Reply from Scott M. O’Grady

The study by Palmer et al. (2011) is the third paper published from our laboratory on the characterization of a human mammary epithelial (HME) cell line that we immortalized by expression of the catalytic subunit of the human telomerase gene (Kim et al. 2002). In our previous study addressing the ion transport properties of these cells (Lee et al. 2007) we showed that the Na+ transport phenotype, P2Y receptor expression profile and lack of anion secretion observed in primary cells matched the results obtained from parallel studies conducted with immortalized HME cells. These findings stand in contrast to isolated mouse and bovine mammary epithelial cells that exhibit both ENaC-dependent Na+ transport and secretagogue-stimulated anion secretion (Blaug et al. 2001, 2003; Schmidt et al. 2001; Quesnell et al. 2007). In response to Dr Shennan's first comment on the origin of HME cells, as stated in our earlier study (Lee et al. 2007), we do not have conclusive proof that the cells originate from the duct. However, HME cells were obtained from a 51-year-old woman, an age where the process of lobular involution significantly reduces the number of secretory alveoli associated with the terminal portions of the ducts (Radisky & Hartmann, 2009). Moreover, lobular alveoli are thought to be the anatomic structures that give rise to breast cancer and in human mammary epithelial cancer cells (MCF-7 cells), purinergic stimulation increases CFTR-dependent anion transport (Flezar & Heisler, 1993). In contrast, HME cells do not exhibit anion secretion in response to purinergic receptor stimulation or to increases in intracellular Ca2+ or cAMP, and CFTR expression is extremely low; thus HME cells do not possess transport characteristics that are typically associated with fluid-secreting epithelia. However, ENaC-dependent Na+ absorption is characteristic of duct epithelial cells found in other exocrine glands such as salivary glands and sweat ducts (Cook et al. 2002; Reddy & Quinton, 2005). With respect to the second comment raised in Dr Shennan's letter, we would like to point out that the experiments described in Palmer et al. (2011) and Lee et al. (2007) were conducted with HME cells grown in media containing hydrocortisone, insulin, epidermal growth factor and bovine pituitary extract as a source for lactogenic hormones. Information about the composition of mammary epithelial growth media (MEGM) is explicitly stated in the Materials section of the manuscript. Our results showing that stimulation with ATP or UTP produces mobilization of intracellular Ca2+ is entirely consistent with what is known about P2Y receptor signalling pathways and is similar to the results obtained with ATP stimulation of MCF-7 cells (Flezar & Heisler, 1993). We suggest that the lack of Ca2+ mobilization following ATP stimulation in lactating mouse acini and bovine mammary cells referenced by Dr Shennan may reflect species differences that could be related to distinct P2Y receptor expression patterns and/or second messenger coupling. As for Dr Shenann's third comment about the previous studies by Linzell and Peaker (Linzell & Peaker, 1971a,b;), it is certainly possible that the transport characteristics of HME cells may not reflect the in vivo ion permeability properties of the duct epithelium. However, it is equally possible that the results obtained by Linzell and Peaker in goats do not necessarily represent the permeability characteristics of human duct epithelium. A possible application of the results obtained from our studies that might provide added insight about the permeability properties of human duct epithelium would be to use ENaC and KCa3.1 channel expression as possible biomarkers for duct epithelial cells. Future experiments could then employ immunohistochemical techniques using sections of human breast tissue to investigate the cellular distribution of these channels, which could be helpful in clarifying the role of the duct in determining the ionic composition of milk. The fourth comment in Dr Shennan's letter suggests an interesting experiment that we have not performed. It would not be surprising that lowering the ionic strength of the apical solution would produce an increase in transepithelial resistance since transcellular ion movements would be reduced under these conditions. However, there may also be effects on expression or activity of membrane transporters, or possibly the paracellular pathway, that could be important in determining the overall permeability of the duct epithelium. Therefore, we would agree that future experiments examining the effects of milk constituents such as lactose on transport function would be worthwhile and perhaps expand the utility of cultured cell models in understanding the regulation of epithelial transport mechanisms that contribute to the ionic composition of milk.