The functional and molecular entities underlying amino acid and peptide transport by the mammary gland under different physiological and pathological conditions.

This review describes the properties and regulation of the membrane transport proteins which supply the mammary gland with aminonitrogen to support metabolism under different physiological conditions (i.e. pregnancy, lactation and involution). Early studies focussed on characterising amino acid and peptide transport pathways with respect to substrate specificity, kinetics and hormonal regulation to allow a broad picture of the systems within the gland to be established. Recent investigations have concentrated on identifying the individual transporters at the molecular level (i.e. mRNA and protein). Many of the latter studies have identified the molecular correlates of the transport systems uncovered in the earlier functional investigations but in turn have also highlighted the need for more amino acid transport studies to be performed. The transporters function as either cotransporters and exchangers (or both) and act in a coordinated and regulated fashion to support the metabolic needs of the gland. However, it is apparent that a physiological role for a number of the transport proteins has yet to be elucidated. This article highlights the many gaps in our knowledge regarding the precise cellular location of a number of amino acid transporters within the gland. We also describe the role of amino acid transport in mammary cell volume regulation. Finally, the important role that individual mammary transport proteins may have in the growth and proliferation of mammary tumours is discussed.

Placental sulphate transport: a review of functional and molecular studies.

Sulphate is required by the feto-placental unit for a number of important conjugation and biosynthetic pathways. Functional studies performed several decades ago established that sulphate transport in human placental microvillus and basal membrane vesicles was mainly via a DIDS-sensitive anion-exchange mechanism. In contrast, no evidence was found for Na⁺-dependent transport. Studies performed using isolated human placental tissue confirmed anion-exchange as the main mechanism. More recently, molecular studies have established the presence of anion-exchange proteins which could play a role in transplacental sulphate movement. However, the presence of transcripts for NaS2 has been reported and has prompted the suggestion that Na⁺-sulphate cotransport may play an important role in maternal-fetal sulphate transport. This article reviews our present knowledge of placental sulphate transport, both functional and molecular, and attempts to form a model based on the available evidence.

The effect of a hyposmotic shock and purinergic agonists on K+(Rb+) efflux from cultured human breast cancer cells.

The effect of a hyposmotic shock and extracellular ATP on the efflux of K(+)(Rb(+)) from human breast cancer cell lines (MDA-MB-231 and MCF-7) has been examined. A hyposmotic shock increased the fractional efflux of K(+)(Rb(+)) from MDA-MB-231 cells via a pathway which was unaffected by Cl(-) replacement. Apamin, charybdotoxin or removing extracellular Ca(2+) had no effect on volume-activated K(+)(Rb(+)) efflux MDA-MB-231 cells. An osmotic shock also stimulated K(+)(Rb(+)) efflux from MCF-7 cells but to a much lesser extent than found with MDA-MB-231 cells. ATP-stimulated K(+)(Rb(+)) efflux from MDA-MB-231 cells in a dose-dependent fashion but had little effect on K(+)(Rb(+)) release from MCF-7 cells. ATP-stimulated K(+)(Rb(+)) efflux was only inhibited slightly by replacing Cl(-) with NO(3)(-). Removal of external Ca(2+) during treatment with ATP reduced the fractional efflux of K(+)(Rb(+)) in a manner suggesting a role for cellular Ca(2+) stores. Charybdotoxin, but neither apamin nor iberiotoxin, inhibited ATP-stimulated K(+)(Rb(+)) release from MDA-MB-231 cells. Suramin inhibited the ATP-activated efflux of K(+)(Rb(+)). UTP also stimulated K(+)(Rb(+)) efflux from MDA-MB-231 cells whereas ADP, AMP and adenosine were without effect. A combination of an osmotic shock and ATP increased the fractional efflux of K(+)(Rb(+)) to a level greater than the sum of the individual treatments. It appears that the hyposmotically-activated and ATP-stimulated K(+) efflux pathways are separate entities. However, there may be a degree of 'crosstalk' between the two pathways.

Activation and inactivation of volume-sensitive taurine efflux from rat mammary gland.

