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Uptake of choline by rat mammary-gland epithelial cells.

ABSTRACT: The neonatal mammal requires especially large amounts of choline to sustain growth. Much of this choline is derived from the newborn's only source of food, milk. The concentration of choline in rat milk [182 +/- 24 microM (S.E.M.)] was much higher than that in maternal serum (11.6 +/- 0.9 microM), suggesting that a mechanism capable of concentrating choline into milk must exist. We characterized choline uptake by mammary epithelial cells (the site of milk production) of the lactating rat. We observed two uptake processes, one saturable and obeying Michaelis-Menten kinetics, and the other non-saturable and linear. At physiological blood choline concentrations, the saturable component of choline uptake predominated. The saturable component had Kapp. = 35 +/- 16 microM, and Vmax. = 1.24 +/- 0.19 nmol/h per mg of protein. Saturable uptake of choline was inhibited by hemicholinium-3. Ca2+ was required for uptake, but Mg2+ was not. Replacement Na+ with K+, Li+ or sucrose inhibited transport. Ouabain did not inhibit choline uptake. Choline concentration in epithelial cells was 67.7 +/- 1.9 nmol/g wet wt. at the start of incubation at 37 degrees C and rose to 80.9 +/- 6.5 nmol/g wet wt. over 30 min. Much of the choline accumulated by the mammary gland (in the presence of endogenous concentrations of choline) remained in the form of choline (50 +/- 1.2%), phosphatidylcholine (12 +/- 2.3%), lysophosphatidylcholine (0.1 +/- 0.03%), betaine (7 +/- 0.3% and phosphocholine (6 +/- 0.5%). In addition, we isolated 25 +/- 1.2% of choline-derived radiolabel in an unidentified compound.

SUBMITTER: Chao CK

PROVIDER: S-EPMC1135035 | biostudies-other | 1988 Aug

REPOSITORIES: biostudies-other

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Project description:INTRODUCTION: Parity-identified mammary epithelial cells (PI-MECs) are an interesting cellular subset because they survive involution and are a presumptive target for transformation by human epidermal growth factor receptor 2 (HER2)/neu in mammary tumors. Depending on the type of assay, PI-MECs have been designated lobule-restricted progenitors or multipotent stem/progenitor cells. PI-MECs were reported to be part of the basal population of mammary epithelium based on flow cytometry. We investigated the cellular identity and lineage potential of PI-MECs in intact mammary glands. METHODS: We performed a quantitative and qualitative analysis of the contribution of PI-MECs to mammary epithelial cell lineages in pregnant and involuted mammary glands by immunohistochemistry, fluorescence-activated cells sorting (FACS), and quantitative polymerase chain reaction. PI-MECs were labeled by the activation of Whey Acidic Protein (WAP)-Cre during pregnancy that results in permanent expression of yellow fluorescent protein. RESULTS: After involution, PI-MECs are present exclusively in the luminal layer of mammary ducts. During pregnancy, PI-MECs contribute to the luminal layer but not the basal layer of alveolar lobules. Strikingly, whereas all luminal estrogen receptor (ER)-negative cells in an alveolus can be derived from PI-MECs, the alveolar ER-positive cells are unlabeled and reminiscent of Notch2-traced L cells. Notably, we observed a significant population of unlabeled alveolar progenitors that resemble PI-MECs based on transcriptional and histological analysis. CONCLUSIONS: Our demonstration that PI-MECs are luminal cells underscores that not only basal cells display multi-lineage potential in transplantation assays. However, the lineage potential of PI-MECs in unperturbed mammary glands is remarkably restricted to luminal ER-negative cells of the secretory alveolar lineage. The identification of an unlabeled but functionally similar population of luminal alveolar progenitor cells raises the question of whether PI-MECs are a unique population or the result of stochastic labeling. Interestingly, even when all luminal ER-negative cells of an alveolus are PI-MEC-derived, the basal cells and hormone-sensing cells are derived from a different source, indicating that cooperative outgrowth of cells from different lineages is common in alveologenesis.

Project description:IntroductionBreast cancer is thought to arise in mammary epithelial stem cells. There is, therefore, a large amount of interest in identifying these cells. The breast is a complex tissue consisting of two epithelial layers (an outer myoepithelial/basal layer and an inner luminal epithelial layer) as well as a large non-epithelial component (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and myocytes). The definitive identification of a mammary epithelial stem cell population is critically dependent on its purity. To date, this has been hampered by the lack of suitable markers to separate out the two epithelial layers, and to remove contaminating non-epithelial cells.MethodsMouse mammary glands were dissociated and stained with CD24. Cells were sorted into separate populations based on CD24 expression and assessed for luminal epithelial and myoepithelial/basal markers by direct fluorescent microscopy and real time PCR. The stem/progenitor potential of these cell populations was assessed in vivo by cleared mammary fat pad transplantation.ResultsThree populations of CD24 expressing cells were identified: CD24Negative, CD24Low and CD24High. Staining of these cells with cytokeratin markers revealed that these populations correspond to non-epithelial, myoepithelial/basal and luminal epithelial cells, respectively. Cell identities were confirmed by quantitative PCR. Cleared mammary fat pad transplantation of these cell populations revealed that extensive mammary fat pad repopulation capacity segregates with the CD24Low cells, whilst CD24High cells have limited repopulation capacity.ConclusionDifferential staining of mammary epithelial cells for CD24 can be used to simultaneously isolate pure populations of non-epithelial, myoepithelial/basal and luminal epithelial cells. Furthermore, mammary fat pad repopulation capacity is enriched in the CD24Low population. As separation is achieved using a single marker, it will be possible to incorporate additional markers to further subdivide these populations. This will considerably facilitate the further analysis of mammary epithelial subpopulations, whilst ensuring high purity, which is key for understanding mammary epithelial stem cells in normal tissue biology and carcinogenesis.

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Uptake of choline by rat mammary-gland epithelial cells.

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