Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures

The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N-methyl-D-aspartate (NMDA) receptors, (2) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine (MK-801), an NMDA receptor antagonist, was able to block part of the toxicity. The activation of NMDA receptors is most likely a secondary effect resulting from glutamate release upon kainate or domoate stimulation. 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-ethyle nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization of kainate receptors led to an increased neurotoxicity upon stimulation with kainate or domoate. In neurons cultured for 12 days in vitro a small but significant neurotoxic effect was observed when stimulated with agonist in the presence of MK-801 and GYKI 53655. This indicates that the toxicity is produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures.     

Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice

L-glutamate, the neurotransmitter of the majority of excitatory synapses in the brain, acts on three classes of ionotropic receptors: NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptors. Little is known about the physiological role of kainate receptors because in many experimental situations it is not possible to distinguish them from AMPA receptors. Mice with disrupted kainate receptor genes enable the study of the specific role of kainate receptors in synaptic transmission as well as in the neurotoxic effects of kainate. We have now generated mutant mice lacking the kainate-receptor subunit GluR6. The hippocampal neurons in the CA3 region of these mutant mice are much less sensitive to kainate. In addition, a postsynaptic kainate current evoked in CA3 neurons by a train of stimulation of the mossy fibre system is absent in the mutant. We find that GluR6-deficient mice are less susceptible to systemic administration of kainate, as judged by onset of seizures and by the activation of immediate early genes in the hippocampus. Our results indicate that kainate receptors containing the GluR6 subunit are important in synaptic transmission as well as in the epileptogenic effects of kainate.     

Selective loss of [3H]kainic acid and [3H]AMPA binding in layer VI of frontal cortex in Huntington's disease

Excitatory amino acid neurotoxicity has been proposed to cause the neostriatal neuronal degeneration of Huntington's disease (HD); N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainate receptors have been hypothesized to play important roles in this process. We have recently reported a loss of neurons in layer VI of the cerebral cortex in HD. Using quantitative autoradiographic methods, we have now measured NMDA, AMPA, and kainate receptor binding in the frontal cerebral cortex of the brains of controls and individuals with HD. We find no change in NMDA receptor binding but a selective decrease in kainate and AMPA receptor binding in layer VI. These data suggest that cerebral cortical neurons possessing kainate or AMPA receptors may be selectively vulnerable in individuals with HD.     

In-vitro characterization of YM872, a selective, potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL(-1) in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 +/- 0.0024 microM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 +/- 0.14 microM), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 microM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 microM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 +/- 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-microM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 +/- 0.031 microM, and blocked 300-microM kainate-induced neurotoxicity with an IC50 value of 1.02 microM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.     

Differential assembly of coexpressed glutamate receptor subunits in neurons of rat cerebral cortex

In the rat, subunits of the glutamate receptor family fall into three pharmacologically distinct groups: alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid preferring receptors (Glu R1-4), kainate preferring receptors (Glu R5-7, KA 1, KA 2), and N-methyl-D-aspartate preferring receptors (NMDA R1, NMDA R2A-2D). In the present study, we demonstrate immunocytochemically that the majority of neurons in rat cerebral cortex coexpress members of all three groups of glutamate receptor subunits, Glu R2/3, Glu R5/6/7, and NMDA R1. Using immunoaffinity purified or immunoprecipitated alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate and N-methyl-D-aspartate receptors, we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors containing Glu R1-4, kainate receptors containing Glu R6, Glu R7, and KA 2 and N-methyl-D-aspartate receptors containing NMDA R1 each form distinct protein complexes that do not share subunits. Our data indicate that a mechanism exists which allows for the specific assembly of selected glutamate receptor subunits into functionally and structurally distinct heteromeric receptors.     

Decahydroisoquinolines: novel competitive AMPA/kainate antagonists with neuroprotective effects in global cerebral ischaemia

In the present studies, we have evaluated the activity of a series of glutamate receptor antagonists from the decahydroisoquinoline group of compounds both in vitro and in vivo. Compound activity at alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptors was assessed using ligand binding to cloned iGluR2 and iGluR5 receptors and on responses evoked by AMPA and N-methyl-D-aspartate (NMDA) in the cortical wedge preparation. In vivo, compounds were examined for antagonist activity electrophysiologically in the rat spinal cord preparation and in the gerbil model of global cerebral ischaemia. Compounds tested were LY293558, which has been shown to protect in models of focal cerebral ischaemia, LY202157 (an NMDA antagonist), LY246492 (an NMDA and AMPA receptor antagonist), LY302679, LY292025, LY307190, LY280263, LY289178, LY289525, LY294486 (AMPA/kainate antagonists) and LY382884 (an iGluR5 selective antagonist). Results obtained support a role for AMPA receptors in cerebral ischemia. LY377770 (a mixed AMPA/iGluR5 antagonist and active isomer of LY294486) demonstrated good neuroprotection with a 2-h time window and may therefore be useful in the treatment of ischaemic conditions.     

