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Physiology of Lactation

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 Advanced Drug Delivery Reviews 55 (2003) 629–641
www.elsevier.com / locate / addr

Mammary physiology and milk secretion

James L. McManaman*, Margaret C. Neville
Department of Physiology, University of Colorado Health Sciences Center, Denver, CO 80262, USA
Received 11 June 2002; accepted 22 January 2003

Abstract

The presence of drugs or other potentially toxic materials in milk is an obvious public health risk, especially to infants and
neonates. There is also increasing concern that human breast cancer is principally epigenetic in origin and results from
environmentally produced lesions. Little is known about the mechanisms by which toxic substances enter milk or mammary
tissue but knowledge of these processes is important to toxicologists and researchers involved in drug design and
metabolism. Five general pathways have been described for transport of proteins, lipids, ions, nutrients and water into milk.
Four of these pathways are transcellular, involving transport across at least two membrane barriers; the fifth is paracellular
and allows direct exchange of interstitial and milk components. Solute transport by these pathways is mediated by a diverse,
and complex array of transport and secretory processes that are regulated by hormonal, developmental, and physiological
factors. Current research is beginning to define the mechanisms underlying some of these processes, however the regulation
and coordination of solute transport mechanisms remains poorly understood. In this article we review our current
understanding of the normal solute transport and secretory processes involved in milk production, and discuss potential
regulatory mechanisms.
 2003 Elsevier Science B.V. All rights reserved.

Keywords: Differentiation; Morphogenesis; Secretion; Transport; Ions; Lipid; Glucose; Amino acids

Contents

1. Introduction ............................................................................................................................................................................ 630

2. Functional anatomy of the lactating mammary gland.................................................................................................................. 630
2.1. Cell biology of the lactating alveolar cell ........................................................................................................................... 630
2.2. Solute transport and secretion pathways ............................................................................................................................. 632
2.2.1. Exocytotic pathway (I) ........................................................................................................................................... 632
2.2.2. Lipid secretion pathway (II).................................................................................................................................... 632
2.2.3. Transcytotic pathways (III) ..................................................................................................................................... 633
2.2.4. Membrane transport pathways (IV) ......................................................................................................................... 633
2.2.4.1. Ion transport.............................................................................................................................................. 633
2.2.4.2. Glucose transport....................................................................................................................................... 633

*Corresponding author. Department of Physiology, Box C240, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Tel.: 1 1-303-315-7093; fax: 1 1-303-315-8110.
E-mail addresses: jim.mcmanaman@uchsc.edu (J.L. McManaman), peggy.neville@uchsc.edu (M.C. Neville).

0169-409X / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved.
doi:10.1016 / S0169-409X(03)00033-4
630 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641

2.2.4.3. Amino acid transport ................................................................................................................................. 633

2.2.4.4. Other agents.............................................................................................................................................. 633
2.2.5. Paracellular transport pathway (V) .......................................................................................................................... 634
3. Mammary gland differentiation and milk secretion .................................................................................................................... 634
3.1. Morphogenesis ................................................................................................................................................................ 634
3.2. Secretory differentiation and activation .............................................................................................................................. 634
3.3. Changes in milk composition during secretory activation .................................................................................................... 635
3.4. Effects of milk stasis on secretion and milk composition ..................................................................................................... 636
4. Hormonal regulation of secretory activation and transport processes ........................................................................................... 636
4.1. Paracellular transport........................................................................................................................................................ 637
4.2. Exocytotic secretion ......................................................................................................................................................... 637
4.3. Lipid secretion ................................................................................................................................................................. 637
4.4. Monovalent ion transport .................................................................................................................................................. 637
4.5. Glucose transport ............................................................................................................................................................. 638
4.6. Amino acid transport ........................................................................................................................................................ 638
5. Conclusions ............................................................................................................................................................................ 638
Acknowledgements ...................................................................................................................................................................... 639
References .................................................................................................................................................................................. 639