A knowledge of volume-sensitive solute transport in mammary cells is important in light of evidence that mammary cell metabolism is regulated by the cellular hydration state. In this report we have examined volume-sensitive taurine and K+ (Rb+) transport by lactating rat mammary tissue. A hyposmotic shock increased taurine efflux from rat mammary tissue: taurine release returned to a basal level upon transferring the tissue back to an isosmotic medium. However, the time taken to activate taurine efflux was less than the time taken to inactivate taurine release. A second subsequent osmotic challenge also increased taurine release but to a lesser extent than the first osmotic shock. A similar pattern was observed for bumetanide-insensitive, volume-activated K+ (Rb+) release from mammary tissue explants suggesting that taurine and K+ efflux are acting in concert to regulate mammary cell volume. An abrupt hyposmotic shock increased taurine efflux from mammary explants to a greater extent than a gradual reduction in the osmolality of the incubation medium. Increasing extracellular K+ increased taurine release via a pathway sensitive to niflumic acid, which suggests that activation of volume-sensitive taurine efflux does not require a change in the ionic strength of the incubation medium or a decrease in intracellular osmolality. A hyposmotic shock also stimulated taurine efflux from rat mammary acini. In contrast, a hyposmotic challenge had no effect on taurine uptake measured under sodium-free conditions. Hyposmotically induced taurine efflux was not dependent upon extracellular calcium. The results suggest that taurine and K+ transport may allow mammary cells to volume-regulate and consequently help to control mammary metabolism.

L-leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics, regulation by estrogen and molecular identity of the transporter.

The transport of L-leucine by two human breast cancer cell lines has been examined. L-leucine uptake by MDA-MB-231 and MCF-7 cells was via a BCH-sensitive, Na+ -independent pathway. L-leucine uptake by both cell lines was inhibited by L-alanine, D-leucine and to a lesser extent by L-lysine but not by L-proline. Estrogen (17beta-estradiol) stimulated L-leucine uptake by MCF-7 but not by MDA-MB-231 cells. L-leucine efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH in a dose-dependent fashion. The effect of external BCH on L-leucine efflux from both cell types was almost abolished by reducing the temperature from 37 to 4 degrees C. There was, however, a significant efflux of L-leucine under zero-trans conditions which was also temperature-sensitive. L-glutamine, L-leucine, D-leucine, L-alanine, AIB and L-lysine all trans-stimulated L-leucine release from MDA-MB-231 and MCF-7 cells. In contrast, D-alanine and L-proline had little or no effect. The anti-cancer agent melphalan inhibited L-leucine uptake by MDA-MB-231 cells but had no effect on L-leucine efflux. Quantitative real-time PCR revealed that LAT1 mRNA was approximately 200 times more abundant than LAT2 mRNA in MCF-7 cells and confirmed that MDA-MB-231 cells express LAT1 but not LAT2 mRNA. LAT1 mRNA levels were higher in MCF-7 cells than in MDA-MB-231 cells. Furthermore, LAT1 mRNA was more abundant than CD98hc mRNA in both MDA-MB-231 and MCF-7 cells. The results suggest that system L is the major transporter for L-leucine in both MDA-MB-231 and MCF-7 cells. It is possible that LAT1 may be the major molecular correlate of system L in both cell types. However, not all of the properties of system L reflected those of LAT1/LAT2/CD98hc.

Indoleamine 2,3-dioxygenase activity and L-tryptophan transport in human breast cancer cells.

The activity and expression of indoleamine 2,3-dioxygenase together with L-tryptophan transport has been examined in cultured human breast cancer cells. MDA-MB-231 but not MCF-7 cells expressed mRNA for indoleamine 2,3-dioxygenase. Kynurenine production by MDA-MB-231 cells, which was taken as a measure of enzyme activity, was markedly stimulated by interferon-gamma (1000 units/ml). Accordingly, L-tryptophan utilization by MDA-MB-231 cells was enhanced by interferon-gamma. 1-Methyl-DL-tryptophan (1 mM) inhibited interferon-gamma induced kynurenine production by MBA-MB-231 cells. Kynurenine production by MCF-7 cells remained at basal levels when cultured in the presence of interferon-gamma. L-Tryptophan transport into MDA-MB-231 cells was via a Na(+)-independent, BCH-sensitive pathway. It appears that system L (LAT1/CD98) may be the only pathway for l-tryptophan transport into these cells. 1-Methyl-D,L-tryptophan trans-stimulated l-tryptophan efflux from MDA-MB-231 cells and thus appears to be a transported substrate of system L. The results suggest that system L plays an important role in providing indoleamine-2,3-dioxygenase with its main substrate, L-tryptophan, and suggest a mechanism by which estrogen receptor-negative breast cancer cells may evade the attention of the immune system.

Functional and molecular characteristics of system L in human breast cancer cells.

