PHYSIOLOGICAL REVIEWS
Vol. 80, No. 3, July 2000
Printed in U.S.A.
Transport of Milk Constituents by the Mammary Gland
D. B. SHENNAN AND M. PEAKER
Hannah Research Institute, Ayr, Scotland
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I. Introduction
II. Experimental Methods Used to Study Mammary Gland Solute Transport
III. Routes of Secretion
A. Description of routes
B. Solute transport via the paracellular pathway
IV. Sodium, Potassium, and Chloride Transport
A. Carrier mechanisms for Na⫹, K⫹, and Cl⫺
B. Na⫹, K⫹, and Cl⫺ channels in the apical membrane
C. Effects of prolactin on Na⫹, K⫹, and Cl⫺ transport
V. Relations Between Sodium, Potassium, and Chloride Transport and Water Movements
VI. Phosphate Transport
VII. Calcium Transport
VIII. Citrate Transport
IX. Iodide Transport
X. Stretch-Sensitive Ion Transport Mechanisms
XI. Choline and Carnitine Transport
XII. Glucose Transport
A. Glucose transporters
B. D-Glucose transport across the apical membrane
C. Transport of D-glucose across the Golgi membrane
D. Control of D-glucose transport
XIII. Amino Acid Transport
A. Amino acid transporters
B. Amino acid transport across the apical membrane of mammary secretory cells
C. Control of amino acid transport
D. Volume-sensitive amino acid transport
E. Peptide transport
F. Does ␥-glutamyl transpeptidase play a role in mammary amino acid transport?
XIV. Fatty Acid Uptake
XV. Coordination and Control
Shennan, D. B., and M. Peaker. Transport of Milk Constituents by the Mammary Gland. Physiol Rev 80: 925–951,
2000.—This review deals with the cellular mechanisms that transport milk constituents or the precursors of milk
constituents into, out of, and across the mammary secretory cell. The various milk constituents are secreted by
different intracellular routes, and these are outlined, including the paracellular pathway between interstitial fluid and
milk that is present in some physiological states and in some species throughout lactation. Also considered are the
in vivo and in vitro methods used to study mammary transport and secretory mechanisms. The main part of the
review addresses the mechanisms responsible for uptake across the basolateral cell membrane and, in some cases,
for transport into the Golgi apparatus and for movement across the apical membrane of sodium, potassium, chloride,
water, phosphate, calcium, citrate, iodide, choline, carnitine, glucose, amino acids and peptides, and fatty acids.
Recent work on the control of these processes, by volume-sensitive mechanisms for example, is emphasized. The
review points out where future work is needed to gain an overall view of milk secretion, for example, in marsupials
where milk composition changes markedly during development of the young, and particularly on the intracellular
coordination of the transport processes that result in the production of milk of relatively constant composition at
a particular stage of lactation in both placental and marsupial mammals.
www.physrev.physiology.org
0031-9333/00 $15.00 Copyright © 2000 the American Physiological Society
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I. INTRODUCTION
various mammary gland preparations, effects on transport may be observed. However, it is difficult to determine
whether a hormone is having a primary, direct effect on a
particular transport system or whether the effect is due to
the overall response of the cell, to increase the degree of
differentiation, for example, or simply to prolong its survival. Therefore, caution must be exercised in interpreting
the individual effects of hormones on transport systems in
such highly integrated differentiation, secretion, and
transport systems.
II. EXPERIMENTAL METHODS USED TO STUDY
MAMMARY GLAND SOLUTE TRANSPORT
The first physiological studies on the mechanism of
milk secretion were done using conscious undisturbed
animals. Although the range of measurements that can be
made is limited, the major advantage is that artifacts are
not introduced as part of the experimental procedure of
removing tissue and trying to maintain it alive and undamaged. It is relatively easy to measure transepithelial solute
gradients across the lactating mammary gland, and moreover, because of the slow rates of milk secretion, it is
possible to make some meaningful electrophysiological
measurements; the effect of changing the luminal contents on the transepithelial potential difference can be
readily tested, for example (27).
Many studies on the mechanism of solute transport
have been done using mammary tissue slices, otherwise
termed fragments and explants. Explants are still a popular choice because they are easily and rapidly prepared;
they do not require biochemical treatment, such as collagenase digestion, which could possibly alter the properties of the alveolar cells. Although explants are a mixed
cell population, the vast majority, at least in those prepared from lactating tissue, are secretory cells. The alveolar nature of mammary tissue means that explants will
give a good measure of transport across the basolateral
membranes of the secretory cells particularly in experiments employing short time courses. However, the relatively large extracellular space of mammary tissue explants together with the presence of large unstirred fluid
layers places limitations on the design of experiments.
Isolated mammary epithelial cells and acini are an alternative to explants to study membrane transport. Like
explants, they are relatively easy to prepare; however, the
isolation process requires that the cells be exposed to
digestive agents. The extracellular space is minimal compared with that associated with explants, but it is difficult
to distinguish between transport occurring at the apical
and basolateral membranes in both these preparations.
There is also the possibility that dissociation of mammary
tissue leads not only to a loss of polarity but also decreases cell viability.
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The last Physiological Reviews article on the mechanism of milk secretion was written in 1971 (118) at a time
when morphological information from electron microscopy was being combined with physiological and biochemical knowledge to provide the basis of our current
understanding of milk secretion at the cellular level. Many
of the hypotheses on the secretory mechanisms for the
various milk constituents have survived more extensive
work and amplification at the cellular and molecular levels, and it is not our intention to cover this older ground
other than in outline. Rather, our intention is to extend
the explanatory reductionist approach to examine the
transport and secretory mechanisms for the major milk
components or their precursors and their integration
within the economy of the secretory cell.
The mammary gland is, in a number of respects, an
unusual exocrine gland. Secretion, once initiated at about
the time of parturition, is continuous but relatively slow in
terms of fluid volume output per unit time. The secretion,
milk in full lactation, is a complex mixture arising from
different secretory pathways across, within, and sometimes between the mammary epithelial cells, comprising
membrane-bound fat droplets, casein micelles, and an
aqueous phase that usually contains lactose and sometimes complex carbohydrates, minerals in various ionic or
bound forms, other proteins, and a bewildering array of
soluble components, some of which may be biologically
active in the young consuming the milk (78, 165). Another
but related unusual feature is that secretion is stored
within the lumen of the gland, either in the secretory
alveoli or duct system, until it is removed at intervals
(varying from several hours in many mammals, to once
daily in rabbits, to once every 2 days in tree shrews, and
to once a week in some pinnipeds) by the sucking young
(46).
Studies in a number of species have shown that while
there may be species-specific changes in milk composition with stage of lactation corresponding to growth
phases of the young (a phenomenon particularly marked
in marsupials), milk composition is markedly resistant to
environmental, including nutritional, changes, whereas
the quantity of milk secreted shows considerable plasticity (162).
The control of the rate of milk secretion will not be
covered in detail, except for the effects of prolactin and
cell stretch, since it has been reviewed extensively in
recent years, particularly the autocrine control of milk
secretion that acts to match supply by the mother to
demand by the young within each individual mammary
gland within the hormonal milieu of lactation that maintains the cells in a secretory state (163, 168, 169, 247–249).
The mammary gland is controlled by a number of
hormones and local factors. When these are added to
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MAMMARY TRANSPORT MECHANISMS
⫹
FIG. 1. K
(Rb⫹) uptake by milk-fat-globule membrane vesicles
(squares) and skim milk membrane vesicles (circles) prepared from
bovine milk in the absence (open symbols) and presence (solid symbols)
of valinomycin. Previously, it has been argued that both of these membrane preparations originate from the apical aspect of mammary secretory cells. It is apparent that skim milk membrane vesicles are permeable to K⫹ in the absence and presence of valinomycin. In contrast,
milk-fat-globule membranes only display a significant permeability to K⫹
in the presence of valinomycin. Data are means ⫾ SE (n ⫽ 3). The
results suggest that skim milk membranes may be a better marker for
the apical membrane of mammary secretory cells than milk-fat-globule
membranes. [Adapted from Shennan (195).]
these membranes (195, 196) (Fig. 1). There is the possibility that the milk-fat-globule membrane is derived from
intracellular membranes rather than the apical membrane
(228, 253). On the other hand, membrane vesicles prepared from bovine skim milk, which are distinct from
milk-fat-globule membranes, are able to transport solutes
into an intravesicular compartment (196). Skim milk
membrane vesicles do appear to originate from the apical
membrane (172).
In ending this section, it should be noted that the
relatively complex morphology of the mammary gland
precludes the use of several techniques to study solute
transport. For example, the mammary epithelium is not
flat and therefore, unlike frog skin and toad bladder,
cannot be mounted in Ussing chambers.
III. ROUTES OF SECRETION
A. Description of Routes
There are five major, known routes of secretion
across the mammary secretory epithelium from the blood
side to milk, four transcellular and one paracellular (116,
118) (Fig. 2): 1) membrane route, 2) Golgi route, 3) milk
fat route, 4) transcytosis, and 5) paracellular route.
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The perfused lactating mammary gland has been successfully employed to characterize solute transport systems in both guinea pig and rat. This technique allows the
transport properties of the blood-facing aspect of the
mammary epithelium to be characterized under nearphysiological conditions. One major assumption is that
the endothelium does not constitute a major barrier to
solute movement between blood and the interstitium.
Recent work suggests that this is a reasonable assumption, since sucrose (an extracellular marker) has a larger
dilution space than albumin (an intravascular marker) in
the lactating bovine mammary gland (176). The major
drawback is that the skills required to achieve a successful preparation take time to acquire. The perfused mammary gland, used in conjunction with the rapid, pairedtracer dilution technique (255), has been successfully
employed to study amino acid transport.
Cultured mammary cells have also been used to
study mammary transport processes. For example, cells
cultured on collagen gels to form monolayers and subsequently mounted in Ussing chambers have allowed the
polarity of the mammary epithelium to be examined.
However, there is justifiable concern that cultured mammary epithelial cells are not fully active in terms of secretion, although an exception to this may be mammary
epithelial cells cultured on a reconstituted basement
membrane. Cells grown in this manner form polarized
acinar structures that enclose a hollow, sealed lumen and
secrete in a vectorial fashion (13, 42, 89). These acini,
termed mammospheres, may prove particularly useful for
characterizing transport across the basolateral membranes of secretory cells. Unfortunately, a technique that
allows purified basolateral membrane vesicles to be prepared from mammary secretory cells has not yet been
developed.
Membrane fractions isolated from milk have been
used in attempts to study the transport properties of the
apical (luminal) membrane. Wooding et al. (254) identified membrane-bound cytoplasmic droplets in goat’s milk
containing fat and protein particles, mitochondria, and
Golgi apparatus (but no nuclei); further studies showed
that the droplets were capable of triacylglycerol biosynthesis (44). Because the cytoplasmic fragments originate
from the apical aspect of mammary secretory cells, it is
reasonable to assume that the surrounding membrane is
indeed representative of the apical membrane. It has been
shown that cytoplasmic droplets transport ions into an
osmotically active space (213).
Because the milk fat globule becomes enveloped by
membrane during its extrusion from the cell, the milk-fatglobule membrane has long been assumed to be representative of the apical membrane of the secretory cell.
However, the finding that bovine milk-fat-globule membrane vesicles are impermeable to K⫹ casts doubt on the
accepted origin or maintenance of original properties of
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1) In the membrane route, substances may traverse
the apical cell membrane (and for those directly derived
from blood, the basolateral membrane). Examples are
water, urea, glucose, Na⫹, K⫹, and Cl⫺ (116).
2) In the Golgi route, secretory products are transported to or sequestered by the Golgi apparatus and secreted into the milk space by exocytosis. Examples are
casein, whey proteins, lactose, citrate, and calcium (116,
118, 267).
