Activation of purinergic receptors by extracellular nucleotides raises intracellular calcium ([Ca²⁺]_i) (Burnstock, 1997). We have previously shown expression of mRNA for the purinergic P2Y receptor subtypes, P2Y2, 4 and 6 in human placenta (Roberts et al. 2001). Both UTP (P2Y2 and P2Y4 selective) and 5BrUTP (P2Y6 selective) stimulate efflux of ⁸⁶Rb from cytotrophoblast cells (Roberts et al. 2003), which is thought to occur via Ca²⁺-activated K⁺ channels. In this study we aimed: (i) to determine the effect of P2Y2, 4 or 6 receptor activation on [Ca²⁺]_i in cytotrophoblast cells; and (ii) to examine P2Y2 and 4 protein expression in human placental trophoblast.

Cytotrophoblast cells were isolated from term placenta, collected following informed consent and in accordance with local ethical guidelines, as previously described (Greenwood et al. 1996). Cells were seeded onto glass cover-slips and maintained in culture for 18hrs. For measurement of [Ca²⁺]_i cells were loaded with fura 2-AM for 30 min at 37 °C and treated for 2 min with either 10 µM UTP or 500 µM 5BrUTP. Experiments were carried out both in the presence (+Ca; 1.8 mM Ca²⁺) and absence (0Ca; no added Ca²⁺ + 0.5 mM EGTA) of extracellular Ca²⁺. The results are expressed as the peak % increase in 340:380 nm ratio above baseline. Expression of P2Y2 and P2Y4 receptor protein was assessed by Western blotting using commercially-available primary antibodies (Alomone). (P2Y6 antibody unavailable).Both UTP and 5BrUTP significantly elevated [Ca²⁺]_i in cytotrophoblast cells in +Ca and 0Ca (Fig. 1). Western blotting identified P2Y2 protein in first trimester and term placenta, and cytotrophoblast cells. P2Y4 was not detected in any placental tissue or trophoblast cell samples (n = 3).The increase in [Ca²⁺]_i with UTP and 5BrUTP in the absence of extracellular Ca²⁺ suggests the activation of P2Y2, 4 and 6 receptors, which mobilise Ca²⁺ from intracellular stores. We have been unable to detect P2Y4 protein and therefore speculate that extracellular uridine nucleotides activate P2Y2 and possibly P2Y6 receptors in the human placenta.

This work was supported by the MRC