Miguel A. Chinchetru

Neurturin responsiveness requires a GPI-linked receptor and the Ret receptor tyrosine kinase

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor (GDNF). Both factors promote the survival of a variety of neurons, and GDNF is required for the development of the enteric nervous system and kidney. GDNF signals through a receptor complex consisting of the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)-linked receptor termed GDNFR-α. Here we report the cloning of a new GPI-linked receptor termed NTNR-α that is homologous with GDNFR-α and is widely expressed in the nervous system and other tissues. By using microinjection to introduce expression plasmids into neurons, we show that coexpression of NTNR-α with Ret confers a survival response to neurturin but not GDNF, and that coexpression of GDNFR-α with Ret confers a survival response to GDNF but not neurturin. Our findings indicate that GDNF and neurturin promote neuronal survival by signalling through similar multicomponent receptors that consist of a common receptor tyrosine kinase and a member of a GPI-linked family of receptors that determines ligand specificity.

Alternative Splicing of a 51-Nucleotide Exon that Encodes a Putative Protein Kinase C Phosphorylation Site Generates Two Forms of the Chicken γ-Aminobutyric AcidAReceptor β2 Subunit

Abstract: Complementary DNAs that encode two forms of the chicken ‐γ‐aminobutyric acid type A (GABAA) receptor β2 subunit have been isolated. These polypeptides differ by the presence (β2L) or absence (β2S) of 17 amino acids, which contain a possible target for phosphorylation by protein kinase C, in the large intracellular loop between the third and fourth membrane‐spanning domains. The extra sequence in the chicken β2L subunit is not found in previously published GABAA receptor β2‐subunit sequences. Analysis of genomic DNA has revealed that the two β2‐subunit mRNAs arise by alternative splicing of a novel 51‐nucleotide exon. Although the two β2‐subunit transcripts appear to be present in 1 ‐day‐old chick brain at similar steady‐state levels, we have been unable to detect an mRNA for the long form of the β2 subunit in either the bovine or the rat. Because the various GABAA receptor genes are thought to have arisen by duplication of a common ancestor, our data, taken together with that on the γ2 subunit, which occurs in two forms that arise by alternative splicing of a 24‐nucleotide exon, suggest that the coding region of the primordial gene or one of its very early descendants contained 10 exons, not nine as previously thought.

Molecular Cloning of α1d-Adrenergic Receptor and Tissue Distribution of Three α1-Adrenergic Receptor Subtypes in Mouse

Abstract: A partial cDNA encoding most of the third intracellular loop of the mouse α1d‐adrenergic receptor subtype was amplified from hippocampus by reverse transcription‐polymerase chain reaction (RT‐PCR) using degenerate oligodeoxynucleotide primers. This DNA fragment was used as a probe to isolate an α1d‐adrenergic receptor cDNA from a mouse brain cDNA library. The deduced amino acid sequence encodes a potential protein of 562 amino acids, and northern hybridization of poly(A)+ RNA isolated from mouse brain detected a single 3.0‐kb transcript. Partial cDNA fragments of the α1b‐ and α1a‐adrenergic receptor subtypes were also amplified from mouse brain and sequenced. Analysis of the mRNA expression by RT‐PCR indicated that the α1‐adrenergic receptors are widely distributed in mouse tissues. The α1d subtype is expressed in brain areas such as hippocampus, striatum, and brainstem and also in many extracerebral tissues, such as lung, liver, heart, kidney, and spleen. The α1a subtype is also expressed in many tissues, whereas the α1b subtype has a more restricted expression, with high levels in striatum, brainstem, and diencephalus.

The autoradiographic perspective of central benzodiazepine receptors: A short review

1. We reviewed studies performed to characterize central benzodiazepine binding sites. 2. An overview of the different radioligands used to characterize BZ1 and BZ2 binding sites and a mapping of these central benzodiazepine sites are described. 3. Saturation studies carried out by autoradiogram quantification also are reviewed. 4. The specific use of the autoradiographic technique to carry out studies on ontogeny, development, and phylogeny is discussed, as well as studies performed using this technique on some diseases and experimental conditions, such as drug treatments or chemical and mechanical lesions.

Effect of Chronic Ethanol Treatment on the γ-Aminobutyric Acid-Mediated Enhancement of [3H]Flunitrazepam Binding in Rat Cortex and Hippocampus

Abstract: The mechanism by which ethanol affects the γ‐aminobutyric acid (GABA)/benzodiazepine complex is not clear. It is known that ethanol enhances the CI− influx mediated by the GABAA receptor complex, and although chronic ethanol administration does not change the KD or Bmax for [3H]flunitrazepam binding, some reports have suggested that it could modify the modulation of benzodiazepine binding produced by GABA. In the present work, we studied the effect of chronic ethanol treatment on the modulation by GABA of [3H]flunitrazepam binding, using light microscopic autoradiography. This technique allows the measurement of densities of benzodiazepine receptors in different brain areas, the visual cortex and hippocampus, which appear to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. We found enhancement of benzodiazepine binding by GABA at concentrations of >10−6M for the various cortical and hippocampal areas studied from both control and ethanol‐treated animals; this enhancement peaked at 10−4M GABA but decreased at 10−3M GABA. We found a clear effect of ethanol treatment on the modulatory properties of GABAA receptor, in both cortex and hippocampus, although only in cortex were the differences statistically significant between control and ethanoltreated animals.

