Effects of pH changes on systems ASC and B in rabbit
ileum
Bjarne Gyldenløve
Munck and
Lars Kristian
Munck
Department of Medical Physiology, The Panum Institute, University of
Copenhagen, DK-2200 Copenhagen; and Department of Internal Medicine,
Køge Hospital, DK-4600 Koge, Denmark
 |
ABSTRACT |
Influx of
D-aspartate
(D-Asp),
L-glutamate
(L-Glu), and serine (Ser) across
the brush-border membrane of the intact mucosa from rabbit ileum has
been examined. L-Glu influx is
chloride independent and completely sodium dependent.
D-Asp and
L-Glu share a transport system
with a maximum transport rate of 1 µmol · cm
2 · h
1
and an apparent affinity constant
(K1/2) of
~0.3 mM. The function of this transport system is pH insensitive
between pH 5.65 and 8.2, and bipolar amino acids do not affect the way
in which the transport system handles
D-Asp and
L-Glu. The characteristics of
this transport system match those of system
X
AG. L-Glu and Ser share a
transporter for which the inhibitor constant (Ki) of
L-Glu against Ser decreases from
54 to 10 mM when pH is reduced from 7.2 to 5.65, while the maximum rate
of transport remains unaffected at ~10
µmol · cm
2 · h
1.
The Ki values (5 mM) of Ser against L-Glu influx
and the L-Glu-sensitive contribution to Ser influx (0.8 µmol · cm
2 · h
1
at 1 mM Ser) are the same at both pH values. The
L-Glu-sensitive transport of Ser
together with the contribution of system
bo,+ account for ~50% of Ser
influx at pH 7.2. The remaining 50% can be ascribed to system B. Transport of Ser by system B is reduced by >95% at pH 5.65. At pH
7.2 Ki of Ser
against transport of leucine (Leu) by system B is 18 mM and
Ki of Leu against
transport of Ser is 1.7 mM. The low-affinity transport of
L-Glu and the
L-Glu-sensitive transport of Ser
are performed by an equivalent of system ASC. Supplementary experiments
using the jejunum confirm the validity of these results for a major
portion of the rabbit small intestine.
anionic amino acids; biological transport; amino acids; brush-border membrane; intestine
 |
INTRODUCTION |
THE INVOLVEMENT OF SEVERAL systems in mediated
intestinal transport of amino acids was demonstrated by experiments
with sacs of everted small intestine, primarily from the golden hamster (37). Evidence was also presented suggesting active
transport of L-glutamate and
L-aspartate, but the rapid
transamination of these amino acids made the evidence inconclusive
(40). Sodium-dependent transport of
L-glutamate and
L-aspartate was first described
for rabbit jejunum and ileum, leading to estimates of affinity constant (K1/2) values
of 7 and 5 mM for L-glutamate
and L-aspartate, respectively,
for influx across the brush-border membrane (BBM) (32).
L-Glutamate uptake by segments
of the chicken small intestine was described in terms of one
high-affinity process and a large, nonsaturable contribution (12),
while uptake by isolated chicken enterocytes was described as the
result of two transport processes, one with a high affinity
(K1/2 = 16 µM)
and one with a low affinity
(K1/2 = 4.1 mM)
(38, 39). In BBM vesicles (BBMV) from the rat small intestine, the
K1/2 value of
uptake of D-aspartate and
L-glutamate was found to be
between 1 and 2 mM (5); in addition,
L-glutamate uptake appeared to
be partly chloride dependent (6). Uptake of
L-glutamate by BBMV from the
human jejunum measured at concentrations between 0.005 and 0.2 mM
corresponded to a
K1/2 of 90 µM
(9). More recent studies of
D-aspartate and
L-glutamate uptake by rabbit
jejunal BBMV have clearly demonstrated the presence of a high-affinity carrier (K1/2 = 60 and 80 µM for L-glutamate
and D-aspartate, respectively),
and, at pH 6.0, a low-affinity carrier
(K1/2 = 7 mM) for
L-glutamate (14).
The latter is not accessible to D-aspartate, while data on
inhibition of L-glutamate uptake
by L-aspartate correspond to an
inhibitor constant
(Ki) of ~75
mM (14). The characteristics described for the high-affinity
transporter correspond to those described for system
X
AG (3). Transport by the
low-affinity system was observed at pH 6.0 but was undetectable at pH
8.0. It could be inhibited by most bipolar amino acids, with alanine
and serine being as effective as leucine and methionine. These
characteristics correspond to those described for system ASC (36). It
was, however, proposed that the low-affinity transport of
L-glutamate was served by a protonated form of the transport system variously described as "carrier of neutral amino acids," system NBB, and, most recently, system Bo. Data on pH-sensitive uptake of glutamate by
Xenopus laevis oocytes injected with
cDNA from human placenta and rabbit small intestine were interpreted
similarly (10, 11).
The aim of the present study was to test the proposal of chloride
dependence of L-glutamate
transport and to examine whether the characteristics of
D-aspartate and
L-glutamate transport across the
luminal membrane of the intact rabbit intestinal epithelium were
similar to those described for rabbit jejunal BBMV (13, 14). Of prime
interest were the relative capacities of the two transport systems, the
unique capacity of bipolar amino acids to
cis-stimulate the vesicle uptake of
D-aspartate, the effects of pH
on a high-capacity system, and the identity of this system. The
detailed study was performed using the distal ileum from the rabbit,
which with this technique has the highest transport capacity (25, 26).
To allow comparison with the results previously reported for jejunal
BBMV (13, 14), we also examined the most important points using sheets
of the jejunum.
 |
MATERIALS AND METHODS |
Animals and Materials
Female albino rabbits with a body weight of 2,500-3,000 g were
raised and maintained with free access to water and food and killed by
intravenous phenobarbital. For examination of chloride dependence and
sodium activation of
L-glutamate transport in rabbit ileum, the solutions were made from a buffer (pH 7.2) composed of (in
mM) 140 Na+, 8 K+, 2.6 Ca2+, 1 Mg2+, and 140 Cl
or 140 isethionate, 8 phosphate, and 1 SO2
4. For the sodium
activation experiments
N-methyl-D-glucamine
(NMDG)-HCl was substituted for sodium chloride. For all
other experiments, 20 mM MES-Tris (pH 5.65 and pH 7.2) or 20 mM
HEPES-Tris (pH 8.2) were used instead of phosphate for buffering pH.
