Chris Watters' Research Interests
I'm interested in learning how calcium gets into milk. More than a decade ago my children told me the answer to this question very matter-of-factly: "Cows put it there!" Since then I've been investigating just how they (and mice and other mammals) accomplish this remarkable feat, working in collaboration with Middlebury undergraduates and colleagues at the University of Colorado Health Sciences Center, the Hannah Research Institute (Scotland) and most recently, the Ion Channel Group of the University of Vermont (UVM).
Calcium serves two very important but very different biological functions. Hydroxyapatite, an insoluble complex of calcium and phosphate, is the major component of vertebrate skeletons, and secondly, very small perturbations in cytoplasmic calcium regulate many cellular activities (1). Normal cells maintain very low calcium relative to those found in both the extracellular milieu and endoplasmic compartments, and appropriate extracellular signals rapidly release bursts of calcium into the cytoplasm, which in turn stimulate different cellular events including growth, secretion and muscle contraction. Failure to maintain these various calcium gradients leads to chronically elevated levels of cytoplasmic calcium and inevitably to cellular dysfunction and death (1). Calcium is thus an extremely important nutrient, and many animals have evolved special mechanisms to assimilate this relatively rare mineral. Mammals nourish their newborn with a milk rich in calcium.
How do the epithelial tissues responsible for absorbing calcium in neonates (and their mothers) and for secreting calcium into milk perform these functions without deregulating normal signaling pathways or in the extreme, committing suicide?
Intestinal epithelial cells maintain low calcium concentrations by transporting calcium in association with calbindin, a vitamin-D inducible calcium-binding protein, but mammary epithelia cells (MEC) apparently do not contain this protein (2, 3). In contrast, MEC may assimilate calcium from blood in numerous, small and very short-lived "waves" similar to those found in pancreatic exocrine cells (4). What then happens to these calcium waves? Considering MEC's role in lactation (the production of milk), we know: 1. calcium is secreted primarily complexed with casein, the major milk protein; 2. calcium-casein micelles are formed in the Golgi complex and in secretory vesicles (SV) as part of this protein's post-translational processing (5); and 3. cytoplasmic calcium is taken up by a calcium pump located in the Golgi and SV, which resembles one found in the sarcoplasmic reticulum (6). Given these observations, it is not unreasonable to hypothesize transient calcium waves provide the basis for loading Golgi and secretory vesicles (and casein), in a manner analogous to the loading of the sarcoplasmic reticulum stores (and calsequestrin) in muscle cells. Such waves may be generated directly by calcium channels located in the plasma membrane or indirectly through the release of intracellular, inositol triphosphate-dependent stores which would ultimately be reloaded with extracellular calcium (7). Indeed, spontaneous calcium oscillations have been reported in cultured MEC (8), but it's not clear how such changes are related to calcium uptake and secretion during lactation.
What is the cytoplasmic level of calcium in MEC and does cytoplasmic calcium change during lactation? Our own preliminary data obtained measuring the fluorescense of the calcium-fluorochrome Fura 2 suggested freshly isolated mammary cells from lactating mice exhibit cytoplasmic calcium levels similar to or slightly elevated over those found in other mammalian cells (9). These measurements, however, were made by dual-beam spectrofluorimetry on large populations of cells over periods of tens of minutes, with inadequate temporal and spatial resolution for detecting localized, transient changes in calcium concentration. Clearly they need to be repeated and refined by examining individual cells in culture with modern imaging techniques. And the proximal or acute signal(s) for lactation, which regulate calcium uptake and secretion, need to be identified.
Currently, my students and I are growing mammary secretory cells, using both primary cultures (10) and cultures of CIT3 cells, which are "immortalized" murine MEC derived from the Comma 1D line (11) whose differentiation in vitro can be manipulated hormonally (12). These cultures are then loaded with the calcium fluorochromes Fura 2 or Fluo 3 and observed by fluorescence microscopy with digital imaging and laser-scanning confocal microscopy in collaboration with Mark Nelson at UVM. Nelson's group is investigating the regulation of blood pressure, capillary diameter and smooth muscle tone, and they have recently characterized calcium "sparks" in vascular smooth muscle cells as the basis of smooth muscle relaxation (e.g., 13).
