Synergistic effects of ATP on oxytocin-induced intracellular Ca²⁺ response in mouse mammary myoepithelial cells

Abstract

An increase in intracellular Ca²⁺ ([Ca²⁺]_i) is essential for mammary myoepithelial cells to contract, leading to milk ejection during lactation. In this study, the intracellular signaling leading to the increase in [Ca²⁺]_i in cultured myoepithelial cells from mouse lactating mammary glands was investigated using fura-2 fluorescence ratiometry. [Ca²⁺]_i increased in cultured myoepithelial cells in response to either oxytocin (1 nM) or ATP (10 µM), and the cells then contracted. These [Ca²⁺]_i responses were diminished by treatment with an inhibitor of phospholipase C (≥1 µM U73122). Intracellular application of inositol 1,4,5-trisphosphate (IP₃: 10 or 100 µM) increased [Ca²⁺]_i. Pretreatment with pertussis toxin (PTX: 0.1 or 1 µg/ml) inhibited the [Ca²⁺]_i response to ATP, but had less of an effect on the response to oxytocin. These results indicate that oxytocin and purinergic receptors are coupled to PTX-insensitive and PTX-sensitive G proteins, respectively, and that their activation leads to the increase in [Ca²⁺]_i through the release of Ca²⁺ from IP₃-sensitive intracellular stores via the inositol-phospholipid signaling pathway. Furthermore, we found that the [Ca²⁺]_i responses to oxytocin at physiological doses (0.01–0.1 nM) were augmented in the presence of a sub-responsive dose of ATP (1 µM). The activation of purinergic receptors may facilitate myoepithelial cell contraction in milk-ejection responses.