We have previously demonstrated that extracellular ATP ([ATP]_o) elevates intracellular calcium ([Ca²⁺]_i) and stimulates calcium-activated K⁺ efflux from trophoblast cells (Clarson et al. 2001). The elevation of [Ca²⁺]_i in cytotrophoblast cells is dependent, in part, on extracellular calcium and this dependency increases during cytotrophoblast cell differentiation. ATP can elevate [Ca²⁺]_i via the ligand-gated P2X receptor family which encodes non-selective cation channels (Buell et al. 1996). Seven receptors have been cloned (P2X1-P2X7), all of which are found in human tissue with the exception of P2X6. However, expression of the P2X receptor family has not been examined in trophoblasts. Therefore the aim of this study was to examine the expression of messenger RNA encoding P2X receptors in the human placenta, isolated cytotrophoblast cells at two stages of differentiation and the BeWo choriocarcinoma cell line.

With ethical approval, cytotrophoblast cells were isolated from human term placenta and maintained in culture for 18 h (relatively undifferentiated mononuclear cells) or 66 h (differentiated multinucleate syncytiotrophoblast-like cells) (Clarson et al. 1996). In this study we investigated expression in first (pooled n = 6), second (pooled n = 5) trimester and term placentas (pooled n = 8), cytotrophoblast cells at 18 h (n = 3) and 66 h (n = 3) of culture and BeWo cells (n = 3). Gene-specific primer pairs were used in 35 cycles of RT-PCR to examine mRNA expression of the P2X receptors. Restriction enzyme digests were carried out at 37 °C for 1 h.

PCR products were obtained for all primer pairs in positive control tissue and confirmed by restriction enzyme digest. P2X1 and P2X4 were found to be expressed in all of the placental tissue and cell types studied while P2X3 and P2X5 were not expressed in any. P2X2 was present in term placenta, and the cell samples, while P2X7 was found in first trimester, term placenta, 18 h cytotrophoblast and BeWo cells.

P2X receptors encode non-selective cation channels, which are highly permeable to calcium (Buell et al. 1996). Their expression in the human placenta implicates the P2X receptors in having a functional role in the elevation of [Ca²⁺]_i, in response to stimulation with [ATP]_o. P2X1 and P2X4 are ubiquitously expressed in placental tissue and trophoblast cells. P2X2 and P2X7 show varying expression which suggests their function in regulation of [Ca²⁺]_i may alter with gestational development and cytotrophoblast cell differentiation.

This work was supported by the MRC.