Background and purpose
Little is known about P2Y receptors in cardiac fibroblasts, which represent the predominant cell type in the heart and differentiate into myofibroblasts under certain conditions. Therefore, we have characterized the phenotype of the cells and the different P2Y receptors at the expression and functional levels in neonatal rat non-cardiomyocytes. Experimental approach: Non-cardiomyocyte phenotype was determined by confocal microscopy by using discoidin domain receptor 2, alpha-actin and desmin antibodies. P2Y receptor expression was investigated by reverse transcription-polymerase chain reaction and immunocytochemistry, and receptor function by cAMP and inositol phosphate (IP) accumulation induced by adenine or uracil nucleotides in the presence or absence of selective antagonists of P2Y(1) (MRS 2179, 2-deoxy-N(6)-methyl adenosine 3',5'-diphosphate diammonium salt), P2Y(6) (MRS 2578) and P2Y(11) (NF 157, 8,8'-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-fluoro-3,1-phenylene)carbonylimino]]bis-1,3,5-naphthalene trisulphonic acid hexasodium salt) receptors. G(i/o) and G(q/11) pathways were evaluated by using Pertussis toxin and YM-254890 respectively. Key
Purpose
Little is known about P2Y receptors in cardiac fibroblasts, which represent the predominant cell type in the heart and differentiate into myofibroblasts under certain conditions. Therefore, we have characterized the phenotype of the cells and the different P2Y receptors at the expression and functional levels in neonatal rat non-cardiomyocytes. Experimental approach: Non-cardiomyocyte phenotype was determined by confocal microscopy by using discoidin domain receptor 2, alpha-actin and desmin antibodies. P2Y receptor expression was investigated by reverse transcription-polymerase chain reaction and immunocytochemistry, and receptor function by cAMP and inositol phosphate (IP) accumulation induced by adenine or uracil nucleotides in the presence or absence of selective antagonists of P2Y(1) (MRS 2179, 2-deoxy-N(6)-methyl adenosine 3',5'-diphosphate diammonium salt), P2Y(6) (MRS 2578) and P2Y(11) (NF 157, 8,8'-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-fluoro-3,1-phenylene)carbonylimino]]bis-1,3,5-naphthalene trisulphonic acid hexasodium salt) receptors. G(i/o) and G(q/11) pathways were evaluated by using Pertussis toxin and YM-254890 respectively. Key
Results
The cells (>95%) were alpha-actin and discoidin domain receptor 2-positive and desmin-negative. P2Y(1), P2Y(2), P2Y(4), P2Y(6) were detected by reverse transcription-polymerase chain reaction and immunocytochemistry, and P2Y(11)-like receptors at protein level. All di- or tri-phosphate nucleotides stimulated IP production in an YM-254890-sensitive manner. AMP, ADPbetaS, ATP and ATPgammaS increased cAMP accumulation, whereas UDP and UTP inhibited cAMP response, which was abolished by Pertussis toxin. MRS 2179 and NF 157 inhibited ADPbetaS-induced IP production. MRS 2578 blocked UDP- and UTP-mediated IP responses.