Alcohólica/genética , Enfermedad de Alzheimer/genética , Animales , Modelos Animales de Enfermedad , Etanol/administración & dosificación , Antagonistas de Aminoácidos Excitadores/farmacología , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Glutamato Carboxipeptidasa II/genética , Masculino , Memoria/efectos de los fármacos , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Urea/farmacologíaRESUMEN
The "glutamate" theory of schizophrenia emerged from the observation that phencyclidine (PCP), an open channel antagonist of the NMDA subtype of glutamate receptor, induces schizophrenia-like behaviors in humans. PCP also induces a complex set of behaviors in animal models of this disorder. PCP also increases glutamate and dopamine release in the medial prefrontal cortex and nucleus accumbens, brain regions associated with expression of psychosis. Increased motor activation is among the PCP-induced behaviors that have been widely validated as models for the characterization of new antipsychotic drugs. The peptide transmitter N-acetylaspartylglutamate (NAAG) activates a group II metabotropic receptor, mGluR3. Polymorphisms in this receptor have been associated with schizophrenia. Inhibitors of glutamate carboxypeptidase II, an enzyme that inactivates NAAG following synaptic release, reduce several behaviors induced by PCP in animal models. This research tested the hypothesis that two structurally distinct NAAG peptidase inhibitors, ZJ43 and 2-(phosphonomethyl)pentane-1,5-dioic acid, would elevate levels of synaptically released NAAG and reduce PCP-induced increases in glutamate and dopamine levels in the medial prefrontal cortex and nucleus accumbens. NAAG-like immunoreactivity was found in neurons and presumptive synaptic endings in both regions. These peptidase inhibitors reduced the motor activation effects of PCP while elevating extracellular NAAG levels. They also blocked PCP-induced increases in glutamate but not dopamine or its metabolites. The mGluR2/3 antagonist LY341495 blocked these behavioral and neurochemical effects of the peptidase inhibitors. The data reported here provide a foundation for assessment of the neurochemical mechanism through which NAAG achieves its antipsychotic-like behavioral effects and support the conclusion NAAG peptidase inhibitors warrant further study as a novel antipsychotic therapy aimed at mGluR3.
Asunto(s)
Dopamina/química , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Ácido Glutámico/química , Núcleo Accumbens/metabolismo , Fenciclidina/farmacología , Corteza Prefrontal/metabolismo , Esquizofrenia/fisiopatología , Animales , Antipsicóticos/farmacología , Conducta Animal , Modelos Animales de Enfermedad , Dopamina/metabolismo , Glutamato Carboxipeptidasa II/química , Ácido Glutámico/metabolismo , Masculino , Neurotransmisores/metabolismo , Ratas , Ratas Sprague-Dawley , Esquizofrenia/tratamiento farmacológicoRESUMEN
Immunohistochemical studies previously revealed the presence of the peptide transmitter N-acetylaspartylglutamate (NAAG) in spinal motor neurons, axons and presumptive neuromuscular junctions (NMJs). At synapses in the central nervous system, NAAG has been shown to activate the type 3 metabotropic glutamate receptor (mGluR3) and is inactivated by an extracellular peptidase, glutamate carboxypeptidase II. The present study tested the hypothesis that NAAG meets the criteria for classification as a co-transmitter at the vertebrate NMJ. Confocal microscopy confirmed the presence of NAAG immunoreactivity and extended the resolution of the peptide's location in the lizard (Anolis carolinensis) NMJ. NAAG was localised to a presynaptic region immediately adjacent to postsynaptic acetylcholine receptors. NAAG was depleted by potassium-induced depolarisation and by electrical stimulation of motor axons. The NAAG receptor, mGluR3, was localised to the presynaptic terminal consistent with NAAG's demonstrated role as a regulator of synaptic release at central synapses. In contrast, glutamate receptors, type 2 metabotropic glutamate receptor (mGluR2) and N-methyl-d-aspartate, were closely associated with acetylcholine receptors in the postsynaptic membrane. Glutamate carboxypeptidase II, the NAAG-inactivating enzyme, was identified exclusively in perisynaptic glial cells. This localisation was confirmed by the loss of immunoreactivity when these cells were selectively eliminated. Finally, electrophysiological studies showed that exogenous NAAG inhibited evoked neurotransmitter release by activating a group II metabotropic glutamate receptor (mGluR2 or mGluR3). Collectively, these data support the conclusion that NAAG is a co-transmitter at the vertebrate NMJ.
