ATP-evoked increase in intracellular calcium via the P2Y receptor in proliferating bovine trophoblast cells

Bovine trophoblasts actively proliferate to elongate blastocysts before implantation. The trophoblast at this stage secretes cytokines and starts to differentiate into an endocrine cell (binucleate cell) for successful pregnancy. Intracellular calcium (Ca²⁺]_i) may act as a second messenger in the trophoblast response. In this study, we investigated Ca²⁺]_i signals in a bovine trophoblast cell line (BT-1) using fura-2 fluorescence. We found that an application of ATP (1 M) induced a transient increase in Ca²⁺]_i in BT-1 cells. The ATP-induced increase was not affected by the removal of extracellular Ca²⁺, but was suppressed by suramin (100 M), an antagonist of P2 receptors. Pretreatment with pertussis toxin (0.1 or 1 g/ml) partially inhibited the response to ATP. The order of potency to increase Ca²⁺]_i was ATP=UTP>ADP. ATP-induced Ca²⁺]_i responses preferentially occurred in cells at the periphery of the colony. The reduced responses at the center of the colony were associated with an increase in cell density and decrease in bromodeoxyuridine incorporation. These results indicated that ATP stimulated P2Y receptors coupled to pertussis toxin-sensitive and -insensitive G proteins, leading to an increase in Ca²⁺]_i as a result of release of Ca²⁺ from intracellular stores in BT-1 cells. The occurrence of ATP-induced Ca²⁺]_i signals depended on the cell confluence and reflected the high proliferative activity of the trophoblast cell population.This work was supported by grants from the Bio-oriented Technology Research Advancement Institution (BRAIN), and the Organized Research Combination System in the Science and Technology Agency of Japan. H.N. is a domestic research fellow supported by Japan Society for the Promotion of Science. A.S. is supported by a post-doctoral fellowship from the Japan Science and Technology Corporation.