1. Introduction physiological factors that affect solute transport and

secretion processes.
Milk is a complex mixture whose composition
reflects the activities of distinct secretion and trans-
port processes of the mammary gland and mirrors the 2. Functional anatomy of the lactating
differing nutritional requirements of mammalian mammary gland
neonates. Milk also can be a source of drugs, dietary
metabolites and xenobiotic agents that can possibly The lactating mammary gland is composed of a
harm newborns [1]. The entry of such substances branching network of ducts formed of epithelial cells
into milk is likely to be mediated by the same ending in extensive lobulo-alveolar (LA) clusters that
transport and secretory pathways used by common are the sites of milk secretion (Fig. 1). A single layer
milk solutes, and influenced by the metabolic and of polarized secretory epithelial cells surrounds each
physiological properties of the mammary gland. At alveolus within these clusters. In turn the alveoli are
present there is little detailed information about the surrounded by myoepithelial cells that function in
mechanisms and factors regulating entry of drugs milk ejection, and a vascularized connective-tissue
and xenobiotic agents into milk. Drugs also have the stroma that contains lipid-depleted adipocytes and
potential to alter transport processes in the mammary fibroblasts. Ejection of milk from alveoli and ducts
gland through direct effects or by altering metabo- requires contraction of myoepithelial cells, stimu-
lism or normal developmental processes [2]. In lated by oxytocin released from the posterior pitui-
addition, mammary tissue and milk possess enzymes tary as part of a suckling induced neuroendocrine
capable of activating drugs and xenobiotic agents reflex.
[3–5], and there is increasing recognition that endog-
enous bioactivation processes may contribute to 2.1. Cell biology of the lactating alveolar cell
breast cancer and other diseases through formation of
genotoxic agents in breast tissue and milk [5,6]. The As expected for highly active secretory cells, the
aim of this chapter is to lay the foundation for cytoplasm of lactating alveolar cells is filled with
understanding the origin and transport of drugs and numerous mitochondria and an extensive rough
xenobiotic agents in milk by providing an overview endoplasmic reticulum network. In addition there is a
of the cell biology and physiology of milk formation well-developed Golgi apparatus, and secretory vesi-
and secretion, and by discussing our current under- cles containing casein micelles are present in the
standing of the normal developmental, hormonal and apical region of the cell [7]. Swelling of trans-most
 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641 631

Fig. 1. Diagram of mammary alveolus and alveolar epithelial cell showing pathways for milk secretion. Milk is secreted by alveolar
epithelial cells into the lumen (arrows). It is then expressed through the ducts by contraction of myoepitheilal cells that surround alveolar and
ductal epithelial cells. The alveolus is surrounded by a well-developed vasculature and a stroma comprising extracellular matrix components,
fibroblasts and adipocytes. The region indicated by the box is expanded to show key structural and transport properties of alveolar cells.
Pathway I depicts exocytotic secretion of milk proteins, lactose, calcium and other components of the aqueous phase of milk. Pathway II
depicts milk fat secretion with formation of cytoplasmic lipid droplets (CLDs) that move to the apical membrane to be secreted as a
membrane bound milk fat globule (MFG). Pathway III depicts vesicular transcytosis of proteins such as immunoglobulins from the
interstitial space. Pathway IV depicts transporters for the direct movement of monovalent ions, water and glucose across the apical and basal
membranes of the cell. Pathway V depicts transport through the paracellular pathway for plasma components and leukocytes. Pathway V is
open only during pregnancy, involution and in inflammatory states such as mastitis. Abbreviations: SV, secretory vesicle; RER, rough
endoplasmic reticulum; BM, basement membrane; N, nucleus; PC, plasma cell; FDA, fat depleted adipocyte; JC, junctional complex
containing the tight and adherens junctions; GJ, gap junction; ME, myoepithelial cell. Redrawn from Ref. [82] with permission.

