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T Branchek

Publications and source records attributed to T Branchek.

At least 19 recordsLinked to original sources
PubMed2006

The Trace Amine 1 receptor knockout mouse: an animal model with relevance to schizophrenia.

Trace amines have been implicated in a number of neuropsychiatric disorders including depression and schizophrenia. Although long known to modulate neurotransmission indirectly through the release of catecholamines, the identification of the Trace Amine 1 receptor (TA1) offers a mechanism by which trace amines can influence synaptic activity directly. TA1 binds and is activated by trace amines such as beta-phenylethylamine and tyramine. Our pharmacological characterization of mouse TA1 showed that, as in rat and primate, amphetamine is an agonist at this receptor but with surprisingly high potency. Without selective ligands for TA1 that do not also possess catecholamine-releasing properties, however, it has not been possible to study its physiological role in the central nervous system. To that end, a line of mice lacking the TA1 receptor was generated to characterize its contribution to the regulation of behavior. Compared with wild-type littermates, TA1 knockout (KO) mice displayed a deficit in prepulse inhibition. Knockout animals, in which the TA1-agonist influence of amphetamine was absent, showed enhanced sensitivity to the psychomotor-stimulating effect of this drug, which was temporally correlated with significantly larger increases in the release of both dopamine and norepinephrine in the dorsal striatum and associated with a 262% increase in the proportion of striatal high-affinity D2 receptors. TA1 therefore appears to play a modulatory role in catecholaminergic function and represents a potentially novel mechanism for the treatment of neuropsychiatric disorders. Furthermore, the TA1 KO mouse may provide a useful model for the development of treatments for some positive symptoms of schizophrenia.

Amphetamine↗
PubMed1999

Characterisation of 5-HT receptors in human coronary arteries by molecular and pharmacological techniques.

5-Hydroxytryptamine (5-HT) can produce both vasoconstrictor and vasorelaxant effects in human coronary arteries and the response to 5-HT can be influenced by the presence of disease. The aim of the present study was to elucidate the 5-HT receptor subtypes responsible for mediating 5-HT-evoked contraction of human coronary arteries using pharmacological, molecular and immunocytochemical approaches. Normal human coronary arteries, with intact endothelium, were mounted in tissue baths, and the vascular responses to 5-HT and 5-HT receptor agonists were studied. The effects of 5-HT1 and 5-HT2 receptor antagonists on these responses were also studied. Expression of messenger ribonucleic acid (mRNA) encoding different 5-HT receptors in human coronary arteries, atrium, ventricle wall and epicardium was determined using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis. The expression of 5-HT1B or 5-HT1D receptor protein was studied using subtype selective antibodies and standard immunocytochemical techniques. The rank order of 5-HT receptor agonist potency in causing vasoconstriction was 5-carboxamido tryptamine, (5-CT) > zolmitriptan = BW183C91 (N10-desmethyl zolmitriptan) = alpha-methyl-5-hydroxytryptamine (alpha-CH3-5-HT) = 5-HT = sumatriptan > 2-methyl-5-hydroxytryptamine (2-CH3-5-HT) = 8-hydroxy-DPAT (8-OH-DPAT). Alpha-CH3-5-HT, 5-CT, 5-HT, zolmitriptan and BW 183C91 were significantly more potent (approximately 3-fold) than sumatriptan and 2-CH3-5-HT, which in turn were more potent than 8-OH-DPAT. Ketanserin and methiothepin (5-HT2 and 5-HT1 receptor antagonists, respectively) caused parallel rightward shifts of the concentration-effect curves to alpha-CH3-5-HT or 5-CT, respectively, without changing the maximum contractile response. In human coronary arteries, atrium. ventricle and epicardium. RT-PCR products corresponding to the human 5-HT2A, 5-HT1B and 5-HT1F receptors were expressed in high levels, mRNAs coding for 5-HT7, 5-HT1A and 5-HT1D receptors were only weakly expressed. No 5-HT1F receptor mRNA was detected. In coronary arteries there was a differential expression of 5-HT1B versus 5-HT1D receptor mRNAs, with 5-HT1B mRNAs being found in greater abundance. Dense 5-HT1B-immunoreactivity was detected on smooth muscle layer within coronary artery, however, 5-HT1D-immunoreactivity was not detected. It is concluded that 5-HT-evoked contraction of human coronary arteries is most probably mediated via the activation of both 5-HT1B and 5-HT2A receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1998

Molecular biology and pharmacology of galanin receptors.

