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. 2003 Jun;23(3):379–400. doi: 10.1023/A:1023644822539

Synergistic Effects of Adenosine A1 and P2Y Receptor Stimulation on Calcium Mobilization and PKC Translocation in DDT1 MF-2 Cells

Bertil B Fredholm 1, Jean W Assender 1, Eva Irenius 1, Noriko Kodama 2, Naoaki Saito 2
PMCID: PMC11530149  PMID: 12825834

Abstract

1. The effect of adenosine analogues and of nucleotides, alone or in combination, on intracellular calcium, accumulation of inositol (1,4,5) trisphosphate (InsP3), and on activation of protein kinase C (PKC) was studied in DDT1 MF2 cells derived from a Syrian hamster myosarcoma. These cells were found to express mRNA for A1 and some as yet unidentified P2Y receptor(s).

2. Activation of either receptor type stimulated the production of InsP3 and raised intracellular calcium in DDT1 MF2 cells. Similarly, the A1 selective agonist N6-cyclopentylade- nosine (CPA) increased PKC-dependent phosphorylation of the substrate MBP4–14 and induced a PKC translocation to the plasma membrane as determined using [3H]-phorbol dibutyrate (PDBu) binding in DDT1 MF-2 cells. However, neither adenosine nor CPA induced a significant translocation of transiently transfected γ-PKC-GFP from the cytosol to the cell membrane. In contrast to adenosine analogues, ATP and UTP also caused a rapid but transient translocation of γ-PKC-GFP and activation of PKC.

3. Doses of the A1 agonist CPA and of ATP or UTP per se caused barely detectable increases in intracellular Ca2+ but when combined, they caused an almost maximal stimulation. Similarly, adenosine (0.6 μM) and UTP (or ATP, 2.5 μM), which per se caused no detectable translocation of either γ- or ε-PKC-GFP, caused when combined a very clear-cut translocation of both PKC subforms, albeit with different time courses. These results show that simultaneous activation of P2Y and adenosine A1 receptors synergistically increases Ca2+ transients and translocation of PKC in DDT1 MF-2 cells. Since adenosine is rapidly formed by breakdown of extracellular ATP, such interactions may be biologically important.

Keywords: adenosine, nucleotides, PKC, smooth muscle, phospholipase

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