Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Expression in animal cells of the 5-HT1A receptor by a vaccinia virus vector system. 153 96

1. 5-Hydroxytryptamine (5-HT) elicited a dose-dependent stimulation of intracellular adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 +/- 0.08 (mean +/- s.e. mean). 2. In order to characterize the 5-HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5-carboxamidotryptamine (5-CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5-CT (pEC50 = 7.81 +/- 0.09) > 5-methoxytryptamine (5-MeOT) (pEC50 = 6.86 +/- 0.36) > 5-HT (pEC50 = 6.68 +/- 0.08). The following compounds, at concentrations up to 10 microM, did not affect basal cyclic AMP levels 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was methiothepin (pKi = 7.98 +/- 0.25) > mesulergine (pKi = 7.58 +/- 0.18) > ritanserin (pKi = 7.20 +/- 0.24) > clozapine (pKi = 7.03 +/- 0.19) > mianserin (pKi = 6.41 +/- 0.19). The following compounds, at concentrations up to 10 microM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5-HT7 receptor subtype-mediated effects. 3. The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 +/- 75% and 572 +/- 63% of basal levels for 5-CT and 5-HT, respectively) compared to brainstem (274 +/- 51% and 318 +/- 46%, respectively) and colliculus astrocytes (244 +/- 15% and 301 +/- 24%, respectively). No significant differences in pEC50 (for either 5-HT or 5-CT) values were observed. 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for the 5-HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5-HT6 receptor also amplified a cDNA fragment from the same samples. 5. From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5-HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally-dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5-HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5-HT6 and 5-HT7 receptor messenger RNA.
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PMID:Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes. 903 57

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H]5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na+ dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised 'neuronal' serotonin transporter.
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PMID:Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles. 969 37

Serotonin (5-HT) is one of the most extensively studied neurotransmitters of the central nervous system. 5-HT is, however, also present in a variety of peripheral tissues including in constituents of the immune system. The function of 5-HT in the immune system has received increasing attention since about 1984, but has been reviewed only once, in 1985. In recent years, modern techniques of molecular biology such as reverse-transcriptase polymerase chain reaction and targeted gene disruption have made it possible to study new important aspects of 5-HT in the immune system. In the first part of the review, we explore whether 5-HT is involved in interactions between the central nervous and immune systems. It emerges that 5-HT may mediate interactions of these two systems by four different pathways. In the second part, we dissect the functional roles of 5-HT in the immune system. We describe the distribution of 5-HT receptors and the 5-HT transporter on immune cells and estimate which levels 5-HT may attain in the extracellular space in physiological conditions and under pathological circumstances such as inflammation, thrombosis, and ischemia. At these 5-HT concentrations, four major functions for 5-HT emerge. These include T cell and natural killer cell activation, delayed-type hypersensitivity responses, production of chemotactic factors, and natural immunity delivered by macrophages. Finally, we discuss promising future avenues to further advance knowledge of the role of 5-HT in the immune system and in neuroimmune interactions.
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PMID:Role of serotonin in the immune system and in neuroimmune interactions. 1008 Aug 56

While homomers containing 5-HT(3A) subunits form functional ligand-gated serotonin (5-HT) receptors in heterologous expression systems (Jackson, M. B., and Yakel, J. L. (1995) Annu. Rev. Physiol. 57, 447-468; Lambert, J. J., Peters, J. A., and Hope, A. G. (1995) in Ligand-Voltage-Gated Ion Channels (North, R., ed) pp. 177-211, CRC Press, Inc., Boca Raton, FL), it has been proposed that native receptors may exist as heteromers (Fletcher, S., and Barnes, N. M. (1998) Trends Pharmacol. Sci. 19, 212-215). We report the cloning of a subunit 5-HT(3B) with approximately 44% amino acid identity to 5-HT(3A) that specifically modified 5-HT(3A) receptor kinetics, voltage dependence, and pharmacology. Co-expression of 5-HT(3B) with 5-HT(3A) modified the duration of 5-HT(3) receptor agonist-induced responses, linearized the current-voltage relationship, increased agonist and antagonist affinity, and reduced cooperativity between subunits. Reverse transcriptase-polymerase chain reaction in situ hybridization revealed co-localization of both 5-HT(3B) and 5-HT(3A) in a population of neurons in the amygdala, telencephalon, and entorhinal cortex. Furthermore, 5-HT(3A) and 5-HT(3B) mRNAs were expressed in spleen and intestine. Our data suggest that 5-HT(3B) might contribute to tissue-specific functional changes in 5-HT(3)-mediated signaling and/or modulation.
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PMID:The pharmacological and functional characteristics of the serotonin 5-HT(3A) receptor are specifically modified by a 5-HT(3B) receptor subunit. 1052 71

