Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 5-hydroxytryptamine3 receptor 5-HT3R has been implicated in gut and cardiac motility and in behavioral disorders. Characteristics of 5-HT3Rs appear to be heterogeneous among species, but human 5-HT3R cDNA has not been identified. We isolated a cDNA encoding 5-HT3R from human hippocampus. The mouse 5-HT3R gene has been reported to generate two alternative splicing isoforms that differ by six amino acids. All of our isolated human clones corresponded to the shorter isoform. Amino acid identities with mouse neuroblastoma N1E-115 and rat brain 5-HT3Rs were 84% for each. Southern blot analysis of human genomic DNA suggested that our cloned transcript encoded a human counterpart for the rodent 5-HT3Rs. This gene was assigned to chromosome 11 using polymerase chain reaction analysis of a human/rodent somatic cell hybrid panel. With the use of Northern blot analysis, 5-HT3R transcripts were identified in human small intestine, colon, and brain regions including hippocampus, amygdala, and striatum. In human heart, 5-HT3R expression was not detectable even with reverse transcriptase-polymerase chain reaction analysis, although it was detectable in mouse heart. Transfection of COS-1 with human 5-HT3R cDNA induced specific binding of the 5-HT3R-selective radioligand [3H]YM060. Human 5-HT3R showed typical characteristics of the 5-HT3R, but its affinity for the 5-HT3R agonist m-chlorophenylbiguanide was much lower than that of rat 5-HT3R. When injected with human 5-HT3R cRNA, the oocytes responded to 5-HT3R agonists with a rapidly developing inward current. The potency of the agonists to induce inward current paralleled that to compete with the radioligand binding, and 2-methyl-5-hydroxytryptamine, a partial agonist for mouse 5-HT3R, was a full agonist for human 5-HT3R. Our data revealed that the 5-HT3R molecule has interspecies differences in both tissue distribution and functional profile.
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PMID:Molecular cloning of human 5-hydroxytryptamine3 receptor: heterogeneity in distribution and function among species. 756 20

NIH-3T3 cells, a nontransformed murine fibroblast cell line previously found to be unresponsive to 5-hydroxytryptamine (5-HT) when cultured in 5-HT-free medium, became responsive to 5-HT, which induced an increase in intracellular calcium concentration. Pharmacological and ligand binding studies showed that NIH-3T3 cells endogenously express a 5-HT2A receptor that, when activated, mobilizes calcium from ionomycin-sensitive intracellular stores via coupling to a pertussis toxin-insensitive pathway. Using reverse transcriptase-PCR cloning and northern blot analysis, the presence of 5-HT2A receptor RNA with a similar nucleotide sequence (99% identity) and molecular size to that of murine brain was detected in NIH-3T3 cells. Responsiveness of the endogenous 5-HT2A receptor in nontransfected cells was completely desensitized after chronic treatment (half-time = 2 h) with 1 microM 5-HT and resensitized on removal of 5-HT. In contrast to NIH-3T3 cells transfected with 5-HT2A receptor cDNA under control of a viral promoter, the long-term agonist-induced functional desensitization in nontransfected NIH-3T3 cells was paralleled by a decrease in both 5-HT2A receptor density and RNA level. These results show that NIH-3T3 cells express an endogenous 5-HT2A receptor that is desensitized by agonist via down-regulation of both receptor number and mRNA. The NIH-3T3 cells provide a novel system for understanding 5-HT2A receptor regulation.
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PMID:Identification of an endogenous 5-hydroxytryptamine2A receptor in NIH-3T3 cells: agonist-induced down-regulation involves decreases in receptor RNA and number. 910 26