The total anion concentration was kept constant within each series of
experiments by varying the concentrations of isethionate,
D-aspartate, and L-glutamate. The concentration
of sodium was kept constant by using both sodium and NMDG salts of
D-aspartate and
L-glutamate. D-Glucose (5 mM) was present in
all solutions, except when measuring the transport of
-methylglucoside. 14C- and
3H-labeled chemicals were
purchased from NEN.
Unidirectional Influx Across BBM
Unidirectional influx across the BBM
(Jmc) of intact
rabbit ileum (0-30 cm from the ileocecal junction) or jejunum
(100-130 cm from the ileocecal junction) was measured as
previously described (27, 32). The excised intestine was mounted
between Lucite plates so that the mucosa was exposed in the bottom of
wells with an area of 0.62 cm2 in
which the solution was oxygenated and stirred by high rates of 100%
O2 flow at 37°C. After
preincubation for 20 min, the tissues were incubated for 0.50 min. The
incubation was stopped by flushing with ice-cold 300 mM
D-mannitol. The exposed tissue
was cut out and extracted for 18 h in 0.1 mM
HNO3. The extract and the
retracted incubation fluid were analyzed in a liquid scintillation
counter (Tri-Carb 2200CA, Packard). The content of
[3H]polyethylene
glycol 4000 or 14C-carboxylated
inulin in the tissue extract was used to correct for extracellular
contamination, and thus corrected, the content of
14C activity or, in the case of
D-aspartate,
3H activity, in the tissue extract
was used to calculate the rate of amino acid influx across the BBM.
It was assumed that the kinetics of unidirectional transport across the
BBM (Jmc)
measured at different substrate concentrations ([A]m) and at
different inhibitor concentrations
([I]m) in the mucosal-bathing solutions could be described as the sum of one or two
saturable Michaelis-Menten processes
(MM1,
MM2) and diffusion
|
(1)
|
where
|
(2)
|
where
P is the diffusive permeability of A
(in cm/h). JAmc is given as
micromoles per square centimeter of mucosal area per hour.
K1/2 and
Ki are given in
millimolar. It is assumed that the sodium activation of
L-glutamate can be described by
|
(2a)
|
where
JL-Glumax
is the maximum rate of transport at the concentration of
L-glutamate chosen, K1/2 is the
concentration of sodium at which half-maximum activation is
accomplished, and nH is the Hill coefficient.
The estimates of transport kinetics were made by nonlinear least
squares fitting to this model of the experimentally determined relationship between
Jmc of A and
[A]m weighted by the
inverse of the standard deviation (SD). The estimates were evaluated by the even distribution of residuals and by calculation of the
2 value with degrees of freedom
(df) being the number of experimental points minus the number of
parameters estimated. The errors of these estimates are 1 SD. The
Ki values were
calculated from the ratios between inhibited and uninhibited fluxes,
assuming that these were described by the sum of a saturable process
conforming to Michaelis-Menten kinetics and diffusion. Errors on fluxes
and estimates of
Ki are standard
errors with the number of observations in parentheses. All influx
results are pooled data from at least two rabbits.
 |
RESULTS |
Chloride and Sodium Dependence of Glutamate Transport
Chloride dependence of transport of anionic amino acids could
complicate measurements of transport at high concentrations unless
parallel increases in total osmotic activities were accepted. Consequently, L-glutamate
influx was measured at 20 µM in rabbit ileum. Unidirectional influx
across the BBM of L-glutamate
measured at 140 mM NaCl was 91.0 ± 11.2 nmol · cm
2 · h
1
(means ± SE of n = 10), 78.5 ± 10.4 nmol · cm
2 · h
1
at 140 mM sodium isethionate, and 0.9 ± 0.2 nmol · cm
2 · h
1
when sodium was substituted with NMDG. Influx of
L-glutamate measured at 1.0 mM
L-glutamate at 140 mM NaCl and
140 mM sodium isethionate in rabbit ileum was 1.15 ± 0.08 and 1.14 ± 0.06 µmol · cm
2 · h
1,
respectively (n = 8). The chloride
independence of L-glutamate influx allowed the use of isethionate instead of chloride, and, whenever L-glutamate or
D-aspartate were used in
concentrations above 10 mM, to reduce the concentration of isethionate
to keep the sum of the concentrations of isethionate and
L-glutamate/D-aspartate constant. The sodium-independent rate of transport
corresponds to a permeability of 0.045 cm/h, which is well within the
range of our estimates of diffusive contributions to influx of amino acids (19, 21, 22). Thus a separate sodium-independent transport of
anionic amino acids is not present in the BBM of rabbit small intestine.
Sodium Activation of Glutamate Transport
The influx of L-glutamate was
measured at 20 µM glutamate at pH 7.2 at concentrations of sodium
between 0 and 140 mM with preincubations and test incubations made at
the same concentrations of sodium (Fig. 1).
Analyzed in terms of Eq.
2a, the results are best
(
2 = 2.896; df = 5) described
by
|
(3)
|
where nH = 1.73 ± 0.25, indicating a
sodium: L-glutamate
stoichiometry of 2:1. As demonstrated below
L-glutamate transport at 20 µM
is almost exclusively by the high-affinity system, so these results
agree with those obtained with BBMV (14).

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Fig. 1.
Kinetics of sodium activation of
L-glutamate
(L-Glu) unidirectional influx
across the brush-border membrane
(Jmc) in rabbit
ileum. Influx of L-Glu was
measured at 0.02 mM L-Glu at 0, 2, 4, 8, 17, 35, 70, or 140 mM sodium chloride, with
N-methyl-D-glucamine
substituted for sodium. Data are means ± SE of 4-6
observations. The curve is best described by
Eq. 3
given in RESULTS.