3) In the milk fat route, milk fat globules are extruded from the apex of the secretory cell surrounded by
membrane (milk-fat-globule membrane); some cytoplasm
is sometimes included (126). Examples are milk fat, lipidsoluble hormones and drugs, some unknown growth factors (in milk-fat-globule membrane) (244), and the product of the ob gene, leptin (214).
4) In transcytosis, vesicular transport involves various organelles, possibly, in some cases also involving
extrusion by route 2 (88, 154). Examples are immunoglobulins during colostrum formation (88), transferrin
(154), and prolactin (154).
5) In the paracellular route, there is direct passage
from interstitial fluid to milk.
In full lactation, routes 1, 2, 3, and, possibly, 4 predominate.
B. Solute Transport Via the Paracellular Pathway
Transcellular transport requires solutes to cross the
constituent plasma membranes of the epithelium,
whereas paracellular transfer involves movement be-
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FIG. 2. Diagram showing the known major
routes of secretion across the mammary epithelium. Routes 1, 2, 3, and 5 correspond to the descriptions given in text. For the sake of clarity,
route 4 (transcytosis) is not shown. [Adapted from
Linzell and Peaker (118).]
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MAMMARY TRANSPORT MECHANISMS
transepithelial electrical resistance and decreased the
transepithelial movements of labeled mannitol and inulin
across monolayers of a mouse mammary cell line (261).
Glucocorticoid-induced formation of mouse mammary
tight junctions involves an increase in the amount of the
tight junction-associated protein ZO-1 and phosphorylation/dephosphorylation events since okadaic acid, a protein serine/threonine phosphatase inhibitor, attenuates
the dexamethasone-induced increase in transepithelial resistance (212). However, mammary development is a
complex series of events, and transforming growth factor
(TGF)-␤ was found to antagonize the effect of dexamethasone (252). There is evidence from in vivo studies that
glucocorticoids may control the permeability of tight
junctions. In late-pregnant goats, an intraluminal injection
of cortisol had marked effects on the composition of fluid
within the lumen: K⫹ concentration increased and Na⫹
decreased, which is consistent with the closing of tight
junctions (224). However, an effect of cortisol on transcellular ion transport cannot be ruled out at this stage.
Hormonal events, other than or as well as a rise in circulating glucocorticoid concentration, appear to be involved
in the final closure near parturition, with the hormonal
events leading to parturition and the onset of copious
milk secretion (lactogenesis stage II, Ref. 68) being
closely linked. For example, in late pregnancy, the goat
mammary gland synthesizes PGF2␣; near term, this production stops. Unilateral treatment with a PGF2␣ analog
prevented the normal changes in milk composition and
increase in the rate of secretion (127). The current view
on the control of tight junctional permeability during
lactogenesis has been well summarized by Nguyen and
Neville (150)
We propose that progesterone inhibits tight junction closure during pregnancy, possibly through or
along with local factors such as TGF-␤ and PGF2␣. As
the level of progesterone drops during parturition or
with ovariectomy, its inhibitory effect is removed,
allowing the mammary epithelial tight junctions to
close. However, glucocorticoid may be required for
final differentiation of the mammary epithelium from
the pregnant animal into the epithelium of the lactating animal with its highly impermeable tight junctions.
Prolactin appears to play a role in the maintenance of
the mammary epithelium in the lactating state and
inhibition of programmed cell death.
The paracellular pathway reappears at the cessation
of lactation. When milk removal was stopped in goats
during late lactation, the key process was determined to
be the local event of stretching the mammary epithelium
by milk accumulating within the lumen of the gland,
leading to a decrease in secretory and metabolic activity
and an increase in the permeability of the tight junctions.
Systemic control by hormones was excluded in this species as was pressure of the accumulated milk simply
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tween the cells via leaky tight junctions. In most species,
solute transport across the lactating mammary epithelium
appears to be mainly transcellular, i.e., the zonulae occludentes are tight junctions. However, there are situations
where a significant flux occurs via a paracellular pathway.
Movement of solutes across the paracellular shunt pathway occurs during pregnancy and, in some species, during late lactation. The early (160) and later work (150) on
this aspect of mammary transport including physiological
and morphological findings have been reviewed, and it is
not our intention to provide a further detailed review
here.
The transepithelial electrical resistance is a good
measure of the tightness (or leakiness) of an epithelium.
However, although it is possible to measure the transepithelial resistance of cultured mammary cell monolayers,
it is more difficult to obtain reliable values of the electrical resistance across the lactating mammary gland in vivo.
A common way of assessing paracellular permeability in
the lactating mammary gland is to measure the solute
content of milk; the concentrations of lactose and K⫹ in
milk decrease (moving down their concentration gradients), whereas those of Na⫹ and Cl⫺ increase when the
junctions become leaky. However, care must be exercised
when relying solely on Na⫹ and K⫹ gradients because a
change may simply reflect an alteration in the transport of
these ions across transcellular pathways. A more preferable way of assessing tight junction permeability in vivo is
to measure the rate of appearance of lactose in plasma or
of an inert disaccharide introduced into plasma in milk
(119, 216).
In general, the paracellular pathway is absent during
lactation and present during pregnancy. However, in the
rabbit during late lactation, when the rate of secretion is
still relatively high, milk lactose and K⫹ concentrations
decrease while those of Na⫹ and Cl⫺ increase. At this
stage, the epithelium is permeable to lactose and sucrose
and more permeable to Na⫹ and Cl⫺ (167). These changes
were reversed by exogenous prolactin (121). Recent evidence suggests that prolactin may also be involved in
some way in maintaining the integrity of the tight junctions (judged by the ability of the gland to maintain solute
concentration gradients) in the rat mammary gland (71).
There is also evidence that a paracellular pathway appears during other physiological states. Characteristic
changes in milk composition were observed in many
goats for 1–2 days up to 4 days before estrus (164), but the
controlling mechanism remains unknown.
There is a major change in the permeability of the
paracellular pathway at the onset of copious milk secretion around the time of parturition (119). There is also
good evidence which suggests that glucocorticoids are
involved in controlling the formation of tight junctions
during mammary development and differentiation in pregnancy. The glucocorticoid dexamethasone increased the
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IV. SODIUM, POTASSIUM, AND CHLORIDE
TRANSPORT
The mammary gland is able to generate and maintain
large Na⫹, K⫹, and Cl⫺ gradients between milk and
plasma. These ions make a substantial contribution to the
osmolality of milk, especially in those species where the
lactose content is relatively low. On the basis of measurements of the concentration gradients between interstitial
or extracellular fluid, intracellular fluid, and milk, and the
electrical potential gradients between these three compartments, Linzell and Peaker in 1971 (117, 118) proposed
a scheme to account for the movement of Na⫹, K⫹, and
Cl⫺ across the lactating mammary epithelium and the
maintenance of milk composition (Fig. 3). In their hypothesis, the main features are that the Na⫹ concentration
inside the cell is kept low, and the K⫹ concentration high,
by the action of a Na⫹-K⫹ pump on the basolateral membrane, whereas these ions are freely distributed between
the interior of the cell and milk according to the electrical
potential difference across the apical membrane. Milk is
electrically positive with respect to the cell, so the concentrations of Na⫹ and K⫹ are lower in milk, but the ratio
of these ions is similar in both compartments, i.e., ⬃3
K⫹:1 Na⫹. The situation for Cl⫺ is, however, different
from that of Na⫹ and K⫹. The concentration of Cl⫺ is
higher inside mammary cells than that expected for an
equilibrium distribution, suggesting that a mechanism exists in both the basolateral and apical membranes which
“actively” pumps Cl⫺ into the cells. Many experimental
observations, at the level of individual transport systems,
supporting the original hypothesis of Linzell and Peaker
have since been made. The relations between lactose
FIG. 3. Profile of ion distribution and membrane potentials (both
measured and calculated) in guinea pig mammary tissue. [Modified from
Linzell and Peaker (118).]
concentration, apical membrane potential difference, and
ionic concentrations have also been considered (160,
159).
A. Carrier Mechanisms for Naⴙ, Kⴙ, and Clⴚ
Three carrier mechanism were postulated in the
scheme of Linzell and Peaker: 1) a Na⫹-K⫹ pump on the
basolateral membrane, 2) an inwardly directed Cl⫺ pump
on the apical membrane, and 3) a similar Cl⫺ transporter
in the basolateral membrane of the secretory cell (117,
118). Evidence for the presence of a Na⫹-K⫹ pump in the
blood-facing aspect of the mammary epithelium is strong,
and several candidates for Cl⫺ pumps have been identified.
Many studies have shown that lactating mammary
tissue from a variety of species displays ouabain-sensitive
Na⫹-K⫹-ATPase activity (242) and ouabain-sensitive Na⫹
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bursting the taut epithelium (69, 161). Similarly, in the
cow, milk stasis leads to an increase in the paracellular
pathway that can be reversed by milking (217). These as
well as other studies indicate an inverse link between
secretory activity and permeability of the tight junctions.
Whether maintenance of impermeable junctions is part of
the secretory activity of the cell, like the synthesis of milk
components, or whether disruption of the junctions is
followed by partial depolarization of the cell which affects
secretory activity, or whether there is signaling from the
junctions to the synthetic and secretory pathways is not
known (150).
The presence of a paracellular pathway may confound studies on milk composition and mammary epithelial permeability. Disruption of the epithelium is evident
in mastitis but also when supraphysiological doses of
oxytocin are given to contract the myoepithelium and
obtain a milk sample. Care must be exercised when interpreting data on milk composition, particularly of the aqueous phase (160).
MAMMARY TRANSPORT MECHANISMS
July 2000
⌬G ⫽ RT ln
冉
关Na ⫹ 兴 i 关K ⫹ 兴 i 关Cl ⫺ 兴 2i
关Na ⫹ 兴 o 关K ⫹ 兴 o 关Cl ⫺ 兴 o2
冊
(1)
where subscripts i and o refer to intracellular and extracellular, respectively. Applying the Na⫹, K⫹, and Cl⫺ concentrations of guinea pig mammary tissue and plasma
(117) to Equation 1 gives a value ⫹0.79RT for G, suggesting that the triple cotransport system operating with a
coupling ratio of 1 Na⫹:1 K⫹:2 Cl⫺ would require an input
of free energy to drive inward transport. Alternatively, a
Na⫹-K⫹-Cl⫺ cotransporter with a stoichiometry of 2
Na⫹:1 K⫹:3 Cl⫺, as found in the squid axon (190), would
have sufficient freeenergy (G ⫽ ⫺1.03RT) associated with
the ion gradients to accomplish inward transport. However, it is clear that further experiments are required to
determine both the stoichiometry and locus of the mammary Na⫹-K⫹-Cl⫺ cotransport mechanism.
Cl⫺/HCO⫺
3 exchange could also be a candidate for a
⫺
Cl pump in lactating mammary tissue (120). For exam-
ple, a Cl⫺/HCO⫺
3 antiport drives the uphill accumulation
of Cl⫺ into smooth muscle cells (2, 50). A DIDS-sensitive
⫺
⫺
anion exchanger that accepts Cl⫺, SO2⫺
4 , I , and NO3 as
substrates has been reported in lactating rat mammary
tissue explants (194, 203). However, it remains to be
shown whether the rat mammary tissue anion-exchanger
transports HCO⫺
3 and, if so, whether it is able to act as a
Cl⫺ pump.