-The increased delivery of serotonin (5-hydroxytryptamine, 5-HT) to the lung aggravates the development of hypoxia-induced pulmonary hypertension in rats, possibly through stimulation of the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). In cultured rat PA-SMCs, 5-HT (10(-8) to 10(-6) mol/L) induced DNA synthesis and potentiated the mitogenic effect of platelet-derived growth factor-BB (10 ng/mL). This effect was dependent on the 5-HT transporter (5-HTT), since it was prevented by the 5-HTT inhibitors fluoxetine (10(-6) mol/L) and paroxetine (10(-7) mol/L), but it was unaltered by ketanserin (10(-6) mol/L), a 5-HT2A receptor antagonist. In PA-SMCs exposed to hypoxia, the levels of 5-HTT mRNA (measured by competitive reverse transcriptase-polymerase chain reaction) increased by 240% within 2 hours, followed by a 3-fold increase in the uptake of [3H]5-HT at 24 hours. Cotransfection of the cells with a construct of human 5-HTT promoter-luciferase gene reporter and of pCMV-beta-galactosidase gene allowed the demonstration that exposure of cells to hypoxia produced a 5.5-fold increase in luciferase activity, with no change in beta-galactosidase activity. The increased expression of 5-HTT in hypoxic cells was associated with a greater mitogenic response to 5-HT (10(-8) to 10(-6) mol/L) in the absence as well as in the presence of platelet-derived growth factor-BB. 5-HTT expression assessed by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization in the lungs was found to predominate in the media of pulmonary artery, in which a marked increase was noted in rats that had been exposed to hypoxia for 15 days. These data show that in vitro and in vivo exposure to hypoxia induces, via a transcriptional mechanism, 5-HTT expression in PA-SMCs, and that this effect contributes to the stimulatory action of 5-HT on PA-SMC proliferation. In vivo expression of 5-HTT by PA-SMC may play a key role in serotonin-mediated pulmonary vascular remodeling.
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PMID:Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin. 1002 7

Serotonin (5-hydroxytryptamine [5-HT]) is a multifunctional amine with wide occurrence in both neural and non-neural tissues, including the lung. The diverse responses to 5-HT are elicited through activation of different 5-HT receptor subtypes. We report the expression and localization of 5-HT receptor 2c subtype (5-HT2c-R) in rat lungs using reverse transcriptase-polymerase chain reaction, nonisotopic in situ hybridization (NISH), and immunohistochemistry. At the messenger RNA (mRNA) level, signal corresponding to approximately 430 base pairs was detected in whole-lung tissue extracts as well as in cultures of isolated alveolar type II cells from fetal and adult rat lung. Using antisense RNA probe for 5-HT2c-R, NISH showed strong positive signal in type II cells. The expression of mRNA signal differed between fetal and adult rat type II cells, with weak, predominantly perinuclear localization in the former and strong cytoplasmic localization in the latter. Immunohistochemistry, using specific monoclonal antibody against 5-HT2c-R, showed perinuclear localization in fetal type II cells; whereas in adult type II cells 5-HT2c-R immunoreactivity was confined mostly to the plasma membrane, as demonstrated by laser confocal microscopy. Identification of 5-HT2c-R expression in alveolar type II cells suggests an important role for this amine in modulating the function of these cells. The differences in cell domain localization between fetal and adult type II cells could indicate developmental regulation of 5-HT2c-R expression in the lung.
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PMID:Expression of serotonin receptor 2c in rat type II pneumocytes. 1034 Sep 36

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.
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PMID:Characterisation of 5-HT receptors in human coronary arteries by molecular and pharmacological techniques. 1037 14

5-HT1-like and 5-HT2 receptors have both been described to mediate contractions to 5-HT in the human umbilical artery (HUA). However, the nature of the 5-HT receptor subtypes is unknown. 2 In isometric force studies with ring preparations of HUA alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) and 5-hydroxytryptamine (5-HT) contracted HUA with pED50 values of 8.04 and 7.74, respectively. In the presence of a subthreshold concentration of another vasoconstrictor sumatriptan and 5-nonyloxytryptamine elicited concentration-dependent contractions with pEC50 values of 7.21 and 7.67, respectively. In the presence of the selective 5-HT1B/D receptor antagonist GR127935, contractile responses elicited by sumatriptan and 5-nonyloxytryptamine were competitively antagonized (pKB 9.01 and 9.02, respectively). In the experiments with 5-HT, GR127935 appeared to be non-competitive with shallow Schild plot slopes. The data were fitted with two linear regression lines and the calculated pKB of the high affinity component (8.90) was comparable to that expected for GR127935 at the 5-HT1B/1D receptor. Several 5-HT2 selective receptor antagonists (spiperone, cyproheptadine, pirenperone) competitively inhibited responses to 5-HT. The selective 5-HT2A antagonist ketanserin against sumatriptan and 5-nonyloxytryptamine behaved as a weak antagonist while against 5-HT demonstrated a competitive antagonism (pKB 8.56). Using specific primers for human 5-HT1B, 5-HT1D and 5-HT2A receptor genes, the reverse transcriptase-polymerase chain reaction revealed mRNA expression of 5-HT1B and 5-HT2A receptors in the HUA. The results suggest that the HUA has a functional population of 5-HT1B and 5-HT2A receptor subtypes which are involved in the contractile response to 5-HT. Contractions mediated by 5-HT1B receptors can be 'uncovered' by exposure to other vasoactive agents.
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PMID:Functional characterization and m-RNA expression of 5-HT receptors mediating contraction in human umbilical artery. 1045 72

