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.

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.

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.

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.

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.

Differences between D- and L-aspartate binding to the Na+-dependent binding sites on glutamate transporters in frozen sections of rat brain.

The Na+-dependent, "high-affinity" transport of L-glutamate (GluT) in brain tissue has become a significant focus of interest, particularly since it has been revealed that abnormalities of GluT may be associated with serious neurological disorders. Using quantitative autoradiography on 3H-sensitive films, we have studied, in thaw-mounted sections of rat brain, the distribution and pharmacology of radioligand binding to sites with characteristics of the substrate-recognition/binding locus on GluT. The technique makes it possible to determine not only the intensity of binding in brain regions but, with a high level of precision, pharmacological constants such as IC50 or nH. [3H]L-aspartate and [3H]D-aspartate are two classical radioligands used in studies of GluT. We have determined IC50 values for the inhibition of [3H]L- and [3H]D-aspartate binding by their non-radioactive counterparts in the cerebral neocortex. hippocampus, striatum, septal nuclei and the cerebellar cortex. The two radioligands did not appreciably differ from each other in their interactions with the binding sites in the forebrain, consistent with all Na+-dependent GluT binding sites in that region having no stereoselectivity for aspartate enantiomers. In the cerebellar cortex, however, the data indicated the presence of a GluT binding site that preferred L- over D-aspartate. These findings contrast with many previous observations and suggest that the pharmacological characteristics of the ligand binding sites on GluT in the mammalian cerebellar cortex may have to be re-assessed and/or a possibility of an existence of (a) hitherto unknown molecule(s) with properties of a glutamate transporter be considered.

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.

Neurotoxicity of NAAG in vivo is sensitive to NMDA antagonists and mGluR II ligands.

N-Acetyl-aspartyl-glutamate (NAAG), an agonist at Group II metabotropic glutamate receptors (mGluR II), also activates the NMDA-type of ionotropic glutamate receptors and, at high micromolar concentrations, has previously been shown to induce neuronal cell death. In the present study we have morphologically quantified the neurotoxic action of intracerebroventricularly administered NAAG on the hippocampal formation and compared it to the action of the selective endogenous NMDA agonist quinolinic acid. Finally, we examined whether the action of NAAG can be modified by NMDA receptor antagonists and mGluR II ligands. NAAG-induced neurodegeneration was found to be less severe than that induced by quinolinate. It was prevented by inhibitors of NMDA receptors and also by an mGluR II agonist (DCG IV) but not by an mGluR II antagonist (EGlu).

(+)- and (-)-cis-2-aminomethylcyclopropanecarboxylic acids show opposite pharmacology at recombinant rho(1) and rho(2) GABA(C) receptors.

The effects of the enantiomers of (+/-)-CAMP and (+/-)-TAMP [(+/-)-cis- and (+/-)-trans-2-aminomethylcyclopropanecarboxylic acids, respectively], which are cyclopropane analogues of GABA, were tested on GABA(A) and GABA(C) receptors expressed in Xenopus laevis oocytes using two-electrode voltage clamp methods. (+)-CAMP was found to be a potent and full agonist at homooligomeric GABA(C) receptors (K:(D) approximately 40 microM: and I:(max) approximately 100% at rho(1); K:(D) approximately 17 microM: and I:(max) approximately 100% at rho(2)) but a very weak antagonist at alpha(1)beta(2)gamma(2L) GABA(A) receptors. In contrast, (-)-CAMP was a very weak antagonist at both alpha(1)beta(2)gamma(2L) GABA(A) receptors and homooligomeric GABA(C) receptors (IC(50) approximately 900 microM: at rho(1) and approximately 400 microM: at rho(2)). Furthermore, (+)-CAMP appears to be a superior agonist to the widely used GABA(C) receptor partial agonist cis-4-aminocrotonic acid (K:(D) approximately 74 microM: and I:(max) approximately 78% at rho(1); K:(D) approximately 70 microM: and I:(max) approximately 82% at rho(2)). (-)-TAMP was the most potent of the cyclopropane analogues on GABA(C) receptors (K:(D) approximately 9 microM: and I:(max) approximately 40% at rho(1); K:(D) approximately 3 microM: and I:(max) approximately 50-60% at rho(2)), but it was also a moderately potent GABA(A) receptor partial agonist (K:(D) approximately 50-60 microM: and I:(max) approximately 50% at alpha(1)beta(2)gamma(2L) GABA(A) receptors). (+)-TAMP was a less potent partial agonist at GABA(C) receptors (K:(D) approximately 60 microM: and I:(max) approximately 40% at rho(1); K:(D) approximately 30 microM: and I:(max) approximately 60% at rho(2)) and a weak partial agonist at alpha(1)beta(2)gamma(2L) GABA(A) receptors (K:(D) approximately 500 micro: and I:(max) approximately 50%). None of the isomers of (+/-)-CAMP and (+/-)-TAMP displayed any interaction with GABA transport at the concentrations tested. Molecular modeling based on the present results provided new insights into the chiral preferences for either agonism or antagonism at GABA(C) receptors.