B. Naⴙ, Kⴙ, and Clⴚ Channels in the
Apical Membrane
The 1971 model of Linzell and Peaker predicts that
the apical membrane of mammary secretory cells possesses Na⫹, K⫹, and Cl⫺ channels. Several lines of experimental evidence support this prediction. First, Blatchford and Peaker (27) have shown that the transepithelial
potential difference across the lactating goat mammary
gland can be altered by varying the Na⫹, K⫹, and/or Cl⫺
concentration in the lumen of the gland in a fashion that
is consistent with the presence of channels for each of
these ions in the apical aspect. However, it was found that
the apical membrane is unable to discriminate between
Na⫹ and K⫹, suggesting that the apical membrane may
express a nonspecific cation channel rather than separate
channels for Na⫹ and K⫹. In this connection, nonspecific
cation channels have been identified in cultured mouse
mammary epithelial cells (72). Second, cytoplasmic fragments isolated from goat’s milk that are surrounded by
apical membrane, transport K⫹(Rb⫹) via barium- and quinine-sensitive channels (213). Third, skim milk membranes, isolated from bovine milk, which appear to be
derived from the apical aspect of secretory cells, express
K⫹ and Cl⫺ channels (196). The channels identified in the
membrane preparations need to be characterized using
electrophysiological techniques.
C. Effects of Prolactin on Naⴙ, Kⴙ, and
Clⴚ Transport
Prolactin was found to decrease the Na⫹ and increase the K⫹ content of mammary tissue taken from
rabbits treated with bromocriptine, a drug that prevents
prolactin release; ouabain inhibited the action (62, 63). In
addition, the unidirectional uptake of K⫹(Rb⫹) by rabbit
mammary explants was stimulated by prolactin via a pathway sensitive to ouabain (61). These observations are
consistent with an effect of prolactin-induced differentiation of the cells on the number of Na⫹-K⫹ pumps present
either induced directly or through an increase in Na⫹
permeability. In this connection it is interesting to note
that prolactin upregulates Na⫹-K⫹-Cl⫺ cotransport in rat
mammary tissue (202). Work using cultured cells supports the claim that prolactin-induced differentiation in-
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and K⫹ transport (3, 61, 62, 117, 194). In addition, there is
good histochemical evidence that Na⫹-K⫹-ATPase is located on the basal and lateral membranes, but not the
apical membranes, of mammary secretory cells (96, 105).
These findings taken together support the scheme of Linzell and Peaker that lactating mammary tissue expresses
a functional Na⫹-K⫹ pump on the blood-facing pole of the
epithelium. Experiments using cultured mammary epithelial cells have provided evidence that the Na⫹-K⫹ pump is
located on the basolateral, but not the apical, membranes
(24 –26). However, there is evidence for Na⫹-K⫹-ATPase
activity on the milk-fat-globule membrane, albeit at a
relatively low specific activity (55).
Na⫹-K⫹-Cl⫺ cotransport could be a candidate for the
⫺
Cl pump in the model of Linzell and Peaker. Na⫹-K⫹-Cl⫺
cotransport has been identified in lactating rat mammary
tissue (194, 203). Thus K⫹ (Rb⫹) transport (both influx
and efflux) by mammary tissue explants is Na⫹ dependent, Cl⫺ dependent, and inhibited by the loop diuretic
furosemide. In addition, Cl⫺ efflux from rat mammary
tissue explants is sensitive to furosemide (203). On the
basis that cotransport activity was detected in explants, it
is predicted that the Na⫹-K⫹-Cl⫺ cotransport system is
situated in the blood-facing aspect of the epithelium. Supporting evidence of Na⫹-K⫹-Cl⫺ expression in mammary
tissue is the finding that bovine milk-fat-globule membrane binds bumetanide (an analog of furosemide) in a
fashion that is dependent on Na⫹, K⫹, and Cl⫺ (166).
The triple cotransporter is able to drive the uphill
transport of Cl⫺ (2). The Na⫹-K⫹-Cl⫺ cotransport system
is electroneutral and is generally assumed to operate with
a stoichiometry of 1 Na⫹:1 K⫹:2 Cl⫺ (43, 79) The freeenergy (⌬G) available to a cotransporter operating with
such a stoichiometry is given by
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volves changes in mammary Na⫹ and K⫹ transport; prolactin increases the mucosal-to-serosal transfer of Na⫹
across monolayers of cultured mouse mammary epithelial
cells via a ouabain-sensitive pathway (24 –26).
V. RELATIONS BETWEEN SODIUM, POTASSIUM,
AND CHLORIDE TRANSPORT AND WATER
MOVEMENTS
VI. PHOSPHATE TRANSPORT
The suckling requires a considerable amount of phosphate for normal growth and development. There are at
least three chemical forms of phosphate in milk: free
inorganic orthophosphate (Pi) in solution as HPO2⫺
and
4
H2PO⫺
4 ; colloidal phosphate, the majority of which is calcium phosphate associated with casein micelles; and casein phosphate (86). For example, of the total phosphate
(20.5 mM) present in goat’s milk, 6.7 mM is Pi, 8.9 mM is
colloidal, and 4.9 mM is casein bound (146). Despite the
complex nature of phosphate in milk, it can be predicted
that milk phosphate is derived from plasma Pi that has to
be transported across the basolateral membranes of the
secretory cells. In accordance with this prediction, the
mammary gland has the capacity to extract large quantities of Pi from blood. For example, it is estimated that the
entire pool of plasma Pi is replaced every 10 min in
lactating rats (30).
The mechanism of Pi transport by the lactating mammary gland has only recently received attention. Pi uptake
by rat mammary tissue explants and the perfused rat
mammary gland occurs predominantly via a Na⫹-dependent process (210).The Na⫹-dependent moiety of Pi up-
take in the latter preparation operates with a Michaelis
constant (Km) of 40 ␮M with respect to the extracellular
Pi concentration. Furthermore, Pi efflux from mammary
tissue can be stimulated by reversing the trans-membrane
Na⫹ gradient. These findings are consistent with the presence of a Na⫹-Pi cotransport system in the basolateral
aspect of the mammary epithelium. Na⫹-Pi cotransport
mechanisms have been identified in a number of epithelia
including the intestine, kidney, and placenta (21, 23, 33),
several of which have been cloned and sequenced (125).
There are, however, several differences between the
properties of the Na⫹-dependent Pi transporter in mammary tissue and those described in other epithelia. First,
Pi uptake by mammary tissue is only weakly inhibited by
structural analogs; arsenate and phosphonoformic acid
(PFA) were found to be weak or ineffective inhibitors of
Pi transport. In contrast, both of these compounds have
been shown to be high-affinity blockers of Na⫹-dependent
Pi transport in other tissues (103). Second, the kinetic
parameters, with respect to Na⫹ activation, are markedly
different; the stimulation of Pi uptake by external Na⫹ did
not exhibit sigmoidal kinetics as found, for example, in
the kidney (125).
Under certain experimental conditions Pi transport in
lactating rat mammary tissue occurs via an anion-exchange system (210). External Pi is able to trans-accelerate Pi efflux via a mechanism sensitive to DIDS. It appears
that the Pi self-exchange system is distinct from the anion
exchanger that accepts Cl⫺ and SO2⫺
4 as substrates (194),
since Pi efflux is not trans-stimulated by Cl⫺ and SO2⫺
4
and Pi does not trans-accelerate SO2⫺
4 efflux (210). Moreover, the finding that the Pi self-exchanger is not Na⫹
dependent suggests that the exchange mechanism is distinct from the Na⫹-Pi cotransport system. However, the
physiological significance of the Pi exchanger remains to
be established.
The finding that Pi in milk is higher than that of
plasma could, at first sight, be taken as evidence that the
apical membrane expresses a Pi transport mechanism.
However, the major pathway for Pi secretion into milk is
believed to be via the Golgi vesicle route. This hypothesis
is based on two important findings. First, Neville and
Peaker (146) found that the apical aspect of the lactating
goat mammary epithelium is almost impermeable to Pi.
Second, the time course of Pi secretion into milk, following an intravascular injection of radiolabeled Pi, was similar to that of casein phosphate and colloidal phosphate.
This finding raises questions about the nature of Pi transport across the Golgi membrane. There is, of course, the
possibility that Golgi secretory vesicles do not need to
transport Pi per se from the cytosol because sufficient Pi
is generated in the Golgi lumen by the hydrolysis of UDP
during lactose synthesis (Fig. 4). However, this mode of Pi
generation will not occur in the Golgi lumen of pinnipeds.
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It is recognized that the synthesis and secretion of
lactose is responsible for the majority of water in milk.
However, this cannot be the whole story, since water still
appears in the milk of those species (i.e., pinnipeds)
which contain little or even no lactose or any other sugar
(151). In a number of secretory epithelia, it is accepted
that the intracellular accumulation of Cl⫺, via Na⫹-K⫹-Cl⫺
cotransport across the basolateral membrane, is the driving force for the secretion of ions and water across the
apical membrane (43, 153). The same process could occur
in the mammary secretory cell, since the necessary gradients exist. Therefore, a situation could arise whereby
the secretion of water by the mammary gland could be
driven partly by the secretion of lactose and partly by the
secretion of ions. Variations in the concentrations of lactose and ions within and between species could be related
to the relative flux between these two mechanisms. This
hypothesis, together with the involvement of paracellular
ion flow in some species, may explain how secretion of
the aqueous phase is achieved in all mammals.
Volume 80
July 2000
MAMMARY TRANSPORT MECHANISMS
VII. CALCIUM TRANSPORT
Milk is a calcium-rich fluid (94). For example, the
total calcium concentration in rabbit’s milk can approach
a concentration of 100 mM. Accordingly, during lactation,
the mammary gland extracts large quantities of calcium
from plasma to meet the requirements of the growing
neonate. Milk calcium, like phosphate, exists in several
chemical forms: free ionized calcium, casein-bound calcium, and calcium complexed to inorganic anions such as
phosphate and citrate. In most milks, calcium associated
with casein micelles comprises the majority of the total
calcium present in milk (144). Neville and Peaker (146)
found that the mammary gland does not transport calcium
in the milk-to-blood direction, suggesting that calcium
cannot cross the apical membrane or tight junctions. Thus
it can be inferred that calcium transport into milk is
transcellular. Furthermore, because the time course of
appearance of labeled milk calcium is similar to that of
casein and lactose, it has been suggested that calcium is
secreted via the Golgi vesicle route (146). It follows that
casein-bound calcium and colloidal calcium, along with
free ionized calcium, are all derived from plasma Ca2⫹,
indicating that Ca2⫹ must be able to cross both the basolateral and Golgi membranes before secretion.
The first question that arises is, What mechanism
transports calcium across the basolateral membrane?
Surprisingly, there is a paucity of information about the
system(s) available for calcium transport across the
blood-facing membrane except for the finding that it is a
temperature-sensitive process that increases during the
transition from pregnancy to lactation (147). Whatever
the nature of the system(s), it must operate with a high
capacity given the large amount of calcium secreted into
milk. Calcium channels would of course fulfill such a role.
However, until the mechanism of calcium uptake by mammary secretory cells is thoroughly studied, the presence
of calcium channels in the basolateral membrane can only
remain a suggestion.
Mammary epithelial cells maintain intracellular Ca2⫹
at a low concentration (56, 75). This observation prompts
the next question, How do mammary epithelial cells maintain a low cytosolic Ca2⫹ concentration while facilitating
a large transcellular calcium flux? A low calcium concentration could be maintained if Ca2⫹ were pumped into
intracellular organelles at a rate that matched the entry of
calcium across the basolateral membranes of the secretory cells. In this connection, there is evidence suggesting
that Golgi membranes express an ATP-driven Ca2⫹ pump
(149, 241, 245). Golgi-derived vesicles are able to accumulate Ca2⫹ by a saturable process (Km ⫽ 0.8 ␮M) that
requires the hydrolysis of ATP (Fig. 4). A Ca2⫹ pump
situated in the basolateral membranes of mammary secretory cells could also act to keep intracellular Ca2⫹ at a low
concentration. However, no evidence for such a transport
system has been presented. In addition, no evidence for a
basolateral Na⫹/Ca2⫹ exchanger, another transport system which could regulate intracellular Ca2⫹, has been
found (143).