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Comparison With Function of BBMV
Our attention was especially drawn to the very low capacity of the
high-affinity transport (14). With a maximum rate of transport of only
1% or less of that of the low-affinity system, it might have
represented an otherwise undetectable contamination by basolateral
membrane vesicles. The cis-effect of
bipolar amino acids on vesicle uptake of
D-aspartate was also intriguing,
partly because of its low specificity and partly because it was related to the effect of leucine and methionine on intestinal transport of
lysine, an effect which at least in the case of transport across the
BBM most likely represents a
trans-effect made possible by the
parallel functions of systems B and
bo,+ (4, 22). Finally, although
lowering the pH is known to stimulate transport of anionic amino acids
in several cellular systems (34, 36), reducing the pH had previously
been seen to reduce intestinal transport of bipolar amino acids (7). A
series of experiments was performed to provide guidance for the further
study of these aspects of intestinal function.
The Concept of Two Transporters
To briefly test the concept of two transporters of anionic amino acids,
we performed paired measurements of influx of 50 mM D-aspartate and 50 mM
L-glutamate at pH 5.65 and 7.2, assuming that this concentration would provide for high degrees of
saturation. At pH 5.65 influx of
L-glutamate and
D-aspartate was 9.67 ± 0.98 and 2.08 ± 0.29 µmol · cm
2 · h
1,
respectively (n = 4 each). At pH 7.2 influx of L-glutamate and D-aspartate was 9.61 ± 0.52 and 2.40 ± 0.34 µmol · cm
2 · h
1,
respectively (n = 4 each). Influx of
L-glutamate was about four times
that of D-aspartate at both pH
values consistent with the presence of two pathways of transport, of
which only one is accessible to both
D-aspartate and
L-glutamate (14). Consequently,
at pH 7.2 paired measurements were made at 1 mM
L-glutamate alone and with 50 mM
D-aspartate or 50 mM
L-glutamate as inhibitors
(n = 6). The rate of transport was
1.03 ± 0.08 µmol · cm
2 · h
1
at 1 mM L-glutamate, which was
reduced to 0.14 ± 0.01 µmol · cm
2 · h
1
when inhibited by 50 mM
L-glutamate and to 0.17 ± 0.03 µmol · cm
2 · h
1
in the presence of 50 mM
D-aspartate. These results agree
with those on BBMV (14) indicating that the transporter of
D-aspartate is shared with
L-glutamate with very similar
affinities. Furthermore, at 1 mM
L-glutamate this common
transporter appears to be responsible for >80% of the total transport.
Effects of pH on Transport Across BBM
The possibility of an effect of pH on transport of anionic amino acids
was examined by using the ability of amiloride at 1 mM to inhibit the
Na+/H+
exchanger and thereby increase pH of the outside microclimate of the
luminal membrane (15). Paired measurements of influx were made at 50 mM
L-glutamate or 1 mM
D-aspartate at pH 7.2 and 140 mM
sodium with or without 1 mM amiloride, with amiloride present in both
preincubation and test incubation (Table
1). A second experiment was performed using
the same procedure, but reducing the sodium concentration to
70 mM sodium to increase the efficiency of amiloride.
Paired measurements were made of
D-aspartate at 0.1 mM
D-aspartate and of
L-glutamate at 0.1 mM
L-glutamate plus 70 mM
D-aspartate at pH 7.2 or at pH
7.2 plus 1 mM amiloride (Table 1). Confirming the observations on BBMV
(14), the results of these measurements (Table 1) demonstrate that the
presumed increase of pH reduced influx of
L-glutamate without affecting
influx of D-aspartate. The
relative effects at 0.1 and 50 mM
L-glutamate suggested that the
effect of decreasing proton concentration might be to decrease the
affinity of L-glutamate for the
transporter not shared with
D-aspartate. This suggestion was
further tested by paired measurements of influx of
D-aspartate and
L-glutamate both at 50 mM.
Influx of L-glutamate at pH 7.2 was 8.13 ± 0.22 µmol · cm
2 · h
1
(n = 8), which was reduced to 4.24 ± 0.35 µmol · cm
2 · h
1
(n = 8) at pH 8.2, whereas
D-aspartate influx at pH 8.2 was
2.85 ± 0.29 µmol · cm
2 · h
1
(n = 8). It is seen that, although
greatly reduced at pH 8.2, the influx of
L-glutamate remains
significantly higher than that of
D-aspartate, indicating
persistent function of the glutamate transporter at pH 8.2.
High concentrations of glutamate might by partial dissipation of the
electrochemical gradient of sodium have unspecific effects. Also, the
effects of amiloride and the effects of changing pH might be
unspecific. These possibilities were examined in two series of paired
measurements. One in which, at 140 mM sodium and 1 mM serine, influx of
serine was measured with and without the presence of 70 mM
L-glutamate at pH 5.65, 7.2, 8.2, and 8.2 with 1 mM amiloride (Table 2).
The same protocol was used in the second series in which 1 mM
-methyl-D-glucoside was used as substrate instead of serine and the influx of this sugar was measured (Table 2). It can be seen (Table 2) that 70 mM
L-glutamate significantly
inhibits transport of serine at pH 5.65 and 7.2 but not at pH 8.2. At
pH 5.65 influx of serine is much reduced compared with pH 7.2, while
the glutamate-sensitive contribution is increased by 67%. Also, the
influx of
-methyl-D-glucoside is reduced at pH 5.65. However, at all the pH values tested, its transport is unaffected by
L-glutamate. Amiloride did not
significantly affect the transport of serine or
-methyl-D-glucoside.
This series of experiments has provided data consistent with the
presence of two transporters of anionic amino acids of which only one
is accessible to D-aspartate.
They demonstrate pH sensitivity of the exclusive glutamate transporter
and suggest an effect of increasing affinity with increasing proton
concentration. This suggestion gained considerable support from the
relatively high efficiency of glutamate as inhibitor of serine
transport at pH 5.65. Unspecific effects of glutamate or amiloride were
not seen.
