Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Expression of the 5-hydroxytryptamine type 2C (5-HT 2C) receptor in NIH/3T3 fibroblasts results in agonist-independent 5-HT2C receptor activation. Some 5-HT2c receptor antagonists decrease this activation and are termed inverse agonists. The present study uses this system to evaluate functional and receptor binding properties of other 5-HT2C receptor antagonists. A number of inverse agonists, including clozapine, and a neutral antagonist (methysergide) were identified in a functional assay. Guanine nucleotides increased the affinity of a radiolabeled inverse agonist ([3H]mesulergine), suggesting that inverse agonists bind the G protein-uncoupled form of the 5-HT2C receptor with high affinity. Competition binding was performed using conditions that separately labeled the G protein-coupled and -uncoupled forms of the receptor. These studies demonstrated that inverse agonists bound the uncoupled form of the 5-HT2C receptor with higher affinity, compared with the G protein-coupled form. Agonists, on the other hand, had higher affinity for the coupled form whereas neutral antagonists had equal affinity for both forms of the receptor. Thus, 5-HT2C receptor neutral antagonists exhibited functional and receptor binding properties consistent with those of classical receptor antagonists. However, 5-HT2C receptor inverse agonists displayed functional and receptor binding properties that were opposite those of agonists.
Mol Pharmacol 1994 Nov
PMID:Reciprocal binding properties of 5-hydroxytryptamine type 2C receptor agonists and inverse agonists. 796 83

Tryptophan hydroxylase (TPH) is the first and presumably rate-limiting enzyme in serotonin (5-HT) biosynthesis. End-product inhibition of rate-limiting enzymes is common and 5-HT is known to inhibit TPH activity in vivo. However, it is not known whether levels of 5-HT could also be involved in the regulation of the TPH gene. In order to determine whether TPH gene regulation is dependent on the 5-HT concentration, 5-HT levels were reduced by the administration of parachlorophenylalanine (PCPA). PCPA is a potent, specific and irreversible inhibitor of TPH activity which drastically reduces 5-HT concentration in the 5-HT neurons and terminals. When PCPA was administered, TPH activity in both cell bodies and nerve terminal areas, was reduced to 10% of control values and recovered to the control levels by day 7 in raphe nucleus, and within 14 days in the hypothalamus. In serotonergic terminal areas, 5-HT could not be detected immunohistochemically at day 1, but slowly recovered within 2 weeks. At all time points examined, aromatic L-amino acid decarboxylase (AADC) levels were not changed either in the cell body or terminal areas. The steady state levels of TPH mRNA estimated by in situ hybridization increased at day 1 and returned to control levels by day 4. AADC message levels were not altered throughout the periods. These data suggest that a decrease in 5-HT concentration may lead to an up-regulation of TPH gene transcription, by an, as yet, unknown mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Brain Res Mol Brain Res 1994 Mar
PMID:Early induction of rat brain tryptophan hydroxylase (TPH) mRNA following parachlorophenylalanine (PCPA) treatment. 801 80

Previous studies from this laboratory have demonstrated that the 5-hydroxytryptamine (5-HT2) receptor subtype is transcriptionally regulated by 5-HT (serotonin) itself in rat myometrial smooth muscle cells. To better understand this transcriptional regulation, we have isolated and characterized the 5'-flanking region of the 5-HT2 receptor gene. Screening of a rat genomic library was accomplished using 5'-directed fragments of 5-HT2 cDNA, and a 5.2-kilobase fragment was isolated. Sequencing demonstrated that the fragment overlapped the 5'-end of the 5-HT2 cDNA by 226 base pairs. Primer extension and RNase protection analyses indicated that three transcriptional start sites, which are common to both rat brain and myometrium, appear to exist and that the 5'-untranslated region of the 5-HT2 receptor cDNA is 1120 base pairs long. Neither classical TATA boxes nor CCAAT sequences were found upstream of any of the start sites identified. Upstream of the dominant start site, however, an initiator consensus sequence, two GC boxes (SP-1 binding sites), and several AP-2 binding sites were identified. Based on this information, a 1.4-kilobase fragment beginning 64 base pairs downstream from the dominant start site was constructed by polymerase chain reaction and ligated into a pCAT vector. Transient transfection of this construct into rat myometrial smooth muscle cells displayed both constitutive and serotonin-induced promoter activity. Serotonin-inducible activity was abolished by a selective 5-HT2 receptor antagonist; however, antagonists selective for other 5-HT receptor subtypes were without effect. Conversely, a selective 5-HT2 receptor agonist completely substituted for serotonin as an inducer. Preliminary deletion experiments indicate that regulation of basal and serotonin-inducible activity likely depends upon different cis elements in the 5-HT2 receptor gene promoter.
Mol Pharmacol 1994 Jun
PMID:Isolation and characterization of the rat 5-hydroxytryptamine type 2 receptor promoter: constitutive and inducible activity in myometrial smooth muscle cells. 802 6

