AJP - Endo Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 291: E1059-E1066, 2006. First published June 20, 2006; doi:10.1152/ajpendo.00062.2006
0193-1849/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/5/E1059    most recent
00062.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by López, A.
Right arrow Articles by Tovar, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by López, A.
Right arrow Articles by Tovar, A. R.

Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in rat mammary gland

Adriana López,1,2 Nimbe Torres,1 Victor Ortiz,1 Gabriela Alemán,1 Rogelio Hernández-Pando,3 and Armando R. Tovar1

1Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; 2Posgrado en Biología Experimental, Universidad Autónoma Metropolitana-Iztapalapa; and 3Departamento de Patología Experimental, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico

Submitted 17 February 2006 ; accepted in final form 10 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Amino acid transport via system A plays an important role during lactation, promoting the uptake of small neutral amino acids, mainly alanine and glutamine. However, the regulation of gene expression of system A [sodium-coupled neutral amino acid transporter (SNAT)2] in mammary gland has not been studied. The aim of the present work was to understand the possible mechanisms of regulation of SNAT2 in the rat mammary gland. Incubation of gland explants in amino acid-free medium induced the expression of SNAT2, and this response was repressed by the presence of small neutral amino acids or by actinomycin D but not by large neutral or cationic amino acids. The half-life of SNAT2 mRNA was 67 min, indicating a rapid turnover. In addition, SNAT2 expression in the mammary gland was induced by forskolin and PMA, inducers of PKA and PKC signaling pathways, respectively. Inhibitors of PKA and PKC pathways partially prevented the upregulation of SNAT2 mRNA during adaptive regulation. Interestingly, SNAT2 mRNA was induced during pregnancy and to a lesser extent at peak lactation. beta-Estradiol stimulated the expression of SNAT2 in mammary gland explants; this stimulation was prevented by the estrogen receptor inhibitor ICI-182780. Our findings clearly demonstrated that the SNAT2 gene is regulated by multiple pathways, indicating that the expression of this amino acid transport system is tightly controlled due to its importance for the mammary gland during pregnancy and lactation to prepare the gland for the transport of amino acids during lactation.

system A; mammary gland; amino acid transport; lactation

MILK PRODUCED BY THE MAMMARY GLAND during lactation is fundamental for the development and growth of the newborn, providing nutrients in the quantity and quality required to maintain metabolic functions. Thus, to fulfill this role, the lactating mammary gland requires, among other nutrients, a large amount of amino acids to sustain different functions, such as synthesis of milk proteins as energy substrates (36) and synthesis of fatty acids (39). Several studies (2, 39) in different species where arteriovenous differences in amino acids across the mammary gland are measured indicate that all amino acids are captured by the gland, with alanine and glutamine the most actively transported. These amino acids are preferentially transported by system A in most tissues (21), and this activity could play a key role in the mammary gland as well as in other tissues (3).

Several studies showed that mammary glands possess characteristics of system A activity (20, 27), as determined by using the nonmetabolizable analog {alpha}-(methylamino)isobutyric acid (MeAIB) (35), such as Na+ dependence, pH sensitivity, preference for the uptake of short-chain neutral amino acids like alanine and serine, as well as the amide amino acids glutamine and asparagine, and relatively lower affinity for threonine and the branched-chain amino acids (10, 15, 21). In addition, system A in mammary gland presents a unique characteristic of this transport system observed in other cell types called adaptive regulation (35). Adaptive regulation occurs when cells are incubated in a medium lacking extracellular amino acid substrates, and as a result there is an increase in system A transport activity. This activity is repressed with actinomycin D or when cells are transferred to an amino acid-rich medium (14).

In recent years, a gene family of sodium-coupled neutral amino acid transporters (SNAT; SCL38 gene family) coding for proteins that possess the classically described system A transport activities, in terms of their functional properties and patterns of regulation, has been cloned (18). Several isoforms have been described and designated SNAT1 (38), SNAT2 or SAT2 (33, 40), and SNAT4 (34). SNAT2 is widely expressed in rat tissues, and its mRNA concentration increases in cultured cells during adaptive regulation or with the addition of cAMP (8). A recent study (22) has demonstrated that the increase of SNAT2 expression in response to amino acid deprivation is associated with the presence of an amino acid response element located in intron 1 of the gene in several species.

