PAX9 gene mutations and tooth agenesis: A review.

Paired box 9 (PAX9) is one of the best-known transcription factors involved in the development of human dentition. Mutations in PAX9 gene could, therefore, seriously influence the number, position and morphology of the teeth in an affected individual. To date, over 50 mutations in the gene have been reported as associated with various types of dental agenesis (congenitally missing teeth) and other inherited dental defects or variations. The most common consequence of PAX9 gene mutation is the autosomal-dominant isolated (non-syndromic) oligodontia or hypodontia. In the present review, we are summarizing all known PAX9 mutations as well as their nature and precise loci in the DNA sequence of the gene. Where necessary, we have revised the loci of the mutations in line with the reference sequence of the PAX9 gene as it appears in the current DNA databases.

The influence of interleukin-1beta on gamma-glutamyl transpepidase activity in rat hippocampus.

Brain infections as well as peripheral challenges to the immune system lead to an increased production of interleukin-1beta (IL-1beta), a cytokine involved in leukocyte-mediated breakdown of the blood-brain barrier. The effects of IL-1beta have been reported to depend on whether the route of administration is systemic or intracerebral. Using 50-day-old male rats, we compared the effects of IL-1beta on brain gamma-glutamyl transpeptidase (GGT; an enzymatic marker of brain capillary endothelium) at 2, 24 and 96 h after either an intravenous (i.v.) injection of 5 microg IL-1beta or an intracerebroventricular (i.c.v. - lateral ventricle) infusion of 50 ng IL-1beta. When the i.v. route was used, the GGT activity underwent small but significant changes; decreasing in the hippocampus 2 h after the i.v. injection, increasing 24 h later and returning to control levels at 96 h. No significant changes in the hippocampal GGT activity were observed at 2 and 24 h following the i.c.v. infusion. The GGT activity in the hypothalamus remained unchanged regardless of the route of IL-1beta administrations. Similar changes in GGT activity were revealed histochemically. The labeling was found mainly in the capillary bed, the changes being most evident in the hippocampal stratum radiatum and stratum lacunosum-moleculare. A transient increase in GGT activity at 24 h, together with a less sharp delineation of GGT-stained vessels, may reflect IL-1beta induced increased turnover of glutathione and/or oxidative stress, that may in turn, be related to altered permeability of the blood-brain barrier in some neurological and mental disorders, including schizophrenia.

A novel splice variant of the Excitatory Amino Acid Transporter 5: Cloning, immunolocalization and functional characterization of hEAAT5v in human retina.

Excitatory Amino Acid Transporter 5 (EAAT5) is abundantly expressed by retinal photoreceptors and bipolar cells, where it acts as a slow glutamate transporter and a glutamate-gated chloride channel. The chloride conductance is large enough for EAAT5 to serve as an "inhibitory" glutamate receptor. Our recent work in rodents has shown that EAAT5 is differentially spliced and exists in many variant forms. The chief aim of the present study was to examine whether EAAT5 is also alternately spliced in human retina and, if so, what significance this might have for retinal function in health and disease. Retinal tissues from human donor eyes were used in RT-PCR to amplify the entire coding region of EAAT5. Amplicons of differing sizes were sub-cloned and analysis of sequenced data revealed the identification of wild-type human EAAT5 (hEAAT5) and an abundant alternately spliced form, referred to as hEAAT5v, where the open reading frame is expanded by insertion of an additional exon. hEAAT5v encodes a protein of 619 amino acids and when expressed in COS7 cells, the protein functioned as a glutamate transporter. We raised antibodies that selectively recognized the hEAAT5v protein and have performed immunocytochemistry to demonstrate expression in photoreceptors in human retina. We noted that in retinas afflicted by dry aged-related macular degeneration (AMD), there was a loss of hEAAT5v from the lesioned area and from photoreceptors adjacent to the lesion. We conclude that hEAAT5v protein expression may be perturbed in peri-lesional areas of AMD-afflicted retinas that do not otherwise exhibit evidence of damage. The loss of hEAAT5v could, therefore, represent an early pathological change in the development of AMD and might be involved in its aetiology.

Na(+)-dependent high-affinity uptake of L-glutamate in cultured fibroblasts.