Galanin was first isolated 15 years ago. Diversity of galanin receptors has been suspected from the study of native tissues and functional responses to galanin and galanin-like peptides in vitro and in vivo. The recent application of molecular biologic techniques to clone galanin receptors has extended this diversity. So far, three galanin receptor subtypes, GALR1, GALR2, and GALR3, have been cloned from both human and rat. Their molecular structure, pharmacologic profiles, tissue distribution, and signal transduction properties have been partially elucidated.

Amino Acid Sequence↗
PubMed1996

[3H]5-hydroxytryptamine labels the agonist high affinity state of the cloned rat 5-HT4 receptor.

We have used the cloned rat 5-HT4 receptor, and determined that the single protein product produced is able to bind both [3H]5-HT and [3H]GR113808 ([1-[(2-methyl sulphonyl) amino] ethyl-4-piperidinyl] methyl-1-methyl-1H-indole-3-carboxylate) with high affinity. The affinities of agonists for the [3H]5-HT (agonist)-labelled receptor were significantly higher than for the [3H]GR113808 (antagonist)-labelled receptor. Furthermore, [3H]5-HT binding was reduced by addition of guanyl nucleotides. These results strongly support the hypothesis that the 5-HT4 receptor displays two interconvertible affinity states (high and low) for agonists, characteristic of many G protein coupled receptors. [3H]5-HT, at the concentration employed, therefore labels the agonist high affinity state of the 5-HT4 receptor in systems in which high densities of this receptor are found.

Affinity Labels↗
PubMed1993

Receptor reserve masks partial agonist activity of drugs in a cloned rat 5-hydroxytryptamine1B receptor expression system.

The present study was undertaken to evaluate the behavior of the rat 5-hydroxytryptamine (5-HT)1B receptor stably expressed in a Y-1 cell line, using both radioligand binding ([125I]iodocyanopindolol) and functional assays (inhibition of forskolin-stimulated cAMP release). The measured EC50 values for agonists were lower than expected from the measured Ki values (e.g., 5-HT, EC50 = 0.49 +/- 0.043, nine experiments; Ki = 5.3 +/- 0.82, four experiments). Furthermore, beta-adrenergic antagonists such as propranolol and pindolol, which have been reported to be partial agonists or antagonists at the 5-HT1B receptors in other systems, were found to be full agonists. To investigate the relationship between receptor occupancy and inhibition of cAMP release (and hence the degree of receptor reserve), we used the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). EEDQ treatment shifted the dose-response curve for 5-HT to the right by 6-7-fold, accompanied by a reduction (30-50%) in maximal response. Analysis of the data by the method of Furchgott revealed a very steep hyperbolic relationship between receptor occupancy and response for 5-HT, with 92 +/- 1.4% (three experiments) receptor reserve at the 50% maximal response level. In contrast to its effect on 5-HT, EEDQ treatment did not significantly shift the dose-response curves for pindolol; rather, only the maximal responses were reduced and a linear relationship was found between receptor occupancy and response for this compound. According to classical receptor theory these data indicate that pindolol is a partial agonist, relative to 5-HT, but because of the high density of 5-HT1B receptors in this system the difference between the intrinsic activities of the two drugs is masked. Therefore, one has to be cautious when interpreting functional data in transfected systems that often display large receptor reserve.

Adrenergic beta-Antagonists↗
PubMed1992

The role of conserved aspartate and serine residues in ligand binding and in function of the 5-HT1A receptor: a site-directed mutation study.

Wild-type and mutant serotonin 1A receptors were transiently expressed in COS-7 cells using the infection-transfection variant of the vaccinia virus/T7 polymerase vector system. The amino acid substitutions in the transmembrane regions, Asp82-->Asn82, Asp116-->Asn116, and Ser198-->Ala198 all resulted in a decrease in affinity for 5-HT by 60-100-fold, without affecting the affinity for the antagonist, pindolol. The binding of agonist to the additional mutant, Thr199-->Ala199, was too weak to be measured, 5-HT induced GTPase activities for all receptors studied. These findings indicate that the residues mutated play an important role in the binding of the agonist and less critical roles in the binding of the antagonist pindolol.

Aspartic Acid↗
PubMed1992

Expression in animal cells of the 5-HT1A receptor by a vaccinia virus vector system.

