Agonist-Like Activity of Antibodies Directed Against the Second Extracellular Loop of the Human Cardiac Serotonin 5-HT 4(e) Receptor in Transfected COS-7 Cells

Abstract

We have previously reported that antipeptide antibodies directed against the second extracellular loop of the cardiac h5-HT4 receptor could block the activation of the L-type Ca channel in human atrial cardiomyocytes. In this paper we investigate the immunological and physiological activity of these antibodies, in a cell system expressing a larger amount of receptors than the atrial cells. The recombinant receptor was expressed at the surface of COS-7 cells under an active form (serotonin, EC50 = 1.81 x 10(-7) M), at a high level (375 +/- 25 fmol receptor/mg total protein) and was able to bind a specific ligand (GR113808) with a high affinity (Kd = 0.28 +/- 0.05 nM). In this system, the same anti-peptide antibodies used for the cardiac cells induced an "agonist-like" effect on the recombinant h5-HT4 receptor. These results are in line with those shown for others G-protein coupled receptors, as adrenoreceptors. In addition, this work showed that the effect of the antibodies is not only dependent on the epitopic region recognised but also on the molecular density and/or the cellular environment of the target receptors. Finally, our results support the hypothesis that the h5-HT4 receptor could be a new target for autoantibodies in patients with atrial arrhythmia.

Polyclonal Antibody Effects on the Human Cardiac 5-HT4(e) Receptors Depend Upon the Expression System

“…The EC 50 value is similar to that recorded in other expression systems (Mialet et al 2000;Bozon et al 2002). Figure 1B (n = 5) shows that the h5-HT 4(c),(e) inverse agonist ML10375 (Blondel et al 1998;Mialet et al 2000) reduced basal-and 5-HT-evoked cAMP.…”

“…It may also depend on the type of tissue and/or the membrane environment (Claesen et al 2000). This study also shows that the effects of the anti-G21V antibody directed against the second extracellular loop of a h5-HT 4 receptor (agonist, Bozon et al 2002), or no effect (this study) could be dependent on the equilibrium constant J.…”

“…We conclude that the anti-G21V antibody had no functional effect alone or in presence of 5-HT upon h5-HT 4(e) receptors expressed in CHO cells. This is a surprising result given that antibodies raised against the second extracellular loop of a number of cardiac muscle GPCRs have been shown to have direct effects either as agonists (Lebesgue et al 1998;Mijares et al 2000;Bozon et al 2002) or antagonists (Sallé et al 2001). In particular, the anti-G21V antibody has been shown to functionally antagonize the 5-HT 4 receptor in human atrial myocytes in presence of 5-HT (Sallé et al 2001).…”

“…The B max for [ 3 H]GR113808 was 347 fmol/mg in CHO cells (Mialet et al 2000) and 375 fmol/mg in COS-7 cells (Bozon et al 2002). The agonist fraction (Pindon et al 2002) is determined by the fraction R * /R total corresponding to the B max of high-affinity [ 3 H]5-HT divided by the B max of [ 3 H]GR113808.…”

“…An antibody (anti-G21V) directed against the second extracellular loop of the human cardiac 5-HT 4 receptor induced agonist-like effects on h5-HT 4(e) receptors when they were expressed in COS-7 cells (Bozon et al 2002). We were then surprised to find that the same antibody (anti-G21V) showed no effect upon the same h5-HT 4(e) receptor when this was expressed in CHO cells.…”