The functional and molecular properties of system L in human mammary cancer cells (MDA-MB-231 and MCF-7) have been examined. All transport experiments were conducted under Na(+)-free conditions. alpha-Aminoisobutyric acid (AIB) uptake by MDA-MB-231 and MCF-7 cells was almost abolished by BCH (2-amino-2-norbornane-carboxylic acid). AIB uptake by MDA-MB-231 cells was also inhibited by L-alanine (83.6%), L-lysine (75.6%) but not by L-proline. Similarly, L-lysine and L-alanine, respectively, reduced AIB influx into MCF-7 cells by 45.3% and 63.7%. The K(m) of AIB uptake into MDA-MB-231 and MCF-7 cells was, respectively, 1.6 and 8.8 mM, whereas the V(max) was, respectively, 9.7 and 110.0 nmol/mg protein/10 min. AIB efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH, L-glutamine, L-alanine, L-leucine, L-lysine and AIB (all at 2 mM). In contrast, L-glutamate, L-proline, L-arginine and MeAIB had no effect. The interaction between L-lysine and AIB efflux was one of low affinity. The fractional release of AIB from MDA-MB-231 cells was trans-accelerated by D-leucine and D-tryptophan but not by D-alanine. MDA-MB-231 and MCF-7 cells expressed LAT1 and CD98 mRNA. MCF-7 cells also expressed LAT2 mRNA. The results suggest that AIB transport in mammary cancer cells under Na(+)-free conditions is predominantly via system L which acts as an exchange mechanism. The differences in the kinetics of AIB transport between MDA-MB-231 and MCF-7 cells may be due to the differential expression of LAT2.

A study of L-leucine, L-phenylalanine and L-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2.

The transport of L-leucine, L-phenylalanine and L-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of L-leucine was inhibited by BCH (65%) and D-leucine (58%) but not by L-proline. The inhibition of L-leucine clearance by BCH and D-leucine was not additive. L-leucine inhibited the peak clearance of radiolabelled L-leucine by 78%. BCH also inhibited the peak clearance of L-phenylalanine (66%) and L-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that L-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.

The effect of hyposmotic and isosmotic cell swelling on the intracellular [Ca2+] in lactating rat mammary acinar cells.

The effect of hyposmotic and isosmotic cell swelling on the free intracellular calcium concentration ([Ca2+]i) in rat mammary acinar cells has been examined using the fura-2 dye technique. Ahyposmotic shock (40% reduction) increased the [Ca2+]i in rat mammary acinar cells in a fashion which was transient; the [Ca2+]i returned to a value similar to that found under isomotic conditions within 180 sec. The increase in the [Ca2+]i was dependent upon the extent of the osmotic shock. The hyposmotically-activated increase in the [Ca2+]i could not be attributed to a reduction in extracellular Na+ or a change in the ionic strength of the incubation medium. Thapsigargin (1 microM) enhanced the hyposmotically-activated increase in the [Ca2+]i. Isosmotic swelling of rat mammary acinar cells, using urea, had no significant effect on the [Ca2+]i. Similarly, a hyperosmotic shock did not affect the [Ca2+]i in rat mammary acinar cells. It appears that the effect of cell swelling on the [Ca2+]i in rat mammary acinar cells depends on how the cells are swollen (hyposmotic vs. isosmotic). This finding may have important physiological implications given that it is predicted that mammary cell volume will change in vivo under isomotic conditions.

Iodide transport in lactating rat mammary tissue via a pathway independent from the Na+/I- cotransporter: evidence for sulfate/iodide exchange.

Although it is beyond doubt that mammary cells accumulate iodide via a Na+-dependent transport mechanism, it is not clear if this is the only pathway for iodide transport in mammary tissue. In view of this, experiments were designed to test for the presence of an anion-exchange pathway which could mediate the transport of iodide into mammary cells; thus, the effect of external iodide on sulfate efflux from rat mammary tissue has been investigated. Iodide trans-stimulated sulfate efflux from mammary tissue explants in a dose-dependent manner: 0.1, 1.0 and 10.0 mM iodide stimulated the fractional release of iodide by 56 +/- 2.2, 166.5 +/- 17.5, and 302.9 +/- 29.8%, respectively. The stimulation of sulfate efflux by external iodide was completely inhibited by DIDS (4.4'-diisothiocyanatostilbene 2,2'-disulfonic acid). Perchlorate (1 mM) also trans-stimulated sulfate efflux in a manner that was inhibited by DIDS. Furthermore, iodide trans-accelerated sulfate efflux from rat mammary acini via a DIDS-sensitive mechanism. The results are consistent with the presence of a DIDS-sensitive anion-exchange mechanism which can accept iodide as a substrate. It appears that the iodide-sulfate exchange mechanism is independent from the sodium-dependent iodide transporter given that sulfate is not a substrate of the latter system. The iodide-sulfate exchanger may operate in parallel with the sodium-dependent iodide transporter to mediate iodide uptake into mammary cells.