Because of the importance of system A activity in the mammary gland during lactation, as well as its role in the control of other amino acid transport systems, it is important to understand the regulation of expression of the SNAT2 isoform in the mammary gland. The results of the present study show that SNAT2 expression in rat mammary gland explants is controlled at multiple levels by a variety of mechanisms that involve PKA and PKC, as well as estradiol. Presumably, all these signals tightly control the expression of SNAT2, depending on the physiological status of the gland during pregnancy and lactation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Nylon membrane filter Hybond N+, deoxycytidine 5'-[{alpha}-32P]triphosphate (3,000 Ci/mmol), and the rediprime DNA labeling system were from Amersham, UK.

Animals. Female Wistar rats with a weight of 200–250 g were obtained from the animal research facility at the Instituto Nacional de Ciencias Médicas y Nutrición. Animals were housed in individual stainless steel cages at 18°C with a 12:12-h light-dark cycle and allowed free access to water and chow diet. Gestational age was determined by vaginal smear to detect spermatozoa. Mammary gland explants were obtained as previously reported (35) from virgin, pregnant, lactating, and postweaning rats. After normal pregnancy and delivery, the litter size was adjusted to 8 pups/dam. Rats at the end of lactation (21 days postpartum) were separated from their pups. Nonpregnant, nonlactacting rats were used as control rats and are referred to as "virgin rats." This study was approved by the Animal Care Committee of the Instituto Nacional de Ciencias Médicas y Nutrición, México, in accordance with international guidelines for the use of animals in research.

Preparation of mammary tissue explants. Mammary tissue explants were prepared as described previously (35). Briefly, rats were anesthetized and then killed by decapitation. The mammary gland was immediately removed and placed at 37°C in 30 ml of Krebs-Ringer bicarbonate buffer, pH 7.4, equilibrated with 95% O2-5% CO2 and contained the following (in mM): 141 NaCl, 5.6 KCl, 3.0 CaCl2, 1.4 KH2PO4, 1.4 MgSO4, 24.6 NaHCO3, and 11 glucose. After removal of the connective tissue, the mammary tissue was diced into 2- to 5-mg explants. The tissue explants were rinsed repeatedly with buffer at 37°C prior to assay.

RNA preparation. Mammary tissue was homogenized in guanidinium buffer containing 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.1 M 2-mercaptoethanol, and 0.5% N-laurylsarcosine with a polytron (PT2000; Kinematica, Lucerne, Switzerland) at the lowest setting. The homogenate was centrifuged at 12,000 g for 15 min at 18°C, and the resulting supernatant was layered onto a CsCl solution containing 5.7 M CsCl and 25 mM sodium acetate, pH 5.2. The CsCl gradient was formed by centrifugation at 113,000 g for 18 h at 18°C to yield total RNA. The RNA was precipitated with 100% ethanol and 3 M sodium acetate, pH 5.2, washed twice in 75% ethanol, and resuspended in formamide. The RNA was quantified by optical density at 260 nm and stored at –80°C until use.

Probe preparation. The probe was prepared by RT-PCR using poly(A)+ mRNA isolated from mammary gland. Poly(A)+ RNA was purified from mammary gland explants by guanidine thiocyanate extraction and ultracentrifugation through a cesium chloride cushion (5) followed by a single round of oligo(dT) cellulose chromatography. The sense primer was 5'-CTACTCATACCCCACGAAGCA-3', which corresponds to nucleotide positions 475–495 in SNAT2 cDNA, and the antisense primer was 5' ACGACTACGCCACTCAGGA-3', which corresponded to nucleotide positions 1824–1842 in SNAT2 cDNA (GenBank accession no. NM181090). RT-PCR was performed using the following PCR amplification conditions: denaturation for 5 min at 95°C, annealing for 1 min at 56.2°C, and extension for 1.30 min at 72°C for 34 cycles; final extension for 7 min at 72°C. The resultant product (1,368 bp) was separated by electrophoresis on a 1.5% agarose-Tris-acetate-EDTA gel containing ethidium bromide and viewed under UV light. Afterward, manual sequencing of the product was performed to establish its molecular identity.

Northern blot analysis. For Northern analysis, 15 µg of RNA were loaded into each lane and were electrophoresed in a 1.0% agarose gel containing 37% formaldehyde. The RNA was then blotted onto nylon membranes (Hybond N+) and cross-linked with a UV crosslinker (Amersham). cDNA probe was labeled with Redivue [{alpha}-32P]dCTP (110 TBq/mmol) using the Rediprime DNA labeling kit. Membranes were prehybridized with rapid-hyb buffer (Amersham) at 65°C for 1 h and then hybridized with the cDNA probe for 2.5 h at 65°C. Membranes were washed once with 2 SSC-0.1% SDS (1 SSC = 0.15 M sodium chloride, 15 M sodium citrate) at room temperature for 20 min and then twice for 15 min with 0.1 SSC-0.1% SDS at 65°C. Digitization of the images and quantitation of radioactivity (cpm) of the bands were done by using the Instant Imager (Packard Instrument, Meriden, CT). Membranes were also exposed to Extascan film (Kodak, Guadalajara, Mexico) at –70°C with an intensifying screen.