Kainate excitotoxicity is mediated by AMPA- but not kainate-preferring receptors in embryonic rat hippocampal cultures

We investigated kainate-induced excitotoxicity in embryonic rat hippocampal cells cultured in a chemically defined medium. Treatment with kainate for 24 h resulted in neuronal death, as assessed by the release of lactate dehydrogenase into the culture media. This neurotoxic effect was kainate dose- and culture age-dependent. EC50 of kainate was 127 +/- 11 microM. 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (f)quinoxaline (NBQX) completely blocked the toxicity, while MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist, also blocked it but not completely. Furthermore, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) attenuated the kainate injury, while the selective and noncompetitive AMPA-preferring receptor antagonist 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzo-diazepine (GYKI 52466) blocked it completely. Concanavalin A (ConA), which potentiates the response to kainate at kainate-preferring receptors, had little effect on kainate toxicity. Further, AMPA alone induced little toxicity, but produced remarkable toxicity when cyclothazide was used to block the desensitization of AMPA-preferring receptors. These results indicate that kainate excitotoxicity in hippocampal cultures is mediated by AMPA- but not kainate-preferring receptors, and that it involves NMDA-receptor-mediated toxicity. The non-desensitizing response at AMPA-preferring receptors may play an important role in kainate-induced excitotoxicity.     

The synaptic activation of the GluR5 subtype of kainate receptor in area CA3 of the rat hippocampus

Two new compounds (LY293558 and LY294486), that antagonize homomeric human GluR5 receptors, were examined against responses mediated by kainate receptors in the CA3 region of rat hippocampal slices. Both compounds (applied at a concentration of 10 microM) antagonized reversibly currents induced by 200 nM kainate. They also antagonized reversibly the synaptic activation of kainate receptors, evoked by high-frequency stimulation of mossy fibres, in the presence of NMDA and AMPA receptor antagonists. These results show that GluR5 subunits are likely to contribute to a kainate receptor on CA3 neurones that mediates responses to both kainate and synaptically-released L-glutamate.     

A new pyrrolyl-quinoxalinedione series of non-NMDA glutamate receptor antagonists: pharmacological characterization and comparison with NBQX and valproate in the kindling model of epilepsy

Antagonists at the ionotropic non-NMDA [AMPA (amino-methyl proprionic acid)/kainate] type of glutamate receptors have been suggested to possess several advantages compared to NMDA (N-methyl-D-aspartate) receptor antagonists, particularly in terms of risk/benefit ratio, but the non-NMDA receptor antagonists available so far have not fulfilled this promise. From a large series of pyrrolyl-quinoxalinedione derivatives, we selected six new competitive non-NMDA receptor antagonists. The basis of selection was high potency and selectivity for AMPA and/or kainate receptors, high in vivo potency after systemic administration, and an acceptable ratio between neuroprotective or anticonvulsant effects and adverse effects. Pharmacological characteristics of these novel compounds are described in this study with special emphasis on their effects in the kindling model of temporal lobe epilepsy, the most common type of epilepsy in humans. In most experiments, NBQX and the major antiepileptic drug valproate were used for comparison with the novel compounds. The novel non-NMDA receptor antagonists markedly differed in their AMPA and kainate receptor affinities from NBQX. Thus, while NBQX essentially did not bind to kainate receptors at relevant concentrations, several of the novel compounds exhibited affinity to rat brain kainate receptors or recombinant kainate receptor subtypes in addition to AMPA receptors. One compound, LU 97175, bound to native high affinity kainate receptors and rat GluR5-GluR7 subunits, i.e. low affinity kainate binding sites, with much higher affinities than to AMPA receptors. All compounds potently blocked AMPA-induced cell death in vitro and, except LU 97175, AMPA-induced convulsions in vivo. In the kindling model, compounds with a high affinity for GluR7 (LU 97175) or compounds (LU 115455, LU 136541) which potently bind to AMPA receptors and low affinity kainate receptor subunits were potent anticonvulsants in the kindling model, whereas the AMPA receptor-selective LU 112313 was the least selective compound in this model, indicating that non-NMDA antagonists acting at both AMPA and kainate receptors are more effective in this model than AMPA receptor-selective drugs. Three of the novel compounds, i.e. LU 97175, LU 115455 and LU 136541, exerted potent anticonvulsant effects without inducing motor impairment in the rotarod test. This combination of actions is thought to be a prerequisite for selective anticonvulsant drug action.     