Uptake of 1 microM [3H]L-glutamate by cultured 3T3 fibroblasts was strongly dependent on extracellular Na+; it was reduced by elevated concentrations of K+ (60 mM) but it was not influenced by variations in the concentration of Ca2+ (0-9.6 mM). D- and L-Asparate, D- and L-threo-3-hydroxyaspartate DL-threo-3-methylaspartate and a few other glutamate derivatives and analogues inhibited the uptake but several close analogues of L-glutamate (including D-glutamate) had no effect, implying that the uptake system is highly structurally selective. The recently identified inhibitor of glutamate uptake in synaptosomal preparations, L-trans-pyrrolidine-2,4-dicarboxylate, was also among the inhibitors. Apparent Km of the uptake was found to be less than 10 microM. The present observations indicate that Na(+)-dependent 'high-affinity' uptake of L-glutamate may appear in structures which are apparently unrelated to glutamatergic synaptic transmission in the CNS.

Na(+)-dependent high affinity uptake of L-glutamate in primary cultures of human fibroblasts isolated from three different types of tissue.

Cultured human fibroblasts isolated from embryonic muscle, skin and peripheral nerve tissues were found to accumulate [3H]L-glutamate by a Na(+)-dependent uptake process strongly inhibited by several glutamate/aspartate analogues including D- and L-aspartate, D- and L-threo-3-hydroxyaspartate and L-trans-pyrrolidine-2,4-dicarboxylate but not D-glutamate. It was also reduced by elevated concentrations of K+, Rb+ and Cs+. The values of Km's were 5-20 microM, well within the 'high affinity' region. Variations in the capacity (Vmax) of [3H]L-glutamate uptake did not correlate with the origin (muscle, skin or nerve tissue) of the fibroblasts. The uptake characteristics suggest that it is mediated by a transport system similar to that commonly observed only in brain tissue.

Morphology and ultrastructure of rat hippocampal formation after i.c.v. administration of N-acetyl-L-aspartyl-L-glutamate.

N-Acetyl-L-aspartyl-L-glutamate (NAAG) is one of the most abundant neuroactive compounds in the mammalian CNS. Our recent observations have suggested that NAAG administered into rat cerebral ventricles can cause neuronal death by apparently excitotoxic mechanisms that can be antagonized by the N-methyl-D-aspartate-receptor blockers and by ligands of metabotropic glutamate receptor of Group II. Therefore, the principal aim of the present study has been to use quantitative morphology, electron microscopy and terminal deoxynucleotidyl transferase-mediated biotin dUTP nick-end labeling to study a dose- and time-dependence as well as regional distribution of neurodegeneration in hippocampi of rats after the intraventricular infusion of 0.25 micromol NAAG/ventricle and of equimolar doses of L-glutamate (L-GLU) and N-acetyl-L-aspartate (NAA), breakdown products of NAAG. The degenerative changes were observed after the infusion of 0.25 and 1.25 micromol of NAAG/ventricle, but not when a dose of 0.05 micromol of NAAG/ventricle was injected into each lateral cerebral ventricle. With a dose of 0.25 micromol of NAAG/ventricle the number of degenerated neurons reached a maximum on the fourth day after the infusion. The neuronal damage following bilateral administration of 0.25 micromol of NAAG/lateral cerebral ventricle exhibited features of a delayed neuronal degeneration, expressed mainly in the layer of dentate granule neurons. The degeneration was characterized on the basis of ultrastructural appearance and DNA-fragmentation. The morphological changes caused by L-glutamate and NAA were much smaller than those observed after the administration of NAAG and displayed a different pattern of regional distribution. The present findings suggest that NAAG can cause a loss of hippocampal neurons in vivo, apparently resulting from the neurotoxicity of NAAG itself.

Autoradiography of P2x ATP receptors in the rat brain.

1. Binding of a P2x receptor specific radioligand, [3H]-alpha,beta-methylene adenosine triphosphate ([3H]-alpha,beta-MeATP) to sections of rat brain was reversible and association/dissociation parameters indicated that it consisted of two saturable components. Non-specific binding was very low (< 7% at 10 nM ligand concentration). 2. The binding was completely inhibited by suramin (IC50 approximately 14-26 microM) but none of the ligands specific for P2y receptors such as 2-methylthio-adenosine triphosphate (2-methyl-S-ATP) and 2-chloro-adenosine triphosphate (2-C1-ATP) nor 2-methylthio-adenosine diphosphate (2-methyl-S-ADP) a ligand for the P2 receptor on blood platelets ('P2T' type) produced strong inhibitions except for P1,P4-di(adenosine-5')tetraphosphate (Ap4A). 3. Inhibitors of Na+,K(+)-dependent adenosine triphosphatase (ATPase) ouabain, P1-ligand adenosine and an inhibitor of transport of, respectively, adenosine and cyclic nucleotides, dilazep, had no effect. 4. The highest density of P2x binding sites was found to be in the cerebellar cortex but the binding sites were present in all major brain regions, especially in areas known to receive strong excitatory innervation.