Asunto(s)
Dipéptidos/farmacología , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Unión Neuromuscular/química , Neurotransmisores/farmacología , Animales , Dipéptidos/análisis , Agonistas de Aminoácidos Excitadores/farmacología , Glutamato Carboxipeptidasa II/análisis , Inmunohistoquímica , Lagartos , Neuronas Motoras/química , Neuronas Motoras/fisiología , N-Metilaspartato/farmacología , Unión Neuromuscular/fisiología , Potasio/farmacología , Terminales Presinápticos/química , Receptores Colinérgicos/análisis , Receptores de Glutamato Metabotrópico/análisisRESUMEN
BACKGROUND: Metabotropic glutamate receptors (mGluRs) have been identified as significant analgesic targets. Systemic treatments with inhibitors of the enzymes that inactivate the peptide transmitter N-acetylaspartylglutamate (NAAG), an mGluR3 agonist, have an analgesia-like effect in rat models of inflammatory and neuropathic pain. The goal of this study was to begin defining locations within the central pain pathway at which NAAG activation of its receptor mediates this effect. RESULTS: NAAG immunoreactivity was found in neurons in two brain regions that mediate nociceptive processing, the periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM). Microinjection of the NAAG peptidase inhibitor ZJ43 into the PAG contralateral, but not ipsilateral, to the formalin injected footpad reduced the rapid and slow phases of the nociceptive response in a dose-dependent manner. ZJ43 injected into the RVM also reduced the rapid and slow phase of the response. The group II mGluR antagonist LY341495 blocked these effects of ZJ43 on the PAG and RVM. NAAG peptidase inhibition in the PAG and RVM did not affect the thermal withdrawal response in the hot plate test. Footpad inflammation also induced a significant increase in glutamate release in the PAG. Systemic injection of ZJ43 increased NAAG levels in the PAG and RVM and blocked the inflammation-induced increase in glutamate release in the PAG. CONCLUSION: These data demonstrate a behavioral and neurochemical role for NAAG in the PAG and RVM in regulating the spinal motor response to inflammation and that NAAG peptidase inhibition has potential as an approach to treating inflammatory pain via either the ascending (PAG) and/or the descending pain pathways (PAG and RVM) that warrants further study.
Asunto(s)
Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Inflamación/enzimología , Sustancia Gris Periacueductal/enzimología , Analgésicos/uso terapéutico , Animales , Formaldehído/toxicidad , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Masculino , Bulbo Raquídeo/enzimología , Neuralgia/tratamiento farmacológico , Neuralgia/enzimología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
A substantial body of data was reported between 1984 and 2000 demonstrating that the neuropeptide N-acetylaspartylglutamate (NAAG) not only functions as a neurotransmitter but also is the third most prevalent transmitter in the mammalian nervous system behind glutamate and GABA. By 2005, this conclusion was validated further through a series of studies in vivo and in vitro. The primary enzyme responsible for the inactivation of NAAG following its synaptic release had been cloned, characterized and knocked out. Potent inhibitors of this enzyme were developed and their efficacy has been extensively studied in a series of animal models of clinical conditions, including stroke, peripheral neuropathy, traumatic brain injury, inflammatory and neuropathic pain, cocaine addiction, and schizophrenia. Considerable progress also has been made in defining further the mechanism of action of these peptidase inhibitors in elevating synaptic levels of NAAG with the consequent inhibition of transmitter release via the activation of pre-synaptic metabotropic glutamate receptor 3 by this peptide. Very recent discoveries include identification of two different nervous system enzymes that mediate the synthesis of NAAG from N-acetylaspartate and glutamate and the finding that one of these enzymes also mediates the synthesis of a second member of the NAAG family of neuropeptides, N-acetylaspartylglutamylglutamate.
