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Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor.

ABSTRACT: A cDNA encoding the full-length 5-HT(1D) receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig 5-HT(1D) receptor coding sequence oligonucleotide primers in reverse transcription-polymerase chain reaction (RT - PCR). The 5' and 3' ends of the porcine 5-HT(1D) receptor cDNA were verified by inverse PCR. Sequence analysis of porcine 5-HT(1D) receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having 92% homology with the human 5-HT(1D) receptor and 88 - 90% homology with other species homologues. The porcine 5-HT(1D) receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos-7 cells. Radioligand binding assays using either [(3)H]-5-CT or [(3)H]-GR125743 on Cos-7 cell membranes showed that pK(i) values of 14 serotonin ligands were highly correlated with those obtained with the human 5-HT(1D) receptor. Nonetheless, a selective antagonist at the human 5-HT(1D) receptor, BRL15572, only poorly recognized the porcine homologue. Using membranes from cells co-expressing the porcine 5-HT(1D) receptor and rat G(alphail)Cys(351) Ile protein, it was shown that 5-HT and zolmitriptan increased, while ketanserin decreased basal [(35)S]-GTPgammaS binding. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 8. 46+/-0.08) agreed with its affinity in displacing the radioligands [(3)H]-5-CT and [(3)H]-GR125743 (pK(i): 8.38+/-0.15 and 8.67+/-0.08, respectively). In conclusion, we have established the cDNA sequence and pharmacology of the cloned porcine 5-HT(1D) receptor. This information would be useful in exploring the role of divergent amino acid residues in the receptor-ligand interaction as well as the role of 5-HT(1D) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

SUBMITTER: Bhalla P

PROVIDER: S-EPMC1572407 | biostudies-literature | 2000 Nov

REPOSITORIES: biostudies-literature

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Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor.

Bhalla P P Sharma H S HS Wurch T T Pauwels P J PJ Saxena P R PR

British journal of pharmacology 20001101 5

A cDNA encoding the full-length 5-HT(1D) receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig 5-HT(1D) receptor coding sequence oligonucleotide primers in reverse transcription-polymerase chain reaction (RT - PCR). The 5' and 3' ends of the porcine 5-HT(1D) receptor cDNA were verified by inverse PCR. Sequence analysis of porcine 5-HT(1D) receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having ...[more]

PMID: 11053216

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Project description:Activation of neurons in the anterolateral bed nucleus of the stria terminalis (BNST(ALG)) plays an important role in mediating the behavioral response to stressful and anxiogenic stimuli. Application of 5-HT elicits complex postsynaptic responses in BNST(ALG) neurons, which includes (1) membrane hyperpolarization (5-HT(Hyp)), (2) hyperpolarization followed by depolarization (5-HT(Hyp-Dep)), (3) depolarization (5-HT(Dep)) or (4) no response (5-HT(NR)). We have shown that the inhibitory response is mediated by activation of postsynaptic 5-HT(1A) receptors. Here, we used a combination of in vitro whole-cell patch-clamp recording and single cell reverse transcriptase polymerase chain reaction (RT-PCR) to determine the pharmacological properties and molecular profile of 5-HT receptor subtypes mediating the excitatory response to 5-HT in BNST(ALG) neurons. We show that the depolarizing component of both the 5-HT(Hyp/Dep) and the 5-HT(Dep) response was mediated by activation of 5-HT(2A), 5-HT(2C) and/or 5-HT(7) receptors. Single cell RT-PCR data revealed that 5-HT(7) receptors (46%) and 5-HT(1A) receptors (41%) are the most prevalent receptor subtypes expressed in BNST(ALG) neurons. Moreover, 5-HT receptor subtypes are differentially expressed in type I-III BNST(ALG) neurons. Hence, 5-HT(2C) receptors are almost exclusively expressed by type III neurons, whereas 5-HT(7) receptors are expressed by type I and II neurons, but not type III neurons. Conversely, 5-HT(2A) receptors are found predominantly in type II neurons. Finally, bi-directional modulation of individual neurons occurs only in type I and II neurons. Significantly the distribution of 5-HT receptor subtypes in BNST(ALG) neurons predicted the observed expression pattern of 5-HT responses determined pharmacologically. Together, these results suggest that 5-HT can differentially modulate the excitability of type I-III neurons, and further suggest that bi-directional modulation of BNST(ALG) neurons occurs primarily through an interplay between 5-HT(1A) and 5-HT(7) receptors. Hence, modulation of 5-HT(7) receptor activity in the BNST(ALG) may offer a novel avenue for the design of anxiolytic medications.

Dataset Information

Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor.

Publications

Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor.

Similar Datasets

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