Ca2⫹ uptake by mammary tissue may be a hormonally regulated process. Ca2⫹ accumulation by cultured
mouse mammary explants, presumably via the basolateral
membranes, is stimulated by 1,25-(OH)2D3, the hormonal
form of vitamin D, in a dose-dependent fashion (137), and
Ca2⫹ transfer into goat’s milk is increased by parathyroid
hormone-related protein (15). However, in both of these
examples, the target mechanism is unknown.
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FIG. 4. A schematic diagram of the mechanisms that may be involved in the transport and metabolism of phosphate and Ca2⫹ in
mammary secretory cells. Phosphate is transported across the basolateral membrane via a (Na⫹-Pi) cotransport mechanism and possibly by an
anion-exchange system. Pi is formed within the lumen of the Golgi
during the synthesis of lactose as a consequence of the hydrolysis of
UDP-galactose. In addition, Pi is generated during the process of casein
phosphorylation. The mechanism of Ca2⫹ uptake across the basolateral
membrane has not been precisely identified but may be a Ca2⫹ channel.
Stretch-activated Ca2⫹ channels may also provide a pathway for Ca2⫹
uptake. Ca2⫹ is pumped across Golgi membranes by a Ca2⫹-ATPase. Pi
and Ca2⫹ are transported into milk along with lactose, casein, and
citrate after exocytosis of Golgi-derived vesicles at the apical aspect of
the secretory cells. LS, lactose synthase; CK, casein kinase; A, ADPase.
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D. B. SHENNAN AND M. PEAKER
VIII. CITRATE TRANSPORT
IX. IODIDE TRANSPORT
The concentration of iodide in milk can be 20- to
30-fold higher than that found in maternal plasma (32),
matching the requirements of the suckling for the synthesis of thyroid hormones. Iodide transport was investigated relatively early because of the contamination of
land and milk supplies by radioactive iodine isotopes
from nuclear explosions or accidents. In addition, it was
thought that an understanding of the mammary iodide
transport mechanism could help limit the transfer of radiolabeled iodide into the food chain via milk after environmental contamination.
The iodide transport system in the thyroid gland has
recently been cloned (47). It is a Na⫹-dependent mechanism sensitive to anions such as perchlorate and thiocyanate (155). A body of evidence suggests that mammary
epithelial cells express a similar iodide transport mechanism. The lactating mammary gland has the ability to
accumulate iodide into milk against a high concentration
gradient by a system that is inhibited by perchlorate and
thiocyanate (31, 32, 53, 114). Iodide uptake by mammary
tissue explants prepared from midpregnant mice is a Na⫹dependent process stimulated by prolactin (184, 185).
Moreover, mRNA encoding for the Na⫹-dependent iodide
transporter has been identified in the lactating human
mammary gland (215).
Although the Na⫹-dependent mechanism is probably
the predominant pathway for iodide uptake by mammary
cells, there is evidence that iodide also enters via a DIDSsensitive anion-exchange pathway; sulfate efflux from rat
mammary tissue can be stimulated by extracellular iodide
(194).
X. STRETCH-SENSITIVE ION TRANSPORT
MECHANISMS
Milk accumulation in the alveolar lumen leads to
distension of the secretory alveoli, and cellular deformation may play an important role in determining secretory
activity during the cessation of lactation. An increase in
intramammary pressure at the end of lactation has been
shown to reduce the rate of milk secretion in the goat
(161), suggesting that, in addition to local, autocrine
chemical control of milk secretion by the milk protein
FIL, mammary distension could also play a part in controlling the rate of milk secretion in response to demand
by the young (48, 163, 168, 169, 247–249).
Cell distension has marked effects on mammary
gland ion transport. Enomoto et al. (58) found that mechanical stimulation of cultured mouse mammary cells,
induced by touching with a blunt microelectrode, resulted
in a short depolarization (1– 8 s) followed by a prolonged
(10 – 40 s) hyperpolarization. At present, the locus of the
stretch-sensitive channels (apical and/or basolateral) on
the mammary epithelial cells is not known. The mechanism underlying the depolarization remains to be determined, but it has been established that the hyperpolarization can be attributed to the activation of quininesensitive, Ca2⫹-dependent K⫹ channels (58, 59, 72).
Accordingly, mechanical stimulation of cultured mouse
mammary cells leads to an increase in the intracellular
Ca2⫹ concentration. However, the increase in the Ca2⫹
concentration is not confined to the touched cell but also
spreads to surrounding cells, even to those that have no
direct physical contact with the stimulated cell, suggesting that a paracrine factor may be involved (60, 73). There
is evidence that ATP is released from cultured mammary
cells upon mechanical stimulation (73). In this connection, extracellular ATP, acting via P2-purinergic receptors,
has been shown to increase intracellular Ca2⫹ in human
breast tumor cells (70). Cell swelling, induced by hyposmotic shock, increases the intracellular Ca2⫹ concentration in acini isolated from lactating mouse mammary
gland (221). The effect consists of a rapid and large transient increase in intracellular Ca2⫹ followed by a sustained plateau phase. The increase in Ca2⫹ is due to
increased Ca2⫹ influx from the incubation medium rather
than release from intracellular stores. The nature and
precise locus of the Ca2⫹ transport pathway is unknown,
but it is insensitive to nifedipine (221). Moreover, it is not
yet known if the primary stimulus is membrane stretch or
a change in the cellular hydration state.
Based on these still sparse observations, we hypothesize that stretch induces ATP release into the milk or
interstitial space, where it interacts with neighboring cells
leading to an increase in calcium entry into these cells.
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Citrate exists in the aqueous phase of milk in a
variety of chemical forms such as calcium citrate⫺, citrate2⫺, and citrate3⫺. The relative proportion of each
chemical form depends on factors such as H⫹, Ca2⫹, and
Mg2⫹ and thus citrate can be considered as an important
milk buffer. The lactating goat mammary epithelium is
impermeable to citrate in both directions, suggesting that
citrate is synthesized within the secretory cells and released into milk after exocytosis of Golgi vesicles (116).
In this connection, a Golgi-enriched fraction isolated from
the bovine mammary gland accumulated citrate in a fashion sensitive to temperature, suggesting that a citrate
transport system may be present in the Golgi apparatus
membrane (267). The putative citrate transport mechanism in the Golgi membranes remains to be fully characterized.
Volume 80
July 2000
MAMMARY TRANSPORT MECHANISMS
XI. CHOLINE AND CARNITINE TRANSPORT
activity of the Na⫹-dependent L-carnitine transport system was found to decrease as lactation progressed (201).
L-Carnitine efflux from lactating rat mammary tissue
is a slow process that is Na⫹ independent and cannot be
stimulated by external L-carnitine. However, cell-swelling,
induced by a hyposmotic shock, increases the fractional
release of radiolabeled L-carnitine from rat mammary tissue (201). The possibility that volume-activated L-carnitine release is mediated via the volume-sensitive amino
acid transport pathway should be investigated.
XII. GLUCOSE TRANSPORT
The concentration of lactose in the milk of most
species is relatively high, and lactose is one of the major
milk osmolytes; for example, the concentrations in human and bovine milk are, respectively, 204 and 140 mM
(158). Lactose is synthesized within the lumen of the
Golgi apparatus by an enzyme complex collectively
known as lactose synthase. Exocytosis of the Golgi secretory vesicles then occurs at the luminal side of the cell.
Golgi and apical membranes of mammary secretory cells
are impermeable to lactose; therefore, because these
membranes are freely permeable to water, the volume of
milk secreted is closely related to the rate of lactose
synthesis.
The lactating mammary gland has a high demand for
D-glucose because it is the obligate precursor for lactose
synthesis and, depending on species, is also used to synthesize lipids and amino acids (110, 115). Glucose transport across both the basolateral and Golgi membranes of
the secretory cells is therefore an important process in
milk secretion.
A. Glucose Transporters
1. GLUT1 expression
Lactating mammary tissue transports D-glucose via a
facilitative transport system similar, if not identical, to
GLUT1. Amato and Loizzi (4) found that guinea pig mammary tissue slices are capable of transporting 2-deoxyglucose, a substrate of GLUT1 type transporters (74), via a
system inhibited by cytochalasin B. Rat mammary acini
also transport 2-deoxyglucose and 3-O-methyl-D-glucose
via a temperature-sensitive and saturable mechanism (Km
for 2-deoxyglucose ⫽ 16 mM) that is inhibited by cytochalasin B and phloretin (225). Carrier-mediated D-glucose
uptake with characteristics of GLUT1 has also been described in mouse mammary acini (177); thus 3-O-methylD-glucose uptake by mouse mammary cells is via a saturable (Km ⫽ 14 mM), cytochalasin B-sensitive pathway.
In accordance with the flux studies quoted in the
previous paragraph, it has been shown that rat mammary
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The organic cation choline is required by the suckling
for normal growth and development including the synthesis of ACh, phosphatidylcholine, and sphingomyelin (258).
The concentrations of choline (and its metabolites) in
rat’s milk and plasma are 1.5 mM and 12 ␮M, respectively,
suggesting that lactation may put a considerable strain on
the maternal choline stores (259). The concentration of
free choline in rat’s milk, 182 ␮M, is consistent with active
choline transport. Lactating rat mammary epithelial cells
possess a high-affinity (Km ⫽ 35 ␮M), Na⫹-dependent
choline carrier that can be inhibited by hemicholinium-3
(41). Rat mammary epithelial cells can maintain high
concentrations of choline (41), suggesting that the Na⫹dependent carrier is situated in the basolateral membrane
and that the transepithelial choline gradient is generated
at the basal membrane of the mammary cell.
L-Carnitine (4-N-trimethylammonia-3-hydroxybutanoate) is essential for the transport of long-chain fatty acids
between cell compartments. For example, the formation
of long-chain acylcarnitines from their respective coenzyme A esters enables the acyl moieties to cross the
mitochondrial membrane where they are subjected to
␤-oxidation (256). L-Carnitine-dependent oxidation of
long-chain fatty acids is particularly important for brain
development in the neonate. The transport of L-carnitine
into milk is physiologically significant because the newborn has a limited capacity for L-carnitine biosynthesis. In
rats, the lactating female depletes its reserves of L-carnitine through secretion into milk; the rate of L-carnitine
secretion is high during the first 3– 4 days of lactation but
thereafter falls as the young develop the capacity to synthesize L-carnitine (186). In the early stages of lactation,
the L-carnitine concentration in rat milk is almost an order
of magnitude greater than that of plasma (320 vs. 35 ␮M),
suggesting that mammary epithelial cells possess a carrier
for L-carnitine. In accordance with this prediction, a concentrative, Na⫹-dependent L-carnitine carrier has been
identified in mammary explants isolated from rats during
the early phase of lactation (201, 257). The carrier operates with a Km of 132 ␮M and maximum velocity (Vmax) of
100 pmol 䡠 h⫺1 䡠 mg intracellular water⫺1. If, as expected,
the carrier is situated on the basolateral membrane of the
alveolar cells it will not be saturated with plasma L-carnitine under physiological conditions. However, the Km
value obtained with explants is probably an overestimate
because of the presence of unstirred fluid layers. The rat
mammary L-carnitine carrier is not stereospecific, since
D-carnitine is an effective cis-inhibitor. In addition, acetylcarnitine, but not choline or taurine, inhibits L-carnitine
uptake by rat mammary explants. These characteristics
suggest that the mammary tissue L-carnitine carrier is
similar to that of renal brush-border membrane vesicles,
neuroblastoma cells, and JAR cells (140, 174, 182). The
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D. B. SHENNAN AND M. PEAKER
2. GLUT4 expression
No evidence has been found for the insulin-sensitive
transporter GLUT4 in lactating rat mammary
epithelial cells (35, 40, 124). However, GLUT4 mRNA is
expressed in mammary tissue taken from virgin and pregnant rats, but expression decreases as gestation
progresses, suggesting that GLUT4 is expressed in mammary adipocytes rather than mammary epithelial cells (35,
40). No detectable expression of GLUT4 mRNA is evident
in lactating bovine mammary tissue (263).
