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Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR la and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both 125 I-MK-801 binding assays and electrophysiological measures. Binding of 125 I-MK-801 to cells transfected with NR 1a/2B is saturable with a K D of 440 pM. The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK-801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, 125 I-MK-801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1 a/2A. NMDA-induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits

Levels of mRNA for the major subunits of the GABAA receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. alpha 1, beta 1, beta 2, beta 3, and gamma 2s were found to be the predominant subunits in the anterior lobe, whereas alpha 2, alpha 3, beta 1, beta 3, gamma 2s, and gamma 1 were the predominant subunits expressed in the neurointermediate lobe. alpha 5, alpha 6, and delta subunits were not detectable. Hill and Scatchard analysis of [3H] muscimol binding to anterior and neurointermediate lobe membranes showed high-affinity binding sites with dissociation constants of 5.6 and 4.5 nM, respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central-type benzodiazepine antagonist [3H]Ro 15-1788 bound to a high-affinity site with a dissociation constant of 1.5 nM in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218,872 displacement of Ro 15-1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue-specific expression of particular GABAA receptor subunits and receptor heterogeneity within individual cells in the pituitary.

Neurons grown in culture die when they are exposed to high concentrations (0.1-1 mM) of the neurotransmitter L-glutamate. A similar phenomenon may occur in the mammalian brain during ischemia and other injuries that cause excessive glutamate release. Activation of N-methyl-D-aspartate (NMDA) receptors and the consequent Ca2+ influx are thought to play a critical role in the process of neuronal toxicity. Events subsequent to the Ca2+ influx are not well understood. We have discovered that nonneuronal kidney cells expressing NMDA receptors after DNA transfection undergo cell death unless they are protected by drugs that block the NMDA receptor ion channel. Furthermore, transfected cells expressing a mutated NMDA receptor that conducts less Ca2+ are less vulnerable to cell death. In addition, we find that even though several active forms of NMDA receptors can be synthesized in these cells after transfection with different cloned subunits, not all receptor types are equally toxic. These experiments suggest that Ca2+ influx through NMDA channels may be toxic to nonneuronal cells and that the NMDA receptor expression may be the major neuron-specific component of excitotoxicity.

Developmental changes in the levels of N-methyl-D-aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.

Muscle tension studies of guinea-pig ileum longitudinal muscle-myenteric plexus preparations suggest that the N-methyl-D-aspartate (NMDA) receptor may be present in the enteric nervous system. Therefore, we investigated the expression of a gene for the NMDA receptor in guinea-pig taenia coli. The gene product was amplified using polymerase chain reaction (PCR) and its synthesis localized using in situ hybridization. A NMDA receptor PCR product from the myenteric plexus was demonstrated with nearly identical sequence characteristics to that in the brain. In situ hybridization studies identified myenteric neurons which express NMDA receptor messenger RNA. Demonstration of the genetic expression of the NMDA receptor supports a role for glutamate as a neurotransmitter in the enteric nervous system.

Lead has long been recognized as a toxic material and its chronic effects on humans and its immediate effect on the environment have been extensively studied. Initially, lead poisoning of workers was the chief concern, but in the last 3 decades studies have been carried out to identify the effects of long‐term exposure to low levels of lead. Deposition to and accumulation in the environment have also been examined. Lead is derived from a number of urban point sources, but also comes in significant quantities from paint and motor car exhausts. This has ensured that lead is widely distributed in the urban scene, and it is persistent. Initially, elevated blood levels in chidren were usually considered to be due to ingestion of lead‐based paints. More recently, lead‐contaminated dust and soils have been recognized as potential sources. Any program that aims to determine environmental lead levels, or intends to identify potential sources of lead that may be absorbed by humans, must take account of res...

We compared gamma-aminobutyric acid (GABA)-activated currents and their modulation by benzodiazepines in cultured human cells transfected with complementary desoxyribonucleic acid (cDNA) encoding different GABAA receptor subunits. Flunitrazepam, a benzodiazepine agonist which potentiates GABA responses in both neurons and astrocytes was only effective in receptors containing the gamma 2 subunit (alpha 1 beta 1 gamma 2 and alpha 5 beta 1 gamma 2). The beta-carboline methyl-4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM) decreased GABA-activated currents in receptors composed of alpha 1 beta 1 gamma 1 and alpha 1 beta 1 gamma 2 subunits but increased GABA-activated currents in receptors containing the alpha 5 subunit (alpha 5 beta 1 gamma 1 and alpha 5 beta 1 gamma 2). These results strongly suggest that flunitrazepam and DMCM do not act on isosteric sites and that differences in the responsiveness of GABAA receptors to these compounds are based on different subunit compositions of GABAA receptors.

