Susan E. Handel

Activation of exocytosis by GTP analogues in adrenal chromaffin cells revealed by patch‐clamp capacitance measurement

The role of GTP‐binding proteins in exocytosis in bovine adrenal chromaffin cells was examined using patch‐clamp capacitance measurement. Internal dialysis with the non‐hydrolysable GTP analogue guanosine 5′‐[βγ‐imido]triphosphate and xanthosine triphosphate (XTP) activated a capacitance increase. Exocytosis triggered by XTP was blocked by guanosine 5′‐[β‐thio]diphosphate (GDPβS) but Ca2+‐induced exocytosis was unaffected. The capacitance increase due to XTP could not be explained by Ca2+ mobilisation since Ins(1,4,5)P3 and caffeine did not mimic the response. Chromaffin cells appear to possess a Ca2+‐independent pathway for exocytosis that involves GTP‐binding proteins. The magnitude of the response to XTP suggested that GTP analogues stimulate both exocytosis and recruitment of secretory granules.

Annexin II (calpactin I) in the mouse mammary gland: immunolocalisation by light- and electron microscopy

SummaryTo elucidate the putative role of annexin II (calpactin I) in the secretory function of mammary tissue its immunolocalisation in the mammary gland of pregnant and lactating mice was investigated by light- and electron microscopy using the immunoperoxidase technique. A low level of fairly uniform annexin II staining was evident throughout the gland despite its mixed composition during pregnancy. In lactating tissue it was revealed that apparently mature alveoli contained a concentration of annexin II staining outlining their epithelium. The staining was localised by immuno-electron microscopy to the apical membrane of these alveolar epithelial cells and their microvillar extentions. There was also an apparent association of annexin II with vesicles of a range of sizes located near, or actually fused with, the apical membrane. Many of the small, stained vasicles could clearly be identified as casein-containing vesicle while the large vesicles were apparently associated with either casein granules or possibly lipid. The appearance of a selective concentration of annexin II in apparently actively secreting mammary epithelial cells, as revealed in this study, is consistent with a possible structural and/or functional role for this protein at the membranes participating in the secretion of protein and possibly lipid from these secretory cells.

The secretory pathway for milk protein secretion and its regulation

Following their differentiation in response to lactogenic hormones during pregnancy and after parturition, mammary epithelial cells synthesise and secrete high levels of milk proteins including the caseins, α-lactalbumin, whey acidic protein, lactoferrin and transferrin. During lactation in the mouse, the caseins are by far the major newly synthesised proteins detectable within mammary epithelial cells. The characteristics of protein secretory pathways have been studied intensively in recent years but relatively little work has been carried out on the cell biology of milk protein secretion or its regulation. Milk protein secretion is of interest due to its physiological importance, from the point of view of the study of a high-efficiency secretory pathway and because of increasing interest in the use of transgenic manipulation of mammary gland gene expression to allow modification of milk composition, including the secretion of pharmaceutical proteins. In this chapter we will review what is known about the protein secretory pathway in lactating mammary cells and focus on possible physiological sites of regulation within the secretory pathway based on our studies on isolated lactating mouse mammary acini.

Autocrine regulation of milk protein secretion

The mammary gland is an attractive target for genetic manipulation. Direct injection into the pronucleus of mammalian eggs is now a routine procedure for introduction of new gene sequences into the germline, and gene constructs containing regulatory sequences from milk protein genes allow targetting of transgene expression specifically to the lactating mammary gland. In this way, foreign genes, or modified endogenous genes, can be expressed at high levels in mammary epithelial cells and the gene product secreted in milk. This approach has been used for production of human non-milk proteins including anti-haemophilic factor IX (Clark et al., 1989) and α1 antitrypsin (Wright et al., 1991) in transgenic sheep milk, and has led to description of such animals as “bioreactors” (Hennighausen, 1990; Carver et al., 1993), such is their level and stability of production. The same approach has also been used to obtain high-level expression of heterologous milk proteins. For example, a construct containing the ovine s-lactoglobulin structural gene and its 5′ and 3′ regulatory sequences was transcribed efficiently in mammary tissue of lactating transgenic mice (Simons et al., 1987; Wilde et al., 1992).