(198). 1) The efflux of 3-O-methyl-D-glucose from lactating
rat mammary tissue explants is stimulated by reversing
the transmembrane Na⫹ gradient. This finding is consistent with a Na⫹-dependent D-glucose transporter operating in the reverse mode or the inhibition of D-glucose
(re)uptake via a Na⫹-dependent mechanism. 2) mRNA
extracted from the rat mammary gland contains a 4-kb
transcript that hybridizes with the cDNA for the rabbit
intestinal Na⫹-dependent D-glucose transporter (SGLT1).
3) A protein reacting specifically with an antibody raised
against a synthetic hydrophilic nonadecapeptide corresponding to a proposed extramembranous loop of the
rabbit intestinal SGLT1 has been found in Western blots
of rat mammary cell membranes. 4) RT-PCR, using primers derived from the sequence of lamb intestinal SGLT1,
generated a product (670 bp) from total rat mammary
RNA identical in size to that obtained from intestinal RNA
(211). Moreover, the sequence was identical to the corresponding region of intestinal SGLT1. In addition, the
ovine mammary gland also expresses a SGLT1 type Dglucose transporter; the sequence is 1,995 bp in length and
has the potential to code for a protein containing 664
amino acids with a sequence identical to that of the lamb
intestinal SGLT1 (211). SGLT1 mRNA has also been found
in lactating bovine mammary tissue (265).
The precise locus and functional significance of the
mammary SGLT1-like transporter remains to be determined. If it is situated in the basolateral aspect of the
mammary epithelium, then it may operate in parallel with
GLUT1 to provide mammary acinar cells with D-glucose.
However, an intracellular or apical location cannot be
ruled out at this stage.
D-glucose
3. GLUT2, GLUT3, and GLUT5 expression
Lactating rat mammary epithelial cells do not appear
to express GLUT2 or GLUT5 (35, 40). Similarly, lactating
bovine mammary tissue does not express GLUT2 mRNA.
Although low levels of GLUT3 and GLUT5 mRNA are
found in the bovine mammary gland, the cellular location
(i.e., epithelial or stromal) of these transcripts in the gland
is not known (263).
4. SGLT1 expression
There is evidence for the presence of a Na⫹-dependent glucose transporter in lactating rat mammary tissue
B.
D-Glucose
Transport Across the
Apical Membrane
D-Glucose, along with a number of other monosaccharides, is present in milk of most species at a much
lower concentration than that found in plasma (65). The
available experimental evidence is consistent with the
presence of a D-glucose transport system on the apical
membrane of mammary secretory cells. Indeed, it has
been suggested that milk D-glucose concentrations can be
taken as a measure of the intracellular concentration (65,
142). D-Glucose and galactose introduced into the lactating goat mammary gland via the teat canal are rapidly lost
from the milk space. The finding that fructose does not
cross the mammary epithelium, even after 16 h, suggests
that D-glucose and galactose cross the mammary epithelium via a transcellular route and not via a paracellular
pathway (64). This conclusion is supported by the finding
that radiolabeled 3-O-methyl-D-glucose introduced into
the milk space of the goat mammary gland via the teat
enters venous blood at a faster rate than radiolabeled
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tissue expresses mRNA that hybridizes with a cDNA
probe for GLUT1 as well as a protein that reacts with an
antibody raised against the COOH terminus region of the
human erythrocyte glucose GLUT1 transporter (35, 124).
Immunofluorescence and immunoelectron studies have
shown that GLUT1 expression is on the basolateral aspect
of the lactating rat mammary epithelium (40, 222), consistent with the finding that 2-deoxyglucose transport occurs at the blood-facing aspect of the intact mammary
gland (226). There are also several reports of GLUT1
expression, both at the mRNA and protein level, in lactating bovine mammary tissue (262–264).
Although the available evidence suggests that GLUT1
mediates the majority of D-glucose uptake by lactating
mammary tissue, this conclusion would be strengthened
if sensitivity to phloretin and cytochalasin could be demonstrated in the perfused lactating mammary gland and if
the sequence of the lactating mammary tissue GLUT1
mRNA were known. It is notable, however, that guinea pig
mammary tissue apparently has more than one type of
saturable transport system for D-glucose (4). Thus cytochalasin B and phloretin do not completely inhibit the
saturable component of 2-deoxyglucose uptake by guinea
pig mammary tissue explants. In addition, a significant
proportion of 2-deoxyglucose uptake by guinea pig mammary tissue slices is via a nonsaturable pathway. Therefore, it appears that GLUT1 may not be the sole mechanism for glucose transport across the basolateral
membrane of mammary secretory cells.
Volume 80
July 2000
MAMMARY TRANSPORT MECHANISMS
C. Transport of D-Glucose Across the
Golgi Membrane
D-Glucose must cross the Golgi membrane to reach
the site of lactose synthesis. A Golgi vesicle fraction
prepared from lactating rat mammary tissue was found to
transport D-glucose; it was postulated that a pore was
involved (246). However, the precise nature of D-glucose
transport across the Golgi membrane remains to be established, although one report indicates that Golgi membranes possess a protein similar to GLUT1. Therefore, it is
possible that GLUT1 transports D-glucose to the site of
lactose synthesis; alternatively, Golgi-derived GLUT1 proteins may be destined for the plasma membrane (124).
D. Control of D-Glucose Transport
Given the important role of D-glucose in the process
of milk secretion, it is of fundamental importance that the
factors that control the transport of D-glucose by mammary epithelial cells are elucidated. However, it is surprising to find that our knowledge of this important area is
relatively patchy. Nevertheless, there are a number of
findings which suggest that D-glucose uptake by mammary epithelial cells is influenced by the stage of mammary development and lactation (and, therefore, by inference, prolactational hormones like prolactin, and growth
hormone) as well as nutritional status.
1. Ontogeny and hormonal control
Lactose synthesis and glucose uptake by the mammary gland increase abruptly at the time of parturition
and decline rapidly as the gland stops secretion before
involution (49, 69). Accordingly, the transport of 3-Omethly-D-glucose by isolated mouse mammary cells in-
creased during progression from the pregnant to the midlactating state (177). A sharp decline in uptake was
observed in the immediate postlactational period. A
change in the Vmax but not the Km of 3-O-methyl-D-glucose
uptake suggests that the number of transporters is affected, rather than the affinity of the carrier, by the stage
of lactation. The expression of GLUT1 mRNA and protein
fall within 24 h of litter removal in the rat (40). These
observations suggest that the differences in D-glucose
transport by mammary tissue during development, lactation, and involution reflect the expression and function of
GLUT1 under the prevailing hormonal milieu of these
physiological states.
Milk secretion in the rat is controlled by both prolactin and growth hormone. Fawcett et al. (66) investigated
the effect of these hormones on the expression of GLUT1
protein in mammary secretory cell membranes isolated
from lactating rats receiving various treatments for 48 h.
Administration of bromocriptine reduced GLUT1 in mammary membranes, as would be expected. In contrast,
GLUT1 levels in membranes from animals that had been
treated with an anti-rat growth hormone serum were not
significantly altered. However, when the two treatments
were combined, GLUT1 expression was reduced to a level
below that found with bromocriptine treatment alone.
The results suggest that prolactin and growth hormone
act synergistically to maintain GLUT1 transporter expression in rat mammary gland plasma membranes. A role for
prolactin in the regulation of GLUT1 expression in cultured mouse mammary epithelial cells has also been reported (87). Prolactin stimulates 2-deoxyglucose uptake
by mammary gland explants isolated from midpregnant
mice (170). Although the target mechanism was not identified, it can be predicted, on the basis of experiments
using rats, that the prolactin effect is on GLUT1.
Zhoa et al. (264) found that administration of bovine
growth hormone releasing factor, but not bovine growth
hormone itself, increased by 21% the amount of GLUT1
mRNA in the lactating bovine mammary gland. However,
the physiological significance is unclear, since the increase in message was not accompanied by a rise in
GLUT1 protein in mammary cell membranes.
The uptake of 2-deoxyglucose by the lactating rat
mammary gland in vivo was found in one study to be
enhanced by insulin (226). This observation is difficult to
reconcile with the failure of insulin to produce acute
stimulation of 3-O-methyl-D-glucose transport by isolated
lactating mouse mammary epithelial cells (177). If insulin
does have a direct effect on D-glucose transport by lactating mammary epithelial cells, then the target must be a
transporter other than GLUT4 (see sect. XIIA). It is possible that insulin stimulates D-glucose uptake by the rat
mammary gland as a result of altering the magnitude of
the transmembrane D-glucose gradient rather than by affecting the transport proteins directly (34).
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sorbitol (65). The nature and significance of D-glucose
transport across the apical membrane remains to be determined. If D-glucose is able to cross the apical membrane of rat mammary epithelial cells, then the available
evidence suggests that the pathway is one other than
GLUT1 because GLUT1 protein could not be detected on
the apical membrane (40).
In contrast to most species (i.e., eutherian mammals), the Tammar wallaby can, in late lactation, maintain
higher concentrations of glucose and galactose in milk
than those in plasma (133, 134). This finding raises interesting questions about the nature of D-glucose and galactose transport across the apical membrane of the mammary epithelium in this species and other marsupials. For
example, why aren’t these monosaccharides rapidly lost
from the milk space as would be predicted if the apical
membrane possessed a facilitative transport mechanism?
937
938
D. B. SHENNAN AND M. PEAKER
2. Effects of starvation
XIII. AMINO ACID TRANSPORT
The lactating mammary gland has a large demand for
amino acids to meet the requirements of milk protein
synthesis. For example, a dairy cow secreting 35 l milk/
day (by no means exceptional) with a protein content of
3.3% needs over 1 kg/day of amino acids to sustain milk
protein synthesis. The first studies on amino acid transport by the lactating mammary gland focused on arteriovenous differences in amino acid concentrations and
combining these with measurement of mammary blood
flow to establish whether amino acids were the blood
source of milk proteins and then whether their quantitative uptake was sufficient to account for milk output of
protein (129). Many studies in goats, cows, sheep, pigs,
guinea pigs, and rats have shown that the arteriovenous
differences across the gland are substantial, especially for
some of the essential amino acids (82, 119, 129, 136, 240).
This extraction of amino acids from blood is generated by
amino acid transport across the basolateral membranes of
the secretory cells. Despite the importance of amino acids
to milk secretion and mammary metabolism, it was some
time before the cellular mechanisms responsible for
amino acid uptake by the mammary gland received the
attention they deserved. Although our current understanding of mammary amino acid transport systems and
their regulation lags behind what is known in other tissues, it is now becoming clear that the lactating mammary
gland possesses amino acid transport systems analogous
to those described in other organs such as the intestine,
kidney, and placenta (209). However, it appears that some
of the mammary transport systems may have unique kinetic properties and mechanisms of control.
A. Amino Acid Transporters
1. Na⫹-dependent transport of neutral amino acids
There is good evidence for system A-like transport of
neutral amino acids in lactating mouse, rat, and bovine
mammary tissue (Table 1). System A is a Na⫹-dependent
transport mechanism that prefers short-chain neutral
amino acids as substrates and has the distinguishing feature of being able to tolerate N-methylated amino acids
(14). Mouse and rat mammary tissue explants transport
aminoisobutyric acid (AIB) via a pathway that is both Na⫹
dependent and sensitive to methylaminoisobutyric acid
(MeAIB), a characteristic of system A (145, 204). However, the substrate specificity of system A in rodent mammary tissue for naturally occurring amino acids remains
to be established. The lactating bovine mammary gland
also appears to express system A; mammary explants
transport MeAIB in a Na⫹-dependent fashion (18).