A cDNA encoding a protein with 70% amino acid identity to the previously characterized gamma-aminobutyric acidA (GABAA) receptor alpha-subunits was isolated from a rat brain cDNA library by homology screening. As observed for alpha 1-, alpha 2-, and alpha 3-subunits, coexpression of this new alpha-subunit (alpha 5) with a beta- and gamma 2-subunit in cultured cells produces receptors displaying high-affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing alpha 2-, alpha 3- or alpha 5-subunits have low affinities for several type I-selective compounds. However, alpha 5-subunit-containing receptors have lower affinities for zolpidem (30-fold) and Cl 218 872 (three-fold) than measured previously using recombinantly expressed type II receptors containing either alpha 2- or alpha 3-subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.

Previous studies in brain and recombinant NMDA receptors have observed heterogeneity in NMDA-sensitive glutamate binding site. We further characterized the glutamate site assembled from NRla, NR2A, and NR2B NMDA receptor subunits using L-[ 3 H]glutamate and [ 3 H]CGP 39653 binding assays. In contrast to earlier reports, we demonstrate a unique pharmacology for the NR2A subunit alone, which has high affinity for agonists but low affinity for competitive antagonists compared with heteromeric combinations of NR1a + NR2A and NR1a + NR2B. Similar to previous reports, we find unequal antagonist affinity between heteromeric combinations of NR1a + NR2A and NR1a + NR2B. However, unlike earlier reports, we describe two binding components within each heteromeric transfection that more closely resemble data obtained for binding to brain membranes. In addition, we show Mg 2+ can alter [ 3 H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [ 3 H]CGP 39653-labeled site between the two heteromeric combinations. Agonist inhibition of [ 3 H]CGP 39653 binding revealed differences between the heteromeric combinations as well as within each heteromeric combination, the latter of which more closely resembled results from brain. These results further determine components of the agonist and antagonist binding sites of the NMDA receptor as well as suggest additional possible mechanisms of heterogeneity of the glutamate site in the brain.

Benzodiazepines are widely prescribed anxiolytics and anticonvulsants which bind with high affinity to sites on the GABAA receptor/Cl- channel complex and potentiate the effect of the neurotransmitter GABA (gamma-aminobutyric acid). The heterogeneity of benzodiazepine recognition sites in the central nervous system was revealed by studies showing different classes of GABAA receptor subunits (classes alpha, beta and gamma) and variant subunits in these classes, particularly in the alpha-class. Expression of recombinant subunits produces functional receptors; when certain alpha-variants are coexpressed with beta- and gamma-subunits the resulting receptors have pharmacological properties characteristic of GABAA-benzodiazepine type I or type II receptors. The alpha-variants are differentially expressed in the central nervous system and can be photoaffinity-labelled with benzodiazepines. Here we report a novel alpha-subunit (alpha 6) of cerebellar granule cells. We show that recombinant receptors composed of alpha 6, beta 2 and gamma 2 subunits bind with high affinity to the GABA agonist [3H]muscimol and the benzodiazepine [3H]Ro15-4513 but not the other benzodiazepines or beta-carboniles. The same distinctive pharmacology is observed with GABAA receptors from rat cerebellum immunoprecipitated by an antiserum specific for the alpha 6 subunit. We conclude that this alpha-subunit is part of a cerebellar receptor subtype, selective for Ro15-4513, an antagonist of alcohol-induced motor incoordination and ataxia.

Pregnane steroids, particularly 3 alpha-hydroxylated metabolites of progesterone, are known to have rapid and profound effects on brain excitability. Recent evidence suggests that the gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor-Cl- ionophore complex may mediate these actions. The data further suggest that these steroids modulate the complex through a novel site independent of other known sites on the complex. The hypothesis that this site is on the GABA(A)-benzodiazepine receptor-Cl- ionophore complex is tested in the present study by determining its presence on transiently expressed GABAA-benzodiazepine receptors.