elements of the Golgi network results from the
osmotic influx of water due to the high concen-
trations of lactose synthesized within this compart-
ment [8]. Lactating alveolar cells have well-de-
veloped synthetic capabilities for lipid and possess a
unique mechanism for lipid secretion [9] that results
in the secretion of triglyceride droplets surrounded
by a specialized milk fat droplet membrane [7,9].
Epithelial cells are connected to each other through
an apical junctional complex composed of adherens-
and tight-junctional elements [10,11] that function to
inhibit direct paracellular exchange of substances
between vascular and milk compartments during
lactation. The basal side of alveolar epithelial cells
contacts myoepithelial cells and the basement mem-
brane, which separates the epithelial compartment
from the stroma and the vascular system. Thus, there
are a number of potential barriers to the transfer of
632 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641
exogenous substances from blood or stromal cells to
milk: (1) vascular or stromal membranes; (2) the
basement membrane; (3) basal epithelial membranes,
(4) paracellular junctional complexes, (5) Golgi
membranes and (6) apical epithelial membranes.
2.2. Solute transport and secretion pathways
Solutes can enter milk through both transcellular
and paracellular routes that can be divided into five
general pathways (Fig. 1). Two pathways exist for
secretion of endogenously generated substances:
aqueous solutes including the major milk proteins,
oligosaccharides, and nutrients such as lactose, cit-
rate, phosphate and calcium are secreted through an
exocytotic pathway (pathway I) that is similar to
exocytotic pathways found in other cell types [12].
Lipids (primarily triglycerides) and lipid-associated
proteins are secreted by a process unique to mam-
mary epithelial cells (pathway II) [9]. Two general
transcellular pathways also exist for transport of
exogenous substances to milk. The transcytosis
pathway (pathway III) transports a wide range of
macromolecular substances derived from serum or
stromal cells, including: serum proteins such as
immunoglobulins, albumin and transferrin [13–15];
endocrine hormones such as insulin, prolactin and
estrogen [14,16]; and stromal derived agents such as
secretory immunoglobulin A (sIgA), cytokines and
lipoprotein lipase [17]. In addition, various mem-
brane transport pathways (pathway IV) exist for the
transfer of ions and small molecules, such as glu-
cose, amino acids, and water across basal and apical
plasma membranes [18]. Finally, there is a paracellu-
lar pathway (pathway V) that provides a direct route
for entry of serum and interstitial substances into
milk [11]. Transport through these pathways is
affected by the functional state of the mammary
gland and regulated by direct and indirect actions of
hormones and growth factors. The general cellular
and physiological properties of solute transport
mechanisms comprising these pathways are summa-
rized below.
2.2.1. Exocytotic pathway ( I)
The exocytotic secretion pathway is the primary
mechanism for protein secretion by alveolar cells as
well as for secretion of water, lactose, oligosac-
charides, phosphate, calcium and citrate. Like ex-
ocytotic secretion mechanisms found in other cells
these substances are packaged into secretory vesicles
within the Golgi that are then transported to the
apical region of the cells, where the vesicles fuse
with the apical plasma membrane discharging their
contents into the extracellular space. Unique features
of this pathway in alveolar cells are the presence of
high concentrations of lactose, phosphate, calcium
and citrate (in non-rodent species) within the vesi-
cles. Lactose is synthesized from UDP-galactose and
glucose within the Golgi by lactose synthase. The
high concentrations of lactose present in the Golgi
during lactation lead to osmotic influx of water that
contributes to the fluidity of milk. Calcium originates
from the plasma, entering the alveolar cytoplasm by
a poorly defined process located at the basal mem-
brane. It is transported from the cytoplasm into
secretory vesicles by an ATP-dependent Ca 21 pump
that is found on Golgi and secretory membranes
[19,20]. Within the vesicles calcium forms large
micellar structures with casein and also complexes
with phosphate and citrate to effectively reduce free
Ca 21 levels. The phosphate in secretory vesicles may
be partly derived from the hydrolysis of UDP-galac-
tose during the synthesis of lactose. Citrate is
thought to be generated endogenously within the
cytoplasm of the alveolar cell and transported into
the Golgi lumen by a currently undefined process.
2.2.2. Lipid secretion pathway ( II)
Mammary epithelial cells of most species have
well developed lipid synthetic, storage and secretion
capabilities. Milk lipids, primarily triacylgycerides
and phospholipids, are synthesized in the smooth
endoplasmic reticulum in the basal region of the cell
from precursor fatty acids and glycerol. Newly
synthesized lipid molecules form into small protein
coated storage structures called lipid bodies or
cytoplasmic lipid droplets that coalesce and are
transported to the apical plasma membrane where
they are secreted by a unique budding process [9], as
membrane enveloped structures called milk fat
globules (MFGs). This process occasionally results
in the inclusion of a crescent of cytoplasm within the
membrane-bound globule [21] enabling any sub-
stance contained in the cytoplasm to enter milk. The
milk fat globule is a major neonatal energy source in
 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641
most, if not all species, and the membrane surround-
ing these structures is a source of phospholipids and
prevents fat globules from coalescing into large fat
droplets that might be difficult to expel during
lactation. From the standpoint of potential drug
metabolism within milk, the milk fat globule mem-
brane is known to contain numerous enzymes,
including oxidases, reductases and hydrolases with
relatively high specific activities [22]. In particular,
milk fat globule membranes are highly enriched in
the purine oxidizing enzyme xanthine oxidoreductase
(XOR) [22]. XOR has broad substrate specificity and
is a potent oxygen radical generator [23], and recent
studies have demonstrated its ability to metabolize
xenobiotic agents, including activation of cancer
chemotherapeutics [24].
2.2.3. Transcytotic pathways ( III)
Transcytotic pathways for transport of proteins
and other macromolecules are active in a variety of
epithelial cell types including mammary alveolar
cells [13,15]. These pathways involve endocytic
uptake of substances at the basal membrane, forma-
tion and maturation of endosomes, and sorting to
lysosomes for degradation, or to the apical recycling
compartment for exocytosis at the apical membrane
[15]. Although information about specific trans-
cytosis of substances by mammary alveolar cells is
limited, transcytotic secretion of immunoglobulin A
in rabbit mammary glands has been shown to occur
by a pathway similar to the one outlined above [25].
In addition, limited prolactin and transferrin trans-