Asunto(s)
Dipéptidos/fisiología , Neuropéptidos/fisiología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Lesiones Encefálicas/tratamiento farmacológico , Dipéptidos/genética , Dipéptidos/metabolismo , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Humanos , Hiperalgesia/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Neuropéptidos/genética , Neuropéptidos/metabolismo , Neurotransmisores/fisiología , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/metabolismo , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Esquizofrenia/tratamiento farmacológico , Trastornos Relacionados con Sustancias/terapiaRESUMEN
N-Acetylaspartylglutamate (NAAG) is the most abundant and widely distributed peptide transmitter in the mammalian nervous system. NAAG activates the metabotropic glutamate mGlu(3) receptor at presynaptic sites, inhibiting the release of neurotransmitters, including glutamate, and activates mGlu(3) receptors on glial cells, stimulating the release of neuroprotective growth factors from these cells. Elevated levels of glutamate released from neurons are associated with the pathology of stroke, traumatic nervous system injury, amyotrophic lateral sclerosis, inflammatory and neuropathic pain, diabetic neuropathy and the schizophrenia-like symptoms elicited by phencyclidine. NAAG is inactivated by specific peptidases following its synaptic release. Novel compounds that inhibit these enzymes prolong the activity of synaptically released NAAG and have significant therapeutic efficacy in animal models of these diverse clinical conditions. In this review, we summarize recent studies in these animal models and discuss the mechanisms by which NAAG peptidase inhibitors achieve these effects.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Isquemia Encefálica/tratamiento farmacológico , Neuropatías Diabéticas/tratamiento farmacológico , Dipéptidos/metabolismo , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Dolor/tratamiento farmacológico , Inhibidores de Proteasas/uso terapéutico , Esquizofrenia/tratamiento farmacológico , Animales , HumanosRESUMEN
The neuropeptidase glutamate carboxypeptidase II (GCPII) hydrolyzes N-acetyl-L-aspartyl-L-glutamate (NAAG) to liberate N-acetylaspartate and glutamate. GCPII was originally cloned as PSMA, an M(r) 100,000 type II transmembrane glycoprotein highly expressed in prostate tissues. PSMA/GCPII is located on the short arm of chromosome 11 and functions as both a folate hydrolase and a neuropeptidase. Inhibition of brain GCPII may have therapeutic potential in the treatment of certain disease states arising from pathologically overactivated glutamate receptors. Recently, we reported that certain urea-based structures act as potent inhibitors of GCPII (J. Med. Chem. 2001, 44, 298). However, many of the potent GCPII inhibitors prepared to date are highly polar compounds and therefore do not readily penetrate the blood-brain barrier. Herein, we elaborate on the synthesis of a series of potent, urea-based GCPII inhibitors from the lead compound 3 and provide assay data for these ligands against human GCPII. Moreover, we provide data revealing the ability of one of these compounds, namely, 8d, to reduce the perception of inflammatory pain. Within the present series, the gamma-tetrazole bearing glutamate isostere 7d is the most potent inhibitor with a K(i) of 0.9 nM. The biological evaluation of these compounds revealed that the active site of GCPII likely comprises two regions, namely, the pharmacophore subpocket and the nonpharmacophore subpocket. The pharmacophore subpocket is very sensitive to structural changes, and thus, it appears important to keep one of the glutamic acid moieties intact to maintain the potency of the GCPII inhibitors. The site encompassing the nonpharmacophore subpocket that binds to glutamate's alpha-carboxyl group is sensitive to structural change, as shown by compounds 6b and 7b. However, the other region of the nonpharmacophore subpocket can accommodate both hydrophobic and hydrophilic groups. Thus, an aromatic ring can be introduced to the inhibitor, as in 8b and 8d, thereby increasing its hydrophobicity and thus potentially its ability to cross the blood-brain barrier. Intrathecally administered 8d significantly reduced pain perception in the formalin model of rat sensory nerve injury. A maximal dose of morphine (10 mg) applied in the same experimental paradigm provided no significant increase in analgesia in comparison to 8d during phase 1 of this pain study and modestly greater analgesia than 8d in phase 2. These urea-based inhibitors of GCPII thus offer a novel approach to pain management.
Asunto(s)
Analgésicos/síntesis química , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Glutamatos/síntesis química , Urea/análogos & derivados , Urea/síntesis química , Analgésicos/farmacología , Animales , Sitios de Unión , Barrera Hematoencefálica/metabolismo , Células CACO-2 , Glutamatos/farmacología , Humanos , Masculino , Dimensión del Dolor , Permeabilidad , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-Actividad , Urea/farmacologíaRESUMEN
The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is inactivated by the extracellular enzyme glutamate carboxypeptidase II. Inhibitors of this enzyme reverse dizocilpine (MK-801)-induced impairment of short-term memory in the novel object recognition test. The objective of this study was to test the hypothesis that NAAG peptidase inhibition enhances long-term (24h delay) memory of C57BL mice. These mice and mice in which glutamate carboxypeptidase II had been knocked out were presented with two identical objects to explore for 10min on day 1 and tested with one of these familiar objects and one novel object on day 2. Memory was assessed as the degree to which the mice recalled the familiar object and explored the novel object to a greater extent on day 2. Uninjected mice or mice injected with saline prior to the acquisition session on day 1 demonstrated a lack of memory of the acquisition experience by exploring the familiar and novel objects to the same extent on day 2. Mice treated with glutamate carboxypeptidase II inhibitors ZJ43 or 2-PMPA prior to the acquisition trial explored the novel object significantly more time than the familiar object on day 2. Consistent with these results, mice in which glutamate carboxypeptidase II had been knocked out distinguished the novel from the familiar object on day 2 while their heterozygous colony mates did not. Inhibition of glutamate carboxypeptidase II enhances recognition memory, a therapeutic action that might be useful in treatment of memory deficits related to age and neurological disorders.