The RNase protection assay (RPA) is an extremely sensitive procedure for detection of messenger RNA (mRNA) in complex sample mixture of total RNA. However, its usefulness has been limited by the requirement for the DNA to be cloned onto an appropriate vector. We have utilized the polymerase chain reaction (PCR) to directly incorporate a T7 RNA polymerase promoter sequence onto the cDNA for the 5-hydroxytryptamine(1B) (5-HT(1B)) receptor. Radiolabeled riboprobe was then synthesized using the PCR product as a template and used in RPA to detect mRNA for 5-HT(1B) receptor in rat brain. The internal control was the beta-Actin mRNA. Due to the simplicity of its design and the lack of need for subcloning, the DNA template synthesis by PCR facilitates the implementation of the RPA. Since the 5-HT(1B) receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters and the protocol described here permits the determination of 5-HT(1B) receptor mRNA levels in the rat cerebellum, striatum, hippocampus and frontal cortex, this protocol is helpful in understanding the involvement of 5-HT(1B) receptors in various physiological phenomena.
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PMID:Application of the polymerase chain reaction to the RNase protection assay for 5-HT(1B) receptor mRNA levels measurement in rat brain tissues. 1059 41

1. Using subtype-selective 5-HT1 receptor agonists and/or the 5-HT1 receptor antagonist GR127935, we characterized in vitro the 5-HT receptor that mediates the contraction of human and bovine cerebral arteries. Further, we investigated which sumatriptan-sensitive receptors are present in human coronary artery by reverse-transcriptase polymerase chain reaction (RT-PCR). 2. Agonists with affinity at the 5-HT1B receptor, such as sumatriptan, alniditan and/or IS-159, elicited dose-dependent contraction in both human and bovine cerebral arteries. They behaved as full agonists at the sumatriptan-sensitive 5-HT1 receptors in both species. In contrast, PNU-109291 and LY344864, selective agonists at 5-HT1D and 5-HT1F receptors, respectively, were devoid of any significant vasocontractile activity in cerebral arteries, or did not affect the sumatriptan-induced vasocontraction. The rank order of agonist potency was similar in both species and could be summarized as 5-HT = alniditan > sumatriptan = IS-159 >>> PNU-109291 = LY344864. 3. In bovine cerebral arteries, the 5-HT1 receptor antagonist GR127935 dose-dependently inhibited the vasoconstrictions elicited by both 5-HT and sumatriptan, with respective pA2 values of 8.0 and 8.6. 4. RT-PCR studies in human coronary arteries showed a strong signal for the 5-HT1B receptor while message for the 5-HT1F receptor was weak and less frequently detected. Expression of 5-HT1D receptor mRNA was not detected in any sample. 5. The present results demonstrate that the triptan-induced contraction in brain vessels is mediated exclusively by the 5-HT1B receptor, which is also present in a majority of human coronary arteries. These results suggest that selective 5-HT1D and 5-HT1F receptor agonists might represent new antimigraine drugs devoid of cerebro- and cardiovascular effects.
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PMID:No contractile effect for 5-HT1D and 5-HT1F receptor agonists in human and bovine cerebral arteries: similarity with human coronary artery. 1071 48

We have isolated from a human genomic library the human 5-hydroxytryptamine 5-HT(5A) and 5-HT(5B) genes. The human 5-HT(5A) gene encodes a protein with similar characteristics to its mouse homologue. When expressed in monkey COS-7 cells, the human 5-HT(5A) receptor displayed a high affinity for tritiated 5-carbamidotryptamine ([3H]5-CT; K(D)=2.8 nM) and iodinated lysergic acid diethylamide ([125I]LSD; K(D)=187 pM). These binding sites displayed the following displacement profile: Ergotamine>Methiothepin>5-CT, Ritanserin>5-HT. Reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the presence of human 5-HT(5A) mRNA in the central nervous system but not in peripheral organs. When expressed in Xenopus oocytes, the 5-HT(5A) receptor was able to couple to the inwardly rectifying K(+) channel, GIRK(1). In contrast to the human 5-HT(5A) gene and the mouse 5-HT(5B) gene, the human 5-HT(5B) gene does not encode a functional protein because its coding sequence is interrupted by stop codons. Our results suggest, therefore, that the 5-HT(5B) receptor has been lost during evolution after the divergence between rodents and primates. The 5-HT(5B) receptor is the first example of a brain-specific protein that is absent in human.
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PMID:Human 5-HT(5) receptors: the 5-HT(5A) receptor is functional but the 5-HT(5B) receptor was lost during mammalian evolution. 1134 85