Specificity of Bipolar Amino Acids as Inhibitors of Glutamate
Transport
If the inhibitory effects of bipolar amino acids on transport of
L-glutamate were caused by
competition for system B, then their
Ki values against
L-glutamate influx should be
identical to their
Ki or
K1/2 values for
system B. Therefore, paired measurements of influx of
L-glutamate were made at pH 7.2, 140 mM sodium, and 1 mM
L-glutamate alone or in the
presence of 10 mM leucine, phenylalanine, or serine. These amino acids
were chosen to cover a relatively wide range of
K1/2 for system
B; 1 mM for leucine (19), 4 mM for phenylalanine (21), and 25 mM for
serine (31). Influx of
L-glutamate was 1.32 ± 0.09 µmol · cm
2 · h
1
in the absence of inhibitor and 1.09 ± 0.03, 0.92 ± 0.04, and 0.91 ± 0.08 µmol · cm
2 · h
1
in the presence of 10 mM leucine,
phenylalanine, and serine, respectively (means ± SE of 6 paired observations). All three inhibitors reduced
influx of L-glutamate to a level
corresponding to transport by the transporter shared with
D-aspartate. The inhibition of
transport by the glutamate transporter must, therefore, be almost
complete and the
Ki values for all
three bipolar amino acids must be well below 10 mM.
A Quest for Cis-Stimulation of
D-Aspartate Influx by Bipolar
Amino Acids
At 0.05 mM D-aspartate,
cis-stimulation of
D-aspartate uptake by jejunal
BBMV was demonstrated, using several bipolar amino acids in
concentrations between 5 and 50 mM (13, 14). Here, influx of
D-aspartate in the distal ileum
and distal jejunum was measured at 140 mM sodium at either 0.05 or 0.10 mM D-aspartate and at pH 5.65 or
7.2 using various bipolar amino acids as inhibitors (Table
3). In these experiments, neither
cis-stimulation nor cis-inhibition was
observed. Recent studies (4, 22) have indicated that
recycling of bipolar amino acids between the cytoplasm and the outside
unstirred water layer by sodium-coupled uptake by system B and exit in
exchange with lysine by system
bo,+ is responsible for the
apparent sodium dependence of lysine influx and for the apparent
cis-stimulation of lysine influx by
leucine and methionine (22). Similarly,
cis-stimulation by bipolar amino acids
present in the unstirred layer might be at hand already in our control
situation, masking any effect of additional exogenous amino acid.
Preincubation at 0 mM sodium will reduce reuptake from the outside
unstirred water layer of amino acids leaking from the enterocytes,
reducing the cytoplasmic pool as source for the outside concentration
of endogenous amino acids during the subsequent period of incubation.
Consequently, to examine the possibility that processes of either
cis- or
trans-stimulation were at hand already
in our control situation, tissues (the ileum) were preincubated at 0 mM
sodium for at least 20 min. After a brief wash at 140 mM sodium, influx
was measured at 140 mM sodium, pH 7.2, and 0.05 mM
D-aspartate with 0 or 10 mM
valine (Table 3). Once again,
cis-stimulation was not demonstrated.
Instead, a small inhibitory effect was seen.
The results presented above suggested that decreasing pH caused an
increase of the affinity of
L-glutamate for the transporter it shares with bipolar amino acids. In addition, the relative efficiencies of leucine, phenylalanine, and serine as inhibitors of
L-glutamate transport questioned
the identity of this common transporter as system B. These questions
were subsequently examined.
Kinetics of D-Aspartate and
L-Glutamate Transport
Kinetics at pH 7.2.
D-Aspartate influx was measured
at 140 mM sodium and at concentrations of
D-aspartate between 0.005 and 50 mM. The results (Fig. 2) are best
(
2 = 1.284; df = 6) described
as
|
(4)
|
D-Aspartate influx
was then measured at 0.05 mM
D-aspartate in the presence of
0-10 mM L-glutamate. The
results (Fig.
3A) correspond to a
Ki of glutamate
of 0.34 ± 0.04 mM.

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Fig. 2.
Kinetics of D-aspartate
(D-Asp) influx in rabbit ileum.
Influx of D-Asp at 140 mM sodium
was measured at 5, 10, 20, 40, 80, 120, 160, 240, or 50,000 µM
D-Asp. Data are means ± SE
of 3-6 observations. The best curve fit is given in
Eq. 4
in RESULTS.
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Fig. 3.
A: inhibitory effect of
L-Glu on
D-Asp influx in rabit ileum.
Influx of D-Asp was measured at
pH 7.2 and 140 mM sodium, at 0.05 mM
D-Asp with 0, 0.25, 0.5, 1.0, 2.5 or 10 mM L-Glu. Data are
means ± SE of 7 observations. Assuming a passive
permeability of 0.017 cm/s and an affinity constant
(K1/2) for
D-Asp of 0.28 mM, the data
correspond to an inhibitor constant
(Ki) for
L-Glu of 0.34 ± 0.04 mM.
B: inhibitory effects of leucine (Leu)
and serine (Ser) on influx of
L-Glu in rabbit ileum.
Influx of L-Glu
was measured at 140 mM sodium, 70 mM
D-Asp, and pH 7.2, at 0.1 mM
L-Glu with 0, 1, 2, 5, 10, or 20 mM Leu ( ) or Ser ( ). Assuming a diffusive contribution to
L-Glu influx of 0.003 µmol · cm 2 · h 1
and a K1/2 for
L-Glu of 54 mM, the data
correspond to a
Ki of 2.0 ± 0.8 and 4.9 ± 0.8 mM for Leu and Ser, respectively.
|
|
L-Glutamate influx was now
measured at 140 mM sodium and concentrations of glutamate between 0.07 and 140 mM. The results of these experiments (Fig.
4) are best
(
2 = 0.267; df = 6) described
as the sum of two saturable processes and diffusion
|
(5)
|

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Fig. 4.
Kinetics of L-Glu influx in
rabbit ileum. L-Glu influx was
measured at 140 mM sodium and pH 7.2 and at 0.07, 0.10, 0.15, 0.20, 0.5, 1.0, 5, 10, 15, 20, 70, or 140 mM
L-Glu. Data are means ± SE
of 7-9 observations. The curve fit is given in Eq. 5 in
RESULTS.
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|
Kinetics at pH 5.65.