Possible expression of functional glutamate transporters in the rat testis.

Neither expression nor functionality is clear in peripheral tissues with the molecular machineries required for excitatory neurotransmitter signaling by L-glutamate (Glu) in the central nervous system, while a recent study has shown that several Glu receptors are functionally expressed in the rat testis. This fact prompted us to explore the possible functional expression in the rat testis of the Glu transporters usually responsible for the regulation of extracellular Glu concentrations in the brain. RT-PCR revealed the expression, in the rat testis, of mRNA for five different subtypes of Glu transporters, in addition to that for particular subtypes of ionotropic and metabotropic Glu receptors. Glutamate transporter-1 (GLT-1) was different in the brain from that in the testis in terms of molecular sizes on Northern and Western blot analyses. In situ hybridization as well as immunohistochemical analysis showed localized expression of glutamate aspartate transporter at interstitial spaces and GLT-1 at elongated spermatids in the rat testis respectively. The expression of mRNA was localized for excitatory amino acid transporter-5 at the basal compartment of the seminiferous tubule in the rat testis. [(3)H]Glu was accumulated in testicular crude mitochondrial fractions in a temperature- and sodium-dependent saturable manner with pharmacological profiles similar to those shown in brain crude mitochondrial fractions. These results suggested that particular subtypes of central Glu transporters for the regulation of extracellular Glu concentrations in the rat testis could be constitutively and functionally expressed.

Effects of variations in ionic concentrations on high affinity uptake of l-glutamate in non-glutamatergic neurons and non-neuronal cells cultured from neonatal rat cortex.

High affinity uptake of l-glutamate in the CNS is known to be strongly dependent on Na(+). However, the stoichiometry and the mechanism of the relationship between Na(+) and l-glutamate at the putative transport site (i.e. whether one or two Na(+) are co-transported with one molecule of l-glutamate and in which order they bind to the site) have been the subject of debate because the results of quantitative studies on Na(+)-dependence of l-glutamate uptake have varied from one experimental model to another. This communication discusses ionic requirements of l-glutamate uptake in two culture systems derived from neonatal rat cortex-one containing no neurons and the other including, in addition to glial cells, GABAergic neurons. The present results are consistent with a model in which one molecule of l-glutamate is co-transported with either one or two Na(+) and no fixed order is required for the binding of Na(+) and l-glutamate to the transport site. Also, in contrast to other studies, l-glutamate uptake was found to be significantly reduced in the absence of Ca(2+) and was not affected by depolarizing concentrations of K(+).

Forty years of amino acid transmission in the brain.

This article is concerned with the discovery that amino acids, particularly L-glutamate and gamma-aminobutyrate (GABA), are central neurotransmitters. The crucial observations that lead to the conclusion that these two amino acids produce most of the synaptic excitation and inhibition in the central nervous system, were made in late 1950's. The combination of neurochemical knowledge and improved electrophysiological techniques was paramount in making these discoveries possible. In particular, the use of specific antagonists in microiontophoretic experiments provided the most decisive evidence. The relationship is also explored between these early findings and those of the present era characterised by extensive use of techniques of molecular biology and the development of drugs against targets identified 30 to 40 years ago.

Ontogeny of K(+)-stimulated release of [(3)H]GABA in rat cerebral cortex studied by a simple technique in vitro.

Ontogenetic development and Ca(2+)-dependence of the K(+)-stimulated release of [(3)H]?-aminobutyric acid (GABA) were studied by two different methods using tissue slices in vitro. The results indicate that, in the developing rat cortex, the K(+)-stimulated release of [(3)H]GABA is initially very low but it develops rapidly during the second and third postnatal weeks. This supports an earlier study which concluded that, during the cortical ontogeny, the ratio of stimulated: resting release of [(3)H]GABA increased at the fastest rate about 9-12 days after the birth, thus preceding the formation of GABAergic synapses by about 10 days. Furthermore, most of the early postnatal release observed in the present experiments is Ca(2+)-independent. An important Ca(2+)-dependent component of the release appears at later developmental stages and it also seems to develop faster than the GABAergic synapses. The present study suggests that the stimulus-coupled release of GABA in the rat cortex profoundly changes during the ontogeny, both quantitatively (the period of rapid development) and qualitatively (with respect to Ca(2+)-dependence). These observations, possibly reflecting changes in the association of GABA release with different structures (e.g. initially axonal growth cones, then neuronal dendrites and only at later stages GABAergic synapses) may be important in the evaluation of the putative role of GABA in synaptogenesis.