Hyposmotically-activated efflux of L-carnitine from a human mammary cancer cell line.

Cell-swelling, induced by a hyposmotic challenge, stimulated the efflux of L-carnitine from a human mammary cancer cell line, MDA-MB-231. The response was dependent upon the extent of the osmotic shock. Hyposmotically-activated L-carnitine efflux was inhibited by the anion transport blocker diiodosalicylate. The efflux of taurine from MDA-MB-231 cells was also stimulated by a hyposmotic shock via a pathway sensitive to diiodosalicylate. L-carnitine efflux from MDA-MB-231 cells was stimulated by isosmotic swelling in a manner which was inhibited by diiodosalicylate. The results suggest that L-carnitine may exit cells via a volume-sensitive pathway: it is possible that L-carnitine efflux may utilize the same pathway as amino acids. The efflux of L-carnitine via this route could have a major effect on the intracellular concentration of L-carnitine and could facilitate transepithelial L-carnitine transport.

Volume-activated K(+)(Rb(+)) efflux in lactating rat mammary tissue.

The effect of cell swelling, induced by a hyposmotic shock, on K(+)(Rb(+)) efflux from lactating rat mammary tissue explants has been studied. A hyposmotic challenge increased the fractional release of K(+)(Rb(+)) from mammary tissue in the absence and presence of the loop-diuretic bumetanide (100 microM). However, the volume-sensitive moiety of K(+)(Rb(+)) efflux was proportionately larger when bumetanide was present in the incubation medium. On the other hand, a hyposmotic shock appeared to reduce the bumetanide-sensitive component of K(+)(Rb(+)) efflux. The increase in K(+)(Rb(+)) efflux, induced by cell swelling, was dependent upon the extent of the hyposmotic challenge. In the presence of bumetanide, substituting Cl(-) with NO(3)(-) reduced the initial increase in volume-sensitive K(+)(Rb(+)) efflux. However, volume-sensitive K(+)(Rb(+)) release was prolonged in the presence of NO(3)(-). Volume-activated K(+)(Rb(+)) efflux from rat mammary tissue explants was inhibited by quinine. Cell swelling increased the intracellular concentration of Ca(2+) in a fashion which depended on the presence of extracellular Ca(2+). However, removing extracellular Ca(2+) did not inhibit volume-activated K(+)(Rb(+)) efflux from rat mammary tissue explants. The results are consistent with the presence of volume-activated K(+) channels in lactating rat mammary tissue. Volume-activated K(+) efflux may play a central role in mammary cell volume regulation.

Transport of milk constituents by the mammary gland.

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.

Volume-sensitive amino acid efflux from a pancreatic beta-cell line.

Cell swelling, induced by a hyposmotic shock, increased the fractional release of taurine from INS-1 cells. Volume-sensitive taurine release was (a) dependent upon the extent of cell swelling; (b) fully reversible; and (c) temperature dependent. Volume-sensitive taurine efflux was independent from the trans-membrane Na(+)-gradient. DIDS markedly inhibited volume-activated taurine efflux but not basal taurine release suggesting that the volume-sensitive pathway is quiescent under isosmotic conditions. Volume-activated taurine release inactivated in the continued presence of a hyposmotic shock. Cell-swelling also increased the fractional release of D-aspartate from INS-1 cells. Volume-activated D-aspartate efflux was inhibited by DIDS, albeit to a lesser extent than volume-sensitive taurine release. It is predicted that volume-sensitive amino acid efflux acts in parallel with other volume-activated transport mechanisms to regulate the volume of insulin-secreting cells.

Regulation of protein synthesis in lactating rat mammary tissue by cell volume.