In agreement with the observed identity of
K1/2 and maximum
rate of uptake of D-aspartate
by BBMV at pH 6 and 8 (14), our data (Table 1) on
D-aspartate influx at pH 8.2, 7.2, and 5.65 did not indicate any pH effect on this function of the
intact epithelium. The kinetics of
D-aspartate transport were,
therefore, not examined at pH 5.65. L-Glutamate influx was measured
at 140 mM sodium and at 0.1-140 mM
L-glutamate. Assuming a passive
permeability to L-glutamate of
0.03 cm2/h, the results (Fig.
5) are best
(
2 = 1.862; df = 8)
described as
|
(6)
|
These results confirm that transport of
D-aspartate is restricted to one
pH-independent, high-affinity transporter, which is shared with
L-glutamate (14).
They demonstrate that the vanishing of the low-affinity transport of
L-glutamate at increasing pH reflects a decrease in affinity alone and that relative to the capacity
of the low-affinity transporter that of the high-affinity system is
more than ten times as high as determined for BBMV.

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Fig. 5.
Kinetics of L-Glu influx in
rabbit ileum at 140 mM sodium and
pH 5.65. Influx of
L-Glu was measured at 0.1, 0.2, 0.44, 0.5, 1.0, 2.5, 5, 10, 15, 35, 70, or 140 mM
L-Glu and 140-0 mM
isethionate. Data are means ± SE of 6 observations. The description
of the best curve fit is given in Eq. 6 in
RESULTS.
|
|
Kinetics of Interactions Between
L-Glutamate, Leucine, and Serine
To restrict transport of
L-glutamate to the low-affinity
transporter, we measured influx at 0.1 mM
L-glutamate in the presence of
70 mM D-aspartate. Because of
the need to use up to 200 mM of glutamate as inhibitor, we measured
influx of serine at 200 mM sodium.
Kinetics at pH 7.2.
Paired measurements of influx of
L-glutamate were made at 140 mM
sodium and 0.1 mM L-glutamate
and 70 mM D-aspartate with 0-20 mM of serine or leucine. Assuming a
K1/2 for
L-glutamate of 54 mM, the
results (Fig. 3B) correspond to a
Ki of 4.9 ± 0.8 and
2.0 ± 0.8 mM for serine and leucine, respectively.
The cited estimate of 25 mM as
K1/2 of serine
for system B was based on measurements that probably included
contributions by systems bo,+ and
Bo,+ (31). To obtain estimates
comparable to those cited for leucine and phenylalanine, we measured
influx of leucine at 140 mM sodium, 100 mM lysine, 0.5 mM leucine,
0-200 mM serine, and 200-0 mM mannitol. In these
measurements, lysine served to exclude leucine from systems bo,+ and
Bo,+, while mannitol served as an
osmotic compensation for serine. The results (Fig.
6) do, therefore, describe the effect of
serine on system B for which they indicate a
Ki of 18 ± 2 mM. To the extent that leucine may be transported by the low-affinity
transporter of L-glutamate this
number overestimates the affinity of serine for system B.

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Fig. 6.
Inhibitory effects of L-Glu on
influx of Ser ( ) and of Ser on influx of Leu ( ) by system B in
rabbit ileum at pH 7.2. Influx of Ser was measured at
200 mM sodium with 0, 35, 50, 70, 100, 140, 150, 175, or 200 mM
L-Glu. With increasing
concentrations of L-Glu, the
concentration of isethionate was reduced from 200 mM to 0 mM. Data are
means ± SE of 4-10 observations. Assuming an
L-Glu-resistant contribution to
Ser influx of 0.800 µmol · cm 2 · h 1
and a K1/2 for
Ser of 5 mM, the data correspond to a
Ki for
L-Glu of 58 ± 9 mM. Influx
of Leu was measured at 0.5 mM Leu with 100 mM lysine and 0, 1, 2, 5, 10, 20 or 100 mM Ser. Data are means ± SE of 7-8 observations.
Assuming a diffusive contribution to Leu influx of 0.025 µmol · cm 2 · h 1
and a K1/2 for
Leu of 1 mM, the data correspond to a
Ki for Ser of
18.2 ± 2.0 mM.
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|
The efficiency of glutamate as inhibitor of influx of serine was
examined in paired measurements at 1 mM serine and 200 mM sodium
with 0-200 mM
L-glutamate (Fig. 6). Assuming a
K1/2 of 4.9 mM
for serine and a glutamate-resistant contribution to influx of serine
of 0.8 µmol · cm
2 · h
1,
the results correspond to a
Ki for glutamate
of 58 ± 10 mM and a glutamate-sensitive transport of 0.8 µmol · cm
2 · h
1.
To examine the assumption that the
L-glutamate-resistant transport
of serine is accomplished by systems B and
bo,+, influx of serine at 1 mM was
measured at 200 mM sodium glutamate and pH 7.2 serine and 0-100 mM
leucine. The results (Fig. 7) demonstrate that at 50 and 100 mM leucine, influx of serine is reduced to the level
of the usual diffusive contributions. On the basis of this assumption
and using 18 mM as the
K1/2 for serine,
the results correspond to a
Ki for leucine of
1.71 ± 0.14 mM. This value does not differ from our previous
estimates of the affinity of leucine for systems B and
bo,+.

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Fig. 7.
Inhibitory effect of Leu on
L-Glu-resistant transport of
Ser. Influx of Ser at 1 mM was measured at 200 mM sodium glutamate and
pH 7.2 Ser and 0-100 mM Leu. With the use of 0.055 µmol · cm 2 · h 1
as the uninhibitable influx and 18 mM as the
K1/2 for Ser, the
results correspond to a
Ki for Leu of
1.71 ± 0.14 mM. The curve depicts this fit.
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Kinetics at pH 5.65.
At pH 5.65 only interactions between serine and anionic amino acids
were examined. Influx of
L-glutamate was measured at 140 mM sodium, 0.1 mM L-glutamate,
and 70 mM D-aspartate in the
presence of 0-20 mM serine (Fig. 8).
Assuming a K1/2
for glutamate of 22 mM, the results correspond to a
Ki for serine of
4.8 ± 0.8 mM.

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Fig. 8.