Preparation of tissue for in situ RT-PCR. The alcohol-fixed mammary gland sections (4 µm) were embedded in paraffin and used for system A mRNA expression determined by in situ RT-PCR. Mammary gland sections were mounted on silane cover slides, deparaffinized for 18 h at 60°C, and sequentially immersed in xylene (30 min at 37°C), absolute ethanol, 50% ethanol, and water. Cells were rendered permeable by incubation for 10 min at room temperature in 0.02% HCl, followed by a 90-s incubation with 0.01% Triton X-100 and then a 30-min incubation at 37°C with 1 µg/ml proteinase K (GIBCO-BRL, Gaithersburg, MD). DNA was removed by incubation for 15 min at 37°C with DNase, 3 U/tissue section (GIBCO-BRL). Reverse transcription was performed at 37°C for 1 h, incubating the tissue in 50 µl of reverse transcriptase buffer (1x, GIBCO-BRL), 0.1 M dithiothreitol, 2 mM dNTP mix (GIBCO-BRL), 0.1 µg/µl of oligo (dT), and 200 U of Moloney murine leukaemia virus reverse transcriptase (GIBCO-BRL). The sections were sealed using the Assembly tool (PerkinElmer, Branchburg, NJ). The RNA was then removed by incubation with 3 U RNase and fixed again by immersion with 4% paraformaldehyde in Sorensen buffer for 3 min at 4°C, and PCR was performed, incubating the tissue sections with 50 µl of 1x reaction buffer (GIBCO-BRL), 2 U of Taq polymerase, 15 mM MgCl2, and dNTPs labeled with digoxigenin (Boehringer Mannheim, Mannheim, Germany). The slides were sealed again and placed in the thermocycler (PerkinElmer). After an initial denaturation at 95°C for 5 min, 35 cycles were performed using an annealing temperature of 56.2°C for 1 min and an extension temperature of 72°C for 1.5 min. PCR products were detected with monoclonal mouse antidigoxigenin antibodies coupled to alkaline phosphatase and tetrazolium nitroblue (Boehringer Mannheim) and counterstained using nuclear fast red. Expression of actin was used as a positive control, and the negative control consisted of performing the whole procedure without the system A primers.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To determine the mechanism(s) of regulation of the SNAT2 gene expression, we studied whether adaptive regulation was accompanied with a concomitant increase of SNAT2 mRNA in mammary gland explants. With this purpose, explants were incubated in an amino acid-free medium for different periods of time ≤300 min. As shown in Fig. 1, SNAT2 mRNA concentration increased steadily during the first 250 min of amino acid deprivation, reaching a 20-fold induction. When incubation of mammary gland explants in amino acid-free medium was prolonged ≤300 min, SNAT2 mRNA concentration began to decay. Interestingly, when mammary gland explants were incubated with an equimolar mixture of the amino acids used by the amino acid transport system A (1 mM each alanine, glycine, and serine), the induction of SNAT2 was suppressed (Fig. 1). However, when explants were incubated with a mixture of large neutral and cationic amino acids (1 mM each phenylalanine, valine, and lysine), a rapid induction of SNAT2 was observed at 50 min, and it was less prolonged compared with explants incubated in an amino acid-free medium (Fig. 1). These results clearly indicated that SNAT2 gene expression in mammary gland explants was susceptible to the presence or absence of small neutral amino acids in the medium. To identify whether the expression of SNAT2 was dependent on RNA synthesis, lactating mammary gland explants were preincubated for 300 min in the presence of 50 µM actinomycin D. As can be seen in Fig. 1, there was no induction of SNAT2 mRNA, indicating that abundance of SNAT2 mRNA was indeed dependent on RNA synthesis during adaptive regulation.


Figure 1
View larger version (44K):
[in this window]
[in a new window]
  		Fig. 1. A: expression of sodium-coupled neutral amino acid transporter (SNAT)2 mRNA in mammary gland explants from lactating rats incubated in an amino acid-free medium ≤300 min or in medium containing a mixture of small neutral [alanine (Ala), glycine (Gly), and serine (Ser)] or large neutral and cationic amino acids [phenylalanine (Phe), valine (Val), and lysine (Lys)] or in an amino acid-free medium containing actinomycin D. B: quantitative analysis of mRNA concentration of the experiments indicated in A. Values were normalized with ribosomal RNA 28S. Values are means ± SE; n = 3.

