Indian Journal of Biochemistry & Biophysics Vol. 38, August 2001. pp. 241-248

Heterogeneity of transport systems for L-glutamine in mouse mammary gland

Rekha Sharma and Yinod K Kansal*

Division of Animal Biochemistry, National Dairy Research Institute. Kamal 132001 Haryana, India

Received 5 JlIne 2000; revised 21 March 2001; accepted 29 March 2001

The characteristics of the transport systems of L-glutamine in lactating mouse mammary gland have been studied. L­glutamine uptake was mediated by three Na+-dependent and one Na+-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na+-dependent uptake exhibited the usual characteristics of system A. The other two Na+­dependent systems, which we have named BCI'-dependent and BCI'-independent, are the new systems identified . These are broad specificity systems and were discriminated on the basis of inhibition analysis. cr dependency and the effect o f preloading mammary ti ssue with amino acids. While L-aspargine inhibited the uptake of L-glutamine via both these broad specificity systems. L-homoserine inhibited the uptake of L-glutamine via only BCr-depe ndent system. The uptake of L­glutamine via the BCI '-independent system was upregul ated by preloading mammary tissue with L-serine, while BCr­dependent system was unaffected. The Na+-i ndependent uptake of L-glutamine was inhibited by 2-aminobicyclo­(2.2.1 )heptane carboxylic acid and other neutral amino acids, and identified as the system L.

Most mammalian cells express a common 'core' set of amino acid transport activities, but these also exhibit wide variations in the type and activity of transport systems. As a result , each cell type is unique with regard to the processes available for amino acid accumulation, which are adapted to its physiological role and metabolic needs I. The lactating mammary gland has a large requirement for amino acids to meet the need of milk protein synthesis. The transport of certain amino acids by the mammary secretory cells could be rate limiting for the synthesis of milk proteins, so a detailed knowledge of individual transport system may help to understand the mechanism and regulation of milk synthesis.

Amino acid transport systems in mammary gland may be divided into two broad categories based on whether they are Na+-dependent or Na+-independent, and each of these categories is subdivided into three groups depending on whether the systems prefer cationic, anionic or zwitter ionic substrates. The zwitter ion preferring transport processes in mammary gland are Na+-dependent system A (ref. 2-5) and Na+­independent systems L( ref. 2-5) and T(ref. 6) . System l is a Na+-independent transporter of cationic amino acids7

. 8 and XAG- is a Na+-dependent system for anionic amino acids9

. A (Na++Cr)-dependent system selective for B-amino acids has been reported in rat

*Author for correspondence Phone:- 0184259124; Fax:- 0184 250042: E-mail: vkk @ndri.hry.nic. in

mammary tissue 10. In addition to these systems, there is yet another class of transporters that have still broader specificity. A Na+-independent system in mouse8 and ratll. 12 mammary gland has been reported to mediate the transport of both zwitter ionic and cationic amino acids. A system that may have broad specificity and is activated by cell swelling has also been documented 13 .

The transport of L-glutamine by lactating mammary ti ssue is of interest because of the reported variations in different systems among various tissues for the uptake of this amino acid. The ASC system mediates the transport of L-glutamine in a variety of cell types such as pigeon erythrocytes, rabbit reticulocytes and Chinese hamster ovary cells 14 . In Ehrlich ascites cells l5 , the systems A, ASC and L have been reported to mediate the uptake of L­glutamine. In addition, system N whose substrate specificity is restricted to L-glutamine, L-aspargine and L-histidine has also been described I6

-17

. Our results show that the uptake of L-glutamine in lactating mouse mammary gland is mediated by four transport systems, two of which are described fo r the first time.

Materials and Methods

Chemicals and buffers L-[U- 14C]gIutamine (Specific activity 250

mCi/mmol) was obtained from Board of Radiation and Isotope Technology, (Mumbai 400 094, India). Amin(oxy)acetic acid, 2-aminobicycJo-(2,2, 1 )heptane-2-

242 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 38, AUGUST 2001

carboxylic acid (BCH), L-homoserine, 2-(methylamino)isobutyric acid (MeAIB) and trans-4-hydroxy-L-proline were from Sigma (St Louis, MO 63178, USA). The other chemicals used were from Himedia, Loba or Sarabhai Merck (Mumbai, India).