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.

Determination of P2X1alpha-sarcoglycan (adhalin) expression levels in failing human dilated cardiomyopathic left ventricles.

This study is concerned with the molecular basis of human idiopathic dilated cardiomyopathy (DCM). This disorder affects the entire heart including both atria and ventricles. It is characterized by a progressive dilatation of the ventricles and loss of contractile power that results in an impaired cardiac output. Changes in cellular levels of dystrophin have been reported in patients with muscular dystrophies (Beckers and Duchenne) which manifest as DCM. However, previous studies using Western blots dos Remedios et al., Electrophoresis 1996, 17, 235-238) of samples of left ventricles from DCM patients showed no abnormalities in dystrophin content. P2X receptors are ATP-gated cation channels located in the sarcolemma. They are upregulated by a factor of about two in the atria of DCM patients compared with nondiseased control samples. A dystrophin-associated protein, alpha-sarcoglycan, has recently been shown to be an ecto-ATPase (an extracellular ATPase) capable of regulating ATP concentrations in the space between the cardiomyocytes. In this report we examine the relationship between changes in P2X1 receptors in left ventricle samples from DCM patients and the concentration of alpha-sarcoglycan. We found no evidence for upregulation of P2X1 receptors nor was the expression of alpha-sarcoglycan significantly altered.

Strategies for studies of neurotoxic mechanisms involving deficient transport of L-glutamate: antisense knockout in rat brain in vivo and changes in the neurotransmitter metabolism following inhibition of glutamate transport in guinea pig brain slices.

This communication briefly reviews characteristics of glutamate transport in the central nervous system and is involved in the aetiology of slow neurodegenerative diseases. Data in the literature suggest that antisense oligonucleotides targeted against glutamate transporters and administered in vivo over a period of days could be used to test the hypothesis. Data from our laboratory have indicated that single intraventricular doses of antisense oligonucleotides can also results in significant reductions in the numbers of substrate binding sites associated with glutamate transporters and may even cause subtle changes in their characteristics. In order to study metabolism in brain tissue, we have used 13C-nuclear magnetic resonance spectroscopy to analyse extracts of slices of guinea pig cerebral cortex exposed to glutamate transport inhibitor L-anti,endo-methanopyrrolidine dicarboxylate (L-a,e-MPDC). The results have shown-for the first time in an experimental model that preserves the relationship between glia and neurones within the context of brain tissue-that inhibition of L-glutamate transport can exert a significant influence on neurotransmitter-related metabolism. These findings suggest that metabolic disturbances caused by deficient glutamate transport could play a significant role in the death of neurones under pathological conditions in vivo.

Development of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder.

