Pharmacological and molecular characterization of functional P2 receptors in rat embryonic cardiomyocytes
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Purinergic receptors activated by extracellular nucleotides (adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP)) are well known to exert physiological effects on the cardiovascular system, whether nucleotides participate functionally in embryonic heart development is not clear. The responsiveness of embryonic cardiomyocytes (E) 12 to P2 receptor agonists by measuring Ca2+ influx did not present response to ATP, but responses to P2 agonists were detected in cardiomyocytes taken from E14 and E18 rats. Photometry revealed that the responses to ATP were concentration-dependent with an EC50 of 1.32 μM and 0.18 μM for E14 and E18 cardiomyocytes, respectively. In addition, other P2 agonists were also able to induce Ca2+ mobilization. RT-PCR showed the presence of P2X2 and P2X4 receptor transcripts on E14 cardiomyocytes with a lower expression of P2X3 and P2X7 receptors. P2X1 and a low level of P2X5 receptor messenger RNA (mRNA) were also expressed at E18. Immunofluorescence data indicated that only P2X2 and P2X4 receptor proteins were expressed in E14 cardiomyocytes while protein for all the P2X receptor subtypes was expressed in E18, except for P2X3 and P2X6. Responses mediated by agonists specific for P2Y receptors subtypes showed that P2Y receptors (P2Y1, P2Y2, P2Y4 and P2Y6) were also present in both E14 and E18 cardiomyocytes. Dye transfer experiments showed that ATP induces coupling of cells at E12, but this response is decreased at E14 and lost at E18. Conversely, UTP induced coupling with five or more cells in most cells from E12 to E18. Our results show that specific P2 receptor subtypes are present in embryonic rat cardiomyocytes, including P2X7 and P2Y4 receptors that have not been identified in adult rat cardiomyocytes. The responsiveness to ATP stimulation even before birth, suggests that ATP may be an important messenger in embryonic as well as in adult hearts.
KeywordsATP P2X receptors P2Y receptors P1 receptors Rat embryo Heart Cardiomyocyte FLIPR Calcium mobilization
Fluorometric imaging plate reader
The authors would like to thank Dr. Chrystalla Orphanides and Dr. Gillian E. Knight for excellent editorial assistance. We also thank Sam Ranasinghe for technical assistance on FLIPR. Dr. Coutinho-Silva was supported by a fellowship from Wellcome Trust. This work was supported by funds from the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico do Brasil (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Programa de Núcleos de Excelência (PRONEX) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
Conflict of interest
The authors claim no conflict of interest.
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