Asunto(s)
Eliminación de Gen , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Glutamato Carboxipeptidasa II/genética , Memoria/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Reconocimiento en Psicología/efectos de los fármacos , Reconocimiento en Psicología/fisiología , Animales , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Técnicas de Inactivación de Genes , Glutamato Carboxipeptidasa II/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Compuestos Organofosforados/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
The group II metabotropic glutamate receptors 2 and 3 (mGluR2 and mGluR3) share sequence homology, common pharmacology and negative coupling to cAMP. We recently discovered that mGluR3 also is negatively coupled through a G-protein to the cGMP transduction pathway in rat cerebellar granule cells and astrocytes. To test the hypothesis that mGluR2 also has access to the cGMP pathway, C6 glioma cells were stably transfected with mGluR2 and mGluR3 cDNA and their coupling to cGMP levels was characterized. In contrast to many other cell lines, C6 has a robust cGMP response that makes it attractive in the study of receptor coupling to this second messenger pathway. Consistent with prior studies, the mGluR3 receptor was negatively coupled to cGMP and this coupling was blocked by PTX. In contrast, mGluR2 agonists failed to reduce sodium nitroprusside stimulated cGMP levels in transfected cell lines where the receptor was negatively coupled to cAMP. These data provide further support for the functional divergence between these two closely related receptors.
Asunto(s)
GMP Cíclico/metabolismo , Neuronas/metabolismo , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Sistemas de Mensajero Secundario/genética , Transducción de Señal/genética , Animales , Línea Celular Tumoral , ADN Complementario/genética , Neuronas/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Toxina del Pertussis/farmacología , Ratas , Sistemas de Mensajero Secundario/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transfección/métodosAsunto(s)
Antipsicóticos/uso terapéutico , Glutamato Carboxipeptidasa II/uso terapéutico , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/toxicidad , Conducta Exploratoria/efectos de los fármacos , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Ratones , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Compuestos Organofosforados/toxicidad , Fenciclidina/toxicidad , Receptores de Glutamato Metabotrópico/deficiencia , Esquizofrenia/inducido químicamente , Esquizofrenia/genéticaRESUMEN
The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) selectively activates group II metabotropic glutamate receptors (mGluRs). Systemic administration of inhibitors of the enzymes that inactivate NAAG results in decreased pain responses in rat models of inflammatory and neuropathic pain. These effects are blocked by a group II mGluR antagonist. This research tested the hypothesis that some analgesic effects of NAAG peptidase inhibition are mediated by NAAG acting on sensory neurite mGluRs at the site of inflammation. Group II mGluR agonists, SLx-3095-1, NAAG and APDC, or NAAG peptidase inhibitors, ZJ-43 and 2-PMPA, injected into the rat footpad reduced pain responses in carrageenan or formalin models. The analgesic effects of SLx-3095-1, APDC, ZJ-43, 2-PMPA and NAAG were blocked by co-injection of LY341495, a selective group II mGluR antagonist. Injection of group II mGluR agonists, NAAG or the peptidase inhibitors into the contralateral rat footpad had no effect on pain perception in the injected paw. At 10-100 microm ZJ-43 and 2-PMPA demonstrated no consistent agonist activity at mGluR2 or mGluR3. Consistent with the conclusion that peripherally administered NAAG peptidase inhibitors increase the activation of mGluR3 by NAAG that is released from peripheral sensory neurites, we found that the tissue average concentration of NAAG in the unstimulated rat hind paw was about 6 microm. These data extend our understanding of the role of this peptide in sensory neurons and reveal the potential for treatment of inflammatory pain via local application of NAAG peptidase inhibitors at doses that may have little or no central nervous system effects.