Effect of vitamin E treatment on N-methyl-D-aspartate receptor at different ages in the rat brain.

A comparative study using membrane homogenate binding, autoradiography, and Western blot assays was carried out to determine the age-related changes in N-methyl-D-aspartate (NMDA) receptors in 4-, 12- and 24-month-old male Wistar rats, treated or not with vitamin E. Vitamin E treatment was 20 mg/kg i.p. daily for 15 days. [(3)H] 5-methyl-10,11-dihydro-5H-dibenzo (a,d) cycloheptan-5,10-imine maleate (MK-801) binding was significantly increased in all areas studied (cortex and hippocampus) at all ages when rats received this treatment. A Western blot study in vitamin-E-treated rats and their controls did not reveal significant differences in the amounts of NR2A, an NMDA receptor subunit widely distributed in the brain mainly in cortex and hippocampus. We conclude that the effect of vitamin E on NMDA receptors is largely age independent. Previous reports and our data have described the presence of age-dependent NMDA receptor changes. The effect of vitamin E in aging is considered to be mediated by free radical scavenging, but from our data, we conclude that this mechanism is not relevant for age-dependent NMDA receptor changes. Our results also support that age or vitamin E treatment have no relevant effects on NR2A subunit, at least until 24 months in rats.

Effect of Ethanol Treatment on Rate and Equilibrium Constants for [3H]Muscimol Binding to Rat Brain Membranes: Alteration of Two Affinity States of the GABAA Receptor

Abstract: Equilibrium binding curves were biphasic in control and ethanol‐treated rats. [3H]Muscimol binds to sites of high (KDA of ∼10 nM) and low (KDB of ∼0.3–0.4 µM) affinity. Chronic ethanol treatment produced a decrease in BmaxA value, and the hyperbolic binding profiles were progressively affected by the chronic and in vitro ethanol treatments, with most of this effect corresponding to the high‐affinity site. IC50 and Ki values were calculated for several competing ligands, using membranes from both control and ethanol‐treated animals. The association and dissociation curves were also biphasic, using a radioligand concentration precluding a significant occupancy of the low‐affinity sites, which suggests the existence of two forms or affinity states of the monoliganded receptor. Chronic ethanol treatment did not produce changes in the values of the dissociation rate constants (fast and slow phases). By contrast, we report for the first time a decrease in the values of the association rate constants, with this decrease being higher for the slow phase. Consequently, the dissociation equilibrium constants are two times higher in chronically ethanol‐treated animals for both phases.

Differential expression of the α1C adrenergic receptor subtype in rat tissues

Three alpha 1 adrenergic receptor subtypes have been identified by molecular cloning techniques. However, the detection of the mRNA for the alpha 1c subtype has not yet been reported in rodent tissues. We have isolated from rat lung, by polymerase chain reaction techniques, a cDNA fragment corresponding to the third intracellular domain of the alpha 1c adrenergic receptor subtype. The nucleotide and deduced amino acid sequences of the cloned fragment presented an 87% and 96% identity, respectively, with those from the bovine receptor. Analysis of the distribution of the receptor mRNA in brain shows highest expression levels in cerebellum and striatum, whereas in non-neural tissues the receptor is mainly expressed in lung and heart. It was also found that the gene sequence which codes for this domain is not interrupted by introns.

Differential effect of chronic ethanol treatment on barbiturate and steroid modulation of muscimol-binding to rat brain cortex

We report the differential alterations produced by chronic ethanol treatment on the modulation, by the barbiturate thiopental and the steroid 5 beta-pregnan-3 alpha-ol-20-one, of the binding of [3H]muscimol to membrane preparations from rat brain cortex. We found a clear barbiturate- and steroid-promoted enhancement of muscimol-binding to membranes in both control and ethanol-treated animals. However, the enhancements were higher in control animals, using the barbiturate, and in ethanol-treated rats, using the steroid, Bmax and Kd values were also differentially affected in control and ethanol-treated animals by the presence of the barbiturate or the steroid.

Immunodetection of serotonin transporter from mouse brain

A DNA fragment encoding amino acid sequences from the amino terminal region of the mouse serotonin transporter was isolated and sequenced. This transporter is widely distributed throughout the mouse brain, as deduced by heminested reverse transcription-polymerase chain reaction (RT-PCR) assays. To identify the serotonin transporter protein, we have developed specific antibodies against a fusion protein containing its amino terminal region, a domain which shows a low degree of homology between the different neurotransmitter transporters. Western blot analysis of mouse brain membranes detected the serotonin transporter as a 71 kDa polypeptide.