 
To study the rate of SNAT2 mRNA decay, explants were preincubated for 200 min in an amino acid-free medium and were then switched to a medium containing a mixture of small neutral amino acids (1 mM each alanine, glycine, and serine) to repress SNAT2 gene expression. A rapid decay of SNAT2 mRNA was observed, and the estimated half-life for the decay was 67 min, indicating that SNAT2 mRNA has a rapid turnover rate. These results indicate that the presence of the amino acid substrates for system A in the medium rapidly repressed the expression of SNAT2 mRNA (Fig. 2).


Figure 2
View larger version (37K):
[in this window]
[in a new window]
  		Fig. 2. A: rate of decay of mammary gland SNAT2 mRNA from rat explants after 200 min of preincubation in an amino acid-free medium that was then incubated ≤2 h in a medium containing a mixture of small neutral amino acids (Ala, Gly, and Ser). B: quantitative analysis of mRNA concentration. Values are means ± SE; n = 3.

 
It has been demonstrated (32) in various tissues that system A activity is upregulated by several hormones. Glucagon and dibutyril-cAMP significantly stimulate system A activity and SNAT2 gene expression in liver cells (15). However, there is evidence that rat mammary gland lacks glucagon receptor (37). We then studied whether SNAT2 mRNA expression in mammary gland explants was responsive to distinct signaling pathways, one coupled to phospholipase C, generating diacylglycerol and inositol 1,4,5-triphosphate, and the other coupled to adenylate cyclase, increasing cAMP levels. To assess whether any of these signaling pathways were involved in regulating system A gene expression in mammary gland, explants were incubated in medium containing alanine, glycine, and serine at 1 mM each, in the presence of 10 µM forskolin or 100 nM PMA. The results showed a strong induction with either compound, indicating that signaling can occur via PKA or PKC (Fig. 3).


Figure 3
View larger version (34K):
[in this window]
[in a new window]
  		Fig. 3. A: effect of 10 µM forskolin or 100 nM PMA on SNAT2 mRNA expression on rat mammary gland explants incubated in a medium containing a mixture of small neutral amino acids (Ala, Gly, and Ser, 1 mM). B: quantitative analysis of SNAT2 mRNA. Values are means ± SE; n = 3.

 
To determine whether stimulation of SNAT2 gene expression during adaptive regulation was mediated via PKA or PKC, mammary gland explants were incubated with the specific inhibitors of PKA or PKC, H89 or GF109203X, respectively, and the inhibitors for adenylate cyclase or phospholipase C, MDL-12330 HCl or ET-18-CH3, respectively, in an amino acid free-medium for 200 min (Fig. 4). As expected, explants incubated in the amino acid free-medium showed an increase in SNAT2 mRNA expression ~5-fold; however, the presence of any of the inhibitors prevented the induction of the SNAT2 gene, indicating that inhibition of both signaling pathways reduced SNAT2 gene expression. As expected, incubation of mammary gland explants in an amino acid-free medium in the presence of forskolin or PMA increased SNAT2 mRNA concentration compared with explants incubated with a medium containing amino acids (Fig. 4).


Figure 4
View larger version (28K):
[in this window]
[in a new window]
  		Fig. 4. A: effect of specific inhibitors of protein kinases A or C and adenylate cyclase and phospholipase C on the expression of SNAT2 mRNA in rat mammary gland explants incubated an amino acid-free medium. B: quantitative analysis of SNAT2 mRNA. Values are means ± SE; n = 3.