Ferrets (Mustela putorius furo) are useful animals for determining anti-emetic activity via 5-HT(3) receptors in vivo. We isolated a cDNA encoding the 5-hydroxytryptamine (5-HT) type 3A receptor subunit (5-HT(3A)) from ferret colon, expressed it in a human embryonic kidney cell line and determined its pharmacological properties. The open reading frame of the isolated cDNA encoded a 483-amino acid protein, corresponding to the shorter splice variant of 5-HT(3A) receptors. Splice variants were no longer detected by reverse transcriptase-polymerase chain reaction. The ferret 5-HT(3A) receptor exhibits a high degree of amino acid sequence identity (>/=80%) to that of other species. Binding studies demonstrated the following rank order of potency for agonists: meta-chlorophenylbiguanide (mCPBG)>2-methyl-5-hydroxytryptamine (2-Me-5-HT)=5-HT, and for antagonists: ondansetron=tropisetron>(+)-tubocurarine>metoclopramide. Electrophysiological studies revealed that mCPBG was a partial agonist and 2-Me-5-HT was an almost fully effective agonist compared to 5-HT.
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PMID:Cloning, expression, and characterization of ferret 5-HT(3) receptor subunit. 1088 8

Little is currently known regarding the electrophysiological response elicited by 5-hydroxytryptamine-7 (5-HT(7)) receptor stimulation in the brain. Previous anatomical studies have shown that the anterior thalamus expresses a high density of 5-HT7 receptors. Therefore, we used whole-cell recording techniques in the in vitro brain slices to examine the effects of serotonin on neurons of the anterodorsal nucleus of the thalamus (ADn). Bath application of 5-HT induces a large membrane depolarization and inward current in neurons of the ADn. Since these cells expressed 5-HT7 receptor mRNA, as determined by single-cell reverse transcriptase-polymerase chain reaction, we pharmacologically characterized the 5-HT receptor mediating this response. We found that the 5-HT1 and 5-HT7 agonists 5-carboxamidotryptamine (5-CT) and 5-methoxytryptamine mimicked the response to 5-HT, whereas the 5-HT2 agonist 2,5-dimethoxy-4-iodoamphetamine did not. Consistent with the involvement of a 5-HT7 receptor, 5-CT was approximately 18 times more potent than 5-HT. Furthermore, administration of the 5-HT(1A) and 5-HT7 agonist 8-hydroxydipropylaminotetralin mimicked and antagonized the effect of serotonin, suggesting it acted as a partial agonist. To determine if either the 5-HT1 or 5-HT7 receptor mediated the 5-HT-induced inward current, we used antagonists. We found that the 5-HT7 ligands ritanserin, methylsergide, LSD, and mesulergine could inhibit the 5-HT-induced inward current, whereas the 5-HT1 antagonist cyanopindolol had no effect. The pA(2) value determined for mesulergine closely approximated that expected for a 5-HT7 receptor. Finally, we found that bath application of the selective antagonist SB-269770 blocks the 5-HT-induced inward current. These results identify the receptor mediating the serotonin-induced membrane depolarization in the ADn as the 5-HT7 subtype.
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PMID:A 5-HT(7) receptor-mediated depolarization in the anterodorsal thalamus. I. Pharmacological characterization. 1125 68