Quantitative receptor autoradiography was used to examine the 5-hydroxytryptamine (5-HT, serotonin) binding sites labelled with serotonin-5-O-carboxymethyl-glycyl-[125I]tyrosinamide ([125I]GTI) in human and guinea-pig brain. Competition experiments using 5-carboxamidotryptamine (5-CT), 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93129) and sumatriptan revealed monophasic displacement curves in various brain regions, suggesting that a homogeneous population of 5-HT1D binding sites was labelled. Displacement of [3H]5-HT (in the presence of 100 nM 8-hydroxy-2(N-dipropylamino)tetralin (8-OH-DPAT) and 100 nM mesulergine) with unlabelled GTI resulted in monophasic competition curves in substantia nigra, globus pallidus and central gray. In contrast, biphasic displacement was observed in hippocampus, nucleus accumbens, claustrum, caudate-putamen and frontal cortex. The distribution of [125I]GTI sites was compared to that of [3H]5-HT binding sites (under so-called '5-HT1D conditions', i.e. in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine, in order to block 5-HT1A and 5-HT1C sites, respectively) in human and guinea-pig brain. Qualitative analysis revealed differences in the distributions of [125I]GTI and [3H]5-HT binding sites. Regions such as CA3 and CA4 of the hippocampus, claustrum and putamen showed [3H]5-HT binding (under '5-HT1D conditions') but no [125I]GTI binding sites, indicating that [3H]5-HT labels besides a GTI sensitive (5-HT1D) receptor population, a non-5-HT1A/1B/1C/1D [3H]5-HT binding site in human and guinea-pig brain. The distribution of these non-5-HT1A/1B/1C/1D [3H]5-HT binding sites was studied with [3H]5-HT under conditions where 5-HT1A, 5-HT1C and 5-HT1D [3H]5-HT binding sites were saturated by the presence of 100 nM 8-OH-DPAT, 100 nM mesulergine and 1 microM GTI. Significant densities of these non-5-HT1A/1B/1C/1D sites were observed in cortical areas, hippocampal structures, nucleus accumbens, amygdala, caudate-putamen and claustrum. It is concluded that [125I]GTI does not label the 5-HT1E binding site, since all competition curves obtained with this radioligand were monophasic. By contrast, [3H]5-HT labels non-5-HT1A/1B/1C/1D [3H]5-HT binding sites, but it remains to be established whether these sites represent a single receptor population.
Brain Res Mol Brain Res 1994 Jan
PMID:A comparative autoradiographic study of 5-HT1D binding sites in human and guinea-pig brain using different radioligands. 816 19

The goal of the present paper was to investigate 5-hydroxytryptamine (5HT)2A and 5HT2C receptor regulation of ion transport in fibroblast cell lines transfected with these receptors. Na+/K(+)-ATPase and Na+/K+/2Cl- cotransport were measured with 86Rb+ as a surrogate for K+ uptake. Serotonin agonists had no effect on Na+/K(+)-ATPase activity in either cell line. Bumetanide, an antagonist of Na+/K+/2Cl- cotransport, almost completely blocked ouabain-insensitive K+ uptake in both cell lines, with an IC50 of about 1 microM. 36Cl- uptake was 2-fold greater than 86Rb+ uptake, consistent with the expected 2:1 stoichiometry. In addition, the Cl-/HCO3- uptake blocker 4,4'-diisothiostilbene-2,2'-disulfonic acid had no effect on Cl- uptake. The 5HT2A/2C receptor agonist (-)-2,5-dimethoxy-4-bromoamphetamine increased ouabain-insensitive K+ uptake, and this effect was blocked by bumetanide. The receptor antagonists mianserin and mesulergine, but not spiperone, blocked (-)-2,5-dimethoxy-4-bromoamphetamine responses in fibroblasts transfected with 5HT2C receptors, and all three antagonists blocked the effects in cells expressing 5HT2A receptors. Ouabain-insensitive 22Na+ uptake was similarly affected. 5HT receptor-related actions were not observed in untransfected parent NIH/3T3 fibroblasts. Thus, we have demonstrated that 5HT2C and 5HT2A receptors are linked to activation of Na+/K+/2Cl- cotransport in transfected fibroblasts. This activity may be a factor in the pharmacological actions of 5HT agonists and antagonists.
Mol Pharmacol 1994 May
PMID:5-Hydroxytryptamine type 2A and 2C receptors linked to Na+/K+/Cl- cotransport. 819 Jan 14