Glutamate decarboxylase solubilized from the rat cerebral cortex by two different concentrations of Triton X-100: effects of glutamate analogues and analysis by SDS-PAGE/western blotting using GAD6 and K2 antibodies.

Analysis of two preparations (containing 0.1% and 0.5% Triton X-100) of glutamate decarboxylase (GAD) by Western blotting using GAD6 and K2 antibodies specifically recognizing two GAD isoenzymes, GAD65 and GAD67, respectively, indicated that the higher concentration of Triton X-100 at best only moderately favoured solubilization of GAD67. Several glutamate analogues were found to be either equally potent or equally inactive as inhibitors of glutamate decarboxylase activities in the two preparations. Among typical ligands for glutamate receptors and transporters, only quinolinic and L-cysteine sulphinic acids were weak inhibitors of GAD. Kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA), 3-((RS)-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), L-threo-3-hydroxy-aspartate, L-trans-pyrrolidine-2,4-dicarboxylate, dihydrokainate, kynurenic acid and N-methyl-D-aspartate were inactive. Even though the activity of glutamate decarboxylase in homogenates of rat cerebral cortex is higher at 0.5% than at 0.1% Triton X-100, structural requirements of the enzyme active site appear to be independent of Triton X-100 concentration. Furthermore, since the less soluble component of the enzyme activity contains about the same ratio of GAD65 to GAD67 as the more soluble one, it does not seem that the fractionation with Triton X-100 can be easily used to separate the two isoenzymes from each other.

Effects of L-trans-pyrrolidine-2,4-dicarboxylate and L-threo-3-hydroxyaspartate on the binding of [3H]L-aspartate, [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoate (CGP 39653), [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainate studied by autoradiography in rat forebrain.

L-trans-Pyrrolidine-2,4-dicarboxylate (L-t-PDC) and L-threo-3- hydroxyaspartate (L-t-3OHA), compounds known to interact strongly with the Na(+)-dependent high affinity uptake of excitatory amino acids in central nervous tissue, were tested as potential inhibitors of binding to glutamate receptors and transport sites in frozen sections of rat brain. [3H] alpha-amino-3-hydroxy- 5-methyl-4-isoxazolepropionate (AMPA), [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and [3H] kainate were used as ligands for the binding sites on the "non-NMDA" classes of glutamate receptors and [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoate (CGP 39653) was used to label NMDA receptor binding sites. The Na(+)-dependent glutamate-uptake site was marked by [3H]L-aspartate. The autoradiograms, obtained by exposing 3H-sensitive film to sections of rat forebrain preincubated with 3H-labelled ligands, were scanned by laser beam and quantified. Distribution patterns of the receptor and transporter sites visualized by the 3H-labelled ligands were compatible with previously published results. [3H]CNQX binding, however, was found to be significantly decreased by Na+.L-t-3OHA was about an order of magnitude stronger than L-t-PDC as an inhibitor of [3H]L-aspartate binding. Neither of the compounds had any important effect at the "non-NMDA" receptor binding sites but L-t-3OHA was a weak inhibitor of [3H]CGP 39653 binding (< 40% at 100 microM). The results suggest that, at low nanomolar concentrations, both compounds are likely to be selective for Na(+)-dependent high affinity glutamate transporter sites. Moreover, L-t-3OHA seems to have a sufficiently high affinity for the site to be almost certainly useful, if available in a 3H-labelled form, as a ligand in autoradiographic studies.

Differences between substrate specificities of l-glutamate uptake by neurons and glia, studied in cell lines and primary cultures.

High affinity uptake of [(3)H]l-glutamate was studied in cultures of continuous cell lines, originating either from mouse neuroblastoma or rat glioma, and in two types of primary cultures containing cerebellar granule cells and astrocytes from cerebral cortex, respectively. In the continuous lines, d- and l-aspartate-4-hydroxamate were found to interact preferentially with the uptake of [(3)H]l-glutamate in glioma cells while l-glutamate-5-hydroxamate and 2-aminoadipate interacted more strongly with [(3)H]l-glutamate uptake in neuroblastoma cells, d-Aspartate-4-hydroxyamate, l-glutamate-5-hydroxamate and 2-aminoadipate were inactive as inhibitors of [(3)H]l-glutamate uptake by either granule cells or astrocytes, grown in primary culture, but several other glutamate analogues, which did not differentiate between neuroblastomal and gliomal uptake of [(3)H]l-glutamate, were somewhat stronger inhibitors of [(3)H]l-glutamate uptake in astrocytes as compared to that in granule cells. However, all of these compounds (N-acetyl-l-glutamate, formimino-l-aspartate, d-homocysteate, l-homocysteate and dl-2-methylglutamate) were only very weak inhibitors and, consequently, it is unlikely that any of them could be useful in experiments with central nervous tissue in vivo or, at least, in brain slices in vitro, attempting to resolve the uptake of l-glutamate into glia- and neuron-localized components.

