Localization of plasma membrane and secretory calcium pumps in the mammary gland

Abstract

Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely via the secretory pathway. However, recent studies suggest that a plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during development. SPCA2 levels increased over 35-fold during lactation with expression localized to luminal secretory cells, while SPCA1 increased only a modest 2-fold and was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1. Our studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation and indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.

Fig. 1

SPCA2 mRNA levels increase more than SPCA1 during murine mammary gland development. SPCA2 and SPCA1 mRNA levels during murine mammary gland development (relative to nulliparous). Bars represent means ± SEM (n = 3–4 mice) and are representative of two independent real-time RT-PCRs. The asterisk (*) denotes a significant difference (p < 0.05) compared to nulliparous gland, using a z-test based on a normal distribution.

Fig. 2

Immunolocalization of calcium ATPases in the lactating mouse mammary gland. Mammary gland sections isolated from lactating mice and immunostained for SPCA2 (A), SPCA1 (B), PMCA2 (C) or PMCA1 (D). All ATPases are shown in green with SMA (red) overlaid with the DAPI (blue) stain. (A) SPCA2. White arrows show cells which do not express SPCA2 outside the acini. (B) SPCA1. White arrows show cells not part of secretory acini that express SPCA1. (C) PMCA2. White arrows show cells which do not express PMCA2 outside the acini. (D) PMCA1. White arrows show cells not part of secretory acini that express PMCA1. Scale bars represent 10 μm in all panels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 3

Localisation of PMCA1 and PMCA2 in SCp2 cells grown in 3D culture. (A) A representative acinus showing staining for PMCA1 (green), α6 integrin (red), and DAPI (blue). (B) The corresponding z–x plane through the acinus described in A. (C) A representative acinus showing staining for PMCA2 (green), a6 integrin (red), and DAPI (blue). (D) The corresponding z–x plane through the acinus described in C. Scale bars represent 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)