The transport of amino acids via system A may, in
part, be responsible for the accumulation of certain neutral amino acids within mammary cells with respect to
plasma (209). In contrast to mouse, rat, and bovine mammary tissue, it appears that the guinea pig mammary gland
does not possess system A activity. This conclusion is
based on the lack of MeAIB-sensitive amino acid transport by the perfused guinea pig mammary gland (131).
Bovine mammary tissue transports AIB via a system
that is Na⫹ dependent but not inhibited by MeAIB (18).
The simplest interpretation of this experimental observation is that bovine mammary tissue possesses system
ASC-like activity. Similarly, it has been concluded that
lactating guinea pig mammary tissue expresses system
ASC-like activity (131). The presence of system ASC in
mouse mammary tissue is a matter of controversy. On the
one hand, Verma and Kansal (231) have shown that methionine may utilize system ASC; on the other hand, Neville et al. (145) found no evidence for AIB transport via
system ASC in mouse mammary tissue. The difference in
results is difficult to reconcile but may reflect metabolism
of methionine in the former study. In this connection, no
evidence for system ASC in rat mammary tissue has been
found (204).
L-Glutamine transport by the lactating rat mammary
gland is partly dependent on the presence of Na⫹ (39).
The Na⫹-dependent component, a low-affinity pathway
that has been localized to the basolateral surface of the
epithelium, is not mediated by system A, since L-glutamine
transport is insensitive to MeAIB. The Na⫹-dependent
moiety of L-glutamine transport remains to be identified,
but a mechanism akin to system N (104) must be a strong
candidate.
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
Overnight starvation of lactating rats reduces 2-deoxyglucose and 3-O-methyl-D-glucose uptake by the mammary gland by ⬎90%. (226); the arteriovenous difference
in D-glucose concentration across the mammary gland is
decreased to a similar extent (156). However, refeeding
starved animals rapidly restores the uptake of glucose
analogs and increases the arteriovenous difference. It is
apparent that the effect of overnight fasting is a consequence of a reduction of D-glucose transport via GLUT1.
Thus starvation decreases cytochalasin B-sensitive 3-Omethyl-D-glucose uptake by isolated lactating mouse
mammary epithelial cells. This reduction in transport occurs as a result of a decrease in the Vmax of uptake and is
accompanied by a decrease in the number of cytochalasin
B binding sites on mammary cell plasma membranes
(175). However, it has been reported that starvation does
not alter the total GLUT1 protein content of lactating rat
mammary tissue, suggesting that the reduction in D-glucose transport induced by starvation is brought about by
the translocation of GLUT1 carriers from the plasma
membrane to an intracellular site (40).
Volume 80
July 2000
TABLE
939
MAMMARY TRANSPORT MECHANISMS
1. Kinetic properties of mammary tissue amino acid transport systems
Amino Acid Transport System
Rat
Experimental
Preparation
Km
112.5 ␮M
Explants
Reference
No.
Vmax
71.3 nmol 䡠 min⫺1 䡠 g cells⫺1
⫺1
⫺1
Rat
Perfused gland
18.1 ␮M
40.3 nmol 䡠 min
Rat
Explants
32.4 ␮M
24.5 nmol 䡠 min⫺1 䡠 g cells⫺1
⫺1
䡠 g tissue
138
139
⫺1
205
197
Rat
Explants
37.5 ␮M
2.5 nmol 䡠 min
Gerbil
Explants
70 ␮M
7.9 nmol 䡠 min⫺1 䡠 g ICW⫺1
䡠 g ICW
⫺1
138
⫺1
Mouse
Mouse
Mouse
Explants
Explants
Explants
2.0 mM
2.7 mM
0.47 mM
197 nmol 䡠 min 䡠 g cells
150 nmol 䡠 min⫺1 䡠 g cells⫺1
18.8 nmol 䡠 min⫺1 䡠 g cells⫺1
145
145
231
Mouse
Explants
0.46 mM
30.0 nmol 䡠 min⫺1 䡠 g cells⫺1
231
Mouse
Explants
0.46 mM
12.4 nmol 䡠 min⫺1 䡠 g cells⫺1
231
⫺1
⫺1
Mouse
Explants
1.67 mM
75.1 nmol 䡠 min
Mouse
Explants
15.75 mM
157.5 nmol 䡠 min⫺1 䡠 g cells⫺1
⫺1
䡠 g cells
⫺1
Mouse
Explants
0.23 mM
31.0 nmol 䡠 min
䡠 g cells
Mouse
Cultured explants
0.75 mM
430 nmol 䡠 min⫺1 䡠 g ICW⫺1
100
100
100
183
Km, Michaelis constant; Vmax, maximum velocity; AIB, aminoisobutyric acid; MeAIB, methylaminoisobutyric acid; BCH, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid.
2. Na⫹-dependent transport of anionic amino acids
In some species, L-glutamate is the most abundant
amino acid in milk protein. In addition, the mammary
gland of some species, notably humans, is able to generate a large transepithelial gradient of free glutamate in
favor of milk (5). In vivo, the mammary gland extracts
large quantities of glutamate from arterial blood (115),
indicating mammary cells do not rely on intracellular
L-glutamate synthesis. In lactating mammary tissue, there
is convincing evidence that L-glutamate and L-aspartate
are transported by a system that is analogous to system
⫺
XAG
(138, 139); the mechanism is of high affinity, is Na⫹
dependent, and interacts only with anionic amino acids.
⫺
Another XAG
-like feature of the transport system in the rat
is that the carrier differentiates between the optical isomers of glutamate but not those of aspartate.
⫺
XAG
-like transport is the predominant, if not only,
mechanism for anionic amino acid transport in rat mammary tissue; no evidence for L-glutamate transport by
⫹
system X⫺
c , a Na -independent transporter which catalyzes the exchange of L-glutamate and L-cystine (11), was
found (139). Studies using the perfused lactating rat mam⫺
mary gland suggest that the system XAG
-like activity is
situated, as predicted, in the basolateral membrane of the
mammary epithelium (138, 139). Although the kinetics
suggest that there is only a single saturable system, a
detailed study of anionic amino acid transport at the
molecular level may reveal that there is more than one
system (although their kinetics will be similar). However,
if it transpires that the Na⫹-dependent moiety of L-glutamate transport in rat mammary tissue is mediated by a
single mechanism, then the mammary system has a pharmacological profile unlike that of the high-affinity, Na⫹dependent systems that have been cloned (EAAC1,
GLAST, and GLT1) and characterized (97, 171, 219). The
evidence for such a difference is based on the effects of
dihydrokainate (DHK) and ␣-aminoadipate (AAD): anionic amino acid transport by mammary tissue is inhibited
by DHK but not AAD (139); EAAC1 is inhibited by AAD
but not DHK; GLT1 is sensitive to both AAD and DHK; and
GLAST is inhibited by neither AAD nor DHK (98, 109).
Another piece of evidence which suggests that the mammary system is different from EAAC1 is the effect of
extracellular amino acids on the efflux of substrates via
the Na⫹-dependent carrier. L-Cysteine is capable of transaccelerating L-glutamate efflux via EAAT3 (260), a human
homolog of EAAC1, whereas it has little or no effect on
amino acid efflux from rat mammary tissue (139). The
mammary gland L-glutamate transport mechanism does
display Na⫹-dependent exchange activity; anionic amino
acids such as L-glutamate, L-aspartate, D-aspartate, and
L-cysteine sulfinate are transported by the carrier operating as an exchanger (139) (Table 2). This mode of operation may allow the uptake of L-glutamate to regulate the
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
Na⫹-dependent L-glutamate uptake
⫺
(system XAG
)
Na⫹-dependent L-glutamate uptake
⫺
(system XAG
)
Na⫹-dependent D-aspartate uptake
⫺
(system XAG
)
Na⫹ ⫹ Cl⫺-dependent taurine uptake
(system ␤)
Na⫹-dependent taurine uptake (possibly
system ␤)
Na⫹-dependent AIB uptake (system A)
Na⫹-independent AIB uptake (system L)
Na⫹-dependent methionine uptake
(system A)
Na⫹-independent, MeAIB-sensitive
methionine uptake (system L)
Na⫹-dependent methionine uptake
(system ASC)
Na⫹-dependent tyrosine uptake (system
unknown)
Na⫹-independent, BCH-insensitive
tyrosine uptake (system T)
Na⫹-independent, BCH-sensitive tyrosine
uptake (system L)
AIB uptake (in presence of prolactin)
Species
940
TABLE
D. B. SHENNAN AND M. PEAKER
2. Effect of external amino acids (at 500 ␮M) on
efflux from lactating rat mammary tissue
D-aspartate
Efflux Rate Constants, min⫺1 ⫻ 10⫺4
Amino Acid
n
Control
⫹ Test
Difference
P Value
L-Glutamate
D-Aspartate
L-Aspartate
7
6
4
4
5
3
5
4
3
75 ⫾ 10
82 ⫾ 12
86 ⫾ 8
58 ⫾ 18
103 ⫾ 9
80 ⫾ 12
87 ⫾ 20
54 ⫾ 11
73 ⫾ 16
362 ⫾ 32
311 ⫾ 32
415 ⫾ 32
369 ⫾ 55
161 ⫾ 14
88 ⫾ 23
105 ⫾ 19
100 ⫾ 14
87 ⫾ 22
287 ⫾ 38
229 ⫾ 43
329 ⫾ 36
311 ⫾ 70
58 ⫾ 9
8 ⫾ 11
18 ⫾ 9
46 ⫾ 3
14 ⫾ 9
⬍0.001
⬍0.01
⬍0.01
⬍0.05
⬍0.01
NS
NS
⬍0.001
NS
CSA
L-Cysteine
L-Leucine
D-Glutamate
DHK
AAD
intracellular concentration of L-aspartate (and vice versa);
however, it is not yet known if the exchange of intracellular amino acids is required for amino acid uptake by the
high-affinity anionic amino acid carrier.
3. Na⫹-dependent transport of ␤-amino acids
Taurine (ethanesulfonic acid), a nonprotein ␤-amino
acid, is present in the milk of a large number of species
(181); in some species, notably pinnipeds, exceptionally
high concentrations are found, 13 mM in the Antarctic fur
seal for example (192). It has been suggested that the high
taurine levels in the milk of certain marine mammals are
related to their high milk fat content (152), since taurine
is required for bile salt synthesis. There is a strong possibility that milk taurine may play an important role in
growth and development of the suckling (90), particularly
in those species whose neonates are not capable of synthesizing taurine. However, taurine is also present in the
milk of species whose young are able to synthesize taurine, suggesting that the transfer of the ␤-amino acid into
milk has other roles.
Lactating rat mammary tissue accumulates taurine to
higher concentrations than plasma, generates a transepithelial concentration gradient with milk concentrations
higher than plasma, and transports radiolabeled taurine
from blood to milk (205, 218, 220). Collectively, these
results suggest that the rat mammary gland possesses an
active transport mechanism for taurine. The predominant
pathway for taurine uptake by rat mammary tissue explants is a high-affinity (Km ⫽ 43 ␮M), Na⫹- and Cl⫺dependent transport mechanism selective for ␤-amino
acids including ␤-alanine and hypotaurine (205). Another
pathway for taurine uptake in rat mammary tissue may
exist, since a small portion of the Na⫹-dependent component is not Cl⫺ dependent (205). The mammary taurine
transport system has similar characteristics to the Na⫹and Cl⫺-dependent taurine transporters that have been
described (and cloned) in other epithelia (122, 178, 229).
However, the molecular identity of the rat mammary taurine transport mechanism(s) remains to be determined.
Maintenance of a high intracellular concentration of taurine by the Na⫹-dependent system may be important in
relation to mammary cell volume regulation; there is good
evidence that taurine (and other amino acids) may play a
role following cell swelling (see XIIID).