The co-infection or infection-transfection variants of the T7 RNA polymerase/vaccinia vector system were used to express 5-HT1ARs in COS-7, BSC-40 and GH3 cells, with co-infection giving ca. 3-fold higher level than infection-transfection. Binding affinities were similar to those of the endogenous 5-HT1AR, with highest affinities for 5-HT and 8-OH-DPAT. Functional properties were demonstrated by assays of agonist-stimulated GTPase activity and its inhibition by pertussin toxin. Immunoblot assays showed expression of the unglycosylated and glycosylated receptor protein in the membrane and, surprisingly, in the cytosolic fractions.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Cloning and expression of a glycine transporter reveal colocalization with NMDA receptors.

A complementary DNA clone encoding a transporter for glycine has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The transporter displays high affinity for glycine (KM approximately 100 microM) and is dependent on external Na+ and Cl-. Northern blot analysis indicates that the distribution of the mRNA encoding the glycine transporter is restricted to the nervous system. In situ hybridization data are consistent with roles for the transporter in both glycine neurotransmission and glycine modulation of N-methyl-D-aspartate (NMDA) receptors in the hippocampus. The identification of this transporter therefore opens the study of the molecular mechanisms underlying both inhibitory glycinergic transmission and NMDA-mediated excitatory transmission.

Amino Acid Sequence↗
PubMed1992

The rat 5-hydroxytryptamine1B receptor is the species homologue of the human 5-hydroxytryptamine1D beta receptor.

The relationship between the serotonin 5-hydroxytryptamine1B (5-HT1B) and 5-HT1D receptors has been the topic of much investigation and speculation since their complementary species distribution was first appreciated. The cloning of genes encoding 5-HT1D receptors has provided tools to investigate this relationship directly. In this study, a rat gene has been cloned that encodes the rat 5-HT1B receptor. Evaluation of the structure of this gene shows that it is a member of the guanine nucleotide-binding protein-coupled receptor superfamily. Comparison of the amino acid sequence of the rat gene with the human 5-HT1D beta gene showed a 93% overall identity and a 96% identity in the transmembrane regions. Comparison of the two sequences revealed zero to two amino acid changes in each of these transmembrane regions, as well as a striking conservation in the connecting loops, indicative of the relationship expected for species homologues of the same gene. The rat gene was expressed transiently in COS-7 cells, and membranes derived from these cells were shown to bind [125I]iodocyanopindolol. The pharmacological profile of this binding site closely matched that of the native rat 5-HT1B receptor (r = 0.95) but not the 5-HT1D receptor (r = 0.07). The cloned rat 5-HT1B receptor was found to couple to the inhibition of adenylate cyclase activity, as expected for a 5-HT1B receptor. These data indicate that, although the 5-HT1B and 5-HT1D receptors are pharmacologically distinct, they are species variants of the same receptor gene, the 5-HT1D beta gene.

Amino Acid Sequence↗
PubMed1990

[3H]-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and [3H] ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor.

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to [3H]ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding the serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both [3H]DOB and [3H]ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] to this system caused a rightward shift and steepening of agonist competition curves for [3H] ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity [3H]DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that [3H]DOB and [3H]ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein. These observations are consistent with the classical view of interconvertible agonist affinity states of GTP-binding protein-coupled receptors and strongly support the "two state" over the "two receptor" model for DOB binding to the 5-HT2 receptor.

DOM 2,5-Dimethoxy-4-Methylamphetamine↗
PubMed1985

The enteric neural receptor for 5-hydroxytryptamine.

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.

5-Hydroxytryptophan↗
PubMed1985

Specific antagonism of enteric neural serotonin receptors by dipeptides of 5-hydroxytryptophan: evidence that serotonin is a mediator of slow synaptic excitation in the myenteric plexus.