Hepes-buffered sodium Ringer phosphate (Na+ plus Cr-buffer) solution contained 106.5 mM NaCl, 5 mM KCl , 5 mM NaHCO), 0.6 mM NaHzP04, 1 mM Na2HP04, 2 mM CaCh, 1 mM MgClz, 20 mM glucose and 20 mM Hepes, and pH adjusted to 7.4 with I M NaOH. Hepes-buffered sodium-free Ringer (Na+-free Cr-buffer) solution contained 113 mM choline chloride, I mM MgS04, 5 mM KHCO), 2 mM CaCh, 20 mM glucose and 20 mM Hepes, and pH adjusted to 7.4 with I M Tris. Hepes-buffered Chloride-free sodi um Ringer phosphate (CIO-free Na+-buffer) solution contained 111.5 mM sodium gluconate, 5 mM KHCO), I mM MgS04, 2 mM calcium gluconate, 0.6 mM NaHzP04, I mM Na2HP04, 20 mM glucose and 20 mM Hepes, and pH adjusted to 7.4 with I M NaOH. Hepes-buffered sodium and Chloride free (Na+- free, Cr-free buffer) solution contained 226 mM mannitol, 5 mM KHCO), 2 mM calcium gluconate, I mM MgS04, 20 mM glucose and 20 mM Hepes, and pH adjusted to 7.4 with I M Tri s. All buffer solutions were equilibrated with air.

Modified buffers were used to study the effect of pH on amino acid uptake. Sodium containing solutions were buffered with NazHP04 and NaHzP04, and sodium-free solution buffered with KzHP04 and KH2P04 at final concentration of 10 mM, otherwise the buffer composition remained the same except that Hepes was omitted.

Allim.a/s and tissue preparation Swiss mice were mai ntai ned on laboratory stock

diet in small animal house of the Institute. Mammary tissue was taken from 10 day post-partum lactating mice. The number of pups with the dam was adjusted to eight within 1 day of parturition . The animals were killed by cervical dislocation and mammary glands were dissected into the buffer solution used for amino ac id uptake studies . Fat and connective tissue were removed and the mammary gland was diced into 0.5-1 mm segments. The tissue was incubated in the same buffer for 30 min at 37°C in a shaking water buth to stab ilize its amino acid content.

Measurement of amino acid transport The amino acid uptake by mammary slices was

determined by incubating tissue (40-70 mg) in the selected buffer containing appropriate concentration

of unlabelled amino acid together with 0.25 IlCilml of labelled amino acid. The final specific activity of radiolabelled L-glutamine in the incubation mixture was 2.5 mCi/mmo!. Amino(oxy)acetic acid (0.5 mM) was included in the buffer to inhibit ce ll amino acid metabolism. Where the concentration of substrate and competing amino acid exceeded 20 mM, isomolarity of the medium was maintained by decreasing the concentration of NaCI or sodium gluconate or choline chloride. Incubations were carried out in a shaking water bath (90 cycles/min) for the period during which the uptake of amino ac id was linear. After incubation, the ti ssue was washed for 15 min with two changes of chilled buffer to remove extracellular rad ioactiviti . Excess moi sture was removed from the ti ssue by blotting it between the leaves of moist Whatman filter paper. The ti ssue was dissolved and radioactivity counted 18 in a liquid scintillation counter (Model 1600 CA TriCarb from Packard, Meriden , CT 06450, USA). Portions of incubation medium were also counted.

