CaR-stimulated transcellular calcium transport in mouse mammary epithelial cells cultured on Matrigel. (a) Fluorescent micrograph of a section through a mammosphere incubated with NHS-LC-biotin and stained with fluorescein-tagged avidin. As one can see, NHS-LC-biotin added to the media is excluded from the lumen, documenting that there is no paracellular leak through the tight junctions between epithelial cells. Lu, lumen; am, apical membrane; bm, basolateral membrane. (b) Same as in a, except that in addition to NHS-LC-biotin, 2.5 mM EGTA was added to the media of the mammosphere cultures. In this instance, the tight junctions became leaky and the NHS-LC-biotin labeled both basolateral and apical membranes. (c) ⁴⁵Ca accumulation within the lumens of mammospheres made from WT mice (white bars) or from BLG-Cre/PTHrP^lox/– mice (black bars) cultured in 1, 5, or 10 mM CaCl₂ or in 1 mM CaCl₂ with 2.5 μM NPS S467 or NPS R467 added. Mammary epithelial cells from the BLG-Cre/PTHrP^lox/– mice did not secrete PTHrP (not shown). As can be seen, extracellular calcium stimulates the accumulation of ⁴⁵Ca in the lumen of mammospheres in a dose-dependent manner, regardless of the presence or absence of PTHrP. Likewise, stimulation of CaR signaling with NPS R467 led to a significant increase in the luminal accumulation of tracer compared with treatment with NPS S467 in both types of cells (WT, P < 0.0001; BLG-Cre/PTHrP^lox–, P < 0.05). Each bar represents the mean of three experiments; error bars represent the SEM.