cytosis have been detected [14] and transfer of
labeled low-density lipoprotein (LDL) from blood to
milk has been reported [26]. Considering that xeno-
biotic agents, including carcinogens and some drugs,
can bind to and be transported by lipoproteins [27],
the transcytotic pathway is a potentially important
route for transfer of these agents to milk.
2.2.4. Membrane transport pathways ( IV)
This category of transport pathways is composed
of several distinct, solute-specific, mechanisms for
transport of monovalent and polyvalent ions and
small molecules such as glucose and amino acids.
Obviously, transcellular transfer of these substances
from blood to milk by these pathways requires the
presence of specific transporters at the basal and
633
apical plasma membranes, or at the basal plasma
membrane and Golgi or secretory membranes. Trans-
port mechanisms for various small molecules have
been identified on these membranes and are summa-
rized below.
2.2.4.1. Ion transport
Transporters or channels for sodium, potassium
and chloride have been identified on the basal and
apical plasma membranes of alveolar cells [18]. In
contrast, calcium, phosphate and iodide transporters
appear to be limited to the basal membrane [28,29].
As indicated above, calcium pump activity has been
demonstrated on Golgi and secretory vesicle mem-
branes, however it is unclear if these membranes
possess transport mechanisms for phosphate or
iodide.
2.2.4.2. Glucose transport
Glucose transport systems have been detected in
the mammary gland at both the apical and basal
plasma membrane, and on Golgi and secretory
vesicle membranes. Two distinct glucose transport
mechanisms have been identified in the mammary
gland; a GLUT1 transporter mechanism [30,31] and
a sodium dependent glucose transporter [32].
GLUT1 appears to mediate glucose transport at the
basal and Golgi membranes, but data from immuno-
fluorescence and 2-deoxyglucose transport studies
suggest that it is does not contribute to glucose
transport at the apical membrane.
2.2.4.3. Amino acid transport
Both sodium-dependent and sodium independent
amino acid transport mechanisms analogous to those
found in other organs have been demonstrated at the
basolateral component of the mammary epithelium
[18,33]. It is unclear at present if apical membranes
have similar transport mechanisms for amino acids,
and it is unknown how amino acids get into milk. It
is also uncertain if the mammary gland has special-
ized systems for transport of intact peptides.
2.2.4.4. Other agents
The presence of higher than expected concen-
trations of certain drugs in milk have raised the
possibility that alveolar cells may have active trans-
port mechanisms for such compounds. In support of
634 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641
this possibility Kari et al. [34] and Toddywalla et al.
[35] demonstrated active transport of the antibiotic
nitrofurantoin across the mammary epithelium in
lactating rats and in cultured mammary epithelial
cells, thus suggesting that membrane transport mech-
anisms may play an important role in the transport of
water-soluble chemicals into milk.
2.2.5. Paracellular transport pathway ( V)
Transport through the paracellular pathway ac-
counts for direct, bi-directional, extracellular move-
ment of both low-molecular-weight substances and
macromolecular solutes between the alveolar lumen
and the interstitial space during pregnancy or with
mastitis or after involution. This pathway is closed
during lactation in humans and most other species
(in the rabbit tight-junction closure may not be
complete) by the presence of very tight tight-junc-
tions between epithelial cells, with the result that
only the transcellular transport pathways (I–IV)
function to transfer solutes to milk.
3. Mammary gland differentiation and milk
secretion
The ability to secrete milk develops during preg-
nancy when the mammary gland is transformed from
a simple ductal tree to a highly efficient exocrine
organ with expansive lobular-alveolar structures.
This transformation is hormonally regulated and
involves changes in both the cellular composition of
the mammary gland and the structural, cellular and
biochemical properties of alveolar cells that are
critical to development of efficient solute transport
and secretory functions.
3.1. Morphogenesis
There is marked proliferation of ductal and alveo-
lar epithelial cells during pregnancy. Studies in mice
showed that ductal cell proliferation, resulting in
formation of side-branches, peaks within the first 5
days of pregnancy [36]. Alveolar cell proliferation,
however, remains elevated from day 6 through day
15 of pregnancy [36]. During this period there is a
marked increase in the number of lobular-alveolar
structures and the loss of the fat pad, implying a
progressive reorganization of the mammary gland
away from a predominately adipose organ to one
with secretory epithelial functions. Gene knockout
experiments have demonstrated that formation of
lobulo-alveolar structures depends on the actions of
progesterone and prolactin receptors functioning
through the STAT5, cyclin D1 and Wnt pathways
[37–39].
3.2. Secretory differentiation and activation
Secretory differentiation of the mammary gland
(lactogenesis) begins around mid-pregnancy in most
species and has been divided into initiation and
activation phases based on differences in the com-
position of mammary secretions, gene expression,
and structural and functional properties of alveolar
cells [7,40]. The initiation phase (previously called
lactogenesis I) is characterized by increased expres-
sion of some milk protein genes and biosynthetic
enzymes, and accumulation of neutral lipid droplets
[7,40]. Alveolar cells become capable of limited
secretion of some milk components during the
initiation phase, which in humans is detected by
measurement of increased concentrations of lactose
and alpha-lactalbumin in the plasma or urine [41,42].
However, in situ analyses of mouse mammary glands
has shown that significant numbers of alveolar cells
do not express milk protein genes during pregnancy
[43], and many alveoli have little or no luminal
volume during pregnancy. Moreover, analysis of the
secretory product extracted from the breasts of
pregnant women showed an absence of casein and
markedly reduced concentrations of lactose. Thus, it
is likely that the secretion that occurs during the
initiation phase of lactogenesis is restricted to a
limited number of alveolar cells with incompletely
developed secretory mechanisms. Copious milk sec-
retion is induced during the secretory activation
phase of differentiation (previously called lac-
togenesis II) that occurs at the end of pregnancy in
rodents and ruminants, and shortly after parturition
in humans. This phase is characterized by homoge-
neous expression of milk protein genes by alveolar
cells [43], induction of additional milk protein gene
and biosynthetic enzyme expression, polarization of
organelles, expansion of mitochondria and RER,
maturation of the Golgi apparatus, and closure of
 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641 635