Calculation of results Results were calculated as nmol am1l10 acid

accumulatedlg mammary cells from measurement of radioactivity and amino ac id concentration in the uptake medium and the extracellular space in mammary ti ssue (determined using eH]sucrose) as the conversion factor3

. MeAIB interacts specifically with the A transport sites which is one component of Na+-dependent transport systems. The uptake of L­glutamine from Na+ plus cr buffer in the presence of MeAIB represents the uptake through a total of MeAIB-insensitive routes, and subtracting from this the uptake from Na+-free buffer would give Na+­dependent, MeAIB-insensitive uptake of L-glutamine. Di fference between L-glutamine uptake from medi a (1) Na+ plus Cr-buffer containing MeAIB and (2) cr­free Na+-buffer containing MeAIB would give L­glutamine uptake through (Na+ + Cn-dependent, MeAIB-insensitive (named as BCIo-dependent) system. Difference between L-glutamine uptake from media (1) Cr-free Na+-buffer containing MeAIB and (2) Na+-free buffer would give L-glutamine uptake through Na+-dependent, Cr-independent, MeAIB­insensitive (named as BCIo-independent) system.

Effect of preloading mammary tissue with amino acid The effect of intracellular accumulation of amino

acids on L-glutamine uptake was studied by loading the mammary slices with selected amino acids prior to uptake studies. The diced mammary tissue was

SHARMA & KANSAL: HETEROGENEITY OF TRANSPORT SYSTEM FOR L-GLUTAMINE 243

incubated with amino acid (10 mM) in Na+ plus cr­buffer for 30 min at 37°C in shaking water bath . The control tissue was incubated in the same buffer without amino acid. At the end of loading period, the tissue slices were washed with buffer (ice-cold) used for amino acid uptake studies.

Statistics Statistical analysis was performed using Student's

unpaired t test and results are expressed as means ± SEM.

Results

Time course of L-glutamille uptake The time course of L-glutamine uptake (for 0.1 mM

external L-glutamine) was studied in Na+ plus cr­buffer for a period of 30 min. The uptake of L­glutamine was linear for 5 min and then it slowly began to level off (Fig. I ). During first 5 min of incubation the radioactivity incorporated into the ti ssue protein (tested as trichloroacetic acid precipitable material) was negligible (0-4% of cellular radioactivity), so an incubation time of 5 min was used for determining the initial rate of L-glutamine uptake in all subsequent experiments.

Effect of transport system A inhibitor on L-glutamine uptake

The effect of MeAIB, a specific inhibitor of transport system A(ref. 19) was studied up to 30 mM

in Na+ plus Cr-buffer. The inhibition of L-glutamine uptake reached maximum at 10 mM MeAIB (Fig.2), so this concentration was used to block L-glutamine uptake through system A in subsequent experiments.

Effect of amino acids on L-glutamine uptake The study of inhibitory effect of one amino acid on

the transport of another amino acid has proved useful for characterization of amino acid transport systems. In order to obtain information on the substrate specificity of Na+-dependent, MeAIB-insensitive route of amino acid transport , the uptake of L­glutamine was tested for possible inhibition by various amino acids and amino acid analogues (Table I). All amino acids except BCH, inhibited the Na+­dependent, MeAIB-insensitive uptake of L-glutamine with the inhibition caused by L-glutamine, L­aspargine and L-arginine being strongest. Na+­independent uptake of L-glutamine from Na+-free cr­buffer was inhibited by all neutral amino acids except hydroxy proline and aspargine. Charged amino acids did not inhibit the Na+-independent uptake of L­glutamine.

Effect of pH on L-glutamine uptake

The effect of decreasing pH on system A and Na+­dependent, MeAIB-insensitive components of L­

glutamine uptake was studied (Fig.3). The system A­mediated uptake of L-glutamine was abolished on lowering the pH of the external medium to 6.5 ,

~400 r--------------------------------------------, CIl

CII (J

CI

--.: 300 E

--"0 E E 200 Q) .¥ co ... Co ::I

~ 100 'E co ... ::I Cl

T

..J O~----~------~------L-----~-------L------~

o 5 10 15 20 25 30

Time, min

Fig.l - Time course of L-glutamine uptake in mouse mammary ti ssue. [Uptakes were measured using an external L-glutamine concentration of 0.1 mM in Na+ plus cr buffer. Values are mean of four determinations with SEM indicated by vertical bars. Where no bar appears the SEM falls within the symbol]