 
These results indicated that SNAT2 expression is regulated by different signal transduction pathways in lactating mammary gland; however, it has not been established whether the SNAT2 gene is expressed differentially during pregnancy and lactation. Thus experiments were designed to determine whether the expression of SNAT2 mRNA changed during pregnancy or lactation. Mammary gland explants from rats at different stages of pregnancy, lactation, and weaning were studied. Northern blot analysis revealed a single band of 4.5 kb corresponding to SNAT2 mRNA in explants of mammary gland derived from pregnant and lactating rats. Furthermore, SNAT2 mRNA concentration increased progressively during pregnancy until day 18 and decreased rapidly near the end of pregnancy. During lactation the pattern of expression of SNAT2 mRNA showed small fluctuation without considerable changes in the first days of lactation. However, there was an increase of SNAT2 mRNA concentration around days 12–16 that coincided with peak milk production (Fig. 5). After postweaning, SNAT2 mRNA decreased rapidly. This finding was strengthened by in situ RT-PCR in mammary gland explants, indicating that lactocytes increased SNAT2 mRNA concentration predominantly during pregnancy (Fig. 6). To understand whether SNAT2 expression was induced by hormonal changes occurring during pregnancy, we performed studies in lactating mammary gland explants incubated in the presence of progesterone, 17beta-estradiol, or both. The addition of progesterone to explants incubated in a medium containing alanine, glycine, and serine at 1 mM each to repress adaptive regulation did not change the expression of SNAT2 mRNA. However, the incubation of explants in the same medium in the presence of 17beta-estradiol markedly increased the concentration of SNAT2 mRNA. The effect of 17beta-estradiol was time dependent, and after 3 h of incubation SNAT2 mRNA increased ~6-fold (Fig. 7). The induction of SNAT2 with 17beta-estradiol was repressed by the addition of the specific inhibitor for estrogen receptor-{alpha}, ICI-182780 M (Fig. 8). These results indicate an additional level of regulation of SNAT2 gene expression involving hormones present during pregnancy.


Figure 5
View larger version (25K):
[in this window]
[in a new window]
  		Fig. 5. Northern blot analysis of SNAT2. A: pattern of expression of SNAT2 mRNA in rat mammary gland during pregnancy, lactation, and postweaning. B: abundance of SNAT2 mRNA estimated by use of the Instant Imager electronic autoradiography system. Values are means ± SE; n = 3.

 

Figure 6
View larger version (144K):
[in this window]
[in a new window]
  		Fig. 6. In situ RT-PCR of SNAT2 mRNA expression in virgin (x200), pregnant (x100), lactating (x100), and postweaning (x100) rat mammary gland explants. SNAT2 mRNA was amplified after reverse transcription, with the primers indicated in MATERIALS AND METHODS.

 

Figure 7
View larger version (22K):
[in this window]
[in a new window]
  		Fig. 7. A and C: effect of 17beta-estradiol or progesterone, respectively, in rat mammary gland explants incubated in a medium containing a mixture of small neutral amino acids (Ala, Gly, and Ser, 1 mM each). B and D: quantitative analysis of SNAT2 mRNA. Values are means ± SE; n = 3. Different letters indicate a significant difference among groups (P < 0.05).

 

Figure 8
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 8. A: inhibition of induction of SNAT2 mRNA concentration by 17beta-estradiol with the specific inhibitor for estrogen receptor-{alpha}, ICI-182780 M. B: concentration of SNAT2 mRNA estimated with the Instant Imager electronic autoradiography system. Values are means ± SE; n = 3. Different letters indicate a significant difference among groups (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study demonstrated that expression of SNAT2 in the mammary gland is regulated by different effectors. As expected, incubation of mammary gland explants in an amino acid-free medium showed adaptive regulation. These results are in agreement with previous reports (35) that show that the activity of system A, measured as uptake of MeAIB into mammary gland explants, increases if tissue is incubated in an amino acid-free medium. The increase in SNAT2 mRNA during adaptive regulation in the mammary gland explants follow kinetics similar to those observed in HepG2 cells or fibroblasts (1, 4, 17). Expression of SNAT2 mRNA occurs rapidly in HepG2 cells after the explants in the amino acid-free medium are incubated (1). The expression was sensitive to the presence of amino acids, mainly the small neutral amino acids, since the mixture of alanine, glycine, and serine prevented the elevation of SNAT2 mRNA concentration, but this reduction was not observed with a mixture of large neutral and cationic amino acids. Adaptive regulation of SNAT2 gene is controlled at the transcriptional level, and a study by Palii et al. (22) determined that a specific amino acid response element is located in the intron 1 and is responsible for regulating SNAT2 expression under conditions of amino acid deprivation.

The estimated half-life of SNAT2 mRNA was ~67 min, indicating the rapid degradation rate of SNAT2 mRNA. This short half-life is in agreement with others estimated by measuring the activity of system A after treatment with glucagon that was between 1.4 and 1.5 h for both in vivo and in vitro studies (6). This rapid turnover allows for tight regulation of utilization of amino acids such as alanine and glutamine, the amino acids more actively transported in the mammary gland (2, 39). Nonetheless, the physiological relevance of adaptive regulation in the mammary gland is not clear, since there is evidence that, although dams consume a low- or an adequate-protein diet, the intracellular pool during the peak of lactation of amino acids, including the amino acid substrates for system A, is not depleted (11).