tight-junction complexes [7,40,44]. These changes in
the cellular and gene expression properties of alveo-
lar cells are reflected in dramatic modifications of the
solute composition of milk and increased secretory
volume (Fig. 2) and indicate coordinated maturation
of secretory mechanisms and alterations in transport
pathways.
3.3. Changes in milk composition during secretory
activation
Changes in the solute composition of milk are an
index of the relative activities of solute synthesis /
transport pathways. Three temporally distinct
changes in milk composition take place at the onset
of lactation. The earliest is a fall in sodium and
chloride and an increase in the lactose concentrations
of milk (Fig. 3). These modifications occur immedi-
ately after delivery and are largely complete by 72 h
postpartum [45]. They precede increases in milk
volume by at least 24 h, and can be explained by
closure of the tight junctions that block the paracellu-
lar pathway. Blocking this pathway prevents lactose,
made by the epithelial cells, from passing from the
lumen of the alveolus to the plasma, and sodium and
chloride from directly entering the lumen from the
interstitial space. These changes result in decreased
concentrations of sodium and chloride, and increased
concentrations of lactose in the mammary secretion.
The increased lactose concentration reflects de-
creased dilution by water entering the lumina with
the ions, rather than an increase in its secretion.
Second, there are transient increases in the rates of
secretion of sIgA and lactoferrin in the milk of
women shortly after delivery (Fig. 3). The con-
centrations of these two important protective proteins
remain high, comprising as much as 10% of milk for
the first 48 h after birth then fall rapidly after day 2,
both from dilution as milk volume secretion in-
creases and from actual decreases in their rates of
secretion, particularly of immunoglobulins. Although
both these proteins are found at high concentrations
in colostrum, they are likely to be secreted by
different mechanisms; as lactoferrin, an endogenous
protein of alveolar cells, is secreted by the exocytotic
pathway (pathway I) [46], while sIgA, a plasma

