[Background] We have previously demonstrated that extracellular ATP induces steroidogenesis via purinergic P2Y receptors (P2YR) in bovine adrenocortical fasciculata cells (BAFC). However, it is not known if extracellular ATP (a very hydrolyzable nucleotide) can survive long enough to act on P2YR during the long incubation period required for BAFC steroidogenesis.
[Purpose] We analyzed the initiation of ATP-induced steroidogenesis in BAFC and its ability to sustain stimulatory levels.
[Materials & Methods] BAFC were aseptically isolated from fresh bovine adrenal cortex and were cultured in Ham‘s F-12 medium supplemented with serum and antibiotics. Three-day primary cultured cells were used for the experiments. Extracellular ATP was quantified by Luciferin-Luciferase assay. Steroidogenesis was determined fluorometrically with cortisol as a standard. cDNA was constructed from BAFC RNA and used for the qPCR templates.
[Results] ATP added to the external culture solution decreased to 11.5% in an hour. The rank order of expression in P2YR subtypes in BAFC was P2Y11 > P2Y6 > P2Y14 > P2Y2 > P2Y1 > P2Y12. No ecto-ATPDase was expressed by the BAFC, but alkaline phosphatase and 5'-nucleotidase were expressed. 
[Conclusion] It is concluded that continuous extracellular ATP stimulation is required for steroidogenesis in BAFC.