In addition to adaptive regulation, several studies have demonstrated that system A activity is induced by glucagon in different cells and tissues (15). Recent evidence (8) demonstrated that cAMP also induces the expression of SNAT2 in cultured cells, and these changes have been associated with an increase in system A activity. We (35) previously demonstrated that the activity of this transport system is also increased by dibutyril cAMP in the mammary gland. The present study clearly demonstrates that the increase of system A transport activity by cAMP is mediated, in part, by an increase in the expression of SNAT2 mRNA. Our results also show that the expression of this gene is activated by adenylate cyclase and phospholipase C-dependent pathways (7). As a result of the activation via both pathways, it is not clear whether SNAT2 gene expression is preferentially activated via PKA or PKC. Surprisingly, our data show that SNAT2 expression is upregulated by both signal transduction pathways. The induction with either pathway was similar, indicating that transcription of SNAT2 is activated in the same fashion via PKA or PKC. There is evidence that, during pregnancy, cAMP levels increase in rodent mammary gland and then fall rapidly after parturation (23), and perhaps this explains, in part, the increase in expression of SNAT2 during pregnancy, although there are other factors that influence the expression of this gene as described below.

Studies measuring amino acid arteriovenous concentration differences across the mammary gland (26, 28) show that uptake of most amino acids, including the amino acids transported by system A, occurs very actively during lactation. However, the pattern of expression of SNAT2 in the mammary gland during pregnancy and lactation has not been determined previously. Our data show that upregulation of SNAT gene expression occurs mainly during pregnancy and during the peak of lactation. These results suggest that SNAT2 gene expression is probably regulated during pregnancy by hormonal changes that occur in this condition. Our data show that SNAT2 gene expression is induced by beta-estradiol but not by progesterone. This is in agreement with previous studies (29, 30) that show an increase in the activity of systems L and A in breast cancer cell lines incubated with beta-estradiol. Induction of system A by estrogens in breast cancer cell lines only occurred in those lines expressing estrogen receptor-{alpha}. During pregnancy, it has been shown (24, 25) that expression of estrogen receptor-{alpha} decreases, but estrogen receptor-beta remains high; however, there is no experimental evidence that associates expression of SNAT2 with the presence of this receptor. More research is needed to understand this interaction. In addition, due to the rapid induction of SNAT2 by beta-estradiol in the mammary gland explants, there is the possibility that this effect could be mediated by a nongenomic action of the estrogen receptor (13, 19). It has been demonstrated (19) that estrogen receptor-{alpha} is also present in the plasma membrane and that binding of beta-estradiol activates cellular signaling systems that involve either G{alpha}s and G{alpha}q proteins. Furthermore, it has been shown (12, 31) that beta-estradiol is able to be activated by a nongenomic mechanism phospholipase C at the membrane and stimulates PKC pathway in osteoblasts (16) and also activates PKA and PKC in neurons altering synaptic transmissions in the cells. Further studies are required to understand whether activation of SNAT2 by beta-estradiol occurs through a genomic or nongenomic mechanism.

