Skip to main content

Advertisement

SpringerLink
Log in

Cloning, pharmacological characterisation and tissue distribution of a novel 5-HT4 receptor splice variant, 5-HT4(i)

  • Original Article
  • Published:
Naunyn-Schmiedeberg's Archives of Pharmacology Aims and scope Submit manuscript

Abstract

5-HT4 receptor pre-mRNA is alternatively spliced in human (h) tissue to produce several splice variants, called 5-HT4(a) to 5-HT4(h) and 5-HT4(n). Polymerase chain reaction (PCR) with primers designed to amplify both 5-HT4(a) and 5-HT4(b) amplified three additional bands in different tissues, two representing different mRNA species both encoding 5-HT4(g) and one representing mRNA for a novel splice variant named 5-HT4(i), cloned from testis and pancreas respectively. Primary and nested PCR detected both 5-HT4(g) and 5-HT4(i) in multiple tissues. Whereas 5-HT4(i), was found in all cardiovascular tissues analysed, 5-HT4(g) was mainly present in atria. However, quantitative RT-PCR indicated 5-HT4(g) expression also in cardiac ventricle. The pharmacological profiles and ability to activate adenylyl cyclase (AC) were compared between four recombinant h5-HT4 splice variants (a, b, g and i) expressed transiently and stably in HEK293 cells. Displacement of [3H]GR113808 with ten ligands revealed identical pharmacological profiles (affinity rank order: GR125487, SB207710, GR113808>SB203186>serotonin, cisapride, tropisetron>renzapride, 5-MeOT>5-CT). In transiently transfected HEK293 cells cisapride was a partial agonist compared to serotonin at 5-HT4(b), 5-HT4(g) and 5-HT4(i) receptors. In membranes from HEK293 cells stably expressing 5-HT4(g) (3,000 fmol/mg protein) or 5-HT4(i) (500 fmol/mg protein), serotonin and 5-MeOT were full agonists while cisapride was full agonist at 5-HT4(g) and partial agonist at 5-HT4(i), probably due to different receptor expression levels. At both 5-HT4(g) and 5-HT4(i), the behaviour of 5-HT4 receptor antagonists was dependent on receptor level. At high receptor levels, tropisetron and SB207710 and to a variable extent SB203186 and GR113808 displayed some partial agonist activity, whereas GR125487 and SB207266 reduced the AC activity below basal, indicating both receptors to be constitutively active. We conclude that the novel 5-HT4(i) receptor splice variant is pharmacologically indistinguishable from other 5-HT4 splice variants and that the 5-HT4(i) C-terminal tail does not influence coupling to AC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2A, B
Fig. 3A, B
Fig. 4
Fig. 5A, B
Fig. 6A, B
Fig. 7A, B
Fig. 8
Fig. 9A–C
Fig. 10

Similar content being viewed by others

References

  • Bach T, Syversveen T, Kvingedal AM, Krobert KA, Brattelid T, Kaumann AJ, Levy FO (2001) 5-HT4(a) and 5-HT4(b) receptors have nearly identical pharmacology and are both expressed in human atrium and ventricle. Naunyn-Schmiedeberg’s Arch Pharmacol 363:146–160

    Google Scholar 

  • Bender E, Pindon A, van Oers I, Zhang YB, Gommeren W, Verhasselt P, Jurzak M, Leysen J, Luyten W (2000) Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant. J Neurochem 74:478–489

    CAS  PubMed  Google Scholar 

  • Blondel O, Vandecasteele G, Gastineau M, Leclerc S, Dahmoune Y, Langlois M, Fischmeister R (1997) Molecular and functional characterization of a 5-HT4 receptor cloned from human atrium. FEBS Lett 412:465–474

    CAS  PubMed  Google Scholar 

  • Blondel O, Gastineau M, Dahmoune Y, Langlois M, Fischmeister R (1998) Cloning, expression, and pharmacology of four human 5-hydroxytryptamine4 receptor isoforms produced by alternative splicing in the carboxyl terminus. J Neurochem 70:2252–2261

    CAS  PubMed  Google Scholar 

  • Bockaert J, Hunzicker-Dunn M, Birnbaumer L (1976) Hormone-stimulated desensitization of hormone-dependent adenylyl cyclase. Dual action of luteinizing hormone on pig graafian follicle membranes. J Biol Chem 251:2653–2663

    CAS  PubMed  Google Scholar 

  • Bruheim S, Krobert KA, Andressen KW, Levy FO (2003) Unaltered agonist potency upon inducible 5-HT7(a) but not 5-HT4(b) receptor expression indicates agonist-independent association of 5-HT7(a) receptor and Gs. Receptors Channels 9:107–116

    Article  CAS  PubMed  Google Scholar 

  • Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    CAS  PubMed  Google Scholar 

  • Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108

    CAS  PubMed  Google Scholar 

  • Claeysen S, Faye P, Sebben M, Lemaire S, Bockaert J, Dumuis A (1997) Cloning and expression of human 5-HT4S receptors. Effect of receptor density on their coupling to adenylyl cyclase. Neuroreport 8:3189–3196

    CAS  PubMed  Google Scholar 

  • Claeysen S, Sebben M, Becamel C, Bockaert J, Dumuis A (1999) Novel brain-specific 5-HT4 receptor splice variants show marked constitutive activity: role of the C-terminal intracellular domain. Mol Pharmacol 55:910–920

    CAS  PubMed  Google Scholar 

  • Eglen RM (1998) 5-HT4 receptors in the brain and periphery. Springer, Berlin Heidelberg New York

