Pregnancy-enhanced Ca2+ responses to ATP in uterine artery endothelial cells is due to greater capacitative Ca2+ entry rather than altered receptor coupling

Uterine artery endothelial cells (UAEC) derived from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes retain pregnancy-specific differences in cell signaling as well as vasodilator production through passage 4. In particular, when P- and NP-UAEC are stimulated with ATP over a 2.5 min recording period, they exhibit similar initial transient peaks in the intracellular free Ca(2+) concentration ([Ca(2+)](i)), but the P-UAEC show a heightened sustained phase. In order to establish whether this was due to an altered subclass of purinergic receptor (P2), both the dose dependency of [Ca(2+)](i) responses to ADP and UTP and the profile of purinergic receptor expression are determined in NP- and P-UAEC. Our findings indicate that while several isoforms of P2X and P2Y receptors are present, it is P2Y2 that is responsible for the ATP-induced initial transient peak in both cell types. We also characterized several key components of the ATP-induced Ca(2+) signaling cascade, including the inositol 1,4,5-trisphosphate receptor and G-proteins, but could not confirm any pregnancy-specific variation in the protein expression that correlated with pregnancy-specific differences in prolonged Ca(2+) signaling. We thus investigated whether such a difference may be inherent to the cell itself rather than specific to the purinergic receptor-signaling pathway. Using thapsigargin (Tg), we were able to demonstrate that the initial Tg-sensitive intracellular pool of Ca(2+)is nearly identical with the capacity in both cell types, but the P-UAEC is nonetheless capable of greater capacitative Ca(2+) entry (CCE) than NP-UAEC. Furthermore, CCE induced by Tg could be dramatically inhibited by 2-aminoethoxydiphenyl borate, suggesting a role for store-operated channels in the ATP-induced [Ca(2+)](i) response. We conclude that changes at the level of capacitative entry mechanisms rather than switching of receptor subtype or coupling to phospholipase C underlies pregnancy adaptation of UAEC at the level of Ca(2+)signaling.