244 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 38, AUGUST 2001

~'60 r-------------------------------------------~ (f)

Q)

()

Cl ...... c: E

In 120

o E c: Q)

"'" 10 ... Q. 80 ::l

Q)

c: E 10 ... ::l

Cl

~ 40 0

• •

5 10 15 20 25 30

MeAIB concentration. mM

Fig.2-Effect of 2-(methy lamino)isobutyric acid on L-glutamine uptake in lactating mouse mammary tissue. [Uptakes were measured using an external L-glutamine concentration of 0.1 mM in Na+ plus cr buffer. Values are mean of four determinations with SEM indicated by verti cal bars. Where no bar appears the SEM fall s within the symbol]

Table I-Effect of amino acids on L-glutamine uptake by lactating mouse mammary tissue [Values. nmol15 min/g cells, are mean ± SEM for four observations. The external

concentration of L-glutamine was 0.1 mM]

Competing amino Na+ plus cr buffer Na+-free buffer Na+-dependent, MeAIB-acid( 10 mM) pluslO mM MeAIB insensitive uptaket

None (control) 77.5 ± 5.0 34.6 ± 2.4 42.9 ±4.8 L-glutamine 23.8 ± 1.1 19.7±1.7- 4.1 ± 1.1 -L-aspargine 42 .2 ± 3.1 33.7 ± 2.5 11.8 ± 4.8-L-histidine 48.4 ± 2.4 18.0 ± 0.7- 30.4 ± 2.6 L-homoserine 38.5 ± 1.7 20.7 ± 1.5- 17.9 ± 1.7-1-4-0H-proline 62.5 ± 3.0 46.8 ± 3.0 15.7±3.4-BCH 73.9 ± 7.1 17.4 ± 1.2- 56.5 ± 7.3 L-arginine 62.7 ± 2.2 49.7 ± 2.4 13.0 ± 3.9-

tDifference between L-glutamine uptakes in media (I) Na+ plus cr buffer plus 10 mM MeAIB and (2) Na+-free buffer. ' The values for the uptakes from Na+-free buffer and for the Na+-dependent, MeAIB­insensitive uptakes were compared with their respective control values for significance (P<O.OI).

whereas, the uptake via Na+-dependent, MeAIB­insensitive system increased significantly at this pH . .,

L-g/utamille uptake ill l11alllll/Qry tissue loader! with amino acids

Prior intracellular accumulation of L-glutamine or serine in mammary cells stimulated Na+-dependent, MeAIB-insensitive uptake of L-glutamine (Table 2). Preloading mammary cells with L-arginine had no effect, whil e preload ing with histidine down regulated

the Na+-dependent, MeAIB-insensiti ve uptake of L­glutamine.

Na+ alld cr dependence of L-glutamille uptake The effect of Na+ and/or cr replacement o n L­

gl utamine uptake was studied in lactating mammary tissue. The results (Table 3) show the presence of two components of Na+-dependent L-glutamine uptake, one dependent on Na+ alone and the other dependent on both Na+ and cr. Since the rates of L-glutamine

SHARMA & KANSAL : HETEROGENEITY OF TRANSPORT SYSTEM FOR L-GLUTAMINE 245

80 en Q)

u Cl -... .£ 60 E In

(5

E ..s 40

Q) ~ co ..... a. ::l Q)

.£ 20 E co ..... ::l

9' -'

0 6 6.5 7 7.5

pH

Fig.3-Effect of pH on the uptake of L-glutamine by lactating mouse mammary tissue. [Uptakes were measured using an external L­glutamine concentration of 0. 1 mM in media containing (I) Na+ and CI; (2) Na+, cr and MeAIB; (3) cr but no Na. The uptake via system A (e ) was determined taking difference of L-glutamine uptake in media I and 2. The Na+-dependent, MeAIB-insensitive (A) uptake was determined taking difference of L-glutamine uptake in media 2 and 3. Each point is a mean ± SEM of six determinations with SEM indicated by vertical bars. Where no bar appears the SEM falls within the symbol. *(p<0.01) significantly different from their control (PH 7.4) values]

