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 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.

Aldosterone acts via an ATP autocrine/paracrine system: The Edelman ATP hypothesis revisited

“…We postulate that this purinergic activation produces the cell contraction that we have observed. A similar mechanism has been reported in myoepithelial cells (19). The contraction profoundly alters the apical membrane topography with the loss of normal microvillar architecture.…”

“…A known modulator of epithelial cell contraction is purinergic stimulation that acts by means of intracellular Ca 2ϩ elevation (19). As already discussed, others have demonstrated that hypotonic-induced sodium transport correlates with ATP release from epithelial cells into the basolateral compartment, and this acts via P2 receptors to stimulate a rise in intracellular Ca 2ϩ (8).…”

Constitutive Coexpression of Nitric Oxide Synthase-1 and Soluble Guanylyl Cyclase in Myoepithelial Cells of Mammary Glands in Mice

“…Although oxytocin is considered to be the most important trigger of contraction of MEC in response to suckling during lactation, MEC might also contract in the resting state following acetylcholine, 5-hydroxytryptamine, bradykinin, arginine-vasopressin and ATP treatment [Nakano et al, 2001]. Consequently, it seems reasonable that a counterregulation mechanism exists for the physiological reduction of the contractile tone in MEC in the lactating as well as in the resting mammary gland.…”

Pathophysiology and Therapeutic Potential of Purinergic Signaling

“…Synergistic effects of ATP and oxytocin in increasing [Ca 2ϩ ] i in mouse mammary myoepithelial cells suggest that activation of purinergic receptors may facilitate myoepithelial cell contraction in the milk ejection response (Nakano et al, 2001). P2Y 2 receptors on apical and basolateral membranes appear to be involved (Blaug et al, 2003).…”