Asunto(s)
Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Neuralgia/tratamiento farmacológico , Compuestos Organofosforados/administración & dosificación , Urea/análogos & derivados , 1-Metil-3-Isobutilxantina/farmacología , Animales , Carragenina , Células Cultivadas , Cerebelo/citología , Cricetinae , Cricetulus , Interacciones Farmacológicas , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Formaldehído/efectos adversos , Lateralidad Funcional , Neuralgia/inducido químicamente , Neuralgia/fisiopatología , Neuronas/efectos de los fármacos , Dimensión del Dolor/métodos , Umbral del Dolor/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Receptores de Glutamato/fisiología , Transfección/métodos , Urea/administración & dosificaciónRESUMEN
Metabotropic receptors may couple to different G proteins in different cells or perhaps even in different regions of the same cell. To date, direct studies of group II and group III metabotropic glutamate receptors' (mGluRs) relationships to second messenger cascades have reported negative coupling of these receptors to cyclic AMP (cAMP) levels in neurons, astrocytes and transfected cells. In the present study, we found that the peptide neurotransmitter N-acetylaspartylglutamate (NAAG), an mGluR3-selective agonist, decreased sodium nitroprusside (SNP)-stimulated cyclic GMP (cGMP) levels in cerebellar granule cells and cerebellar astrocytes. The mGluR3 and group II agonists FN6 and LY354740 had similar effects on cGMP levels. The mGluR3 and group II antagonists beta-NAAG and LY341495 blocked these actions. Treatment with pertussis toxin inhibited the effects of NAAG on SNP-stimulated cGMP levels in rat cerebellar astrocytes but not in cerebellar neurons. These data support the conclusion that mGluR3 is also coupled to cGMP levels and that this mGluR3-induced reduction of cGMP levels is mediated by different G proteins in cerebellar astrocytes and neurons. We previously reported that this receptor is coupled to a cAMP cascade via a pertussis toxin-sensitive G protein in cerebellar neurons, astrocytes and transfected cells. Taken together with the present data, we propose that mGluR3 is coupled to two different G proteins in granule cell neurons. These data greatly expand knowledge of the range of second messenger cascades induced by mGluR3, and have implications for clinical conditions affected by NAAG and other group II mGluR agonists.
Asunto(s)
Astrocitos/fisiología , GMP Cíclico/metabolismo , Neuronas/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Aminoácidos/farmacología , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Cerebelo/citología , Cerebelo/fisiología , Dipéptidos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas de Unión al GTP/metabolismo , Neuronas/efectos de los fármacos , Nitroprusiato/farmacología , Toxina del Pertussis/farmacología , Ratas , Ratas Sprague-Dawley , Xantenos/farmacologíaRESUMEN
Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress neurotransmitter release through selective activation of presynaptic group II metabotropic glutamate receptors. Therefore, strategies to elevate levels of NAAG following brain injury could reduce excessive glutamate release associated with TBI. We hypothesized that the NAAG peptidase inhibitor, ZJ-43 would elevate extracellular NAAG levels and reduce extracellular levels of amino acid neurotransmitters following TBI by a group II metabotropic glutamate receptor (mGluR)-mediated mechanism. Dialysate levels of NAAG, glutamate, aspartate and GABA from the dorsal hippocampus were elevated after TBI as measured by in vivo microdialysis. Dialysate levels of NAAG were higher and remained elevated in the ZJ-43 treated group (50 mg/kg, i.p.) compared with control. ZJ-43 treatment also reduced the rise of dialysate glutamate, aspartate, and GABA levels. Co-administration of the group II mGluR antagonist, LY341495 (1 mg/kg, i.p.) partially blocked the effects of ZJ-43 on dialysate glutamate and GABA, suggesting that NAAG effects are mediated through mGluR activation. The results are consistent with the hypothesis that inhibition of NAAG peptidase may reduce excitotoxic events associated with TBI.