Postnatal development of the distribution of different isoforms of purinergic (P2X) receptors on smooth muscle cells in relation to the development of the innervation of the cells by nerve varicosities in the rat urinary bladder has been determined with immunofluorescence and confocal microscopy. Antibodies against the extracellular domains of the P2X(1) to P2X(6) receptors were used to detect the receptors in the bladder. Several other antibodies were used to identify sympathetic varicosities and Schwann cells. At one day postnatal (D1) there were few strings of varicosities denoting isolated axons, with most axons confined to large nerve trunks. Small size clusters of P2X(1) to P2X(6) receptor subtypes (about 0.4 microm diameter) were observed in the muscle which were independent of each other, and sometimes juxtaposed to the rare isolated varicosity strings. At D4 large numbers of strings of varicosities could be discerned throughout the detrusor. Most of these clouds of small P2X(1) to P2X(6) receptor clusters in their immediate vicinity. Some of these were colocalised with the varicosities, which were of parasympathetic origin as they failed to counter-stain with antibodies to tyrosine hydroxylase. Up to D14 there was a gradual coalescence of many of the isolated P2X(1-6) small receptor clusters so that they became colocalized, often at varicosities. Most of the varicosities in isolated strings possessed receptor clusters at this time. By D21 it was rare to find varicosity strings in the detrusor that were not either in close juxtaposition with P2X small receptor clusters or possessing such clusters in colocalization. However, large numbers of small P2X receptor clusters, many of which consisted of a mixture of isoforms, could be found spatially unrelated to nerve varicosities throughout the detrusor muscle. In the adult, single axons were either coextensive with one or more isoforms of P2X receptor clusters or these were immediately juxtaposed to the axons so that is was rare to find a varicosity that did not possess a receptor cluster. However, different combinations of colocalized P2X receptor isoforms could still be discerned in small clusters unrelated to varicosities. These observations are discussed in relation to the mechanism of formation of the receptor clusters and their migration beneath parasympathetic varicosities during development.

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.

Increase in expression of P2X1 receptors in the atria of patients suffering from dilated cardiomyopathy.

P2X1 receptors are ATP-sensitive ligand-gated cation-selective channels abundant in smooth muscle tissues such as bladder and vas deferens. They have also been detected in the central and peripheral nervous system and in heart tissue. We have earlier reported distinct changes in the expression of the PX1 subtype of P2X receptors in hearts of patients suffering from dilated cardiomyopathy (DCM). The study was, however, based on Western blots from only five DCM samples and three control hearts. Moreover, the antibody was directed against a peptide derived from the sequence of rat P2X1. In the present project we have examined larger groups of both DCM and control hearts (n = 14 and 11, respectively). Furthermore, the antibody used in this paper differs significantly from the one used in our previous report. The present antibody was raised against an 18-residue peptide sequence (Lys 68-84 Val) derived from the human P2X1 sequence. Most of the label in the Western blots was concentrated over a triplet of bands migrating with an apparent Mr of about 45,000. Quantitative densitometry indicated that this band was more strongly expressed (by approximately 80%) in DCM hearts compared with the controls.

Identification and localisation of ATP P2X receptors in rat midbrain.

P2X receptors that are gated by extracellular ATP are among the few known examples of ligand-gated cation-selective channels. There have been seven cloned proteins identified to date as members of the P2X receptor family in a wide range of tissues from the peripheral and central nervous systems and from many species. To determine the distribution of the P2X subtypes in the rat midbrain, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting was combined with immunolocalisation using confocal microscopy. Subtypes P2X1-6 were detected in the periaqueductal gray area and the ependymal layer bordering the ventricle with a widespread distribution.

Analysis of novel P2X subunit-specific antibodies in rat cardiac and smooth muscle.

P2X receptors are cation-selective channels gated by extracellular adenosine triphosphate (ATP). There are relatively few known types of ligand-gated receptors. In vertebrates they include acetylcholine (Ach), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA), glycine, and glutamate as well as ATP. Ach, 5-HT, GABA and glycine ligand-gated receptors are related in evolutionary terms, while glutamate and ATP receptors form separate groups. There have been seven cloned proteins identified to date as members of the P2X receptor family in a wide range of cells and species. We have carried out hydropathy investigations and sequence comparisons of each of the seven subunits in order to examine the putative transmembrane and cysteine-rich extracellular domains. Probable locations of disulphide bridges are consistent with there being two separate extracellular folding domains. Assessment of the putative surface-accessible regions was used to select small localised amino acid segments in nonglycosylated regions for raising antibodies against each of the P2X receptor subunits. To test the specificity of these novel P2X receptor antibodies and their presence in cardiac and smooth muscle, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting was undertaken in homogenised rat heart, bladder, kidney, and vas deferens.

P2X (purinergic) receptor distributions in rat blood vessels.