(3SR,4aRS,6SR,8aRS)-6-(1H-tetrazol-5-yl)decahydroisoquinoline-3-carboxylic acid, a novel, competitive, systemically active NMDA and AMPA receptor antagonist

We report the synthesis and characterization of 6 (LY246492), which is a competitive N-methyl-D-aspartate (NMDA) and 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoic acid (AMPA) receptor antagonist. Tetrazole-substituted amino acid 6 was prepared in four steps from the recently described aldehyde 7. The optical isomers (-)-6 and (+)-6 were obtained from the same sequence of reactions using the corresponding isomers of 7. The compound displaces both NMDA and AMPA receptor binding and antagonizes depolarizations in cortical slices evoked by both NMDA and AMPA. In mice and pigeons, the compound showed antagonism of responses mediated through NMDA and AMPA receptors. Using the resolved optical isomers of 6, both NMDA and AMPA antagonist activities were found to reside in a single isomer, (-)-6.     

Kainate receptors exhibit differential sensitivities to (S)-5-iodowillardiine

Characterization of the role of kainate receptors in excitatory synaptic transmission has been hampered by a lack of subtype-selective pharmacological agents. (S)-5-Iodowillardiine (IW), an analog of willardiine [(S)-1-(2-amino-2-carboxyethyl)pyrimidine-2,4-dione], a heterocyclic amino acid found in Acacia and Mimosa seeds, was previously shown to be highly potent on native kainate receptors in dorsal root ganglion neurons. We examined the responses evoked by IW from recombinant homomeric and heteromeric kainate receptors expressed in human embryonic kidney 293 cells. IW potently elicited currents from glutamate receptor 5 (GluR5)-expressing cells, but showed no activity on homomeric GluR6 or GluR7 receptors. Co-expression of these receptor subunits with KA-2 subunits produced receptors that were weakly sensitive to IW. GluR5/KA-2 receptors had a higher EC50 value than homomeric GluR5 and exhibited a much faster recovery from desensitization. Finally, we found that the IW selectivity for GluR5 compared with GluR6 was determined by amino acid 721, which was previously shown to control alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate sensitivity of these kainate receptor subunits. The pharmacological selectivity and commercial availability of IW suggests that this compound may be of use in characterizing the molecular constituents of native kainate receptor responses.     

Cobalt accumulation in neurons expressing ionotropic excitatory amino acid receptors in young rat spinal cord: morphology and distribution

Excitatory amino acids (EAA) acting on N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiments suggest that NMDA receptors are localized mainly in lamina II while kainate and AMPA receptors are found on both dorsal and ventral horn neurons. However the cell types expressing EAA receptors and their laminar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressing AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusion of hemisected spinal cords of 14 day-old rat pups in vitro with excitatory amino acid receptor ligands in the presence of CoCl2. Cobalt has been shown to enter cells through ligand-gated ion channels in place of Ca2+. Cells which accumulated cobalt ions following activation by ionotropic excitatory amino acid receptors were visualized histochemically. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric acid (D-AP-5) (20 microM) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-selective excitatory amino acid receptor antagonist kynurenic acid (4 mM). Passive opening of the voltage-gated Ca(2+)-channels by KCl (50 mM) did not result in cobalt uptake, indicating that cobalt enters the cells through ligand-gated Ca(2+)-channels. AMPA (500 microM), kainate (500 microM), or NMDA (500 microM) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA superfusion, resulted in cobalt labeling of glial cells. Our results show that the cobalt uptake technique is a useful way to study the morphology and distribution of cells expressing receptors with ligand-gated Ca2+ channels.     

Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness

The present study determines the proportions of unmyelinated cutaneous axons at the dermal-epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal-epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Adelta and Abeta, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive.     

Enhancement of the N-methyl-D-aspartate response in spinal dorsal horn neurons by cAMP-dependent protein kinase

Glutamate-gated ion channels mediate excitatory synaptic transmission in the central nervous system and are involved in synaptic plasticity, neuronal development and excitotoxicity (5,24). These ionotropic glutamate receptors were classified according to their preferred agonists as AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), KA (kainate), and NMDA (N-methyl-D-aspartate) receptors [Trends Pharmacol. Sci., 11 (1990) 25-33]. The present study of NMDA receptor channels expressed in acutely isolated spinal dorsal horn (DH) neurons of young rat reveals that they are subject to modulation through the adenylate cyclase cascade. Whole-cell voltage-clamp recording mode was used to examine the effect of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) on the responses of DH neurons to NMDA. Whole-cell current response to NMDA was enhanced by 8 Br-cAMP, a membrane permeant analog of cAMP or by intracellular application of cAMP or catalytic subunit of PKA.     

Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors

Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[3H]AMPA binding experiments, high expression was seen (Bmax = 0.8-3.8 pmol/mg protein) along with high affinity binding to a single site (Kd, nM+/-S.D.): GluR1o, 32.5+/-2.7; GluR3o, 23.7+/-2.4; GluR1o + GluR3o, 18.1+/-2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues > L-glutamate > quinoxaline-2,3-diones > kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3o vs. GluR1o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3o and heteromeric receptors than at homomeric GluR1o, suggesting that its efficacy and/or desensitisation properties are different at GluR1o vs. GluR3o.     

5-(N-oxyaza)-7-substituted-1,4-dihydroquinoxaline-2,3-diones: novel, systemically active and broad spectrum antagonists for NMDA/glycine, AMPA, and kainate receptors

A group of 5-aza-7-substituted-1,4-dihydroquinoxaline-2,3-diones (QXs) and the corresponding 5-(N-oxyaza)-7-substituted QXs were prepared and evaluated as antagonists of ionotropic glutamate receptors. The in vitro potency of these QXs was determined by inhibition of [3H]-5,7-dichlorokynurenic acid ([3H]DCKA) binding to N-methyl-D-aspartate (NMDA)/glycine receptors, [3H]-(S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding to AMPA receptors, and [3H]kainate ([3H]KA) binding to KA receptors in rat brain membranes. 5-(N-Oxyaza)-QXs 12a-e all have low micromolar or submicromolar potency for NMDA/glycine receptors and low micromolar potencies for AMPA and KA receptors. QXs 12a-e display 2-12-fold selectivity for NMDA/glycine receptors compared to AMPA receptors, and approximately 2-fold difference between AMPA and KA potency. In contrast to other QXs that either show high selectivity for NMDA (such as ACEA 1021) or AMPA (such as NBQX) receptors, these molecules are broad spectrum antagonists of ionotropic glutamate receptors. 7-Nitro-5-(N-oxyaza)-QX (12e) is the most potent inhibitor among 12a-e, having IC50 values of 0.69, 1.3, and 2.4 microM at NMDA, AMPA, and KA receptors, respectively. In functional assays on glutamate receptors expressed in oocytes by rat cerebral cortex poly(A+) RNA, 7-chloro-5-(N-oxyaza)-QX (12a) and 7-nitro-5-(N-oxyaza)-QX (12e) have Kb values of 0.63 and 0.31 microM for NMDA/glycine receptors, and are 6- and 4-fold selective for NMDA over AMPA receptors, respectively. 5-(N-Oxyaza)-7-substituted-QXs 12a-e all have surprisingly high in vivo potency as anticonvulsants in a mouse maximal electroshock-induced seizure (MES) model. 7-Chloro-5-(N-oxyaza)-QX (12a), 7-bromo-5-(N-oxyaza)-QX (12b), and 7-methyl-5-(N-oxyaza)-QX (12c) have ED50 values of 0.82, 0.87, and 0.97 mg/kg i.v., respectively. The high in vivo potency of QXs 12a-e is particularly surprising given their low log P values (approximately -2.7). Separate studies indicate that QXs 12a and 12e are also active in vivo as neuroprotectants and also have antinociceptive activity in animal pain models. In terms of in vivo activity, these 5-(N-oxyaza)-7-substituted-QXs are among the most potent broad spectrum ionotropic glutamate antagonists reported.     

Pharmacological differentiation of kainate receptors on neonatal rat spinal motoneurones and dorsal roots