Astrocytes contain transport systems that are capable of removing various neurotransmitters from the synaptic cleft by transporters present in the plasma membrane. Glial serotonin transporter (SERT) plays an important role in the re-uptake of 5-hydroxytryptamine (5-HT). We examined the pharmacological characterization of 5-HT uptake into rat cortical synaptosomes and cultured rat astrocytes, and the immunodetection of glial SERT proteins using specific site-directed monoclonal antibodies (MoAb). Furthermore, using a reverse transcriptase-polymerase chain reaction (RT-PCR) method, we addressed the expression of SERT mRNA in cultured rat astrocytes. We investigated the inhibitory effects of various monoamine uptake inhibitors on the uptake of [3H]5-HT into cultured astrocytes and cortical synaptosomes. Tricyclic antidepressants (clomipramine and imipramine) as well as selective serotonin re-uptake inhibitors (fluvoxamine, fluoxetine and zimelidine) were very potent inhibitors of [3H]5-HT uptake in both preparations. In contrast, the inhibitory effects of NE uptake inhibitors (nisoxetine and desipramine) and cocaine were weaker than those of 5-HT uptake inhibitors. In addition, dopamine (DA) uptake inhibitors (nomifensine and GBR-12935) exhibited a Ki value in the low micromolar range. The inhibitory potencies were in the order 5-HT uptake inhibitors (clomipramine, fluvoxamine, fluoxetine, imipramine and zimelidine) > NE uptake inhibitors (nisoxetine and desipramine) = cocaine > DA uptake inhibitors (nomifensine and GBR-12935). There was no difference in the order of the inhibitory effects of various monoamine uptake inhibitors between the two preparations. A correlation analysis of the potencies of various monoamine uptake inhibitors in the inhibition of [3H]5-HT into cultured astrocytes and cortical synaptosomes produced a highly significant correlation coefficient of 0.9893 (P < 0.0001). Immunocytochemical staining using anti-SERT MoAb in cultured astrocytes revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or golgi membranes, showed a considerable level of immunoreactivity. Extracts of astrocytes and synaptosomes from the cortex were immunoblotted with anti-SERT MoAb. SDS-PAGE/Western blots indicate that anti-SERT MoAb recognized two bands of 120 and 73 kDa in both preparations. RT-PCR demonstrated that astrocytes in cultured expressed mRNA for the cloned SERT protein, which has been characterized as the neuronal SERT. These pharmacological experiments indicate that this uptake process takes place through glial SERT that is very similar to neuronal SERT. Furthermore, the present data also indicate that the presence of the mRNA and protein for the neuronal SERT were established in cultured rat astrocytes, and the polypeptide portion of SERT in astrocytes and frontal cortex could be the same gene product.
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PMID:Pharmacological characterization and visualization of the glial serotonin transporter. 1131 48

By using the reverse transcriptase polymerase chain reaction technique, the expression of 5-hydroxytryptamine (5-HT) receptor subtype mRNAs in the rat lumbar dorsal root ganglion (DRG) was investigated following unilateral injection of complete Freund's adjuvant (CFA) into the rat hind paw. The results showed that 5-HT(1A), 5-HT(1B), 5-HT(1D), 5-HT(1F), 5-HT(2A), 5-HT(3), 5-HT(4), 5-HT(5A) and 5-HT(7) receptor subtypes were present in the rat lumbar DRG. CFA injection resulted in a significant increase in mRNA level of 5-HT(1A), 5-HT(1B), 5-HT(1F), 5-HT(2A), 5-HT(3), 5-HT(4) and 5-HT(7) receptor subtypes and a marked induction of 5-HT(2C) subtype mRNA in the DRG. The present results suggest the important roles for these 5-HT receptor subtypes in generating peripheral nociceptive signaling and provide evidence to elucidate the mechanism of 5-HT in nociception.
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PMID:Changes of the expression of 5-HT receptor subtype mRNAs in rat dorsal root ganglion by complete Freund's adjuvant-induced inflammation. 1143 94


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