The distribution of purinergic (P2X1 and P2X2) receptors on smooth muscle cells in relation to autonomic nerve varicosities in rat blood vessels has been determined using immunofluorescence and confocal microscopy. P2X1 and P2X2 receptors were visualised using rabbit polyclonal antibodies against the extracellular domain of the receptors and varicosities visualised using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2X1 receptor clusters were observed on the smooth muscle cells of mesenteric, renal, and pulmonary arteries as well as in the aorta and in veins: a large approximately elliptical cluster 1.32+/-0.21 microm long and 0.96+/-0.10 microm in diameter; and a smaller spherical cluster with a diameter of 0.32+/-0.05 microm. The latter occurred throughout the media of arteries of all sizes, whereas the former were restricted to the adventitial surface of the media and to endothelial cells, except for the pulmonary artery, in which large receptor clusters were found throughout the media of the vessel. At the adventitial surface, the large clusters are in general located beneath SV2 labelled varicosities. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2X and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2X receptor clusters. P2X2 receptors were located on nerves and on endothelial cells. They were also found in low density on the smooth muscle cells in the media. These observations are discussed in relation to the mechanism of purinergic transmission to the smooth muscle cells of blood vessels.

Glutamate transport in cultures from developing avian cerebellum: presence of GLT-1 immunoreactivity in Purkinje neurons.

Immunocytochemical studies indicated that Purkinje cells cultured from chick embryonic cerebellum (embryonic day 8) strongly express a glutamate transporter EAAT2 cloned from human brain (GLT-1 in rat brain). At both 7 days and 14 days in culture, Purkinje neurons accumulated 1 microM [3H]L-glutamate via a potent "high-affinity" transport system that could be inhibited by D- and L-threo-3-hydroxyaspartate (D- and L-t-3OHA) and by L-trans-pyrrolidine-2,4-dicarboxylate (L-t-PDC). The order of potency of the three inhibitors was L-t-PDC approximately L-t-3OHA > D-t-30HA. Only the value of IC50 (concentration causing 50% inhibition) for D-t-3OHA significantly changed between 7 days (116 microM) and 14 days in culture (40 microM). All nH approximately 1, except in the case of the inhibition by D-t-3OHA at 14 days in culture (nH = 0.57), indicating the possible appearance of heterogeneity of the transport sites at later stages of culturing. Chronic inhibition of L-glutamate transport by L-t-PDC resulted in major changes in the morphology of Purkinje cells; particularly, the neurites almost completely regressed.

Ligand recognition sites on P2X receptors studied by quantitative autoradiography of [3H]alpha,beta-methylene-ATP binding in rat brain.

The specificity of alpha, beta-methylene-ATP for P2X receptor binding sites in the CNS has been examined by testing the effects of several ATP analogues and other ATP-related substances on the binding of 10 nM [3H]alpha,beta-methylene-ATP to 20 microns thick sections of fresh-frozen rat brain. The labelling of the putative P2X receptor binding sites by [3H]alpha,beta-methylene-ATP was evaluated by quantitative densitometry. [3H]alpha,beta-methylene-ATP binding was strongly inhibited by two close ATP analogues, 3'-O-(trinitrophenyl)-adenosine-5'-triphosphate and beta,gamma-imido-ATP (IC50 2.5 microM). beta,gamma-Methylene-ATP was, however, less potent (< 50% inhibition at 25 microM). Inosine-5'-triphosphate, guanosine-5'-triphosphate, uridine-5'-triphosphate, and cytidine-5'-triphosphate were practically inactive up to concentrations of 100 microM. Periodate oxidised ATP and 1, N6-etheno-ATP produced < 50% inhibition at 100 and 500 microM concentrations, respectively. Cations (K+, Rb+, Cs+, and Mg2+ at 5 mM and Na+ at 150 mM) reduced [3H]alpha,beta-methylene-ATP binding by no more than 50%. Several agents known to interact with Ca2+- and/or ATP-related cationic channels (Cd2+, glibenclamide, dantrolene, nifedipine, and thapsigargin) had no effect. We conclude that [3H]alpha,beta-methylene-ATP at low nanomolar concentrations binds to a site that has very strict structural requirements and is pharmacologically similar to ATP P2X receptors.