5-hydroxytryptamine-4 (5-HT4) receptors have been proposed to contribute to the generation of atrial fibrillation in human atrial myocytes, but it is unclear if these receptors are present in the hearts of small laboratory animals (e.g. rat). In this study, we examined presence and functionality of 5-HT4 receptors in auricular myocytes of newborn rats and their possible involvement in regulation of gap junctional intercellular communication (GJIC, responsible for the cell-to-cell propagation of the cardiac excitation). Western-blotting assays showed that 5-HT4 receptors were present and real-time RT-PCR analysis revealed that 5-HT4b Was the predominant isoform. Serotonin (1 mu M) significantly reduced cAMP concentration unless a selective 5-HT4 inhibitor (GR113808 or ML10375, both 1 mu M) was present. Serotonin also reduced the amplitude of L-type calcium currents and influenced the strength of GJIC without modifying the phosphorylation profiles of the different channel-forming proteins or connexins (Cxs), namely Cx40, Cx43 and Cx45. GJIC was markedly increased when serotonin exposure occurred in presence of a 5-HT4 inhibitor but strongly reduced when 5-HT2A and 5-HT2B receptors were inhibited, showing that activation of these receptors antagonistically regulated GJIC. The serotoninergic response was completely abolished when 5-HT4, 5-HT2A and 5-HT2B were simultaneously inhibited. A 24 h serotonin exposure strongly reduced Cx40 expression whereas Cx45 was less affected and Cx43 still less. In conclusion, this study revealed that 5-HT4 (mainly 5-HT4b), 5-HT2A and 5-HT2B receptors coexisted in auricular myocytes of newborn rat, that 5-HT4 activation reduced cAMP concentration, I-CaL and intercellular coupling whereas 5-HT2A or 5-HT2B activation conversely enhanced GJIC. (C) 2009 Elsevier Inc. All rights reserved.