Inhibitory effect of Ser on
L-Glu influx in rabbit ileum at
140 mM sodium and pH 5.65. L-Glu
influx was measured at 0.1 mM
L-Glu and 70 mM
D-Asp with 0, 1, 2, 5, 10, or 20 mM Ser. Data are means ± SE of 4 observations. Assuming a diffusive
contribution to L-Glu influx of
0.003 µmol · cm 2 · h 1
and a K1/2 for
L-Glu of 22 mM, the data
correspond to a
Ki for Ser of 4.8 ± 0.8 mM.
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Influx of serine was measured at 200 mM sodium and 1 mM serine in the
presence of 0-200 mM
L-glutamate (Fig.
9). Assuming a
K1/2 of 4.8 mM
for serine and a glutamate-resistant transport of serine of 0.2 µmol · cm
2 · h
1,
the results correspond to a
Ki for glutamate
of 9.9 ± 1.5 mM and a glutamate-sensitive serine transport of 0.8 µmol · cm
2 · h
1.
Thus both the affinity of serine for the glutamate transporter and the
magnitude of its glutamate-sensitive transport are independent of pH.
In contrast, at pH 5.65 the glutamate-resistant flux of serine is
reduced to a level within the range observed for transport of bipolar
amino acids by system bo,+,
indicating that transport by system B is eliminated at pH 5.65. This
interpretation was examined in paired measurements of serine influx at
pH 5.65 and 7.2 at 140 or 0 mM sodium. Reducing sodium from 140 to 0 mM
reduced serine influx at pH 5.65 from 1.34 ± 0.06 to 0.15 ± 0.01 µmol · cm
2 · h
1
(n = 6) and at pH 7.2 from 2.27 ± 0.18 to 0.31 ± 0.03 µmol · cm
2 · h
1
(n = 6). Thus at both pH values
sodium-independent serine influx is close to the glutamate-resistant
influx seen at pH 5.65 (see Table 2). This supports the notion that at
pH 5.65 transport of serine is restricted to system
bo,+, the low-affinity transporter
of L-glutamate, and diffusion.

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Fig. 9.
Inhibitory effects of L-Glu and
L-Asp on Ser influx in rabbit
ileum at 200 mM sodium and pH 5.65. Influx of Ser was measured at 1 mM
Ser with 0, 50, 100, 150 or 200 mM
L-Glu ( ). Data are means ± SE of 4 observations. Assuming a
L-Glu-resistant contribution to
Ser influx of 0.2 µmol · cm 2 · h 1
and a K1/2 of Ser
of 5 mM, the data correspond to a
Ki for Glu of 9.9 ± 1.5 mM. Influx of Ser was measured at 1 mM Ser with 0, 2, 5, 10, 25, 50, 100, 150, 175, or 200 mM
L-Asp ( ). Data are means ± SE of 4-8 observations. With the above assumptions, the data
correspond to a
Ki for
L-Asp of 79 ± 5 mM.
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The data obtained with rabbit BBMV indicate that at pH 6.0 Ki of
L-aspartate against the
low-affinity transport of
L-glutamate is ~75 mM. To
allow comparison with these results, we measured influx of serine at 1 mM at pH 5.65 and 200 mM sodium with 0-200 mM
L-aspartate present. Assuming an
L-aspartate-resistant
contribution to the transport of serine of 0.2 µmol · cm
2 · h
1,
the results correspond to a
Ki of
L-aspartate of 79 ± 5 mM and an L-aspartate-sensitive influx
of 0.8 µmol · cm
2 · h
1.
Jejunal Transport of
D-Aspartate,
L-Glutamate, and Serine
In the rabbit small intestine, system
Bo,+ is present only in the ileum
(25). Influx of other amino acids is characterized by an aborally
increasing capacity with constant affinity (23, 25-27).
Conceivably, the differences between the present study and those
performed with jejunal BBMV (13, 14) might reflect this topographic
variation, while the possibility of
cis-stimulation was excluded by the
data in Table 3. The questions as to the relative capacities of the two
transporters of anionic amino acids and the pH effects on transport of
glutamate and serine were dealt with in the following two series of experiments.
The pH effect and the question of relative capacities were examined in
paired measurements of influx of
D-aspartate and
L-glutamate at 140 mM sodium and
50 mM of the amino acids at pH 5.65 and 8.2. Influx of
L-glutamate was 5.51 ± 0.32 and 3.07 ± 0.33 µmol · cm
2 · h
1
at pH 5.65 and pH 8.2, respectively
(n = 6). Influx of
D-aspartate was 1.73 ± 0.24 and 1.52 ± 0.40 µmol · cm
2 · h
1
at pH 5.65 and 8.2, respectively (n = 6). It is seen that increasing pH from 5.65 does not affect the influx
of D-aspartate, which is
significantly exceeded by the influx of
L-glutamate at both pH values.
At pH 5.65 the difference between the two fluxes is 3.8 µmol · cm
2 · h
1,
which assuming identical diffusive permeabilities of
D-aspartate and
L-glutamate represents the
function of the high-capacity system. Assuming a diffusional
permeability of 0.03 cm/h, the mediated transport of
D-aspartate is 0.7 µmol · cm
2 · h
1,
corresponding to ~20% of transport of
L-glutamate by its low-affinity transporter.
The pH effect on transport of serine was examined by paired
measurements at 200 mM sodium and 5 mM serine with 0 or 200 mM L-glutamate at pH 7.2 and 5.65 (Table 4). Under these conditions influx of
serine was increased also in the distal ileum. As with the distal ileum
(Tables 4 and 2), at pH 5.65 glutamate reduces the jejunal influx of
serine to a level close to that expected for system
bo,+ together with diffusion (21),
and L-glutamate is a more
efficient inhibitor at pH 5.65 than at pH 7.2.
 |
DISCUSSION |
The present study confirmed the observations of anionic amino acid
transport by the rabbit small intestine as being served by a high- and
a low-affinity system and of proton stimulation of the latter system
(13, 14). In contrast, cis-stimulation by bipolar amino acids of the high-affinity system could not be confirmed, and while the ratio between the capacities of the two systems was ~1:200 with BBMV (14), it was ~1:10 with the intact epithelium. Nor was the specificity of interactions between bipolar amino acids and the low-affinity transport consistent with system B
being responsible for this function.