Table 2-Uptake of L-glutamine by lactating mouse mammary gland slices preloaded with amino acids. [Prior to uptake measurement, mammary tissue slices were loaded with selected amino acid as described in Materials and Methods. The external concentration of L-glutamine

was 0.1 mM. Values, nmol/5 minlg cells, are mean ± SEM for six observations]

Amino acid loaded Na+ plus cr buffer Na+-free buffer Na+-dependent, MeAIB-+10 mM MeAIB insensitive uptaket

None (control) 87.1 ± 3.0 46.8 ± J.7 40.3 ± 2.4 L-glutamine 112.1 ± 7.0 59.2 ± 3.3 53.0 ± 5.3' L-serine 120.4 ± 2.5 35.0 ± 1.0 73.6 ± 2.7" L-histidine 73.1 ± 3.4 44.7 ± 1.8 28.9 ± 3.4" L-arginine 76.2 ± 3.0 35.8 ± 1.7 40.7 ± 3.0

tOifference between L-glutamine uptakes in media (I) Na+ plus cr buffer plus 10 mM MeAIB and (2) N a + -free bu ffer. Values for the Na+-dependent, MeAIB-insensi tive uptakes were compared with their control value for significance (*p<0.05; **p,<O.OI).

Table 3 - Na+ and Crdependence of the uptake of L-glutamine by lactating mouse mammary tissue. [Values, nmol/5 min/g cells, are mean ± SEM for seven

observations. The external concentration of L-glutamine was 0.1 mM]

Medium

Na+plus Cr-buffer Cr-free Na+-bulfer Na+-free buffer Cr-free, Na+-free buffer Uptake components

Uptake

120.3 ± 3.1 58.6 ± 1.5 16.7 ±0.7 18.3 ± 0.9

(Na+ +CI")-dependentt 61.7 ± 1.6 Na+-dependent, Cr-independent* 41.9 ± 0 .6

t Oifference between L-glutamine uptake in media Na+ pillS Cr-buffer and Cr-free Na+-buffer. tOifference between L-glutamine uptakes in media Cr-free Na+ buffer and Na+­free bu ffer.

246 INDIAN J. BIOCHEM. BIOPHYS .• VOL. 38. AUGUST 2001

uptake from Na+-free, Cr-buffer and Na+-free, cr­free buffer were similar, Na+-independent uptake was independent of Cr.

these components of L-glutamine uptake, the cr­dependent component being more sensiti ve. L­homoserine diminished the uptake of L-glutamine via Cr-dependent system but not via Cr-independent system. The two components of Na+-dependent. MeAIB-insensitive uptake of L-glutamine, di scriminated on the basis of cr requirement, hereafter will be referred as to BCl--dependent and BCl--independent systems. BCH did not modify the uptake of L-glutamine mediated by e ither of the Na+­dependent, MeAIB-insensitive systems.

Effect of all/ino acid starvatioll. on L-glutamine uptake Table 4 describes changes in L-glutamine uptake

due to starvation of lactating mouse mammary ti ssue in amino acid-free medium. The rate of L-glutamine uptake via Na+-dependent, MeAIB-insensitive system deceased to less then one-sixth of the control in mammary ti ssue following starvation for amino acids for 2 hr.

Substrate specificity of c r-dependent and cr­il/dependell.t components of Na+ -dependell.t, MeAIB­insell.sitive uptake of L-glutamine

To determine whether the Cr-dependent and cr­independent components of Na+-dependent, MeAIB­insensitive uptake of L-glutamine represent the function of a si ngle transport system or two different en tities, we studied the capacity of selected amino ac ids to inhibit the e two systems (Table 5). Among the amino acids tested, L-aspargine inhibited both

Effect of amino acid loading Oil BCr-dependent all.d BCl--ill.dependent systems of L-glutamine uptake

L-glutamine uptake was measured after loading mammary gland explants with L-serine or L-arginine (Table 6). The uptake of L-glutamine via BCr­dependent system was not affected by preloading mammary tissue with either L-serine or L-arginine, whereas BCl--independent system mediated uptake increased by preloading mammary tissue with L­senne.

