Isolation of the serotoninergic 5‐HT4(e) receptor from human heart and comparative analysis of its pharmacological profile in C6‐glial and CHO cell lines

• Published in: British journal of pharmacology 2000-02, Vol.129 (4), p.771-781
• Main Authors: Mialet, Jeanne, Berque‐Bestel, Isabelle, Eftekhari, Pierre, Gastineau, Monique, Giner, Mireille, Dahmoune, Yamina, Donzeau‐Gouge, Patrick, Hoebeke, Johan, Langlois, Michel, Sicsic, Sames, Fischmeister, Rodolphe, Lezoualc'h, Frank
• Format: Article
• Language: English
• Subjects: 5‐HT4 ligands; adenylyl cyclase; Alternative Splicing; Amino Acid Sequence; Animals; Antibody Specificity; atrial arrhythmia; benzamides; Binding, Competitive; Biological and medical sciences; CHO Cells - metabolism; Cloning, Molecular; Cricetinae; Glioma - genetics; Glioma - metabolism; G‐protein coupled receptors; Heart Atria - chemistry; Human; Humans; inverse agonism; Medical sciences; Molecular Sequence Data; Myocardium - chemistry; Neuropharmacology; Neurotransmitters. Neurotransmission. Receptors; Organ Specificity; Pharmacology. Drug treatments; Rats; Receptors, Serotonin - biosynthesis; Receptors, Serotonin - genetics; Receptors, Serotonin - isolation & purification; Receptors, Serotonin - physiology; Receptors, Serotonin, 5-HT4; Reverse Transcriptase Polymerase Chain Reaction; Serotonin Antagonists - pharmacology; Serotonin Receptor Agonists - pharmacology; serotoninergic receptors; Serotoninergic system
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0703101

RT–PCR technique was used to clone the human 5‐HT4(e) receptor (h5‐HT4(e)) from heart atrium. We showed that this h5‐HT4(e) receptor splice variant is restricted to brain and heart atrium. Recombinant h5‐HT4(e) receptor was stably expressed in CHO and C6‐glial cell lines at 347 and 88 fmol mg−1 protein, respectively. Expression of h5‐HT4(e) receptors at the cell membrane was confirmed by immunoblotting. The receptor binding profile, determined by competition with [3H]‐GR113808 of a number of 5‐HT4 ligands, was consistent with that previously reported for other 5‐HT4 receptor isoforms. Surprisingly, we found that the rank order of potencies (EC50) of 5‐HT4 agonists obtained from adenylyl cyclase functional assays was inversely correlated to their rank order of affinities (Ki) obtained from binding assays. Furthermore, EC50 values for 5‐HT, renzapride and cisapride were 2 fold lower in C6‐glial cells than in CHO cells. ML10302 and renzapride behaved like partial agonists on the h5‐HT4(e) receptor. These results are in agreement with the reported low efficacy of the these two compounds on L‐type Ca2+ currents and myocyte contractility in human atrium. A constitutive activity of the h5‐HT4(e) receptor was observed in CHO cells in the absence of any 5‐HT4 ligand and two 5‐HT4 antagonists, GR113808 and ML10375, behaved as inverse agonists. These data show that the h5‐HT4(e) receptor has a pharmacological profile which is close to the native h5‐HT4 receptor in human atrium with a functional potency which is dependent on the cellular context in which the receptor is expressed. British Journal of Pharmacology (2000) 129, 771–781; doi:10.1038/sj.bjp.0703101