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 microM), and the cells then contracted. These [Ca2+]i responses were diminished by treatment with an inhibitor of phospholipase C (> or = 1 microM U73122). Intracellular application of inositol 1,4,5-trisphosphate (IP3: 10 or 100 microM) increased [Ca2+]i. Pretreatment with pertussis toxin (PTX: 0.1 or 1 microgram/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 microM). The activation of purinergic receptors may facilitate myoepithelial cell contraction in milk-ejection responses.