The High-Affinity System
Consistent with the observations made with rabbit BBMV (14), a
component of the passage of
D-aspartate and
L-glutamate across the BBM of
the intact epithelium is characterized by equally high affinities,
insensitivity to changes in pH between 5.65 and 8.2, and to inhibition
by bipolar amino acids, by an activation by sodium indicating a
sodium:L-glutamate stoichiometry
of 2:1, and by independence of the presence of chloride. The difference
by a factor of 3-5 between the present estimates of the affinities of the two anionic amino acids and those made with BBMV is similar to
the difference between previous kinetic estimates for the high-affinity transport of taurine and
-alanine in rabbit intestine (17, 27),
which with respect to affinity and capacity is comparable to the
high-affinity transporter of
D-aspartate and
L-glutamate. Therefore, and
because all our kinetic observations on the low-affinity transport
agree with those made with BBMV, we would expect a
Jmax for
D-aspartate between 0.1 and 0.2 µmol · cm
2 · h
1
and that the model proposed to account for
cis-stimulation by bipolar amino acids
of D-aspartate uptake by BBMV
would apply to its passage across the BBM of the intact epithelium.
With a Jmax of
~1
µmol · cm
2 · h
1
and absence of any cis-stimulation,
these expectations were clearly not fulfilled.
It is well established that effects of cotransport of sodium with
bipolar amino acids and glucose on the electrical potential difference
across the mucosal membrane suffice to cause large mutual inhibition of
uptake by BBMV between these nutrients (16). With the intact epithelium
such inhibition reaches only barely significant levels (19). In the
present study, the passive contribution to transport at 0.05 and 0.1 mM D-aspartate amounts to
<3%, while with the BBMV the passive contribution at 0.05 mM can be
as high as 30% of the total uptake. However, since the
cis-effect seems to be subject to self
inhibition by D-aspartate, it
cannot be accounted for by a potential difference effect on the passive contribution to uptake. An electrostatic interpretation
of the cis-effect would therefore have
the unlikely requirement that sodium-coupled
D-aspartate uptake represents a
net transfer of negative charge. The electrostatic interpretation of
the cis-effect would also require a
similar effect of D-glucose, a
test for which there are no published results. It appears then that the
procedures used to prepare BBMV reduce the capacity of the
high-affinity transporter in a way that can, at least partly, be
remedied by imposing cotransport of sodium and a variety of bipolar
amino acids. Recirculation, using systems B and
bo,+, of the endogenous pool of
bipolar amino acids between the cytoplasm and the outside, unstirred
microclimate seems to be a significant stimulus for lysine influx and
to be responsible for its apparent cis-stimulation by bipolar amino acids
(22). Although preincubation at 0 mM sodium reduced this effect on
lysine transport, it did not affect
D-aspartate influx. Therefore,
it seems unlikely that our failure to demonstrate this
cis-stimulation can be explained by it
being present already under the control conditions of our experiments.
The Low-Affinity System
Stimulation of transport of anionic amino acids by reduction of the
ambient pH has been described for Chinese hamster ovary cells (34),
Ehrlich ascites tumor cells (36), and HeLa cells and
Xenopus laevis oocytes injected with
cDNA from several organs and species, such as mouse testis (35), human
coriocarcinoma cell line (10), and rabbit small intestine (11). The
effect of pH has been ascribed to protonation of the anionic amino acid (33), but mostly to protonation of the transporter (10, 11, 14, 34,
36). The mechanism of the pH influence has, however, not been examined.
The observation that amiloride at pH 7.2 had a relatively much greater
effect on L-glutamate influx at
0.1 mM than at 50 mM suggested that lowering pH led to a decrease of
K1/2 of
L-glutamate for the low-affinity
transport. The present results proved this to be the case (Figs. 4 and
6), leaving the
Jmax unaffected.
The effect of pH changes on transport of bipolar amino acids by the pH-sensitive transporter has not been examined in detail. However, measurements at one concentration in Ehrlich ascites tumor cells (36)
and in a Chinese hamster ovary cell line (34) indicate that for
transport of bipolar amino acids by system ASC the kinetic parameters
are unaffected by lowering pH to 6.0, while transport by system A is
greatly reduced. In these cells system A is the closest equivalent to
the intestinal system B. These results can, therefore, be seen as
consistent with the reduction of alanine transport by the rabbit ileum
seen with decreasing pH (7). In agreement with this interpretation of
the studies on ovary cells and Ehrlich ascites tumor cells, the present
results demonstrate that both affinity for and maximum rate of
transport of serine by the transporter shared with glutamate are
unaffected by the changes in pH used in this study.
The present results provide in addition the important information that
at pH 7.2 L-glutamate can
eliminate only one-half or a little less of the total transport of
serine. This raises the question of the identity of at least two
transporters involved in serine transport. At pH 7.2 system
bo,+ can account for only
25-33% of the
L-glutamate-resistant transport of serine. System Bo,+ does not
function in the absence of chloride (24), and the imino acid carrier is
inaccessible to serine (20). Thus only system B is left as purveyor of
the L-glutamate-resistant,
sodium-dependent transporter of serine. This conclusion is supported by
the difference between a
Ki for serine of
5 mM against the transport of
L-glutamate and of 18 mM against
leucine transport by systems bo,+
and B as well as by the
Ki of 1.7 mM for
leucine against the L-glutamate-resistant transport
of serine. These estimates do not differ from previous estimates of the
affinity for systems bo,+ and B. It follows that it is not system B but system ASC that is the
transporter shared by
L-glutamate and serine. This
conclusion is supported by the difference between a
Ki of 5 mM for
serine against L-glutamate
transport and its
Ki of 18 mM
against the chloride-independent and lysine-resistant transport of
leucine. It is also consistent with the observation on BBMV and
cDNA-injected HeLa cells and Xenopus
laevis oocytes that alanine, serine, and threonine have
as high or higher affinities than leucine and methionine as inhibitors
of the low-affinity L-glutamate
transport by these cellular systems (10, 11, 14). In contrast, in the
rabbit small intestine the affinities of leucine and methionine for
system B by far exceed those of alanine, serine, and threonine (19, 21,
22, 31, 34). In addition, the relatively very high degree of
L-glutamate inhibition of
phenylalanine uptake by BBMV observed at pH 6.0 can be explained partly
by the higher affinity of
L-glutamate and partly by the
decrease in transport by system B seen at reduced pH. In particular,
this nearly complete inhibition of phenylalanine uptake led us
previously (27, 28) to believe that system B was responsible for the
low-affinity transport of L-glutamate and therefore to
discard system ASC as a BBM transporter, since direct evidence for its
presence there had not been published.