The aim of the present study was to investigate the effects of melatonin on non-adrenergic, non-cholinergic (NANC) relaxant neurotransmission in the gastrointestinal tract, which is mainly mediated by nitrergic and peptidergic mechanisms. Melatonin (10(-7)-10(-3) M) had no effect on the basal tonus of the rat gastric fundus smooth muscle. Relaxant responses following electrical stimulation(40 V; 0.5 ms pulse duration; 10 s stimulation duration) under NANC conditions on a 5-hydroxytryptamine (5-HT, 10(-7) M) contraction plateau were elicited at frequencies in the range of 0.5-16 Hz. Melatonin significantly reduced these inhibitory NANC responses (16 Hz without melatonin: -103 +/- 6.3%; melatonin 10(-5) M: -80.4 +/- 7.5%; melatonin 10(-4) M: -39.1 +/- 17.1%). Intracellular recording was carried out in a mouse colonic preparation. Electrical neural stimulation of the mouse colonic neurons caused biphasic intracellular hyperpolarization in smooth-muscle cells. The initial fast component is apamin-sensitive, and the following slow component is dependent on nitrergic mechanisms, as it is abolished in the presence of NG-nitro-L-arginine (L-NNA). Melatonin significantly reduced the nitric oxide-dependent slow component of neurally transmitted hyperpolarization, whereas the initial fast component was left unchanged. In a synaptosomal preparation of the enteric nervous system of rat intestine, enzymatic nitric oxide synthase (NOS) activity was significantly reduced by melatonin at concentrations ranging from 10(-7) to 10(-4) M (basal preparation including cofactors: 61.2 +/- 9.4 fmol/mg; melatonin 10(-4) M: 39.2 +/- 6.9 fmol/mg). Reverse transcriptase-polymerase chain reaction (RT-PCR) studies were conducted to investigate the melatonin receptors (mt(1), MT(2) and MT(3)) present in the esophagus, stomach and ileum of the rat. The presence of mt1 mRNA expression alone, but not of mRNA expression for MT(2) or MT(3), was demonstrated in the tissues. In conclusion, this study demonstrates that melatonin reduces the functional inhibitory NANC response. It shows that this effect may be the result of a reduction of the nitrergic component of the smooth-muscle inhibitory junction potential (IJP) and related to direct inhibition of NOS activity in enteric synaptosomes. The presence of mt1 receptor transcripts adds supportive evidence for a possible physiological role of melatonin within the enteric nervous system.
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PMID:Melatonin reduces non-adrenergic, non-cholinergic relaxant neurotransmission by inhibition of nitric oxide synthase activity in the gastrointestinal tract of rodents in vitro. 1215 44

Serotonin plays important physiological functions at the intestinal level. However, nothing is known concerning its inactivation mechanisms in the human intestine. So, the aim of this work was to characterize the uptake of serotonin at the apical and basolateral membranes of human intestinal epithelial (Caco-2) cells. Uptake of [3H]serotonin at the apical membrane of Caco-2 cells was specific and Na+-, Cl--, and potential-dependent. It was concentration dependently inhibited by several monoamines (with the following rank order of potency: serotonin >> dopamine > or = noradrenaline) and tricyclic and nontricyclic antidepressants (with the following rank order of potency: fluoxetine > desipramine > cocaine > GBR 12909). In contrast, it was not affected by corticosterone (0.01-100 micro M) and was only partially inhibited by decynium-22 (0.001-10 micro M). Transepithelial apparent permeability (Papp) to [3H]serotonin in the apical-to-basolateral direction was reduced by desipramine (0.4 micro M) and fluoxetine (0.02 micro M), and it was not Na+-dependent nor affected by corticosterone (100 micro M). Uptake of [3H]serotonin at the basolateral membrane of Caco-2 cells was Na+-dependent and reduced by desipramine (0.4 micro M) and fluoxetine (0.02 micro M), and it was not affected by corticosterone (100 micro M). The Papp to [3H]serotonin in the basolateral-to-apical direction was reduced by desipramine (0.4 micro M) and fluoxetine (0.02 micro M), and it was not affected by Na+ omission or by corticosterone (100 micro M). Reverse transcriptase-polymerase chain reaction indicates that mRNA of the neuronal serotonin transporter (SERT) is present in Caco-2 cells and in human small intestine. In conclusion, these results suggest that human intestinal epithelial Caco-2 cells functionally express SERT, both at their apical and basolateral cell membranes.
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PMID:Uptake of serotonin at the apical and basolateral membranes of human intestinal epithelial (Caco-2) cells occurs through the neuronal serotonin transporter (SERT). 1268 18


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