5-Hydroxytryptamine (5HT)1C and 5HT2 receptors display paradoxical down-regulation when exposed to receptor antagonists in vivo, a property that is unique to these two subtypes of serotonin (5HT) receptors. Because of the absence of cell culture model systems, the mechanisms involved in this paradoxical down-regulation have been difficult to explore. The present study focuses on the regulation of 5HT1C receptors in primary cultures of rat choroid plexus epithelial cells. Exposure of the epithelial cell cultures to 100 nM mianserin, a receptor antagonist, or (-)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane, an agonist, for 72 hr caused a loss of 5HT1C receptor binding sites, as determined by [3H]mesulergine binding to crude membrane preparations. No significant changes in Kd values were observed. Neither the agonist nor antagonist caused a significant change in binding sites after 24 hr. A solution hybridization assay was used to determine whether the down-regulation by mianserin or (-)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane was accompanied by a decrease in the steady state level of 5HT1C receptor mRNA. These studies showed that neither treatment caused an alteration in the levels of 5HT1C receptor mRNA. Thus, it is possible to reproduce the in vivo regulatory effects of drugs on 5HT1C receptors in choroid plexus epithelial cells in culture, including the atypical down-regulation by receptor antagonists. Using this cell culture model system, indirect transynaptic effects and decreases in receptor mRNA levels have been ruled out as mechanisms accounting for the down-regulation.
Mol Pharmacol 1993 Oct
PMID:5-hydroxytryptamine1C receptor density and mRNA levels in choroid plexus epithelial cells after treatment with mianserin and (-)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane. 823 22

Serotonin (5-hydroxytryptamine or 5-HT) is an important biogenic amine that functions as both a neurotransmitter and a hormone in the central nervous system (CNS) and the periphery. We report here the isolation of a cDNA from the OK cell that encodes a serotonin receptor (OKc1). When expressed in cultured cells, it displayed the pharmacological profile and negative coupling with adenylyl cyclase characteristic of a 5-HT1B receptor subtype. Similar to the cloned rodent 5-HT1B receptors, it had high affinity for the beta-adrenergic ligand [125I]iodocyanopindolol, because of the presence of an asparagine instead of a threonine residue in the seventh transmembrane region. The ligands used displayed the following rank order of potencies: cyanopindolol > RU24969 > methiothepin > serotonin > sumatriptan > methysergide > 8-OH-DPAT > isoproterenol. This profile correlates well (r = 0.97) with the native OK cell 5-HT1B receptor. When OKc1 is compared to the rat, mouse, and human 5-HT1B receptors, it has an amino acid sequence identity of 82%, but it is only 54% identical to the human 5-HT1D receptor.
Mol Pharmacol 1994 Jan
PMID:The cloning and expression of an OK cell cDNA encoding a 5-hydroxytryptamine1B receptor. 830 76