Table 4 - Effect of amino ac id starvation on L-glutamine uptake by lactating mouse mammary ti ssue. [Values, nmol/5 minlg cells, are mean ± SEM for four observations.

The ex ternal concentration of L-glutamine was 0.1 mM]

Na+ plus cr buffer + 10mM MeAIB Na+-free buffer Na+-dependent. MeAIB-insensiti ve uptake~

Control

78.2 ± 2.7 33.8 ± 1.7 44.5 ± 1.7

Starvedt

42.6 ±4.6 35.5 ± 2.5 7. 1 ± 2.5 '

tprior to uptake measu rement ti ssue slices were incubated in respective buffers withou t amino acids for 2hr. ~ Oifference between L-glutamine uptake in media (I ) Na+ plus cr buffer plus 10 mM MeAIB and (2) Na +-free buffer. ' Value for Na+-dependent. MeA IB -i nsensiti ve uptake was compared with its control value for significance (p<0.0 I).

TableS- Effect of amino acids on the BCr-dependent and BCr-independent systems of L-glutami ne. [Values, nmol/5 min/g cell s are mean ± SEM for eight observations. The external concentration of

L-glutamine was 0.1 mM]

Competing amino acids None L-aspargine L-homoserine BCH

Na+ plus Crbu ffer + 10 mM MeAIB 69.4 ± 7.5 32.5 ± 2.0 52.6 ± 7.2 57.4 ± 8.9 cr free. Na+ buffer + 10 mM MeAIB 38.8 ± 0.9 26.6 ± 0.6 33.4 ± 3.5 29.2 ± 2.1 Na+ free buffer 20.9 ± 1.3 12.2 ± 1.1 14.0 ± 0.7 12. 1 ± 0.8 BCr -dependent t 27.7±4.5 6.0±1.3" 19.6±2.5' 28.2±6.1 Bcr- independent ~ 18.2 ± 1.0 14.4 ± 1.0' 19.5 ± 2.5 16.2 ± 1.4 tOifference between L-glutamine uptakes in media ( I) Na+ plus cr buffer containing MeAIB and (2) cr free. Na+ buffer containing MeAIB . *Oifference between L-glutamine uptake in media ( I) cr free. Na+ buffer containing MeAIB and (2) Na+ free buffer. Va lues for the Iransport systems were compared with their respecti ve cont rol values for signifi cance (*p<0.05; **p<O.O I ).

)

SHARMA & KANSAL : HETEROGENEITY OF TRANSPORT SYSTEM FOR L-GLUTAMINE 247

Table 6-Effect of pre loading lactating mouse mammary tissue with amino acids on BCI--dependent and BCI--independent systems of L-glutamine uptake. [Values, nmol/5 min/g cells, are mean ± SEM for

etght observations. The external concentration of L-glutamine was 0.1 mM. Prior to uptake measurement. mammary ti ssue slices were loaded with selected amino acid as described in Materials and Methods]

Amino acid loaded

Na+ plus cr buffer + 10 mM MeAIB Cr-free, Na+-buffer + 10 mM MeAIB Na + -free buffer BCr -dependent t BCI--i ndependent*

None 124.9 ± 3.1 72.5 ± 2.4 24.6 ± 1.7 53.4 ± 3.0 47.9± 1.4

L-serine 152.2 ± 6.2 91.0 ± 3.0 30.7 ± 1.9 61.2±3.1 60.3±2.1 *

L-arginine 123.0 ± 9.5 71.9±2.4 29.8 ± 3.0 51.3 ± 3.9 42.1 ± 2.2

t Oifference between L-glutamine uptakes in media (I) Na+ plus cr buffer containing MeAIB and (2) cr free Na+ buffer containing MeAIB. +Oifference between L-glutamine uptake in media ( I) cr free Na+ buffer containing MeAIB and (2) Na+ free buffer. Values for the transport systems were compared with their respective control values for significance (p<O.OI).