High affinity uptake of cAMP in rat brain: Inhibition by coronary vasodilators dilazep and hexobendine.

High affinity uptake of cAMP by slices of rat cerebral cortex was found to be inhibited by specific coronary vasodilators dilazep and hexobendine, but it was insensitive to a wide variety of centrally-acting drugs. Experiments using subcellular fractionation of cortical homogenates indicated preferential association of cAMP uptake with fractions rich in synaptosomes. Furthermore, autoradiographical studies showed that cAMP uptake by cerebellar slices was particularly strong in the molecular layer which is known to have a relatively high density of synaptic terminals. The present results suggest that high affinity uptake of cAMP is located in the vicinity of synaptic contacts, probably in nerve terminals. The data on the effects of drugs, especially the inhibition by hexobendine and dilazep, offer a pharmacological means to investigate a possible role of cAMP uptake in synaptic transmission.

Quantitative autoradiography of Na+-dependent [3H]L-aspartate binding to L-glutamate transporters in rat brain: structure-activity studies using L-trans-pyrrolidine-2,4-dicarboxylate (L-t-PDC) and 2-(carboxycyclopropyl)-glycine (CCG).

Sodium-dependent binding of [3H]L-aspartate was studied in thaw-mounted horizontal sections of fresh-frozen (i.e. not fixed) rat brain. After the incubation with [3H]L-aspartate, the sections were exposed against a 3H-sensitive film and the resulting autoradiograms were evaluated by quantitative densitometry. Effects of several inhibitors were examined and their potency expressed as IC50 and nH. Together with previously published data, the present study supports the view that [3H]L-aspartate binding to fresh-frozen sections of rat brain represents interaction of the radioligand with the substrate-binding sites on glutamate transporters. The most potent inhibitors were (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and (2S,4R)-4-methylglutamate. In contrast, L-anti,endo-3,4-methanopyrrolidine dicarboxylate (L-a,e-MPDC) was about an order of magnitude less potent. Only subtle regional variations in the characteristics of inhibitors of [3H]L-aspartate binding were detected. It is not certain whether these differences reflect regional variations in the distribution of individual glutamate transporters or regional peculiarities in their pharmacological characteristics. In particular, (2S,4R)-4-methylglutamate, shown previously to differentiate between GLT-1 (principal glutamate transporter in the forebrain) and GLAST (expressed mainly in the cerebellum), did not strongly differentiate between the binding of [3H]L-aspartate in forebrain and cerebellum. Computer-assisted molecular modelling using selected glutamate analogues with restricted conformation (L-trans-pyrrolidine-2,4-dicarboxylate and four isomers of 2-(carboxycyclopropyl)-glycine: L- and D-CCG I, L-CCG III and L-CCG IV) identified at least one area of unfavourable steric interaction. We conclude that the quantitative autoradiographic studies using [3H]L-aspartate or other transporter-specific ligands, will be a useful tool to study the pharmacology of substrate binding sites on glutamate transporters in the mammalian brain in situ.

Type A and B gaba receptors mediate inhibition of acetylcholine release from cholinergic nerve terminals in the superior cervical ganglion of rat.

The effects of ?-amino-n-butyric acid (GABA), (+)bicuculline, isoguvacine and 3-(4-chlorophenyl)-4-aminobutyrate [(+/-)baclofen] on the K-induced release of [(3)H]acetylcholine (ACh) were studied in the superior cervical ganglia of the rat in vitro. GABA and isoguvacine inhibited [(3)H]ACh release and these inhibitions were reversible by (+)bicuculline. Furthermore, the release of [(3)H]ACh was also inhibited by (+/-)baclofen. In receptor-binding studies, binding of [(3)H]GABA to membrane preparations from the superior cervical ganglia was inhibited by both (+/-)baclofen and (+)bicuculline. It is concluded that the inhibitory effect of GABA on the release of ACh can be mediated by GABA(A)(bicuculline-sensitive) and by GABA(B) (baclofen-activated) receptors. Our findings are compatible with the existence of a non-synaptic GABAergic inhibitory system involving GABA(A) and GABA(B) receptors on cholinergic nerve terminals in the superior cervical ganglion of rat.