In earlier work with the Nelson group, characterizing ion channels in the plasma membranes of freshly isolated MEC and mammary acini (14), I found no evidence of voltage-stimulated calcium influx, and it's likely any calcium waves would require the opening of a ligand-gated channel. Similar patch-clamping studies of cultured MEC performed in parallel with fluorescence imaging should address this matter. We are also examining mammary myoepithelial cells for calcium waves and sparks, because these cells resemble smooth muscle cells and are responsible for the oxytocin-induced contractions that release milk from the mammary gland during suckling (15).
My students and I are also interested in characterizing the cellular basis for mammary involution, the complex process whereby weaning not only curtails milk production but leads to the widespread death of MEC. Much is now known about programmed cell death (apoptosis) in other systems, and we have observed this phenomenon in cultured MEC and are investigating what hormonal cues may be involved.
Recently, Middlebury College completed a new Science Center (Bicentennial Hall), and we moved into the center in Fall, 1999. Bicentennial Hall contains modern, climate-controlled teaching and research laboratories, and specifically, a large interior suite provided with clean, well-filtered air for advanced instruction and student research in cell culture, patch-clamping and digital imaging microscopy. I look forward to continuing these various projects in collaboration with Middlebury undergraduates in our new facilities.
1. Rasmussen, H., P. Barrett, J. Smallwood, W. Bollag, and C. Isales, 1990. Calcium ion as intracellular messenger and cellular toxin. Environm. Health Persp. 84:17-25.
2. Lindell, A. C., 1995. Analysis of mammary gland epithelial cell cytosolic Ca2+-binding proteins using the dye "Stains-All". Senior Honors Thesis, Middlebury College, 52 pp.
3. Normand, D. 1966. Intracellular calcium homeostasis in the secretory cells of mouse mammary gland tissue. Senior Honors Thesis, Middlebury College, 63 pp.
4. Thorn, P., A. M. Lawrie, P. M. Smith, D. V. Gallacher, & O. H. Petersen. 1993. Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol triphosphate. Cell. 74:661-668.
5. Neville, M. C. and C. D. Watters, 1983. The secretion of calcium into milk. J. Dairy Res. 66:371-380.
6. Watters, C. D., 1984. A calcium-stimulated adenosine triphosphatase in Golgi-enriched membranes of lactating murine mammary tissue. Biochem. J. 224:39-45.
7. Berridge, M. J., 1997. Elementary and global aspects of calcium signaling. J. Physiol. 499:291-306.
8. Furuya, K., K. Enomoto and S. Yamagishi, 1993. Spontaneous calcium oscillations and mechanically and chemically induced calcium responses in mammary epithelial cells. Pflugers Arch. 422:295-304.
9. Watters, C.D., T. J. Murphy* and J. L. Bass*, 1990. Determination of cytoplasmic calcium concentrations in secretory cells isolated from lactating murine mammary glands using Fura 2. J. Cell Biol. 111:469a.
10. Camfield, R. A., 1997. Toward optimal primary culture conditions for mouse mammary epithelial cells. Senior Honors Thesis, Middlebury College, 52 [+16] pp.
11. Danielson, K. G., C. J. Oborn, E. M. Durban, J. S. Butel, and D. Medina, 1984. Epithelial mouse mammary cell line exhibiting normal morphogenesis in vivo and functional differentiation in vitro. P.N.A.S., USA. 81:3756-3760.
12. Neville, M. C., 1996, personal communication.
13. Nelson, M. T., H. Cheng, M. Rubart, L. F. Santana, A. D. Bonev, H. J. Knot, & W. J. Lederer, 1995. Relaxation of arterial smooth muscle by calcium sparks. Science. 270:633-637.
14. Watters, C. D., J. Patlak & M. T. Nelson, 1994. Ion channels in the plasma membranes of murine mammary epithelial cells. Biophys. J. 66:A217.
15. Soloff, M. S., J. Chakraborty, P. Sadhukhan, D. Senitzer, M. Wieder, M. A. Fernstrom & P. Sweet, 1975. Purification and characterization of mammary myoepithelial and secretory cells from the lactating rat. Endocrinol. 106:887-897.