Fig. 2. The rate of secretion of milk volume and macronutrients in milk during the first 8 days postpartum. Data were derived from a study
of 12 multiparous women who weighed their infants before and after every feed for the first 7 days postpartum and gave frequent milk
samples. These data illustrate the high level of coordination required to produce milk of consistent composition during early lactation.
(Taken from Ref. [47], with permission).
636 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641
Fig. 3. Changes in the concentration of certain milk components
during the first week post-partum. (A) Time course of changes in
the lactose, chloride and sodium concentrations contrasted with
the mean milk volume transfer to the infant. Changes in these milk
components begin immediately postpartum and are complete at
least 24 h prior to the achievement of a steady state in milk
volume. As described in the text the decrease in sodium and
chloride and increase in lactose concentration reflect closure of the
tight junctions between the mammary epithelial cells. (Adapted
from Ref. [45], with permission). (B) Changes in the concen-
tration of secretory IgA and lactoferrin during the onset of
lactation in women (data replotted from Ref. [83], with permission
of the Pediatric Clinics of North America).
derived protein, is secreted by receptor mediated
transcytosis (pathway III) [25]. In addition, the peak
secretion rate of lactoferrin occurs at the same time
as that of lactose and the major milk proteins, while
sIgA secretion peaks 1 day earlier [45] (Fig. 3),
suggesting that the exocytotic and transcytosis path-
ways are regulated differently during early lactation.
The third phase occurs about 36 h postpartum and
is associated with massive and concerted increases in
milk volume and the rates of synthesis and / or
secretion of almost all the components of mature
milk including, but not limited to, lactose, protein
(mainly casein), lipid, calcium, sodium, magnesium,
potassium, citrate, glucose and free phosphate. Con-
sidering that the secretion of these substances in-
volves the actions of several distinct transport path-
ways and biosynthetic processes, such tightly
synchronized increases implies the presence of a
common activation switch and mechanisms for coor-
dinating their activities.
3.4. Effects of milk stasis on secretion and milk
composition
Incomplete milk removal is known to reduce milk
volume in both women and dairy animals [47–49].
Studies in goats showed that decreased milk yield
was due to inhibition of secretion and that acute
reduction of milk removal sharply increased con-
centrations of lactose and a-lactalbumin in blood and
sodium and serum albumin in milk [50], indicating
increased paracellular permeability. Importantly, it
was shown that chemically increasing tight-junction
permeability by isoosmotically injecting the calcium
chelators EGTA [51] or citrate [52] into the lumens
of goats also decreased milk yield, thus mature
secretory function appears to require closure of tight-
junctions and consequently closure of paracellular
transport.
4. Hormonal regulation of secretory activation
and transport processes
The fall in progesterone at parturition is generally
agreed to be required for the onset of milk secretion,
and activation of milk protein gene expression and
biosynthetic processes [40,53]. In humans it is
known that removal of the placenta, the source of
progesterone, is necessary for the initiation of milk
secretion, and it has been reported that the retention
of placental fragments with the potential to secrete
progesterone delays lactation [54]. Exogenous pro-
gesterone has been shown to prevent lactose and
lipid synthesis in mammary glands of pregnant rats
[53] and cattle [55] after removal of the ovary—the
source of progesterone in these species. Progesterone
has also been shown to suppress b-casein expression
in the rat mammary gland during pregnancy [56,57]
and the fall in progesterone levels at parturition are
linked to increased b-casein synthesis [58]. In gener-
al the mechanism by which the fall in progesterone
induces secretory activity and the extent to which
 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641
this decline affects individual secretory and transport
pathways remain largely unknown. However, it is
increasingly clear that other hormones, in particular
prolactin and cortisol [44], must be present in
appropriate concentration if a decline in progesterone
is to trigger secretory activation.
4.1. Paracellular transport
By following the appearance of 14 C-sucrose in
blood after periodic injections into mammary ducts
of pregnant animals, Nguyen et al. [44] demonstrated
that tight junction closure was triggered by a fall in
progesterone, but both glucocorticoids and activation
of the prolactin receptor by either prolactin or
placental lactogen were necessary for closure to
