Type I and type II GABAA-benzodiazepine receptors produced in transfected cells.

GABAA (gamma-aminobutyric acid A)-benzodiazepine receptors expressed in mammalian cells and assembled from one of three different alpha subunit variants (alpha 1, alpha 2, or alpha 3) in combination with a beta 1 and a gamma 2 subunit display the pharmacological properties of either type I or type II receptor subtypes. These receptors contain high-affinity binding sites for benzodiazepines. However, CL 218 872, 2-oxoquazepam, and methyl beta-carboline-3-carboxylate (beta-CCM) show a temperature-modulated selectivity for alpha 1 subunit-containing receptors. There were no significant differences in the binding of clonazepam, diazepam, Ro 15-1788, or dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) to all three recombinant receptors. Receptors containing the alpha 3 subunit show greater GABA potentiation of benzodiazepine binding than receptors containing the alpha 1 or alpha 2 subunit, indicating that there are subtypes within the type II class. Thus, diversity in benzodiazepine pharmacology is generated by heterogeneity of the alpha subunit of the GABAA receptor.

Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei.

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.

Transient expression shows ligand gating and allosteric potentiation of GABAA receptor subunits.

Human gamma-aminobutyric acid A (GABAA) receptor subunits were expressed transiently in cultured mammalian cells. This expression system allows the simultaneous characterization of ligand-gated ion channels by electrophysiology and by pharmacology. Thus, coexpression of the alpha and beta subunits of the GABAA receptor generated GABA-gated chloride channels and binding sites for GABAA receptor ligands. Channels consisting of only alpha or beta subunits could also be detected. These homomeric channels formed with reduced efficiencies compared to the heteromeric receptors. Both of these homomeric GABA-responsive channels were potentiated by barbiturate, indicating that sites for both ligand-gating and allosteric potentiation are present on receptors assembled from either subunit.

Functional chloride channels by mammalian cell expression of rat glycine receptor subunit.

Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.

Neurosteroids act on recombinant human GABAA receptors.

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.

Sequence and expression of human GABAA receptor alpha 1 and beta 1 subunits.

The deduced amino acid sequences of cDNA clones encoding human GABAA receptor alpha 1 and beta 1 subunits are presented. The human subunits display very high levels of sequence identity with the corresponding bovine receptor subunits. The cloned human GABAA receptor subunits induce the formation of GABA-gated chloride channels when expressed in mammalian cells.

SB-205384: a GABA(A) receptor modulator with novel mechanism of action that shows subunit selectivity.

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA(A) receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods. 2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA(A) currents in oocytes expressing human GABA(A) subunits. This temporal effect was observed for the alpha3beta2gamma2 subunit combination with little effect in subunit combinations containing either alpha1 or alpha2. 3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 microM drug was seen on the alpha3beta2gamma2 subunit combination. 4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA(A) currents in oocytes expressing the alpha3beta2gamma2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the gamma2 subunit compared to those containing the gamma1. 5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the alpha3beta2gamma2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.

The expression of a NMDA receptor gene in guinea-pig myenteric plexus.

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.

A steroid recognition site is functionally coupled to an expressed GABA(A)-benzodiazepine receptor.

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.

Differential benzodiazepine pharmacology of mammalian recombinant GABAA receptors.

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.

Dopamine regulates expression of the glandular-type kallikrein gene at the transcriptional level in the pituitary.

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.

Primary aortoduodenal fistula: successful diagnosis and treatment.

We have presented a case of aortoenteric fistula, which was successfully repaired. This diagnosis must be considered in gastrointestinal bleeding of uncertain cause, even if the typical risk factors are absent. If the correct diagnosis can be made promptly, surgical repair of the fistula is possible.

Gamma-aminobutyric acidA receptor alpha 5-subunit creates novel type II benzodiazepine receptor pharmacology.

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.

gamma-Aminobutyric acid type A receptor point mutation increases the affinity of compounds for the benzodiazepine site.

Recombinantly expressed gamma-aminobutyric acid type A (GABAA) receptors consisting of alpha 1, beta 2, and gamma 2 subunits contain a binding site for benzodiazepines that differs in its properties from that of alpha 3 beta 2 gamma 2 receptors. Amino acid substitutions between the GABAA receptor alpha subunits were analyzed for their effect on the binding of compounds to the benzodiazepine site. By converting ever smaller regions of the alpha 3 subunit sequence to that of the alpha 1 subunit, we show that a single substitution (glycine for glutamic acid) increases the affinity for several compounds approximately 10-fold without changing the affinity for nonselective compounds. Hence, the identified amino acids may interact directly with the ligand and define part of the benzodiazepine binding sites in these receptors.

High affinity agonist binding to cloned 5-hydroxytryptamine2 receptors is not sensitive to GTP analogs.

