Molecular cloning, pharmacological properties and tissue distribution of the porcine 5-HT(1B) receptor
| Autor: | P, Bhalla, H S, Sharma, X, Ma, T, Wurch, P J, Pauwels, P R, Saxena |
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| Rok vydání: | 2001 |
| Předmět: |
DNA
Complementary Pyridines Swine Molecular Sequence Data Gene Expression Sulfur Radioisotopes Tritium Binding Competitive Radioligand Assay Sequence Homology Nucleic Acid Animals Tissue Distribution Amino Acid Sequence RNA Messenger Cloning Molecular In Situ Hybridization Cerebral Cortex Base Sequence Dose-Response Relationship Drug Sequence Homology Amino Acid Sequence Analysis DNA Recombinant Proteins Serotonin Receptor Agonists Guanosine 5'-O-(3-Thiotriphosphate) Receptors Serotonin Benzamides COS Cells Papers Receptor Serotonin 5-HT1B Serotonin Antagonists Sequence Alignment |
| Zdroj: | British journal of pharmacology. 133(6) |
| ISSN: | 0007-1188 |
| Popis: | Using a combination of RT - PCR and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-HT(1B) receptor cDNA derived from porcine cerebral cortex. Sequence analysis revealed 1170 bp encoding an open reading frame of 390 amino acids showing a 95% similarity with the human 5-HT(1B) receptor. The recombinant porcine 5-HT(1B) cDNA was expressed in monkey Cos-7 cells and its pharmacological profile was determined by radioligand binding assay using [(3)H]-GR125743. The affinities of several agonists (L694247ergotamineor =5-carboxamidotryptamine=dihydroergotamine=5-HTCP122638=zolmitriptansumatriptan) and putative antagonists (GR127935methiothepinSB224289ritanserinketanserinor =BRL15572) correlated highly with those described for the recombinant human 5-HT(1B) receptor. In membranes obtained from cells co-expressing the porcine 5-HT(1B) receptor and a mutant G(alphao)Cys(351)Ile protein, 5-HT and zolmitriptan increased, while the 5-HT(1B) receptor antagonist SB224289 decreased basal [(35)S]-GTPgammaS binding, thus showing inverse agonism. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 7.64+/-0.04) agreed with its affinity in displacing the antagonist [(3)H]-GR125743 (pK(i): 7.36+/-0.07). The 5-HT(1B) receptor mRNA was observed by RT-PCR in several blood vessels, cerebral cortex, cerebellum and trigeminal ganglion. In situ hybridization performed in frontal cerebral cortex sections revealed the expression of 5-HT(1B) receptor mRNA in pyramidal cells. In conclusion, we have cloned and established the amino acid sequence, ligand binding profile and location of the porcine 5-HT(1B) receptor. This information may be useful in exploring the role of 5-HT(1B) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine. |
| Databáze: | OpenAIRE |
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