Serotonin 5-hydroxytryptamine (5-HT)2 receptors are implicated in the etiology of mental disease and depression. Drugs that interact with the 5-HT2 receptor are used therapeutically to treat such illnesses, and their mechanisms of action are of great interest. In this study 5-HT2 receptor-ligand interactions were examined by site-directed mutagenesis in which three aspartic acid to asparagine mutants (Asn-120, Asn-155, and Asn-172) were created and expressed in NIH3T3 cells. The Asp-120 to asparagine mutant exhibited the same affinity for 125I-lysergic acid diethylamide (125I-LSD) as did the wild-type receptor and showed a decreased and GTP-insensitive binding affinity for the agonists 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (approximately 10-fold) and the antagonists ketanserin and mianserin (approximately 10-fold) but not spiperone. The mutation also abolished agonist-stimulated formation of [3H]polyphosphoinositides (PI). The Asn-155 mutant showed reduced binding affinity for 125I-LSD (Kd, 2.8 nM versus 0.6 nM for the wild-type receptor) and had reduced affinity for agonists (approximately 30-fold) and for antagonists (14-75-fold). However, the Asn-155 receptor retained GTP sensitivity and the ability to stimulate PI formation. The Asn-172 mutant retained the wild-type Kd value for 125I-LSD, exhibited only approximately 5-fold reduced affinity for 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane while retaining GTP-sensitive agonist binding showed no change in affinity for ketanserin, and had a small decrease in mianserin and spiperone binding (approximately 6-fold). The Asn-172 receptor also retained the ability to form PI. These results indicate that Asp-120 is necessary for allosteric activation of the guanine nucleotide-binding protein. Asp-155 is necessary for high affinity binding, probably by acting as a counterion for the amine group of the ligand. The different effects of the three mutations on ketanserin, mianserin, and spiperone binding affinity suggest that these antagonists may share overlapping but different binding domains. The information provided by this study may facilitate the design of therapeutic site-selective compound based on the structure of the 5-HT2 receptor.
Mol Pharmacol 1993 Jun
PMID:Site-directed mutagenesis of the serotonin 5-hydroxytrypamine2 receptor: identification of amino acids necessary for ligand binding and receptor activation. 831 24

Serotonin (5-hydroxytryptamine; 5-HT) has recently been shown to induce collagenase production in myometrial smooth muscle cells (Jeffrey et al. (1991) J. Cell. Physiol. 146, 399-406) by activating transcription of the collagenase gene (Wilcox et al. (1992) J. Biol. Chem. 267, 20752-20757) following an interaction with the 5-HT2 receptor (Rydelek-Fitzgerald et al. (1993) Mol. Cell. Endocrinol. 91, 67-74). These studies were performed to investigate factors controlling the regulation of 5-HT2 receptors in these cells. Northern blot analysis indicates that serotonin increases levels of 5-HT2 receptor mRNA in cells by approximately 4-fold. Detectable increases in mRNA levels occur within 2 h after administration of serotonin with maximal levels occurring after 12 h. The 5-HT2 receptor antagonists, ketanserin and spiperone, inhibit the serotonin-mediated increase in receptor mRNA. Selective 5-HT2 receptor agonists ((+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI) and quipazine) mimic the effect of serotonin, whereas 5-HT1 and 5-HT3 receptor agonists ((+/-)-8-hydroxydipropylaminotetralin (8-OH-DPAT), 1-(3-chlorophenyl)piperazine dihydrochloride (mCPP), m-phenylbiguanide) have no effect. These data demonstrate that serotonin induces an increase in 5-HT2 receptor mRNA by interacting with the 5-HT2 receptor itself. Nuclear run-on analysis revealed that serotonin increases the initiation of 5-HT2 receptor mRNA synthesis. Moreover, the protein synthesis inhibitor, cycloheximide, prevents the induction of the mRNA for the receptor, demonstrating that serotonin-dependent increases in 5-HT2 receptor transcription require de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1993 Apr
PMID:Serotonin-mediated 5-HT2 receptor gene regulation in rat myometrial smooth muscle cells. 831 29

Regional expression and antidepressant drug-induced regulation of mRNA encoding the serotonin (5-HT) transporter were studied in rat brain. While 5-HT transporter mRNA is abundantly expressed in the midbrain raphe complex, lower concentrations were also found in frontal cortex, hippocampus, and neostriatum using a combination of reverse transcriptase-polymerase chain reaction (RT-PCR), Southern hybridization, and sequence analysis. Long-term administration of antidepressants which inhibit 5-HT reuptake, but not monoamine oxidase inhibitors or 5-HT receptor agonists, decrease 5-HT transporter mRNA steady-state concentrations. Based on these observations, we conclude that (1) mRNA coding for the 5-HT transporter is present in several brain areas associated with ascending HT pathways, and (2) chronic treatment with reuptake inhibiting antidepressants may be associated with regulation of the 5-HT transporter at the level of gene expression which may contribute to the neuroadaptive mechanisms that likely underlie their therapeutic efficacy.
Brain Res Mol Brain Res 1993 Jan
PMID:Regional brain expression of serotonin transporter mRNA and its regulation by reuptake inhibiting antidepressants. 838 6


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