Regional distribution and pharmacological characteristics of [3H]N-acetyl-aspartyl-glutamate (NAAG) binding sites in rat brain.

Autoradiographical studies revealed that 10 nM [3H]N-acetyl-aspartyl-glutamate (NAAG) labelled grey matter structures, particularly in the hippocamus, cerebral neocortex, striatum, septal nuclei and the cerebellar cortex. The binding was inhibited by (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)-glycine (DCG IV), an agonist at group II metabotropic glutamate receptors (mGluR II). (RS)-alpha-Methyl-4-tetrazolylphenylglycine (MTPG), (RS)-alpha-cyclopropyl-4-phosphonoglycine (CPPG) and (RS)-alpha-methylserine-O-phosphate monophenyl ester (MSOPPE), all antagonists at mGluR II and mGluR III, also inhibited [3H]NAAG binding. Other inhibitors were (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD), a broad-spectrum mGluR agonist with preference for groups I and II and the mGluR I agonists/mGluR II antagonists (S)-3-carboxy-4-hydroxyphenylglycine (3,4-CHPG) and (S)-4-carboxy-3-hydroxyphenylglycine (4,3-CHPG). Neither the mGluR I specific agonist (S)-dihydroxyphenylglycine nor any of the ionotropic glutamate receptor ligands such as kainate, AMPA and MK-801 had strong effects (except for the competitive NMDA antagonist CGS 19755, which produced 20-40% inhibition at 100 microM) suggesting that, at low nM concentrations, [3H]NAAG binds predominantly to metabotropic glutamate receptors, particularly those of the mGluR II type. Several studies have indicated that NAAG can interact with mGluR II and the present study supports this notion by demonstrating that sites capable of binding NAAG at low concentrations and displaying pharmacological characteristics of mGluR II exist in the central nervous tissue. Furthermore, the results show that autoradiography of [3H]NAAG binding can be used to quantify the distribution of such sites in distinct brain regions and study their pharmacology at the same time.

Autoradiography of [3H]alpha,beta-methylene-ATP binding sites in medulla oblongata and spinal cord of the rat.

Excitatory purinoceptors of P2x-type have long been known to exist on smooth muscle cells, but recently it has been shown that they are also involved in synaptic transmission in the CNS. We have used a P2x-specific agonist, alpha,beta-methylene-ATP, as a 3H-labelled radioligand, to study the distribution and characteristics of P2x receptor-binding sites in the spinal cord and medulla oblongata. Using auto-radiographic techniques, [3H]alpha,beta-methylene-ATP binding was found throughout the grey matter of the spinal cord with small areas of above-average density of binding sites in the marginal zone and substantia gelatinosa at all spinal levels and in the central grey matter of the thoracic spinal cord. In the medulla, [3H]alpha,beta-methylene-ATP binding was found to be strong in all cranial nerve nuclei, particularly those known to receive primary sensory fibres. We have found that the binding of [3H]alpha,beta-methylene-ATP in the spinal cord and medulla was inhibited by a broad-spectrum P2-receptor antagonist suramin (IC50 approximately 27 microM). This is in accordance with the data obtained previously in the forebrain and cerebellum. There was, however, no inhibition of [3H]alpha,beta-methylene-ATP binding by another close analogue of alpha,beta-methylene-ATP and P2x ligand beta,gamma-methylene-ATP (10 microM). The latter result is discussed in terms of possible involvement of Ca2+ in the binding of [3H]alpha,beta-methylene-ATP to P2x receptors in the CNS.

Sensitivity of the binding sites on glutamate transporters to neurotoxic agents.

Autoradiography of [3H]L-aspartate binding to sections of rat brain was used to study the sensitivity of Na(+)-dependent glutamate transporters to neurotoxic agents such as Zn2+, NH4+, oxygen-containing free radicals and mercuric chloride. Only mercuric chloride was a strong inhibitor in cerebral neocortex, hippocampus, neostriatum, thalamus and cerebellar cortex. It is concluded that the substrate-binding sites on Na(+)-dependent glutamate transporters are relatively resistant to direct effects of Zn2+, NH4+ and free radicals but they may depend on the structural integrity of thiol bonds. Direct inhibitory effect of mercury on the binding site could significantly contribute to its long-term neurotoxicity.