It is not clear why expression of SNAT2 increases during pregnancy and decreases during the first part of lactation. Amino acids are needed for lactocyte formation during pregnancy but also for synthesis of milk lipids and proteins during lactation. This change in expression could also be associated with the possibility that the mammary gland is preparing the milk-producing cells for lactation by synthesizing amino acid transporters during pregnancy for lactation. It has been shown (9) that system A protein in skeletal muscle is located in vesicles that can recycle to the plasma membrane to increase activity in response to insulin. Perhaps the synthesis of system A protein is increased and the new synthesized transporter located in intracellular vesicles and cycled to the membrane after receiving a hormonal signal that occurs during lactation. We are conducting studies to detect whether in fact this event takes place.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) Grant 212226-5-25637M.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. R. Tovar, Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición, Vasco de Quiroga No 15, Mexico City, 14000, Mexico (e-mail: artovar{at}quetzal.innsz.mx)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bain PJ, LeBlanc-Chaffin R, Chen H, Palii SS, Leach KM, and Kilberg MS. The mechanism for transcriptional activation of the human ATA2 transporter gene by amino acid deprivation is different than that for asparagine synthetase. J Nutr 132: 3023–3029, 2002.[Abstract/Free Full Text]
  2. Barber T, Triguero A, Martinez-Lopez I, Torres L, Garcia C, Miralles VJ, and Vina JR. Elevated expression of liver gamma-cystathionase is required for the maintenance of lactation in rats. J Nutr 129: 928–933, 1999.[Abstract/Free Full Text]
  3. Bussolati O, Dall'Asta V, Franchi-Gazzola R, Sala R, Rotoli BM, Visigalli R, Casado J, Lopez-Fontanals M, Pastor-Anglada M, and Gazzola GC. The role of system A for neutral amino acid transport in the regulation of cell volume. Mol Membr Biol 18: 27–38, 2001.[ISI][Medline]
  4. Gazzola RF, Sala R, Bussolati O, Visigalli R, Dall'Asta V, Ganapathy V, and Gazzola GC. The adaptive regulation of amino acid transport system A is associated to changes in ATA2 expression. FEBS Lett 490: 11–14, 2001.[CrossRef][ISI][Medline]
  5. Glisin V, Crkvenjakov R, and Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13: 2633–2637, 1974.[CrossRef][Medline]
  6. Handlogten ME and Kilberg MS. Induction and decay of amino acid transport in the liver. Turnover of transport activity in isolated hepatocytes after stimulation by diabetes or glucagon. J Biol Chem 259: 3519–3525, 1984.[Abstract/Free Full Text]
  7. Hansen LH, Gromada J, Bouchelouche P, Whitmore T, Jelinek L, Kindsvogel W, and Nishimura E. Glucagon-mediated Ca2+ signaling in BHK cells expressing cloned human glucagon receptors. Am J Physiol Cell Physiol 274: C1552–C1562, 1998.[Abstract/Free Full Text]
  8. Hatanaka T, Huang W, Martindale RG, and Ganapathy V. Differential influence of cAMP on the expression of the three subtypes (ATA1, ATA2, and ATA3) of the amino acid transport system A. FEBS Lett 505: 317–320, 2001.[CrossRef][ISI][Medline]
  9. Hyde R, Peyrollier K, and Hundal HS. Insulin promotes the cell surface recruitment of the SAT2/ATA2 system A amino acid transporter from an endosomal compartment in skeletal muscle cells. J Biol Chem 277: 13628–13634, 2002.[Abstract/Free Full Text]
  10. Hyde R, Taylor PM, and Hundal HS. Amino acid transporters: roles in amino acid sensing and signalling in animal cells. Biochem J 373: 1–18, 2003.[CrossRef][ISI][Medline]
  11. Jansen GR, Schibly MB, Masor M, Sampson DA, and Longenecker JB. Free amino acid levels during lactation in rats: effects of protein quality and protein quantity. J Nutr 116: 376–387, 1986.[Abstract/Free Full Text]
  12. Kelly MJ, Lagrange AH, Wagner EJ, and Ronnekleiv OK. Rapid effects of estrogen to modulate G protein-coupled receptors via activation of protein kinase A and protein kinase C pathways. Steroids 64: 64–75, 1999.[CrossRef][ISI][Medline]
  13. Kelly MJ and Levin ER. Rapid actions of plasma membrane estrogen receptors. Trends Endocrinol Metab 12: 152–156, 2001.[CrossRef][ISI][Medline]
  14. Kilberg MS, Pan YX, Chen H, and Leung-Pineda V. Nutritional control of gene expression: how mammalian cells respond to amino acid limitation. Annu Rev Nutr 25: 59–85, 2005.[CrossRef][ISI][Medline]
  15. Kilberg MS, Stevens BR, and Novak DA. Recent advances in mammalian amino acid transport. Annu Rev Nutr 13: 137–165, 1993.[CrossRef][ISI][Medline]
  16. Le Mellay V, Grosse B, and Lieberherr M. Phospholipase C beta and membrane action of calcitriol and estradiol. J Biol Chem 272: 11902–11907, 1997.[Abstract/Free Full Text]
  17. Ling R, Bridges CC, Sugawara M, Fujita T, Leibach FH, Prasad PD, and Ganapathy V. Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A. Biochim Biophys Acta 1512: 15–21, 2001.[Medline]