In view of the finding that gerbil milk contains a high
concentration of taurine (⬃5 mM) (181), the uptake of taurine by lactating mammary tissue from the gerbil has also
been investigated (197). Taurine uptake by gerbil mammary
tissue explants, like that of the rat, is mediated by a highaffinity (Km⫽ 70 ␮M), Na⫹-dependent transport system that
is selective for ␤-amino acids. However, the Na⫹-dependent
moiety of taurine uptake is not Cl⫺ dependent, and anions
⫺
⫺
such as NO⫺
3 and SCN can substitute for Cl (197). In this
⫺
⫹
connection, a Cl -independent, Na -dependent taurine
transport system has been found in cultured human kidney
cells (95); however, this mechanism has a low affinity (Km⫽
1 mM) for taurine. Similarly, a Na⫹-dependent taurine transport system that is not coupled to Cl⫺ has been identified in
rabbit proximal kidney tubule (29).
4. Na⫹-independent transport of neutral amino acids
System L, a well-studied system for neutral amino
acids that is sensitive to 2-aminobicyclo-[2,2,1]-heptane-2carboxylic acid (BCH), has been identified in lactating
mouse, guinea pig, bovine, and rat mammary tissue (18,
131, 145, 206, 231) (Table 1). This conclusion is based on
the finding that mammary tissue from these species exhibits BCH-sensitive, Na⫹-independent transport of neutral amino acids. System L in mammary tissue appears, on
the basis of cis-inhibition studies, to have a wide substrate specificity and a low strereospecificity (145, 206).
This mechanism may be the single most important pathway for the uptake of the essential neutral amino acids by
the lactating gland. Studies using the perfused lactating
guinea pig and rat mammary gland preparations have
shown that system L is situated in the blood-facing aspect
of the mammary epithelium (39, 131). There is evidence to
suggest that in bovine mammary tissue system L acts as
an exchanger: intracellular cycloleucine is able to transstimulate the uptake of cycloleucine by bovine mammary
explants. Similarly, L-methionine uptake by mouse mammary explants via system L is trans-accelerated by intracellular L-methionine (99). However, AIB efflux from rat
mammary tissue is not trans-stimulated by substrates of
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D-Aspartate efflux was measured into a medium containing (in
mM) 135 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 10 glucose, and 20 Tris-MOPS,
pH 7.4, and then to a similar medium supplemented with a “test” amino
acid at 500 ␮M. CSA, L-cysteinesulfinate; DHK, dihydrokainate; AAD,
aminoadipate. Only those amino acids that inhibited L-Glu and D-Asp
uptake, with the exception of DHK, were effective at trans-stimulating
⫹
D-Asp efflux, suggesting that the high-affinity Na -dependent carrier is
able to act as an exchanger as well as a cotransport system. The finding
that DHK did not markedly stimulate D-Asp efflux indicates that DHK is
a nontransported inhibitor. [Data from Millar et al. (139).]
Volume 80
July 2000
MAMMARY TRANSPORT MECHANISMS
5. Cationic amino acid transport
The transport of cationic amino acids by mammary
tissue is of particular interest because it has been shown
in several species that L-arginine is taken up by mammary
tissue in excess of its requirement for milk protein synthesis (45). It has been suggested that L-arginine is used as
a precursor for amino acids whose uptake is less than that
required for milk protein synthesis as well as for urea
(115, 130). In addition, L-arginine is needed by mammary
epithelial cells for the synthesis of nitric oxide (112). The
transport of cationic amino acids by lactating bovine and
rat mammary tissue does not depend on Na⫹ (17, 208).
L-Lysine and L-arginine (and ornithine) share a pathway
for transport in bovine mammary tissue that is not cisinhibited by MeAIB and L-histidine (but possibly by c-Leu)
(17). However, the results of these experiments must be
interpreted with caution, since the effects of the amino
acids were not tested on the initial rate of L-lysine uptake.
Nevertheless, Baumrucker (17) concluded that the cationic amino acid transport in bovine mammary tissue is
mediated via system y⫹. L-Lysine and L-arginine also share
a pathway for transport in lactating rat mammary tissue,
but it is evident, on the basis of cis-inhibition studies, that
neutral amino acids also interact with the L-lysine carrier
(208). Thus a wide range of neutral amino acids are able
to reduce L-lysine uptake by mammary tissue explants.
The finding that L-lysine is trans-stimulated by L-leucine
and L-glutamine (even in the absence of extracellular
Na⫹) suggests that neutral amino acids are actually transported by the cationic amino acid carrier rather than
simply acting as inhibitors. It is interesting to note that
Neville et al. (145) found that L-arginine inhibited AIB
uptake by lactating mouse mammary tissue. The cationic
amino acid carrier that interacts with neutral amino acids
has also been found using the perfused rat mammary
gland, suggesting that it is located in the basolateral membrane of the secretory cells (38). The available evidence
suggests that the cationic amino acid transport system
that interacts with neutral amino acids in lactating rat
mammary tissue is not system y⫹. However, the uptake of
cationic amino acids by rat mammary tissue may be mediated by more than one pathway as the kinetics of Llysine transport have not been thoroughly examined. The
molecular identity of the rat mammary tissue cationic
amino acid transport mechanism remains to be identified,
but there is the possibility that the system may be related
to one of the amino acid transport systems (bo⫹, y⫹L) that
accepts both cationic and neutral amino acids as substrates (22, 52). On a cautionary note, it must be borne in
mind that metabolism of L-lysine (particularly in the experiments employing explants) may have confounded the
interpretation of the results.
B. Amino Acid Transport Across the Apical
Membrane of Mammary Secretory Cells
To date, the amino acid transport systems that have
been identified in mammary tissue probably reside in the
blood-facing aspect of the mammary epithelium. However, the fact remains that the milk of some species
contains significant quantities of free amino acids. A striking example is the milk of pinnipeds, where the total
concentration of free amino acids is in the range 12–21
mM (192). Invariably, the most abundant free amino acids
in milk are taurine and L-glutamate (181, 192). Although
paired measurements of free amino acids in milk and
plasma from single animals are lacking, it would appear
that some amino acids are concentrated in milk. For
example, L-glutamate is concentrated in human milk (5).
There is the possibility that amino acids can cross the
apical membrane via specific carriers, but no studies have
been reported.
C. Control of Amino Acid Transport
Amino acid uptake by lactating mammary tissue is
regulated by a variety of factors including prolactin, milk
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system L (206). Likewise, L-phenylalanine egress from rat
mammary tissue, in the absence of extracellular Na⫹, is
not significantly affected by supplementing the incubation
medium with system L substrates (D. B. Shennan, unpublished data). If it is assumed that system L in rat mammary
tissue is identical to system L in bovine and mouse mammary glands, then the finding that amino acid uptake, but
not efflux, is trans-stimulated suggests that system L in
mammary tissue operates with asymmetrical kinetics.
This form of asymmetry between the internal and external
face of the carrier favors amino acid retention by the
mammary epithelium particularly after a postprandial increase in plasma amino acids. In this connection, a sulfate
carrier with asymmetrical kinetics for trans-stimulation
between its internal and external face has been described
in human placental brush-border membrane vesicles (36).
However, there is a distinct possibility that system L in rat
mammary tissue may not be able to act as an exchange
mechanism, suggesting that there may be tissue-specific
variants of the transport mechanism. This is already apparent with respect to substrate specificity given that
system L in some tissues (e.g., the human placenta) does
not transport AIB (57).
System T, a Na⫹-independent mechanism originally
identified in human erythrocytes (189), has been described in mouse mammary tissue (100). Thus the portion
of L-tyrosine uptake by mouse mammary tissue explants
that is not dependent on extracellular Na⫹ and is not
inhibited by BCH has been ascribed to system T. This
mechanism prefers aromatic amino acids (L-tyrosine, Ltryptophan, and L-phenylalanine) as substrates.
941
942
D. B. SHENNAN AND M. PEAKER
D. Volume-Sensitive Amino Acid Transport
Most cells regulate their volume after swelling or
shrinking. Perturbations induced by anisosmotic conditions and substrate accumulation activate membrane
transport mechanisms (85, 106). Swelling activates the
release of K⫹, Cl⫺, and organic osmolytes such as amino
acids (especially taurine); this solute efflux together with
osmotically obliged water returns cells to their normal
volume. Shrinkage has the opposite effect, leading to the
uptake of solutes such as Na⫹, K⫹, Cl⫺, and amino acids
and, consequently, water.
Mammary tissue has a relatively high intracellular
free amino acid pool (93, 207, 209). In particular, the
concentrations of nonessential amino acids are higher
than in plasma or milk. It appears that mammary tissue
may utilize amino acids to regulate volume after swelling
because exposing rat mammary explants to hyposmotic
conditions increased the efflux of radiolabeled taurine
and glycine in a rapid and reversible manner (207). This
efflux is seen in the perfused rat mammary gland, suggesting that the amino acids were exiting via the basolateral membrane of the secretory cell (39). However, the
magnitude of the contribution of amino acids to mammary cell volume regulation is not known.
It is not yet known if the amino acids whose transport
is affected by cell swelling utilize one or more pathways. The
simplest explanation of the available evidence is that amino
acids, such as taurine and glycine, exit mammary tissue via
a single pathway. Volume-sensitive taurine efflux from mammary tissue may be channel mediated, since the process is
relatively temperature insensitive and does not exhibit
trans-stimulation (207). However, the exact identity of the
mammary tissue pathway is unknown. Apparently, volumeactivated taurine efflux from some, but not all, cell types is
via volume-activated anion channels. For example, it is beyond doubt that taurine is able to traverse anion channels in
Madin-Darby canine kidney, C6 glioma, and inner medullary
collecting duct cells (9, 28, 92). The finding that volumeactivated taurine efflux is also inhibited by anion transport
blockers such as DIDS, 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB), and niflumic acid has also been used to
support the claim that amino acids use volume-activated
anion channels (81, 107, 108, 157, 191). However, care must
be exercised in interpreting studies employing anion transport inhibitors because these compounds are notoriously
nonspecific (37). Interestingly, volume-sensitive taurine efflux from lactating rat mammary is inhibited by DIDS, NPPB,
and niflumic acid, albeit at relatively high concentrations.
This could, at first sight, be taken as evidence that volumesensitive taurine efflux from mammary tissue utilizes anion
channels. However, Shennan and co-workers (200, 207)
found that the effluxes of radiolabeled I⫺ and D-aspartate,
substrates of volume-activated anion channels, were not
increased when mammary explants were subjected to a
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stasis, and starvation. Most of the studies have involved
measurement of arteriovenous differences, sometimes
combined with blood flow determinations, and it was not
possible to identify which transport systems were affected. Nevertheless, it is possible in a few cases to be
almost certain what pathways would have been involved;
for example, if a manipulation affected the extraction of
anionic amino acids, then it can safely be assumed that
the effect was on the high-affinity, Na⫹-dependent, anionic amino acid transporter given that this is the only
pathway available for this class of amino acids.
Because prolactin is of major importance to milk
secretion in rats, it is not surprising that full secretory
activity stimulated by prolactin is associated with
changes to amino acid transport. Treating rats with bromocriptine during peak lactation, to block prolactin release, reduced the arteriovenous concentration difference
across the mammary gland for all amino acids with the
exception of L-aspartate, L-glutamate, and L-valine. However, the arteriovenous differences were restored (but not
for L-threonine, L-isoleucine, and L-leucine) by exogenous
prolactin (234). AIB uptake by mammary tissue explants
from rats treated with bromocriptine was reduced; the
reduction in AIB uptake is attributable to amino acid
transport systems A and L (206). Similarly, c-Leu transport by cultured rat mammary acini isolated from lactating rats during late lactation can be stimulated by prolactin, a finding consistent with an increase in system L
(187). Amino acid transport by mammary tissue taken
from midpregnant mice also responds to prolactin; AIB
transport was increased but could be inhibited by cycloheximide and actinomycin D (183).
