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J A Gingrich

Publications and source records attributed to J A Gingrich.

15 recordsLinked to original sources
PubMed2001

Dissecting the role of the serotonin system in neuropsychiatric disorders using knockout mice.

RATIONALE: The serotonin system has an important role in the modulation of several processes relevant to psychiatry such as anxiety, affect, aggression, and drug abuse. This review summarizes the recent progress in elucidating the function of the serotonergic system using knockout mice. This review while not exhaustive, highlights recent findings of relevance to psychopharmacology. OBJECTIVES: To familiarize the reader with the technique and the findings from serotonergic knockout mice. METHODS: Information included in this review was drawn from our own experience in this field and relevant publications from other investigators. RESULTS: We have focused on three main themes that have emerged from studies with mice bearing single-gene mutations of serotonergic genes: anxiety, aggression, and drug abuse. Mice lacking the 5-HT1A have been found to be more anxious in several behavioral paradigms. Elevated levels of aggression have been reported in mice lacking the monoamine oxidase A and the 5-HT1B receptor genes. The mice lacking the 5-HT1B receptor have also been reported to exhibit an increased vulnerability to cocaine. The molecular basis of this enhanced vulnerability has been linked to compensatory changes in the nucleus accumbens. These results and their correlation with pharmacological studies will be discussed. CONCLUSION: Mice lacking key components of the serotonin system have provided us with important animal models of genetic vulnerability to conditions such as anxiety disorders, aggression, and drug abuse. Ongoing research with these mice may help elucidate the mechanistic functioning of this complex system.

Animals↗
PubMed2000

The broken mouse: the role of development, plasticity and environment in the interpretation of phenotypic changes in knockout mice.

With the advent of gene knockout technology has arisen the problem of how to interpret the resulting phenotypic changes in mice lacking specific genes. This problem is especially relevant when applied to behavioral phenotypes of knockout mice, which are difficult to interpret. Of particular interest are the roles of development and compensatory changes, as well as other factors, such as the influence of the gene knockout on nearby genes, the effect of the genetic background strain, maternal behavioral influences, and pleiotrophy.

Animals↗
PubMed1995

Developmental changes in the differential expression of two serotonin 5-HT3 receptor splice variants in the rat.

PCR was used to isolate identical partial cDNA clones encoding a serotonin 5-HT3 receptor subunit from rat nodose and superior cervical ganglia. The amino acid sequence predicted from these clones, extending from the putative transmembrane domain I to the stop codon, demonstrated a 93% homology with the 5-HT3 receptor A (R-A) subunit cloned from NCB 20 hybridoma mouse neuroblastoma/Chinese hamster embryonic brain cells. Comparison of the sequences of the rat gene and cDNA encoding this subunit revealed a five amino acid deletion, GSLLP, located within the putative second intracellular loop of the receptor subunit. This deletion was shown to occur at an intron/exon junction. Therefore, alternative splicing was probably responsible for the presence of short (5-HT3 R-As) and long (5-HT3 R-AL) forms of 5-HT3 R-A mRNA in these ganglia. PCR experiments, with specific primers located upstream and downstream of the GSLLP deletion, were used to detect reverse transcribed 5-HT3 R-A mRNAs. A short fragment (92 bp), corresponding to the deleted form, and a long fragment (107 bp), corresponding to the nondeleted form, were amplified from various regions of the CNS and peripheral ganglia of the rat, as well as from NG108-15 hybridoma cells. In the adult rat, the ratio of the two forms varied very little from one tissue to another, the long form corresponding to only approximately 10% of the total 5-HT3 R-A mRNA. Study of their respective distributions during ontogeny demonstrated a differential expression of the short and long forms in some tissues during late embryonic development, at embryonic day 17 (E17) or E20.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1993

Molecular cloning, stable expression and desensitization of the human dopamine D1b/D5 receptor.

The sub-family of dopamine D1-like receptors is now known to be comprised of at least two members: the originally cloned D1 receptor (herein referred to as the D1a receptor) and a related receptor referred to as the D1b, D1 beta or D5 dopamine receptor (herein referred to as the D1b/D5 receptor). Here, we characterize the D1b/D5 receptor expressed transiently in COS-7 cells and permanently in Ltk- cells. Transiently expressed human D1b/D5 receptors bind the D1 specific ligand [125I]SCH 23982 saturably and with high affinity (KD = 500 pM). Competition for [125I]SCH 23982 binding to rat D1b/D5 and human D1a and D1b/D5 receptors supports the contention that the two D1b/D5 receptors are species homologues. Furthermore, in COS-7 cells, as previously observed, dopamine competes for the binding of [125I]SCH 23982 to human D1b/D5 receptors with a higher affinity than that seen at the human D1a receptor. These results are similar to those seen in Ltk- cells permanently transfected with the human D1b/D5 receptor. In these cells, dopamine competition for [125I]SCH 23982 binding is complex, sensitive to guanine nucleotides and of a higher affinity than that observed for dopamine binding to the human D1a receptor expressed in these same cells. In both D1a and D1b/D5 expressing Ltk- cells, dopamine stimulates adenylyl cyclase with an EC50 of approximately 200 nM. Furthermore, preincubation of Ltk- cells expressing the D1a and D1b/D5 receptors with dopamine results in desensitization of the response of adenylyl cyclase to subsequent agonist stimulation.