The Topographic Problem
The most detailed part of the present study rests on the use of rabbit
distal ileum, although its stepping stone is a study performed with
rabbit jejunal BBMV (14). This was done because the distal ileum is the
most clearly defined section, the easiest to prepare, the most
thoroughly examined (19, 20, 23), and the section in which the amino
acid transporters have the highest capacities (25, 26, 27). The decline
in transport capacity observed with increasing distance from the
ileocoecal junction is larger for system
Bo,+ and for the imino acid
transporter than for system B (25-27). However, one study of transport
of anionic amino acids by the rabbit small intestine presented both
jejunal and ileal observations (33). These data did not indicate a
topographic difference sufficient to account for the differences
between the present results and those reported for BBMV. Nevertheless,
the possibility of such an explanation was examined. Clearly,
cis-stimulation of
D-aspartate influx was not
present in the jejunum. Corrected for diffusive contributions the ratio
between influx of D-aspartate
and L-glutamate at 50 mM was the
same in the jejunum as in the ileum, and the pH effects on influx of
D-aspartate,
L-glutamate, and serine were the
same in the two sections. Thus the differences discussed seem to
reflect methodological differences.
We have previously defined system B as a sodium-dependent,
chloride-independent, lysine-resistant transporter of bipolar amino acids (28). Now the quality of
L-glutamate resistance must be added. It may be of some interest to note that this is the third case
of detection of a transporter different from what was first seen as the
carrier of neutral amino acids (37). The first being system
bo,+ (19, 30) and the next system
Bo,+, initially termed
-alanine
carrier (20). That system ASC does transport bipolar amino acids in
general has some effect on the estimates of their
K1/2 on system B. In the case of leucine, the effect will be a slight overestimate of
K1/2, while for serine (Fig. 6) and probably alanine and threonine
K1/2 will be underestimated.
In Table 5, the present and previously
published (19, 20) estimates of the affinities of the amino acids used
in this study for the amino acid transporters of the luminal membrane of the rabbit small intestine are summarized.
pH Problems
With the intact epithelium the buffering capacity of the molecular
constituents of the membrane surface and the function of the
sodium-proton exchanger prevent absolute control of the pH of the
microclimate at the membrane surface. It is known that with a pH of 7 in the bathing solution the sodium-proton exchanger maintains a
slightly lower pH at the membrane and that this difference can be
greatly reduced when the exchanger is inhibited by 1 mM amiloride (15).
It is likely that the same effect of the exchanger is present at a
solution pH of 8.2, while with a solution pH of 5.65 the pH at the
membrane will deviate less. At this pH 4% of the
L-glutamate and <1% of the
D-aspartate will exist in the
bipolar form.
Comparison With Other Studies
The presence of a low-affinity transport of
L-glutamate
(K1/2 = 7 mM) and
L-aspartate
(K1/2 = 5 mM) was
previously indicated by data on influx of these amino acids in the
rabbit ileum (32). The conclusiveness of this study was, however,
limited by the use of a too narrow (2.5-25 mM) concentration
range. A study of glutamate uptake by BBMV prepared from human jejunum (9) was conducted using concentrations below 1 mM. The results reflected the function of a high-affinity system with characteristics similar to those described for system
X
AG. Also, transport of
L-aspartate and
L-glutamate across monolayers of human Caco-2 cells (29) seems to reflect the function of a system X
AG alone. In the rat a common
transporter has been described for
L-glutamate and
D-aspartate with
K1/2 values
between 1 and 2 mM for uptake by BBMV used with concentrations not
exceeding 5 mM (6). However, using the rat intestinal cryptlike cell
line IEC-17, L-glutamate
transport systems have been described with characteristics
corresponding to systems X
AG and
ASC (18). Whether these two transporters are both present in the
luminal membrane of the mature enterocyte in its proper location is not
known. In the isolated chicken enterocyte a high-affinity system
(K1/2 = 16 µM)
and a low-affinity system
(K1/2 = 4.1 mM)
have been described (38, 39). However, except for the
K1/2 values, the
two systems have identical characteristics; in particular they both
accept D-aspartate. Rather than
being an equivalent of system ASC, the lower-affinity system might as well be a system X
AG located on
the basolateral part of the enterocyte membrane.
Perspectives
The presentation of this study may give the impression of too much
confidence in the case of intact epithelia as opposed to the use of
BBMV. It clearly remains, however, for further studies to determine
whether cis-stimulation reflects a
methodological artifact specific for the BBMV preparation and whether
the present technique overestimates the capacity of the high-affinity
system X
AG relative to the
low-affinity system ASC. However, it is evident that a proper
understanding of the function of the intestinal BBM rests on the use of
several independent methods. It has previously been demonstrated that
intestinal transport functions expressed in Xenopus
laevis oocytes do not all necessarily belong in the BBM
of the enterocyte (22). In the present study, it is demonstrated that
even when intestinal transport functions do belong there, they must be
classified with a glance to older observations on intact epithelia.
The possibility that the present estimate of the relative capacity of
system X
AG is correct has
functional implications. Although a transport of anionic amino acids
with a capacity of ~0.1
µmol · cm
2 · h
1
seems of limited use to an animal, a 10 times greater capacity with a
relatively high affinity could be essential. This is especially important since other amino acids do not compete for use of system X
AG, while all bipolar amino acids
efficiently compete for transport by system ASC.
 |
ACKNOWLEDGEMENTS |
This study was supported by the Novo Nordic foundation.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: B. G. Munck, Dept. of Medical Physiology,
The Panum Institute, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark.
Received 28 April 1998; accepted in final form 6 October 1998.
 |
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