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内容記述 |
Mammary myoepithelial cells in mouse lactating glands were isolated and cultured to characterize the intracellular calcium (Cai2+) signalling leading to contraction. The Cai2+ was measured using the ratio of fura-2 fluorescence (F340/F360) with an image analysis system, and the contraction was simultaneously monitored by the changes of fluorescence intensity (F360).<br /> The first step of this study was to establish an isolation method of myoepithelial cells from the mammary gland. The new isolation method consisted of (1) the removal of connective tissue around the alveoli by the Dispase-collagenase method, (2) the dissociation of myo- and secretory epithelial cells from the alveoli by pipetting in 0.02% EDTA phostphate-buffered saline (PBS), and (3) the isolation of myoepithelial cells by Percoll density gradient centrifugation. Isolated myoepithelial cells were clearly distinguished from secretory epithelial cells by morphological and physiological characteristics.<br /> Cultured myoepithelial cells assumed irregular spindle shapes and contracted well in response to oxytocin. F-actin stained with NBD-phallacidin was observed in dense bundles along the longitudinal axis in the cytoplasm. At the ultrastructual level, abundant bundles of microfilaments were prominent in the cytoplasm. In myoepithelial cells thin and thick filaments existed but not arranged in regular arrays in contrast to skeletal and cardiac muscles. Caveolae, the invagination of plasma membrane, were arranged in rows parallel to bundles of microfilaments, and associated with smooth endoplasmic reticulum. The immunofluorescence pattern of caveolin-1, a principal component protein of caveolae, was found to correspond to caveolae. Thus, using the new method he could isolate and culture myoepithelial cells that responded to oxytocin. Cultured myoepithelial cells showed well organized contractile features like smooth muscle cells.<br /> Oxytocin (≧0.1 nM) induced an increase in Cai2+(F340/F360) and contraction monitored by F360 in the central region of the cells. The increase in Cai2+ was transient even in the presence of oxytocin, while contraction followed the increase in Cai2+ and was persistent. The oxytocin-induced Cai2+ increase in the Ca2+ -free solution (0.5 mM EGTA) was nearly identical to that in normal Ringer's solution. The result suggests that oxytocin increases Cai2+ by releasing Ca2+ from intracellular stores and thereby induces contraction. The following results are consistent with intracellular stores being the primary source of Ca2+ for oxytocin-induced Cai2+ increase: (1)Nifedipine (1O μM) did not affect the oxytocin-induced increase in Cai2+, and (2) oxytocin did not induce the increase in Cai2+ after an application of thapsigargin (1 μM), an inhibitor of endoplasmic reticulum Ca2+-ATPase. Caffeine (1 or 10 mM), an activator of Ca2+-induced Ca2+ release (CICR), did not induce the change in Cai2+, and ryanodine (10 μM) did not affect the oxytocin-induced increase in Cai2+. These results suggest that CICR is not involved in myoepithelial contraction.<br /> Various substances were examined whether they induce the Cai2+ increase and contraction in myoepithelial cells. Bradykinin (≧1O nM), acetylecholine (≧10 μM), arg-vasopressin (≧1 nM) and ATP (≧10 μM) induced the increase in Cai2+ and contraction, while noradrenaline (100 μM), histamine (100 μM), endothelin-1 (100 nM), endothelin-3 (10 nM), substance P (100 nM) and ansiotensin-II (10 μM) did not have any effects on Cai2+. The Cai2+ responses to bradykinin, acetylcholine, arg-vasopressin and ATP were not affected in the Ca2+-free solution. These results indicate that some neuroactive and vasoactive substances act on myoepithelial cells and induce the increase in Cai2+ by releasing Ca2+ from intracellular stores.<br /> He investigated effects of nucleotides on myoepithelial cells in more detail because extracellular nucleotides released from mechanically stimulated secretory epithelial cells are known to play a key role in propagating the intercellular Ca2+ wave in these cells. ATPinduced an increase in Cai2+ and contraction in about 80% of myoepithelial cells. The Cai2+ response to ATP was not affected by Ca2+ removal but was suppressed by suramin (100 μM) , an antasonist of ATP receptors. The order of potency of nucleotides to increase Cai2+ was ATP = ADP > UTP > UDP. These results suggest that ATP receptors in myoepithelial cells may be of the P2Y type.<br /> Application of ATP (1 μM) plus oxytocin (≦ 100 pM) induced the increase in Cai2+ and contraction, although each of the stimulants did not have any effect on Cai2+ at the doses. The result suggests that ATP may ensure myoepithelial cell contraction induced by oxytocin at physiological concentrations (about 50 pM).<br /> In the lactating mammary gland, secretory epithelial cells suffer dual mechanical stress from the pressure of milk accumulated in the alveoli during milk secretion and the contraction of myoepithelial cells upon milk ejection. Mechanically stressed secretory epithelial cells may provide extracellular nucleotides to myoepithelial cells and allow to initiate and potentiate oxytocin-induced milk ejection. Thus, in addition to the hormonal action of oxytocin, extracellular ATP may work as a paracrine mediator to enhance or regulate the milk ejection process. |
要旨・審査要旨 / Abstract, Screening Result (320.9 kB)