Discussion Our results show that Na+-dependent routes

account for a major portion of L-glutamine uptake in lactating mouse mammary gland. The Na+-dependent uptake of L-glutamine is heterogenous. The MeAIB­sensitive component of L-glutamine uptake exhibited the usual characteristics of system A in this study. The decrease in system A activity with the decrease in pH of the external medium was similar to that reported in other tissuesI5.2o.2 1 .

Possible candidates for Na+-dependent, MeAIB­insensitive component of L-glutamine uptake are the ASC and broad specificity systems. Both are Na+­dependent and insensitive to MeAIB(ref. 22, 23) . The ASC system has been shown to be a major component of Na+-dependent transport of amino acids in rabbit intestine, rabbit reticulocytes, pigeon erythrocytes, rat hepatocytes, cultured human fibroblasts, HTC hepatoma cells and Chinese hamster ovary cells l4

.

Broad specificity systems Band BO, unlike system A or ASC, accept almost all neutral amino acids and have been described in brush border membranes of

123 24 d' . 125 26 . I I' I II f . rena . an lI1testlOa . eplt le ta ce s 0 a vanety of vertebrates. System BO.+ characterized in mouse blastocysts mediates the uptake of both cationic and neutral amino acids27

Sharing an amino acid carrier should result in competition between substrate amino acids presented simultaneously to the carrier. When this was done, both neutral and charged amino acids caused inhibition of L-glutamine uptake through the Na+­dependent, MeAIB-insensitive component. Inhibition by L-homoserine and t-4-0H-L-proline suggests the system ASC type activity. The ASC system has a preference for linear dipol ar amino acids, such as L-

alanine, L-serine and L-cysteine, and has relatively higher affinity for amino acids containing a distal hydroxyl group28. However, the presence of a system other than ASC is indicated by inhibition of the Na+­dependent, MeAIB-insensitive component of L­

glutamine uptake by L-arginine, as this amino acids is known to be excluded by the ASC system31. Inhibition of Na+-dependent, MeAIB-insensitive system(s) mediated uptake of L-glutamine by neutral as well as cationic amino acids suggests the presence of broad specificity system(s), although the uptake via this route was not affected by BCH, known to interact with broad specificity system in mouse blastocysts I.

The Na+-dependent, MeAIB-insensitive uptake of L-glutamine was not significantly affected by L­histidine, suggesting for the absence of system N in mouse mammary tissue, since L-histidine is a known preferred substrate of this system32. Absence of system N is confirmed by the observation that the Na+-dependent, MeAIB-insensitive uptake of L­

glutamine was increased at low pH (6.0) of the extracellular medium, whereas, the N system activity is known to diminish with the decrease in extracellular pH (ref. 16,28). Further, the uptake of L­glutamine via the Na+-dependent, MeAIB-insensitive systems was inhibited following starvation of mouse mammary tissue for amino acids. System N, on the other hand, is known to be upregulated by starvation of cells for its substrate amino acids33

. 34.

Na+-dependent, MeAIB-insensitive uptake of L­

glutamine was resolved into two components namely BCl--dependent and BCl--independent. These two systems showed difference in their response to inhibition caused by competing amino acids, and by preloading mammary tissue with L-serine.

248 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 38, AUGUST 2001

Na + -independent uptake of L-glutamine was inhibited by BCH, a specific system L inhibitor35

, and other neutral amino acids except t-4-0H-proline. Although the L system transports a large number of neutral amino acids~(i in mammalian cells, it excludes proline25

. Therefore it is concluded that the Na+­independent uptake of L-glutamine was mediated by the L system. Although the Na+-independent broad specificity system has been reported in lactating mouse mammary ti ssueS, it does not contribute to L­glutamine uptake, since the Na+-independent uptake of L-glutamine was not inhibited by L-arginine.

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