Asunto(s)
Lesiones Encefálicas/metabolismo , Dipéptidos/agonistas , Inhibidores Enzimáticos/farmacología , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Animales , Ácido Aspártico/metabolismo , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/fisiopatología , Citoprotección/efectos de los fármacos , Citoprotección/fisiología , Dipéptidos/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos/uso terapéutico , Líquido Extracelular/efectos de los fármacos , Líquido Extracelular/metabolismo , Glutamato Carboxipeptidasa II/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Masculino , Microdiálisis , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/prevención & control , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Neurotoxinas/antagonistas & inhibidores , Neurotoxinas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The peptide transmitter N-acetylaspartylglutamate (NAAG) is present in millimolar concentrations in mammalian spinal cord. Data from the rat peripheral nervous system suggest that this peptide is synthesized enzymatically, a process that would be unique for mammalian neuropeptides. To test this hypothesis in the mammalian CNS, rat spinal cords were acutely isolated and used to study the incorporation of radiolabeled amino acids into NAAG. Consistent with the action of a NAAG synthetase, inhibition of protein synthesis did not affect radiolabel incorporation into NAAG. Depolarization of spinal cords stimulated incorporation of radiolabel. Biosynthesis of NAAG by cortical astrocytes in cell culture was demonstrated by tracing incorporation of [3H]-glutamate by astrocytes. In the first test of the hypothesis that NAA is an immediate precursor in NAAG biosynthesis, [3H]-NAA was incorporated into NAAG by isolated spinal cords and by cell cultures of cortical astrocytes. Data from cerebellar neurons and glia in primary culture confirmed the predominance of neuronal synthesis and glial uptake of NAA, leading to the hypothesis that while neurons synthesize NAA for NAAG biosynthesis, glia may take it up from the extracellular space. However, cortical astrocytes in serum-free low-density cell culture incorporated [3H]-aspartate into NAAG, a result indicating that under some conditions these cells may also synthesize NAA. Pre-incubation of isolated spinal cords and cultures of rat cortical astrocytes with unlabeled NAA increased [3H]-glutamate incorporation into NAAG. In contrast, [3H]-glutamine incorporation in spinal cord was not stimulated by unlabeled NAA. These results are consistent with the glutamate-glutamine cycle greatly favoring uptake of glutamine into neurons and glutamate by glia and suggest that NAA availability may be rate-limiting in the synthesis of NAAG by glia under some conditions.
Asunto(s)
Ácido Aspártico/análogos & derivados , Sistema Nervioso Central/metabolismo , Dipéptidos/biosíntesis , Enzimas/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Ácido Aspártico/biosíntesis , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacocinética , Células Cultivadas , Sistema Nervioso Central/citología , Cerebelo/citología , Cerebelo/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Medio de Cultivo Libre de Suero/farmacología , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacocinética , Glutamina/metabolismo , Glutamina/farmacocinética , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Cloruro de Potasio/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , TritioRESUMEN
The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar V(max) and K(m) values (1.4 micro m and 54 pmol/min/mg; 3.5 micro m and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG.
Asunto(s)
Encéfalo/enzimología , Glutamato Carboxipeptidasa II/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Membrana Celular/enzimología , Células Cultivadas , Clonación Molecular , Cricetinae , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glutamato Carboxipeptidasa II/genética , Concentración de Iones de Hidrógeno , Metales/farmacología , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/biosíntesis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia , Médula Espinal/enzimología , TransfecciónRESUMEN
Phencyclidine (PCP) administration elicits positive and negative symptoms that resemble those of schizophrenia and is widely accepted as a model for the study of this human disorder. Group II metabotropic glutamate receptor (mGluR) agonists have been reported to reduce the behavioral and neurochemical effects of PCP. The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective group II agonist. We synthesized and characterized a urea-based NAAG analogue, ZJ43. This novel compound is a potent inhibitor of enzymes, glutamate carboxypeptidase II (K(i) = 0.8 nM) and III (K(i) = 23 nM) that deactivate NAAG following synaptic release. ZJ43 (100 microM) does not directly interact with NMDA receptors or metabotropic glutamate receptors. Administration of ZJ43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior, and head movements. To test the hypothesis that this effect of ZJ43 was mediated by increasing the activation of mGluR3 via increased levels of extracellular NAAG, the group II mGluR selective antagonist LY341495 was co-administered with ZJ43 prior to PCP treatment. This antagonist completely reversed the effects of ZJ43. Additionally, LY341495 alone increased PCP-induced motor activity and head movements suggesting that normal levels of NAAG act to moderate the effect of PCP on motor activation via a group II mGluR. These data support the view that NAAG peptidase inhibitors may represent a new therapeutic approach to some of the components of schizophrenia that are modeled by PCP.
Asunto(s)
Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Actividad Motora/efectos de los fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/tratamiento farmacológico , Conducta Estereotipada/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Masculino , Fenciclidina , Ratas , Ratas Sprague-Dawley , Esquizofrenia/inducido químicamente , Urea/análogos & derivados , Urea/síntesis química , Urea/farmacologíaRESUMEN
Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.