Adenylyl Cyclases↗
PubMed1992

Identification, characterization, and molecular cloning of a novel transporter-like protein localized to the central nervous system.

During the course of large scale purification of the D1 dopamine receptor from rat brain, a protein of approximately 87,000 daltons (p87) was observed to copurify with the D1 receptor through four chromatographic steps. To characterize the nature of this protein, bovine and rat cDNA clones were isolated and sequenced. The bovine and rat clones were highly conserved (98.5% identity). Each clone possessed an open reading frame of 2226 base pairs encoding a protein of 742 amino acids (calculated MW of 82,500), containing three stretches of peptide sequence obtained from p87 sequence analysis. Comparison of the deduced peptide sequence of this protein with those found in available databanks revealed that it was a novel protein related to the family of nutrient transport proteins from eukaryotes and bacteria, including, the mammalian facilitated glucose transporters, the yeast transporters for maltose, lactose, and glucose, and the proton-driven bacterial transporters for arabinose, xylose, and citrate. In addition p87 also shares with these transporters a similar hydropathicity profile that suggests the presence of 12 transmembrane segments. The mRNA for p87 appears to be localized primarily, if not exclusively, to the central nervous system. Northern blot analysis reveals a message of approximately 4.8 kb in cortex, hippocampus, brain stem, and cerebellum, but no detectable signal in peripheral tissues such as spleen, liver, kidney, lung, heart, or skeletal muscle. Evidence form Western blot analysis and immunohistochemistry suggests that this protein may be expressed in intracellular organelles or the membrane of synaptosomes rather than plasma membrane. Based on its structure and properties, p87 appears to define a new class of transporter-like proteins.

Amino Acid Sequence↗
PubMed1992

Location and molecular cloning of D1 dopamine receptor.

Recently, our laboratory has purified the D1 dopamine receptor 6600 fold to near homogeneity from digitonin solubilized rat striatal membranes using sequential affinity, ion exchange, lectin, and size exclusion chromatographies. The resulting receptor preparations still retained ligand binding activity (-11,000 pmol [3H]SCH 23390 bound per mg/protein) and appeared as a single band at 70-80 kDa on SDS-PAGE. In order to learn more about the sequence and structure of this protein, we recently cloned the gene for a human CNS D1 dopamine receptor. This gene has an open reading frame of 1388 nucleotides and encoded for a protein with a deduced amino acid sequence of 446 residues. When expressed in mammalian cells the cloned D1 receptor had all the ligand binding properties expected for a D1 receptor (SCH 23390 > cis flupenthixol > raclopride and SKF 38393 > apomorphine > dopamine > quinpirole). The cloned D1 receptor was found to stimulate adenylyl cyclase but not phospholipase C. The message for this D1 dopamine receptor was found in caudate, putamen, frontal cortex, and hippocampus, but not in substantia nigra, heart, or kidney. These accomplishments now will allow the pursuit of biochemical studies of the receptor protein as well as investigations into structure/function relationship of the receptor using a molecular biological techniques.

Amino Acid Sequence↗
PubMed1991

Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with the D1A receptor.

Multiple D1 dopaminergic receptor subtypes have been postulated on the basis of pharmacological, biochemical, and genetic studies. We describe the isolation and characterization of a rat gene encoding a dopamine receptor that is structurally and functionally similar to the D1 dopamine receptor. The coding region, which is intronless, encodes a protein of 475 amino acids (Mr 52,834) with structural features that are consistent with receptors coupled to guanine nucleotide-binding regulatory proteins. The expressed protein binds dopaminergic ligands and mediates stimulation of adenylyl cyclase with pharmacological properties similar to those of the D1 dopamine receptor. The gene encoding the human homologue of this receptor subtype is located to the short arm of chromosome 4 (4p16.3), the same region as the Huntington disease gene. In striking contrast to the previously cloned D1 receptor, little or no mRNA for the receptor described here was observed in striatum, nucleus accumbens, olfactory tubercle, and frontal cortex. High levels of mRNA for this receptor were found in distinct layers of the hippocampus, the mammillary nuclei, and the anterior pretectal nuclei, brain regions that have been shown to exhibit little or no D1 dopamine receptor binding. On the basis of its properties we propose that this dopamine receptor subtype be called D1B.

