1.Establishment of heterologous expression model of hSERT in Xenopus laevis oocytes.
Yi-Ying WANG ; Zhu JIN ; Ci-Zhen LI ; Yuan-Mou LIU
Chinese Journal of Applied Physiology 2005;21(4):444-448
AIMTo determine the feasibility of establishing the heterologous expression model of human- serotonin transporter(hSERT or 5-HTT).
METHODScRNA of SERT was transcribed from cDNA, which was cloned in the pOTV vector. Each oocyte of mature xenopus laevis was injected with transcribed cRNA in vivo and incubated at room temperature for 4-9 days. Recording the current induced by 5-HT with voltage clamp technique tested the function of the expressed 5-HT transporter.
RESULTSThe transporter current could be observed in Ringer's solution containing 5-HT, and the 5-HT induced current were concentration-dependent. Norepinephrine and dopamine could not induce the transporter current while the 5-HT induced current could be specifically inhibited by 5-HTT blocker, desipramine.
CONCLUSIONThe results demonstrate that the heterologous expression product in xenopus laevis oocytes is human 5-HT transporter.
Animals ; Carrier Proteins ; genetics ; DNA, Complementary ; genetics ; Female ; Gene Expression ; Models, Animal ; Oocytes ; metabolism ; RNA, Messenger ; genetics ; Serotonin ; metabolism ; Serotonin Plasma Membrane Transport Proteins ; biosynthesis ; genetics ; Xenopus laevis
2.An inhibitory compound against the interaction between Galpha(s) and the third intracellular loop region of serotonin receptor subtype 6 (5-HT(6)) disrupts the signaling pathway of 5-HT(6).
Yun Hee CHOI ; Hatan KANG ; Won Kyu LEE ; Taehyun KIM ; Hyewhon RHIM ; Yeon Gyu YU
Experimental & Molecular Medicine 2007;39(3):335-342
Serotonin receptor subtype 6 (5-HT(6)) is a neurotransmitter receptor, which is involved in various brain functions such as memory and mood. It mediates signaling via the interaction with a stimulatory G-protein. Especially, the third intracellular loop (iL3) of 5-HT(6) and the alpha subunit of stimulatory G protein (Galpha(s)) are responsible for the signaling process of 5-HT(6). Chemical compounds that could inhibit the interaction between the iL3 region of 5-HT(6) and Galpha(s) were screened from a chemical library consisted of 5,600 synthetic compounds. One of the identified compounds bound to Galpha(s) and effectively blocked the interaction between Galpha(s) and the iL3 region of 5-HT(6). The identified compound was further shown to reduce the serotonin-induced accumulation of cAMP in 293T cells transformed with 5-HT(6) cDNA. It also lowered the Ca2+ efflux induced by serotonin in cells expressing 5-HT(6) and chimeric Galpha(s5/q). These results indicate that the interaction between the iL3 of 5-HT(6) and Galpha(s) can be exploited for screening of regulatory compounds against the signaling pathway of 5-HT(6).
Animals
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Calcium/metabolism
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Cell Line
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Cephalosporins/*pharmacology
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Cricetinae
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Cricetulus
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Cyclic AMP/biosynthesis
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GTP-Binding Protein alpha Subunits, Gs/antagonists & inhibitors/*metabolism
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Humans
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Receptors, Serotonin/*drug effects/metabolism/*physiology
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Serotonin/pharmacology
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Serotonin Antagonists/pharmacology
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Signal Transduction
3.Vardenafil Increases Cell Proliferation in the Dentate Gyrus through Enhancement of Serotonin Expression in the Rat Dorsal Raphe.
Tae Soo KIM ; Il Gyu KO ; Yun Hee SUNG ; Sung Eun KIM ; Bo Kyun KIM ; Seung Kook PARK ; Mal Soon SHIN ; Chang Ju KIM ; Sang Jin YOON ; Khae Hawn KIM
Journal of Korean Medical Science 2009;24(6):1099-1104
This study was conducted to evaluate the effects of vardenafil (Levitra), a phosphodiesterase-5 (PDE-5) inhibitor, on cell proliferation in the hippocampal dentate gyrus and on 5-hyroxytryptamine (5-HT, serotonin) synthesis and tryptophan hydroxylase (TPH) expression in the rat dorsal raphe nucleus. Male Sprague-Dawley rats were divided into 6 groups (n=5 in each group): a control group, a 0.5 mg/kg-1 day vardenafil-treated group, a 1 mg/kg-1 day vardenafil-treated group, a 2 mg/kg-1 day vardenafil-treated group, a 1 mg/kg-3 day vardenafil-treated group, and a 1 mg/kg-7 day vardenafil-treated group. 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry was then performed to evaluate cell proliferation in the dentate gyrus. In addition, 5-HT and TPH immunohistochemistry was conducted to evaluate serotonin expression in the dorsal raphe. The results revealed that treatment with vardenafil increased cell proliferation in the dentate gyrus and enhanced 5-HT synthesis and TPH expression in the dorsal raphe in a dose- and duration-dependent manner. The findings demonstrate that the increasing effect of vardenafil on cell proliferation is closely associated with the enhancing effect of vardenafil on serotonin expression under normal conditions.
Animals
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Cell Proliferation/*drug effects
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*Dentate Gyrus/cytology/drug effects/metabolism
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Imidazoles/*pharmacology
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Male
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Phosphodiesterase Inhibitors/*pharmacology
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Piperazines/*pharmacology
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*Raphe Nuclei/cytology/drug effects/metabolism
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Rats
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Rats, Sprague-Dawley
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Serotonin/*biosynthesis
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Sulfones/pharmacology
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Triazines/pharmacology
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Tryptophan Hydroxylase/metabolism
4.Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine..
Bukhtiar H SHAH ; Huma RASHEED ; Ibrahim H RAHMAN ; Amir H SHARIFF ; Fatima L KHAN ; Hina B RAHMAN ; Sara HANIF ; Sheikh A SAEED
Experimental & Molecular Medicine 2001;33(4):226-233
Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.
Diltiazem/pharmacology
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Dose-Response Relationship, Drug
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Drug Synergism
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Estrenes/pharmacology
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Flavones/pharmacology
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Human
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In Vitro
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Indomethacin/pharmacology
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Kinetics
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Mitogen-Activated Protein Kinases/metabolism
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Phosphorylation/drug effects
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Platelet Activating Factor/*pharmacology
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Platelet Activation/drug effects
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Platelet Aggregation/*drug effects/physiology
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Pyrrolidinones/pharmacology
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Serotonin/*pharmacology
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Thromboxane A2/biosynthesis
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Verapamil/pharmacology
5.Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine..
Bukhtiar H SHAH ; Huma RASHEED ; Ibrahim H RAHMAN ; Amir H SHARIFF ; Fatima L KHAN ; Hina B RAHMAN ; Sara HANIF ; Sheikh A SAEED
Experimental & Molecular Medicine 2001;33(4):226-233
Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.
Diltiazem/pharmacology
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Dose-Response Relationship, Drug
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Drug Synergism
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Estrenes/pharmacology
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Flavones/pharmacology
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Human
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In Vitro
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Indomethacin/pharmacology
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Kinetics
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Mitogen-Activated Protein Kinases/metabolism
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Phosphorylation/drug effects
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Platelet Activating Factor/*pharmacology
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Platelet Activation/drug effects
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Platelet Aggregation/*drug effects/physiology
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Pyrrolidinones/pharmacology
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Serotonin/*pharmacology
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Thromboxane A2/biosynthesis
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Verapamil/pharmacology