  • Gerald C, Adham N, Kao HT, Olsen MA, Laz TM, Schechter LE, Bard JA, Vaysse PJ, Hartig PR, Branchek TA, Weinshank RL (1995) The 5-HT4 receptor: molecular cloning and pharmacological characterization of two splice variants. EMBO J 14:2806–2815

    CAS  PubMed  Google Scholar 

  • Kaumann AJ (1994) Do human atrial 5-HT4 receptors mediate arrhythmias? Trends Pharmacol Sci 15:451–455

    CAS  PubMed  Google Scholar 

  • Kaumann AJ, Sanders L (1998) 5-Hydroxytryptamine and human heart function: the role of 5-HT4 receptors. In: Eglen RM (ed) 5-HT4 receptors in the brain and periphery. Springer, Berlin Heidelberg New York, pp 127–148

  • Kaumann AJ, Lynham JA, Brown AM (1996) Comparison of the densities of 5-HT4 receptors, β1- and β2-adrenoceptors in human atrium: functional implications. Naunyn-Schmiedeberg’s Arch Pharmacol 353:592–595

    Google Scholar 

  • Medhurst AD, Lezoualc’h F, Fischmeister R, Middlemiss DN, Sanger GJ (2001) Quantitative mRNA analysis of five C-terminal splice variants of the human 5-HT4 receptor in the central nervous system by TaqMan real time RT-PCR. Brain Res Mol Brain Res 90:125–134

    Article  CAS  PubMed  Google Scholar 

  • Mialet J, Berque-Bestel I, Eftekhari P, Gastineau M, Giner M, Dahmoune Y, Donzeau-Gouge P, Hoebeke J, Langlois M, Sicsic S, Fischmeister R, Lezoualc’h F (2000) 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. Br J Pharmacol 129:771–781

    CAS  PubMed  Google Scholar 

  • Moniotte S, Vaerman JL, Kockx MM, Larrouy D, Langin D, Noirhomme P, Balligand JL (2001) Real-time RT-PCR for the detection of beta-adrenoceptor messenger RNAs in small human endomyocardial biopsies. J Mol Cell Cardiol 33:2121–2133

    Article  CAS  PubMed  Google Scholar 

  • Pesole G, Mignone F, Gissi C, Grillo G, Licciulli F, Liuni S (2001) Structural and functional features of eukaryotic mRNA untranslated regions. Gene 276:73–81

    Article  CAS  PubMed  Google Scholar 

  • Prins NH, Shankley NP, Welsh NJ, Briejer MR, Lefebvre RA, Akkermans LM, Schuurkes JA (2000) An improved in vitro bioassay for the study of 5-HT4 receptors in the human isolated large intestinal circular muscle. Br J Pharmacol 129:1601–1608

    CAS  PubMed  Google Scholar 

  • Salomon Y, Londos C, Rodbell M (1974) A highly sensitive adenylate cyclase assay. Anal Biochem 58:541–548

    CAS  PubMed  Google Scholar 

  • Sambrook J, Fritch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

  • Themmen AP, Hinrichs V, Birnbaumer M (1993) In situ assay of hormone-stimulated adenylyl cyclase in 96-well microtitration plates: an aide to rapid identification of transformed cell clones. J Recept Res 13:69–78

    CAS  PubMed  Google Scholar 

  • Van den Wyngaert I, Gommeren W, Verhasselt P, Jurzak M, Leysen J, Luyten W, Bender E (1997) Cloning and expression of a human serotonin 5-HT4 receptor cDNA. J Neurochem 69:1810–1819

    PubMed  Google Scholar 

  • Vilaro MT, Domenech T, Palacios JM, Mengod G (2002) Cloning and characterisation of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants. Neuropharmacology 42:60–73

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Peter Molenaar at Department of Medicine, University of Queensland, Prince Charles Hospital, Australia, is acknowledged for collection of human ventricular tissue samples. This work was supported by the Norwegian Council on Cardiovascular Diseases, the Norwegian Cancer Society, Anders Jahre’s Foundation for the Promotion of Science, The Novo Nordisk Foundation, The Family Blix foundation and grants from the Norwegian Academy of Sciences and from the University of Oslo. The experiments were performed in accordance with all regulations concerning biomedical research in Norway.

Author information

Authors and Affiliations

  1. Department of Pharmacology, University of Oslo, PO Box 1057, 0316, Blindern, Oslo, Norway

    Trond Brattelid, Kurt A. Krobert, Kjetil W. Andressen & Finn Olav Levy

  2. MSD Cardiovascular Research Center, Rikshospitalet University Hospital, 0027 , Oslo, Norway

    Trond Brattelid, Ane M. Kvingedal, Kurt A. Krobert, Kjetil W. Andressen, Trond Bach & Finn Olav Levy

  3. Institute for Surgical Research, University of Oslo, Rikshospitalet University Hospital, 0027, Oslo, Norway

    Trond Brattelid, Ane M. Kvingedal, Kurt A. Krobert, Kjetil W. Andressen, Trond Bach, Marit E. Hystad & Finn Olav Levy

  4. Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK

    Alberto J. Kaumann

Authors
  1. Trond Brattelid
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ane M. Kvingedal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kurt A. Krobert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kjetil W. Andressen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Trond Bach
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marit E. Hystad
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alberto J. Kaumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Finn Olav Levy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Finn Olav Levy.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brattelid, T., Kvingedal, A.M., Krobert, K.A. et al. Cloning, pharmacological characterisation and tissue distribution of a novel 5-HT4 receptor splice variant, 5-HT4(i) . Naunyn-Schmiedeberg's Arch Pharmacol 369, 616–628 (2004). https://doi.org/10.1007/s00210-004-0919-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00210-004-0919-4

Keywords

Search

Navigation