occur. The mechanisms by which glucocorticoids
and prolactin receptor activation affect tight-junction
permeability are unknown. However, local injections
of cortisol are also known to decrease tight junction
permeability in goats [59] and dexamethasone, a
synthetic glucocorticoid, has been shown to induce
closure of tight junctions in mammary epithelial cell
lines [60,61]. While direct effects of prolactin on
tight junction formation have not been demonstrated,
it is required for survival of alveolar cells and the
maintenance of lactation [40]. Thus, progesterone
may antagonize inductive actions of glucocorticoids,
and or prolactin, on tight junction formation and
interactions between the activities of these two
nuclear receptor hormones, and the prolactin sig-
naling pathway may be critical determinants in the
closure of these structures.
4.2. Exocytotic secretion
Surprisingly little is known about regulation of
this pathway given its prominent role in secretion of
aqueous milk components. In part, this is due to the
complex and intimate linkage between solute synth-
eses, and vesicle formation and secretion, that makes
it difficult to distinguish effects on secretion from
those on synthesis. In addition, there is a lack of
suitable cell culture or in vitro models of mammary
exocytosis. Nevertheless, prolactin appears to be
required for secretory activation in all species, and is
necessary to maintain milk yield in humans and rats
but possibly not ruminants [40]. The basis of these
637
effects, however, is uncertain and may reflect multi-
ple actions of prolactin. For example, evidence
suggests that prolactin promotes alveolar survival,
maintenance of tight-junctions and milk protein and
lactose synthesis [40,62,63]. However, there is evi-
dence that prolactin stimulates casein secretion by
activating the release of arachidonic acid in rabbit
mammary epithelial cells [64]. Disrupting the Golgi
with brefeldin A inhibited casein secretion and
arachidonic acid release, but did not affect prolactin
receptor phosphorylation or interfere with activation
of the a-casein response element [65]. Thus, the
possibility should be considered that prolactin may
activate exocytotic secretion through an arachadonic
acid mediated pathway that is distinct from the one
used to activate gene expression.
4.3. Lipid secretion
As for the exocytotic pathway, little is known
about the regulation of lipid secretion by mammary
alveolar cells due to the lack of a suitable model
system and potential difficulties distinguishing ef-
fects on biosynthesis from effects on secretion. In
particular, given the prominent role of glucose in
both de novo fatty acid synthesis and lactose syn-
thesis in non-ruminants, hormonal regulation of
glucose uptake mechanisms (see Section 4.5) may
significantly affect the amounts of both materials
available for secretion in humans. One study in rats,
however, suggests that different hormones may
regulate lactose and lipid secretion. Flint and Gard-
ner [63] found that bromocriptine depletion of
prolactin significantly decreased lactose concentra-
tion and increased lipid concentration of milk. The
basis of this difference was not defined but the
results suggest that exocytotic and lipid secretion
pathways may be under different hormonal control.
4.4. Monovalent ion transport
Prolactin is an osmoregulator in some species of
fish, birds and amphibians [66] and it has been
shown to induce a ouabain sensitive increase in K 1
and decrease in Na 1 in mammary tissue taken from
rabbits treated with bromocriptine to inhibit prolactin
release [67]. Initially this effect was attributed to
prolactin-induced increases in Na 1 –K 1 pump num-
638 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641
ber during differentiation [18], but recent studies
have shown that prolactin can increase Cl 2 transport
activity in cultured mammary epithelial cells by
JAK2 dependent phosphorylation of Na 1 –K 1 –2Cl 2
co-transporter [68]. Transport of iodide in mammary
tissue also appears to be hormonally regulated.
Iodide uptake and levels of the sodium dependent
idodide symporter (NIS) sharply increase in rat
mammary tissue at parturition [29] and similar
changes in NIS mRNA have been observed in mouse
mammary tissue between pregnancy and lactation
[69]. Prolactin and oxytocin are required to maintain
iodide transport into lactating rat mammary tissue,
but not other tissues, and prolactin has been shown
to induce expression of NIS in explant cultures of
mouse mammary glands [70]. These findings are
consistent with a prolactin-mediated induction of
NIS expression at the onset of lactation. Although
progesterone effects on NIS induction have not been
reported, the abrupt nature of the increase in NIS at
parturition raises the possibility that progesterone
may suppress NIS expression during pregnancy.
4.5. Glucose transport