Research on the role of serotonin (5-hydroxytryptamine, 5-HT) in the function of the enteric nervous system has been impeded by the lack of specific inhibitors of the enteric neural actions of 5-HT. Saturable, reversible, high affinity enteric binding sites for 3H-5-HT have recently been characterized and radioautographically located. Affinity for the 3H-5-HT binding site requires an indole ring substituted with a free hydroxyl group. These 3H-5-HT binding sites have been proposed to be enteric neural 5-HT receptors. This hypothesis was tested in the current study by comparing the ability of compounds to inhibit the binding of 3H-5-HT with their electrophysiologically determined actions on myenteric neurons. 5-Methoxytryptamine did not inhibit the binding of 3H-5-HT to enteric membranes and neither mimicked nor antagonized the effects of 5-HT on the membrane potential of myenteric neurons. Two dipeptides of 5-hydroxytryptophan, N-acetyl- and N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP and N-hex-5-HTP-DP) inhibited the binding of 3H-5-HT (K1 = 0.25 microM for 5-HTP-DP and 1.19 microM for N-hex-5-HTP-DP). 5-HTP-DP applied by pressure microejection or superfusion (10 microM) antagonized the slow postsynaptic depolarization of myenteric neurons evoked by microejection of 5-HT. 5-HTP-DP also blocked the 5-HT-induced presynaptic reduction in amplitude of nicotinic fast synaptic potentials; however, 5-HTP-DP itself did not affect these responses. Moreover, 5-HTP-DP also failed to affect responses of myenteric neurons to microejected substance P, their muscarinic response to acetylcholine, or antidromic action potentials. In contrast, both dipeptides blocked the slow synaptic potentials seen in type II/AH neurons following stimulation of fiber tracts in interganglionic connectives. These data support the hypotheses that enteric 3H-5-HT binding sites are enteric neural 5-HT receptors, that dipeptides of 5-hydroxytryptophan are specific antagonists at these receptors, and that 5-HT is one of the mediators of slow synaptic potentials in the myenteric plexus.

5-Hydroxytryptophan↗
PubMed1984

Enteric receptors for 5-hydroxytryptamine.

[3H]5-hydroxytryptamine [( 3H]5-HT) was used as a radioligand to study enteric 5-HT receptors. Membranes were derived from preparations of longitudinal muscle with adherent myenteric plexus and of mucosa-submucosa dissected from guinea pig and rabbit small intestines. Filtration and radioautographic analyses were used. Specific [3H]5-HT binding was found in both preparations. Binding was saturable and dissociable with equilibrium dissociation constants (Kd) of 2.7 and 1.4 nM, respectively. A kinetic estimate of Kd (1.5 nM) was similar to that determined by saturation analysis and the Hill coefficient approximated unity. Ring-hydroxylation of indoles was found to be a requirement for antagonism of [3H]5-HT binding. On the other hand, substitutions could be made in the aliphatic side chain of tryptamines without destroying the affinity of analogues for the binding sites. The inability of antagonists to displace [3H]5-HT indicated that the binding sites were not muscarinic or nicotinic receptors, alpha- or beta-adrenoceptors, H1 or H2 histamine receptors, dopamine receptors or either the S1 or S2 types of 5-HT receptor that have been found in the brain. Frozen section dry-mount radioautography revealed the [3H]5-HT binding sites to be located in ganglia of the myenteric plexus and at the boundary between the mucosa and submucosa. The similarity between the structure-activity requirements for affinity at the [3H]5-HT binding sites and activation of neural or M receptors for 5-HT in the gut, as well as the characteristics and location of the binding sites suggests that they are enteric neural receptors for 5-HT.

Animals↗
PubMed1984

The development of photoreceptors in the zebrafish, Brachydanio rerio. I. Structure.

Morphological development of photoreceptors in the central retina of the zebrafish, Brachydanio rerio, was studied by using light and electron microscopic techniques. Outer segments (OS) first appeared at 2.5 days postfertilization (d2.5). On d3, synaptic elaborations were seen. By d8, two OS types were present and were identified as cones. The first indication of rod formation was also evidenced at this time, when vitreally positioned nuclei were observed and rodlike cells were infrequently detected in electron micrographs. At d12, the full complement of zebrafish photoreceptors, rods and four cone types, was identified. From this time on cells grew until adult dimensions were reached at d24. These structural observations are consistent with results of functional studies which utilized physiological and behavioral techniques.

Animals↗
PubMed1984

The development of photoreceptors in the zebrafish, brachydanio rerio. II. Function.

The functional development of rod and cone photoreceptors in the zebrafish (Brachydanio rerio) was studied by electroretinographically measuring flicker fusion frequencies. Two to 3 days after fertilization, fish gave little or no response to high intensity stimuli. Increases in sensitivity and flicker resolution were observed after this time. Biphasic response curves typical of adults could be elicited from fish as young as 2 weeks postfertilization, thus indicating a functional divergence of rods and cones. The results are consistent with behavioral and anatomical analyses of zebrafish photoreceptor development.

Animals↗