Transcriptional regulation of GABAA receptor gamma2 subunit gene.

We have cloned the promoter regions of the genes for the mouse and human gamma2 subunits of the type A receptors for gamma-aminobutyric acid (GABA). For the mouse, the two major transcription start sites were at +1 (by definition) and +43, as established by rapid amplification of cDNA ends (RACE) and primer extension. This numbering places the start methionine at +297. There was no TATA or CCAAT box. Both mouse and human sequences have a candidate neuron-restrictive silencer element (NRSE) site in the first intron (+956 in mouse). We made assorted mouse-based promoter/reporter (luciferase) constructs starting from a core extending from -331 to +136, varying sizes at both ends, and including and excluding the putative NRSE and more proximal sequences. These were tested by transient transfection in several neuron-like and non-neuronal cell lines. Both proximal and distal downstream elements appeared to help direct expression to neuron-like cells, the NRSE in the intron, by repression in non-neurons, and a 24-bp portion of the 5' untranslated region starting at +113 (named GPE1) by preferentially promoting expression in neuron-like cells. Cotransfected human NRSF (transcription factor for NRSE) reduced reporter expression in neuron-like cells for constructs containing the NRSE in two locations. In gel mobility shift assays, the mouse gamma2 NRSE and a consensus NRSE both bound in vitro translated NRSF very similarly, and the NRSF gave the same major shifted band with the mouse gamma2 NRSE as was observed with nuclear extracts.

Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the gamma 2L subunit.

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family.

Amino-acid sequences derived from complementary DNAs encoding the alpha- and beta-subunits of the GABA/benzodiazepine receptor from bovine brain show homology with other ligand-gated receptor subunits, suggesting that there is a super-family of ion-channel-containing receptors. Co-expression of the in vitro-generated alpha-subunit and beta-subunit RNAs in Xenopus oocytes produces a functional receptor and ion channel with the pharmacological properties characteristic of the GABAA receptor.

Limbic, hypothalamic, cortical and spinal regions are enriched in receptors for thyrotropin-releasing hormone: evidence from [3H]ultrofilm autoradiography and correlation with central effects of the tripeptide in rat brain.

Light microscopic autoradiographic localization of specific recognition sites for thyrotropin-releasing hormone (TRH) was determined on thin sections of rat brain using the potent analogue [3H](3-Me-His2)-TRH ([3H]MeTRH). Microdensitometric analysis of the relative optical densities of TRH receptor labelling revealed the following brain regional enrichment: lateral and cortical amygdaloid nuclei greater than ventral dentate gyrus greater than n. accumbens greater than medial septum greater than piriform cortex greater than paraventricular thalamic and hypothalamic nuclei greater than preoptic area greater than diagonal band of Broca greater than lateral septum greater than I-IV layers of frontoparietal cortex greater than dorsal hippocampus greater than olfactory tubercle greater than caudate putamen; globus pallidus. In the spinal cord the apparent relative enrichment of TRH receptors was: substantia gelatinosa = central canal gray greater than ventral gray greater than dorsal gray (layers III-VII) greater than white matter. This heterogeneous distribution of TRH binding sites correlated well with our previous data obtained from membrane binding studies. Furthermore, the specific anatomical localization of receptors for TRH in many nuclei was consistent with those loci involved in the mediation of many physiological and behavioural actions of the peptide in rodent brain.

Characterization and autoradiographic localization of TRH receptors in sections of rat brain.

Receptors for thyrotropin-releasing hormone (TRH) in rat brain have been localized autoradiographically by exposing tritium-sensitive film to sections labeled with [3H]3-Me-His2-TRH. Greatest grain density was found over certain nuclei of the amygdala, with considerable density over several other forebrain areas. Properties of TRH receptor binding in frozen sections closely resembled those of receptors in fresh membrane fragments.

Rat brain TRH receptors: kinetics, pharmacology, distribution and ionic effects.

Optimal conditions for measuring receptor binding for thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) have been determined using 3H-labelled [3-Me-His2]TRH [( 3H]MeTRH). Binding assays conducted at 0 degree C for 5-6 h using sodium phosphate- and/or Hepes-buffered tissue resuspensions, with subsequent filtration through Whatman GF/B filters, yielded the best results. Association and dissociation of [3H]MeTRH binding to amygdala membranes were time and temperature dependent. Dissociation kinetics appeared biphasic. Progressive reduction in receptor affinity and capacity and increased radioligand breakdown were observed at elevated temperatures. Bacitracin (25-1000 microM) prevented peptide degradation but inhibited receptor binding (8-37%). Detailed competition experiments using MeTRH and other drugs yielded a pharmacological profile similar to that observed previously in other tissues indicating TRH receptor identification. Highest density of TRH receptors was observed in the retina and numerous limbic areas. Monovalent and divalent cations modulated [3H]MeTRH binding by reducing apparent receptor number.

Biochemical similarity of rat pituitary and CNS TRH receptors.

Chemical properties of receptor binding sites for thyrotropin-releasing hormone (TRH) in rat pituitary, retina, amygdala and hypothalamus were compared by examining the influence of sulfhydryl reagents on specific binding of [3H](3-Me-His2)-TRH ([3H]MeTRH). Dithiothreitol-induced reduction of disulfide bonds, alkylation of thiol residues by N-ethylmaleimide and their oxidation by 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), all produced marked reduction of [3H]MeTRH binding, which could be prevented in part by preincubation with exogenous TRH. In all tissues, concentration-dependent loss of binding activity was observed following exposure to micromolar heavy metals and mono- and divalent cations, with apparent additive effects between cations and DTT and NEM. Most changes appeared to reflect only a decrease in receptor density (Bmax). The similar sensitivity of all tissues to these compounds complements existing evidence for a close resemblance of TRH receptors in the CNS and pituitary.

Muscarinic receptor binding in sheep anterior pituitary.

The existence of typical muscarinic receptors in membranes of sheep anterior pituitary was detected by binding of [3H]quinuclidinyl benzilate ([3H]QNB), a potent and specific muscarinic antagonist. [3H]QNB binding sites in anterior pituitary had an equilibrium dissociation constant of about 20-40 pM, a rate constant for association at 37 degrees C of about 2 x 10(8) M-1 min-1, a rate constant for dissociation at 37 degrees C of about 4 x 10(-3) min-1, and the expected specificities for a variety of cholinergic and other drugs. The concentration of [3H]QNB binding sites in anterior pituitary, about 4-6 pmol/g tissue, was at least twice that in posterior pituitary, but less than a quarter that in the hypothalamus. Since there is no established cholinergic or other innervation of the anterior pituitary, the presence there of typical muscarinic receptors, if we assume that they are functional, suggests that acetylcholine reaches the tissue through the hypophyseal portal circulation, and that it may thus have a role in the regulation of pituitary function.