A complementary DNA (cDNA) encoding a serotonin receptor with 51% sequence identity to the 5HT-1C subtype was isolated from a rat brain cDNA library by homology screening. Transient expression of the cloned cDNA in mammalian cells was used to establish the pharmacological profile of the encoded receptor polypeptide. Membranes from transfected cells showed high-affinity binding of the serotonin antagonists spiperone, ketanserin and mianserin, low affinity for haloperidol (a dopamine D2 receptor antagonist), 8-OH-DPAT as well as MDL-72222 and no detectable binding of [3H]serotonin. This profile is consonant with the 5HT-2 subtype of serotonin receptors. In agreement with this assignment, serotonin increased the intracellular Ca2+ concentration and activated phosphoinositide hydrolysis in transfected mammalian cells. The agonist also elicited a current flow, blocked by spiperone, in Xenopus oocytes injected with in vitro synthesized RNA containing the cloned nucleotide sequences.

A nonrepetitive complementary RNA probe specific for an intervening sequence of the rat pro-opiomelanocortin (POMC) gene primary transcript was used to analyze the hormonal regulation of POMC gene transcription in individual cell nuclei in the rat pituitary by in situ hybridization. This probe recognized only full-length POMC heterogeneous nuclear RNA, as verified by Northern blots of pituitary RNA. When pituitary sections were hybridized with this 3H-labeled POMC intron A probe, silver grains were predominantly localized over the nuclei of cells that expressed POMC in the anterior and intermediate lobes. Adrenalectomy increased both the average grain density over corticotroph nuclei and the number of cells in the anterior pituitary with significant numbers of silver grains over their nucleus. Dexamethasone administration to intact or adrenalectomized rats results in the rapid (within 30 minutes) disappearance of silver grains over the nuclei of corticotrophs in the anterior lobe, suggesting that POMC gene transcription had been inhibited. However, adrenalectomy or dexamethasone administration did not alter the silver grain density over nuclei of intermediate lobe melanotrophs. Thus, this in situ hybridization assay utilizing an intervening sequence-specific POMC probe can measure rapid physiological changes in POMC heterogeneous nuclear RNA in individual cell nuclei.

A glandular-like kallikrein enzyme, a member of a well-characterized family of specific arginyl endopeptidases that may be involved in prohormone processing, has previously been shown to be present in the anterior and neurointermediate lobes of the rat pituitary. We isolated glandular-like kallikrein cDNAs from cDNA libraries prepared from these two tissues. By nucleotide sequence, restriction endonuclease, solution hybridization/nuclease protection, and blot analyses, we showed that, of the 8-10 rat kallikrein-encoding genes, it is the true glandular kallikrein mRNA that is expressed in both pituitary lobes. RNA blot-hybridization analysis of anterior and neurointermediate lobe pituitary RNA revealed a kallikrein mRNA of approximately equal to 900 base pairs. As analyzed by blot-hybridization and solution hybridization/nuclease protection analyses, the true glandular kallikrein mRNA was present at low levels: approximately equal to 0.05% of total mRNA in both male and female neurointermediate lobes. Similar low levels of the glandular kallikrein mRNA were found in the male anterior lobe, whereas the levels were 10- to 15-fold higher in the female anterior lobe. In vivo administration of a dopamine agonist (bromocryptine) or antagonist (haloperidol) caused a decrease or increase, respectively, in the amount of true glandular kallikrein mRNA in the neurointermediate lobe of both sexes that closely paralleled changes in proopiomelanocortin mRNA levels. Bromocryptine decreased and haloperidol increased true glandular kallikrein mRNA levels in the female anterior lobe but had no effect in the male anterior lobe. Nuclear transcription run-on studies showed that the changes in mRNA were due, at least in part, to parallel effects of haloperidol on kallikrein gene transcription. Thus, these studies have demonstrated that the pituitary expresses the glandular-type member of the kallikrein gene family and that dopaminergic compounds elicit changes in kallikrein mRNA, at least in part, by modulating transcription. In the intermediate lobe, regulation of true glandular kallikrein gene expression is parallel to that of proopiomelanocortin gene expression, suggesting that the enzyme may play a physiological role in the production and/or secretion of the proopiomelanocortin peptides in this tissue.

Neurotransmission effected by GABA (gamma-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates. The primary structures of GABAA receptor alpha- and beta-subunits have been deduced from cloned complementary DNAs. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed gamma 2, which shares approximately 40% sequence identity with alpha- and beta-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the gamma 2 subunit with alpha 1 and beta 1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.