Glucose, derived from plasma, is required for
lactation and serves as a substrate for several key
metabolic processes in alveolar cells, including lac-
tose synthesis, where it is an obligate precursor, fatty
acid synthesis in non-ruminants and triglyceride
esterification [71,72]. The glucose transport prop-
erties of lactating mammary tissue also indicate that
GLUT1 is a major glucose transporter [18] and
GLUT1 has been immunolocalized to the basal
membrane of alveolar cells in lactating rat mammary
glands [31]. As indicated above, because lactose is
synthesized from UDP-galactose and glucose by
lactose synthase within the Golgi apparatus, there is
also a requirement for glucose transport across Golgi
membranes and recent studies have demonstrated
co-localization of GLUT1 with the Golgi marker
b-COP in lactating mouse mammary tissue [73].
The factors regulating GLUT1 levels and glucose
transport in alveolar cells are not well understood,
but several lines of evidence suggest that membrane
targeting of GLUT1, possibly mediated by prolactin
in combination with growth hormone or other lac-
togenic agents, may be an important regulator of
function. First, glucose uptake in the mammary gland
and GLUT1 protein levels at the basal-lateral mem-
brane of mammary alveoli increase between preg-
nancy and lactation and decrease when milk secre-
tion ceases [31]. Second, GLUT1 targeting to Golgi
in alveolar cells was shown to correlate with the
onset of secretion and to be lost when secretion was
inhibited [73]. Additional experiments in cultured
mammary epithelial cells showed that targeting of
GLUT1 to Golgi complex was induced by prolactin
and cortisol treatment [74] and raise the possibility
that lactogenic hormones regulate the substrate func-
tions of glucose by affecting GLUT1 targeting.
Finally, both the GLUT1 and GLUT4 transporter
isoforms have been shown to be recruited to the
plasma membrane of adipocytes by a process that is
dependent on activation of PI3 kinase and its direct
down-stream target, the ser-threonine kinase AKT
[75,76]. Phosphorylated (active) AKT levels have
been shown to increase in mammary tissue between
pregnancy and lactation and to decrease when milk
secretion is inhibited [77], and prolactin has been
shown to activate phosphorylation of PI3 kinase and
AKT in cell culture models [78]. Thus, PI3 kinase
and AKT activation may be a common thread in the
regulation of glucose transport in adipocytes and
epithelial cells.
4.6. Amino acid transport
Using arteriovenous differences as an assay it was
shown that bromocriptine treatment reduced trans-
port of all amino acids at the basal membrane of
mammary alveolar cells, and that exogenous prolac-
tin restored transport of most, but not all, amino
acids to their normal values [79]. However, milk
stasis also reduced amino acid transport [80], thus
the effects of bromocriptine and prolactin on amino
acid transport may have been indirect, resulting from
alterations in tight junction permeability. Prolactin
was also shown to increase amino acid uptake in
cultured mammary epithelial cells, but it was unclear
if the effects were directly related to uptake or to
indirect effects on survival and metabolism [81].
5. Conclusions
Significant advances have been made in our
understanding of milk secretion and solute transport
 J.L. McManaman, M.C. Neville / Advanced Drug Delivery Reviews 55 (2003) 629–641
processes, and modern cell and molecular biological
approaches are beginning to make it possible to
define the mechanisms regulating these processes in
greater detail. Not surprisingly, current research
indicates that the regulatory mechanisms are as
interesting and diverse as milk production itself.
Although the transport of some solutes (such as
iodide) appears to be regulated at the level of
transporter expression, many of the transport and
secretory processes appear to be regulated at the
cellular or biochemical level by changes in structure
(e.g., paracellular transport), localization (e.g., glu-
cose transport) or activity (e.g., Na 1 , K 1 , Cl 2 co-
transporter).
Finally, while it is clear that developing detailed
information about individual transport mechanisms is
an important step in understanding how specific
drugs or xenobiotic agents enter milk, it is also
necessary to understand how the activities of the
various transport processes are coordinated with
metabolic processes in order to develop accurate
models of drug transport and metabolism in mam-
mary tissue. Given the complexity of milk, the
number of systems involved in solute transport, and
the potential for cross-talk between hormones and
regulatory mechanisms, understanding the mecha-
nism of this coordination may be the greatest chal-
lenge facing physiologists in the future.
Acknowledgements
Preparation of this article was supported in part by
NIH grant HD19547.
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