Agonists for GTP-binding protein (G protein)-coupled receptors are thought to bind with high affinity to the complex of receptor and G protein. Nonhydrolyzable GTP analogs have been shown to disrupt this complex and reduce the binding affinity for many agonists. Antagonists are thought to bind to the receptor whether or not it is coupled to the G protein, and therefore binding remains unchanged in the presence of GTP analogs. The binding of the serotonin 5-hydroxytryptamine (5-HT)2 receptor agonists serotonin (5-HT) and 4-bromo-2,5-dimethoxyphenylisopropylamine is not affected by the presence of GTP analogs when the cloned 5-HT2 receptor is expressed in the 293 human embryonic kidney cell line. The same receptor expressed in mouse NIH3T3 cells is partially sensitive to GTP analogs. Both cell lines have similar proportions of agonist and antagonist binding sites, and agonist stimulation of both cell lines leads to a robust increase in phosphoinositide hydrolysis. Differences in GTP metabolism in 293 cells is not likely to be the cause of the observed difference in inhibition of agonist binding, because the cloned 5-HT1A serotonin receptor expressed in these cells is sensitive to GTP analogs. The GTP-insensitive agonist binding is best explained by the existence of a G protein-receptor complex in 293 cells that is not sensitive to GTP analogs. Such a G protein-receptor complex may explain the fraction of agonist binding in the brain that is not sensitive to GTP analogs.

Levels of benzodiazepine receptor subtypes and GABAA receptor alpha-subunit mRNA do not correlate during development.

Developmental changes in the pharmacological properties of the GABAA receptor have been suggested to result from changes in the subunit composition of the receptor complex. The nicotinic acetylcholine receptor is structurally related to the GABAA receptor and undergoes a developmental subunit switch at the neuromuscular synapse. To examine the mechanistic similarities between these systems we sought to find whether the changes in GABAA receptor subunits are controlled by changes in messenger RNA levels, as they are for the nicotinic acetylcholine receptor. We found a 10-fold increase in the level of alpha 1-subunit mRNA, and a small increase in levels of GABAA/benzodiazepine receptors from day 1 to day 24 of rat cerebellar development. We also found that the levels of alpha 1-subunit mRNA were higher than the levels of mRNA encoding other alpha subunits at all developmental time points. The low levels of messenger RNA for alpha 2, alpha 3, and alpha 5 subunits are inconsistent with the high levels of type II benzodiazepine binding in the rat cerebellum at birth because these alpha subunits have been shown to form GABAA receptors with type II benzodiazepine binding. These findings are inconsistent with simple models that would explain the developmental differences in GABAA receptor pharmacology simply as a result of changes in alpha-subunit gene expression.

Developmental changes in human gamma-aminobutyric acidA receptor subunit composition.

gamma-Aminobutyric acid (GABA) is the neurotransmitter at most inhibitory synapses in the human central nervous system. The GABAA receptor, a ligand-gated ion channel, is the site of action of benzodiazepines, the most widely prescribed neuroactive drugs. It was recently demonstrated that there are multiple subtypes of GABAA receptors. Studies of rodents have shown that receptor subunits are developmentally controlled. The major alpha subunit of the adult receptor is expressed at low levels before birth. This study, using postmortem human tissue, shows that GABAA receptors are present in significant numbers in the human cerebellum at birth, and the numbers rise threefold by adulthood. Two subtypes of benzodiazepine receptors were detected by binding studies in the neonate, whereas only a single subtype of receptor was detected in the adult cerebellum. Comparison to recombinant human GABAA receptors shows that receptors containing alpha 1 constitute 50% of the receptors at birth and the percentage rises to over 95% by adulthood. In both cerebral cortex and cerebellum, a dramatic rise in alpha 1 messenger RNA was observed during development, suggesting that the complement of GABAA receptors differs in infants and adults. These findings have significant implications for normal neurodevelopment as well as for the understanding and treatment of pathophysiological states such as seizures.

Characterization of glutamate binding sites in receptors assembled from transfected NMDA receptor subunits.

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 NR1a, NR2A, and NR2B NMDA receptor subunits using L-[3H]glutamate and [3H]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 Mg2+ can alter [3H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [3H]CGP 39653-labeled site between the two heteromeric combinations. Agonist inhibition of [3H]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.

Measurement of intracellular calcium using bioluminescent aequorin expressed in human cells.

Changes in intracellular free calcium concentration ([Ca2+]i) are involved in many important physiological responses. Detecting changes in [Ca2+]i is crucial to understanding the physiologic roles of intracellular free calcium. We have characterized changes of [Ca2+]i in human cells transfected with apoaequorin cDNA. When reconstituted in vivo by incubating transfected cells with coelenterazine, aequorin emits light upon binding free calcium and acts as a bioluminescent indicator for calcium. We have used this system to determine the concentration response relationship of serotonin for its receptor. Cells cotransfected with serotonin receptor cDNA and apoaequorin cDNA emitted light upon treatment with serotonin. The light emission responses were saturable and serotonin concentration-dependent, and they were inhibited by serotonin antagonists. Human 293 cells that stably express apoaequorin have been created. This system should facilitate the investigation of [Ca2+]i involvement in physiological and pathophysiological responses.