The objectives of this study, conducted on neonatal rat spinal cord and dorsal roots in vitro, were to characterise the actions of a range of willardiine analogues on GluR5-containing kainate receptors present in dorsal roots, to determine whether GluR5-containing receptors are also present on motoneurones, and to differentiate responses mediated by kainate receptors from those mediated by AMPA receptors on motoneurones. (S)-5-Trifluoromethyl-willardiine, (S)-5-iodowillardiine, (S)-5-iodo-6-azawillardiine and ATPA were found to be potent agonists of kainate receptors on dorsal roots (EC50 values 0.108 +/- 0.002, 0.127 +/- 0.010, 0.685 +/- 0.141 and 1.3 +/- 0.3 microM, respectively) being more potent but of lower efficacy than kainate (EC50 value 14.8 +/- 1.8 microM). (S)-5-Iodo-6-azawillardiine blocked kainate-induced depolarisations of the dorsal root, probably via its desensitising action. Kainate-induced responses of dorsal roots were weakly antagonised by (RS)-3,5-dicarboxyphenylglycine (DCPG) (apparent KD 1.5 +/- 0.4 mM). Kainate receptors containing GluR5 subunits do not appear to be present on motoneurones since (RS)-3,5-DCPG (1 mM) potentiated rather than antagonised kainate-induced depolarisations of motoneurones. Although (S)-5-iodowillardiine (a potent and selective agonist at GluR5-containing kainate receptors) depolarised motoneurones (EC50 value 5.8 +/- 0.6 microM), such depolarisations were antagonised by both (RS)-3,4- and (RS)-3,5-DCPG, which are selective AMPA receptor antagonists at motoneurones, showing a KD value of 73 microM (Schild slope, 0.96 +/- 0.09) and an apparent KD value of 123 +/- 38 microM, respectively. This accords with the previously reported activity of willardiine analogues at AMPA receptors. Since neither (RS)-3,4- nor (RS)-3,5-DCPG antagonised kainate-induced motoneuronal depolarisations but cyclothiazide enhanced and GYK153655 blocked these responses it is possible that a component of the kainate response may be mediated by a population of DCPG-insensitive AMPA receptors on motoneurones. However, it is also possible that a population of kainate receptors other than those containing GluR5 subunits, are responsible for these effects. The new compounds introduced in this study are likely to be useful tools for studying the physiological role of kainate receptors in CNS function.     

Determination of degradation rates of transfer and ribosomal ribonucleic acids in cultured rat hepatocytes by measuring N6-threoninocarbonyladenosine, dihydrouridine, and pseudouridine in medium using high-performance liquid chromatography

The cause of the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) remains unexplained. One potential pathogenetic mechanism is chronic toxicity due to disturbances of the glutamatergic neurotransmitter system, mediated via alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive glutamate receptors. Functional AMPA receptors consist of various combinations of four subunits (designated GluR1-4). The GluR2 subunit is functionally dominant and renders AMPA receptors impermeable to calcium. Most native AMPA receptors in the mammalian central nervous system (CNS) contain the GluR2 subunit and are calcium impermeable. We have investigated the composition of AMPA receptors expressed on normal human spinal motor neurons by in situ hybridization to determine their likely subunit stoichiometry. Highly significant levels of mRNA were detected for the GluR1, GluR3, and GluR4 subunits. However, GluR2 subunit mRNA was not detectable in this cell group. The absence of detectable GluR2 mRNA in normal human spinal motor neurons predicts that they express calcium-permeable AMPA receptors unlike most neuronal groups in the human CNS. Expression of atypical calcium-permeable AMPA receptors by human motor neurons provides a possible mechanism whereby disturbances of glutamate neurotransmission in ALS may selectively injure this cell group.     

Neurotoxin domoic acid produces cytotoxicity via kainate- and AMPA-sensitive receptors in cultured cortical neurones

Domoic acid, a naturally occurring kainoid, has been responsible for several outbreaks of fatal poisoning after shellfish ingestion, and we examined its neurotoxic mechanism in cultured murine cortical neurones. Using observations of neuronal viability and morphology, exposure to domoic acid for 24 h was found to induce substantial concentration-dependent neuronal cell death. Domoic acid-mediated neuronal death was attenuated by the non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-selective antagonist LY293558 ((3S,4aR,6R,8aR)-6-[2-(1H-tetrazol-5-yl)-ethyl]-1,2,3, 4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid), but unaffected by NS-102 (5-nitro-6,7,8,9-tetrahydrobenzo[g]indole-2, 3-dione-3-oxime)--a low-affinity kainate receptor antagonist. Domoic acid was equipotent with (S)-AMPA (EC50 values 3.8 and 3.4 microM respectively); however, (S)-AMPA induced only 50% cell death compared to > 80% cell death induced by domoic acid. Kainate also killed > 80% of cortical neurones; however, domoic acid was about 19 times more potent than kainate (EC50 75 microM). We show the potent neurotoxicity of domoic acid for the first time in a pure neuronal model and indicate that domoic acid acts via high-affinity AMPA- and kainate-sensitive glutamate receptors to produce excitotoxic cell death.