  18. Mackenzie B and Erickson JD. Sodium-coupled neutral amino acid (System N/A) transporters of the SLC38 gene family. Pflügers Arch 447: 784–795, 2004.[CrossRef][ISI][Medline]
  19. Moggs JG and Orphanides G. Estrogen receptors: orchestrators of pleiotropic cellular responses. EMBO Rep 2: 775–781, 2001.[CrossRef][ISI][Medline]
  20. Neville MC, Lobitz CJ, Ripoll EA, and Tinney C. The sites for alpha-aminoisobutyric acid uptake in normal mammary gland and ascites tumor cells. A comparative study of mouse tissues in vitro. J Biol Chem 255: 7311–7316, 1980.[Free Full Text]
  21. Palacin M, Estevez R, Bertran J, and Zorzano A. Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 78: 969–1054, 1998.[Abstract/Free Full Text]
  22. Palii SS, Chen H, and Kilberg MS. Transcriptional control of the human sodium-coupled neutral amino acid transporter system A gene by amino acid availability is mediated by an intronic element. J Biol Chem 279: 3463–3471, 2004.[Abstract/Free Full Text]
  23. Rillema JA. Cyclic nucleotides, adenylate cyclase, and cyclic AMP phosphodiesterase in mammary glands from pregnant and lactating mice. Proc Soc Exp Biol Med 151: 748–751, 1976.[Abstract]
  24. Saji S, Jensen EV, Nilsson S, Rylander T, Warner M, and Gustafsson JA. Estrogen receptors alpha and beta in the rodent mammary gland. Proc Natl Acad Sci USA 97: 337–342, 2000.[Abstract/Free Full Text]
  25. Saji S, Sakaguchi H, Andersson S, Warner M, and Gustafsson J. Quantitative analysis of estrogen receptor proteins in rat mammary gland. Endocrinology 142: 3177–3186, 2001.[Abstract/Free Full Text]
  26. Shennan DB. Mammary gland membrane transport systems. J Mammary Gland Biol Neoplasia 3: 247–258, 1998.[CrossRef][ISI][Medline]
  27. Shennan DB and McNeillie SA. Characteristics of alpha-aminoisobutyric acid transport by lactating rat mammary gland. J Dairy Res 61: 9–19, 1994.[ISI][Medline]
  28. Shennan DB, Millar ID, and Calvert DT. Mammary-tissue amino acid transport systems. Proc Nutr Soc 56: 177–191, 1997.[ISI][Medline]
  29. Shennan DB, Thomson J, Barber MC, and Travers MT. Functional and molecular characteristics of system L in human breast cancer cells. Biochim Biophys Acta 1611: 81–90, 2003.[Medline]
  30. Shennan DB, Thomson J, Gow IF, Travers MT, and Barber MC. L-leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics, regulation by estrogen and molecular identity of the transporter. Biochim Biophys Acta 1664: 206–216, 2004.[Medline]
  31. Shingo AS and Kito S. Estradiol induces PKA activation through the putative membrane receptor in the living hippocampal neuron. J Neural Transm 112: 1469–1473, 2005.[CrossRef][ISI][Medline]
  32. Shotwell MA, Kilberg MS, and Oxender DL. The regulation of neutral amino acid transport in mammalian cells. Biochim Biophys Acta 737: 267–284, 1983.[Medline]
  33. Sugawara M, Nakanishi T, Fei YJ, Huang W, Ganapathy ME, Leibach FH, and Ganapathy V. Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A. J Biol Chem 275: 16473–16477, 2000.[Abstract/Free Full Text]
  34. Sugawara M, Nakanishi T, Fei YJ, Martindale RG, Ganapathy ME, Leibach FH, and Ganapathy V. Structure and function of ATA3, a new subtype of amino acid transport system A, primarily expressed in the liver and skeletal muscle. Biochim Biophys Acta 1509: 7–13, 2000.[Medline]
  35. Tovar AR, Avila E, DeSantiago S, and Torres N. Characterization of methylaminoisobutyric acid transport by system A in rat mammary gland. Metabolism 49: 873–879, 2000.[CrossRef][ISI][Medline]
  36. Tovar AR, Becerril E, Hernandez-Pando R, Lopez G, Suryawan A, Desantiago S, Hutson SM, and Torres N. Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland. Am J Physiol Endocrinol Metab 280: E480–E488, 2001.[Abstract/Free Full Text]
  37. Trottier NL. Nutritional control of amino acid supply to the mammary gland during lactation in the pig. Proc Nutr Soc 56: 581–591, 1997.[CrossRef][ISI][Medline]
  38. Varoqui H, Zhu H, Yao D, Ming H, and Erickson JD. Cloning and functional identification of a neuronal glutamine transporter. J Biol Chem 275: 4049–4054, 2000.[Abstract/Free Full Text]
  39. Vina JR and Williamson DH. Effects of lactation on L-leucine metabolism in the rat. Studies in vivo and in vitro. Biochem J 194: 941–947, 1981.[ISI][Medline]
  40. Yao D, Mackenzie B, Ming H, Varoqui H, Zhu H, Hediger MA, and Erickson JD. A novel system A isoform mediating Na+/neutral amino acid cotransport. J Biol Chem 275: 22790–22797, 2000.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/5/E1059    most recent
00062.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by López, A.
Right arrow Articles by Tovar, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by López, A.
Right arrow Articles by Tovar, A. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.