As would be expected from the arrest of secretion
that occurs in the glands of rats that are sealed to prevent
milk removal, the uptake (as interpreted from arteriovenous differences) of all amino acids was reduced (240).
Similarly, starvation for 24 h decreased the arteriovenous
difference in concentration of all the amino acids in a
reversible manner (237). Consistent with the in vivo observations, it was subsequently shown that the uptake of
radiolabeled AIB by mammary acini isolated from starved
lactating rats is markedly reduced (223, 239). It has been
suggested that the reduction in amino acid transport can
be attributed to an increase in the circulating concentrations of ketone bodies (237). In contrast, in the mouse,
Verma and Kansal (232) found that L-methionine uptake
by mammary explants from animals that had been
starved, for the very long period of 48 h, was markedly
stimulated. It appeared that the increase in uptake can be
attributed to systems A and ASC (but not system L) and
reflects an increase in the Vmax of the transport mechanisms. These results are difficult to reconcile with those
found using the rat but could be associated with the
longer period of starvation.
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MAMMARY TRANSPORT MECHANISMS
hyposmotic challenge, and it was concluded that volumesensitive amino acid efflux from mammary tissue does not
utilize anion channels (Fig. 5). Volume-activated amino acid
efflux independent of anion channels has been demonstrated in other cell types (51, 113, 188). It must be borne in
mind that the experiments of Shennan and co-workers (207,
209) do not rule out the existence of volume-activated anion
channels in rat mammary tissue, only that amino acids do
not appear to use that route. In this connection, Kulpa (111)
has recently shown using the whole cell, patch-clamp recording technique that cultured bovine mammary cells possess volume-sensitive Cl⫺ channels.
E. Peptide Transport
Because of the nature of the digestive system and the
diverse sources of protein available to ruminant animals,
particular diets can be devised that alter the availability of
amino acids. Dairy animals have very large mammary
glands for their body size (83) and, therefore, extract
considerable quantities of amino acids from blood. Early
studies in the goat and cow showed that “the mammary
uptake of essential amino acids is sufficient to account for
the output of their corresponding residues in the protein”
(129). Later, there have been suggestions that the mammary uptake of some essential amino acids is insufficient
to account for their output in milk protein (135). For
example, in lactating goats, L-phenylalanine and L-histidine uptake were found to be insufficient for the output of
these residues in milk. (8). On the basis that these amino
acids cannot be synthesized within mammary cells, it has
been suggested that amino acids derived from circulating
peptides (and/or proteins) may make up the apparent
shortfall. There is good evidence that amino acids derived
from intravenously administered dipeptides can be incorporated into milk proteins in the goat (7, 8). The possible
mechanisms for this incorporation are transport of intact
peptides with intracellular hydrolysis and extracellular
hydrolysis followed by uptake of the free amino acids. In
this connection, Wang et al. (243) claim to have evidence,
albeit indirect, for the transport of intact peptides by
mouse mammary tissue explants; however, the mechanism(s) remains to be characterized.
In a recent study, an attempt was made to quantify
the transport of intact peptides across the basolateral
membrane of rat mammary secretory cells (199). The
transport of dipeptides containing a D-isomer at the NH2
terminus, a configuration which confers resistance to hydrolysis (132), by the perfused lactating rat mammary
gland was examined. This experimental approach demonstrated dipeptide transport by a low-affinity pathway.
However, the rate of peptide transport was low compared
with the uptake of free amino acids (199). The results
suggest that the pathway for dipeptide transport across
the basolateral membrane of rat mammary secretory cells
is different from the peptide transport systems that have
been cloned from the intestine (PepT1) and kidney
(PepT2) (67, 123).
The study by Shennan et al. (199) does not rule out a
role for a peptide transport system such as PepT1 or
PepT2 in the apical membrane of mammary secretory
cells. A specific peptide transporter on the apical aspect
of the epithelium could act as a scavenger system to
transport the products of milk protein hydrolysis back
into secretory cells. It is interesting to note that a favorable pH gradient exists across the apical membrane for
proton-coupled peptide transport, and rabbit mammary
gland has been reported to express mRNA encoding the
high-affinity PepT2 transport system (54). However, the
location of the transporter remains to be established.
It has been suggested that peptides may cross the basolateral membranes of epithelia via an anion transporter
(132). Support for this hypothesis comes from the finding
that a range of aromatic peptides and cephalosporin antibiotics are able to interact with the renal organic anion transporter (230). In this connection, benzylpenicillin, an antibiotic which contains an amide bond, is actively transported
from blood to milk across the lactating caprine mammary
gland via a pathway sensitive to probenecid, an inhibitor of
the renal organic anion transport system (193). Similarly,
probenecid reduces the transfer of benzylpenicillin into
ovine milk (266). Evidence for an organic anion transporter
in mammary tissue is strengthened by the finding that an
analog of p-aminohippuric acid (N4-acetylated p-aminohippuric acid), a model substrate of the kidney organic anion
transport mechanism, is concentrated in bovine and caprine
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FIG. 5. Effect of a hyposmotic shock (307–182 mosmol/kgH2O) on
the fractional release of radiolabeled taurine (Œ) and D-aspartate (F)
from the perfused lactating rat mammary gland. Data are means ⫾ SE
(n ⫽ 3). The results suggest that the volume-activated system is selective
for taurine and is situated in the basolateral aspect of the mammary
epithelium. [From Calvert and Shennan (39).]
943
944
D. B. SHENNAN AND M. PEAKER
milk (179). Active transport of 4-aminoantipyrine, N4-acetylated sulfanilamide, and nitrofurantoin across the bovine,
caprine, and rat mammary gland, respectively, has also been
described (10, 101, 102, 180, 227). There is the possibility that
an organic anion transport system, analogous to the one
expressed in the kidney, may provide a route for small
peptides across the basolateral membrane of mammary secretory cells.
The rat mammary gland is able to hydrolyze peptides
extracellularly followed by uptake of the free amino acids
(199). Thus anionic peptides, presented to mammary tissue explants or the perfused gland, are able to transstimulate D-aspartate efflux. It is envisaged that anionic
amino acids, produced as a consequence of peptidases
that hydrolyze anionic dipeptides extracellularly, stimulate D-aspartate efflux via the high-affinity anionic amino
⫺
acid carrier, system XAG
(199) (Fig 6). It appears that the
rat mammary gland has a large capacity to hydrolyze
anionic peptides, since some dipeptides were almost as
effective as free L-glutamate in eliciting an increase in
D-aspartate efflux. It is possible that the uptake of free
amino acids following the extracellular hydrolysis of plasma-derived peptides could make an important contribution to the supply of certain amino acids for milk protein
synthesis. The exact identity of the peptidases and the
quantitative importance of blood peptides, as opposed to
amino acids, remain to be established.
F. Does ␥-Glutamyl Transpeptidase Play a Role
in Mammary Amino Acid Transport?
Meister (128) proposed that ␥-glutamyl transpeptidase (␥-GT; EC 2.3.2.2.), an enzyme whose substrates
include glutathione (␥-glutamyl-cysteinylglycine) and certain amino acids, is involved in amino acid transport. It
was envisaged that ␥-GT (in conjunction with other enzymes) utilized intracellular glutathione to effect the
transport of extracellular amino acids into the cell. However, the original model was revised on the finding that
the enzymatic site for glutathione was on the extracellular
face (80). The model predicts that following the hydrolysis of extracellular glutathione, the ␥-glutamyl moiety is
transferred to an amino acid to form a ␥-glutamyl-dipeptide that is subsequently taken up by cells. The initial
hypothesis that ␥-GT was involved in amino acid transport was based on the correlation between high rates of
amino acid transport and ␥-GT activity. In this connection, lactating bovine and rat mammary tissue express
␥-GT activity with a specific activity second only to that
found in the kidney (19). In the rat, ␥-GT activity increases
with the onset of lactation and is regulated by prolactin. A
role for ␥-GT in rat mammary tissue amino acid transport
was inferred from the finding that the arteriovenous concentration gradients for those amino acids that are good
substrates of ␥-GT were reduced after the administration
of inhibitors (serine borate or anthglutin) of ␥-GT (233,
235). However, interpretation of these results relies
heavily on the assumption that the inhibitors are specific
for ␥-GT. The hypothesis that ␥-GT has a direct role in
amino acid transfer is weakened in light of the finding that
the turnover of glutathione required to sustain amino acid
transport is much higher than the predicted capacity of
mammary tissue (236). An alternative explanation for the
effect of ␥-GT on amino acid metabolism is that ␥-GT
produces a signal that is capable of regulating amino acid
uptake by rat mammary tissue. The main candidates are
␥-glutamyl-amino acids or 5-oxoproline; the latter compound is formed intracellularly during the metabolism of
␥-glutamyl-amino acids (236, 238). Treating rats with bromocriptine to reduce prolactin secretion inhibited ␥-GT
and decreased the arteriovenous concentration difference
of a number of amino acids. However, amino acid extraction by the rat mammary gland was restored to control
levels by a product of ␥-GT, namely, ␥-glutamyl-glutamine. Similarly, 5-oxoproline was shown to reverse the
bromocriptine-induced decrease in amino acid uptake by
the rat mammary gland.
Kansal and Kansal (99) have cast doubt on a functional relation between the activity of ␥-GT and amino
acid uptake by the lactating mammary gland. They found
that inhibition of ␥-GT had no effect on the uptake of
L-methionine and L-alanine by lactating mouse mammary
tissue explants.
Bovine mammary tissue ␥-GT is able to hydrolyze
extracellular glutathione; the liberated amino acids were
subsequently transported into the intracellular compartment (20). This observation coupled with the finding that
there is an arteriovenous glutathione concentration dif-
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FIG. 6. Proposed scheme for the extracellular hydrolysis of anionic
dipeptides (using L-Glu-L-Ala as an example) and subsequent uptake of
the liberated anionic amino acid via the high-affinity, Na⫹-dependent
anionic amino acid carrier in lactating mammary cells. [From Shennan et
al. (199). Copyright 1998, with permission from Elsevier Science.]
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MAMMARY TRANSPORT MECHANISMS
ference across the bovine mammary gland suggested that
glutathione, derived from red blood cells, may be an
important source of cysteine for milk protein synthesis
(173). Similarly, it has been concluded that the goat mammary gland also utilizes erythrocyte-derived glutathione
as a source of amino acids (6).
XIV. FATTY ACID UPTAKE
XV. COORDINATION AND CONTROL
It is clear from this review of the transport mechanisms operating across the basolateral membrane of the
secretory cell that the degree of coordination between
synthetic, secretory, and transport processes to produce
milk of virtually constant but complex composition must
be very high. Even for protein synthesis, a key question is
how the numerous transport processes for amino acids
act in concert to deliver the fixed-proportion mixture
required.
While efforts will continue to identify which transporters of those available within the genome are expressed by the mammary gland and what systemic and
local factors control that expression and activity after
transcription, a major topic for future research must be
how all the intracellular events are integrated, whether by
quantitatively coordinated gene expression in response to
hormonal or local signals or by cross-talk and feedback
between the various pathways. In this respect, it is interesting to note that the majority of work on intracellular
signaling within the mammary gland has been concerned
with mammary development, because of the obvious relation to breast cancer, rather than with the control of
secretion (250, 251).
It is time for investigations of the coordinated regulation to extend to those changes in the composition of
the secretion that occur with physiological state, the formation of colostrum in late pregnancy, and the shift to
milk production at around the time of parturition, for
example. There are also several major changes in milk
composition during lactation in marsupials, matching the
requirements of the young at different stages in development (76, 84) that should receive increased attention.
Address for reprint requests and other correspondence:
D. B. Shennan, Hannah Research Institute, Ayr, Scotland KA6
5HL (E-mail: [email protected] and/or peakerm@hri.
sari.ac.uk).
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