Amino Acid Sequence↗
PubMed1991

Localization of D1 dopamine receptor mRNA in brain supports a role in cognitive, affective, and neuroendocrine aspects of dopaminergic neurotransmission.

Expression of a D1 dopamine receptor was examined in the rat brain by using a combination of in situ hybridization and in vitro receptor autoradiography. Cells expressing D1 receptor mRNA were localized to many, but not all, brain regions receiving dopaminergic innervation. The highest levels of hybridization were detected in the caudate-putamen, nucleus accumbens, and olfactory tubercle. Cells expressing D1 receptor mRNA were also detected throughout the cerebral cortex, limbic system, hypothalamus, and thalamus. D1 receptor mRNA was differentially expressed in distinct regions of the hippocampal formation. Dentate granule cells were labeled in dorsal but not ventral regions, whereas the subicular complex was prominently labeled in ventral but not dorsal regions. Intermediate to high levels of D1 binding sites, but no hybridizing D1 receptor mRNA, were detected in the substantia nigra pars reticulata, globus pallidus, entopeduncular nucleus, and subthalamic nucleus. In these brain regions, which are involved in the efferent flow of information from the basal ganglia, D1 receptors may be localized on afferent nerve terminals originating in other brain regions. These results indicate that in addition to a role in control of motor function, the D1 receptor may also participate in the cognitive, affective, and neuroendocrine effects of dopaminergic neurotransmission.

Amygdala↗
PubMed1991

Molecular characterization of G-protein coupled receptors: isolation and cloning of a D1 dopamine receptor.

This article summarizes the recent progress our laboratory has made in understanding the molecular characteristics of the D1 dopamine receptor. The D1 dopamine receptor from rat striatum has been purified to near homogeneity using a combination of several chromatographic steps. Furthermore, the gene for the human D1 dopamine receptor has been cloned, sequenced, and expressed. The cloned receptor has all the pharmacologic and biochemical properties of the classical D1 receptor coupled to adenylyl cyclase which has been previously described in the central nervous system.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and expression of the gene for a human D1 dopamine receptor.

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.

Amino Acid Sequence↗
PubMed1990

Dopamine receptor subtypes: beyond the D1/D2 classification.

The D1/D2 dopamine receptor classification is widely accepted. However, intense investigative efforts over the last several years using pharmacological, biochemical and behavioral approaches have produced results that are increasingly difficult to reconcile with the existence of only two dopamine receptor subtypes. Recent developments, including cloning of the cDNAs and/or genes for several members of the large family of G-protein-coupled receptors, have revealed that heterogeneity in the pharmacological or biochemical characteristics of individual receptors often indicates the presence of previously unsuspected molecular subtypes. In this article, Marc Caron and colleagues have assembled the main lines of evidence that suggest the presence of several novel subtypes for both D1 and D2 dopamine receptors and predict that molecular cloning will, in the near future, confirm their existence.

Animals↗
PubMed1990

Molecular characterization of dopamine receptors.

The D1 and D2 dopamine receptors have been biochemically characterized using specific probes based on the subtype selective antagonists SCH 23390 and spiperone, respectively. The D2 dopamine receptor was identified from several tissues by photoaffinity labeling and was purified from bovine anterior pituitary to homogeneity using a combination of affinity, lectin and hydroxylapatite chromatography. A complementary DNA (cDNA) encoding a rat brain D2 dopamine receptor has been cloned via low stringency hybridization using a portion of the beta 2-adrenergic receptor gene as a probe. Photoaffinity crosslinking and affinity chromatography have also been used to identify and purify the rat brain D1 dopamine receptor.

Affinity Labels↗
PubMed1988

Affinity chromatography of the D1 dopamine receptor from rat corpus striatum.

The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific. Adsorption of receptor activity could be blocked in a stereoselective fashion [SCH 23390 greater than SCH 23388; (+)-butaclamol greater than (-)-butaclamol]. The elution of [3H]SCH 23390 activity from the gel demonstrated similar stereoselectivity for antagonist ligands. Agonists eluted receptor activity with a rank order of potency consistent with that of a D1 receptor [apomorphine greater than dopamine greater than (-)-epinephrine much greater than LY 171555 greater than serotonin]. SCH 39111-Sepharose absorbed 75-85% of the soluble receptor activity, and after the gel was washed extensively, 35-55% of the absorbed receptor activity could be eluted with 100 microM (+)-butaclamol with specific activities ranging from 250 to 450 pmol/mg of protein. The affinity-purified receptor retains the ligand binding characteristics of a D1 dopamine receptor. This affinity chromatography procedure should prove valuable in the isolation and molecular characterization of the D1 dopamine receptor.

Animals↗