The effect of changing cell volume on rat mammary protein synthesis has been examined. Cell swelling, induced by a hyposmotic challenge, markedly increased the incorporation of radiolabelled amino acids (leucine and methionine) into trichloroacetic acid (TCA)-precipitable material: reducing the osmolality by 47% increased leucine and methionine incorporation into mammary protein by 147 and 126% respectively. Conversely, cell shrinking, induced by a hyperosmotic shock, almost abolished the incorporation of radiolabelled amino acids into mammary protein: increasing the osmolality by 70% reduced leucine and methionine incorporation into mammary protein by 86 and 93% respectively. The effects of cell swelling and shrinking were fully reversible. Volume-sensitive mammary tissue protein synthesis was dependent upon the extent of the osmotic challenge. Isosmotic swelling of mammary tissue, using a buffer containing urea (160 mM), increased the incorporation of radiolabelled leucine into TCA-precipitable material by 106%. Swelling-induced mammary protein synthesis was dependent upon calcium: removing extracellular calcium together with the addition of EGTA markedly reduced volume-activated protein synthesis. Cell swelling-induced protein synthesis was inhibited by the Ca(2+) ATPase blocker thapsigargin suggesting that volume-sensitive protein synthesis is dependent upon luminal calcium.

Lysine uptake by mammary gland tissue from lactating sows.

Kinetic properties and substrate specificity of the lysine transport system in porcine mammary gland were studied using mammary tissue explants from nine lactating sows. Sodium dependence of lysine uptake was determined by replacing sodium in the medium with choline. Kinetic parameters of lysine uptake were determined using lysine concentrations from 5 microM to 5.12 mM. Competition of lysine uptake by other amino acids was determined using the cationic amino acids, arginine and ornithine, and using other essential amino acids. Transport of lysine was time-dependent and was unaffected by replacing sodium with choline. Lysine uptake occurred by a transport mechanism with a Km of approximately 1.4 mM and a Vmax of 7.9 mmol x kg cell water(-1) x 30 min(-1). Lysine uptake was inhibited by arginine and ornithine and by high concentrations of L-alanine, L-methionine, L-leucine, cycloleucine, and D-lysine, but not by 2-(methylamino)-isobutyric acid. This transport mechanism is the primary system responsible for uptake of cationic amino acids in lactating sow mammary tissue. The relatively high Km, compared with physiological blood concentrations of lysine, indicates that the kinetic properties of the lysine transport system should not be limiting to milk protein synthesis. Transmembrane transport of lysine by lactating sow mammary tissue should be a direct function of plasma concentrations. However, interactions of other amino acids with the uptake system may affect lysine uptake.

Further evidence for the existence of a volume-activated taurine efflux pathway in rat mammary tissue independent from volume-sensitive Cl- channels.

The effect of cell-swelling, induced by a hyposmotic shock, on the fractional loss of taurine, D-aspartate and iodide from lactating rat mammary tissue explants has been examined. In paired experiments, cell-swelling markedly increased the fractional efflux of taurine but not that of D-aspartate. Similarly, in paired experiments, a hyposmotic challenge stimulated taurine release but not iodide efflux. The results suggest that volume-activated taurine efflux from mammary tissue explants is via a pathway independent from volume-sensitive anion channels. It is apparent that the volume-activated taurine efflux pathway in mammary tissue is not the volume-sensitive organic osmolyte-anion channel which has been described in other cells. Therefore, the results of this study together with others in the literature constitute prima facie evidence for the existence of more than one type of swelling-activated pathway which accepts taurine as a substrate.

The regulation of Na(+)-dependent anionic amino acid transport by the rat mammary gland.

The regulation of anionic amino acid transport, using radiolabelled D-aspartate as a tracer, by rat mammary tissue explants has been examined. Na(+)-dependent D-aspartate uptake by mammary tissue increased between late pregnancy and early lactation and again at peak lactation but thereafter declined during late lactation. In contrast, the Na(+)-independent component of D-aspartate uptake by mammary explants did not change significantly with the physiological state of the donor animals. Premature weaning of rats during peak lactation markedly decreased Na(+)-dependent D-aspartate uptake by mammary tissue. In addition, premature weaning also reduced the effect of reversing the trans-membrane Na(+)-gradient on the fractional loss of D-aspartate from mammary tissue explants. Unilateral weaning of rats during peak lactation revealed that milk accumulation per se reduced the Na(+)-dependent moiety of D-aspartate uptake by mammary tissue suggesting that the transport of anionic amino acids is regulated to match supply with demand. Treating lactating rats with bromocryptine reduced D-aspartate uptake by mammary tissue explants suggesting that the transport of anionic amino acids by the rat mammary gland is regulated by prolactin.