Amino acid depletion activates TonEBP and sodium-coupled
inositol transport
Renata
Franchi-Gazzola1,
Rossana
Visigalli1,
Valeria
Dall'Asta1,
Roberto
Sala1,
Seung Kyoon
Woo2,
H. Moo
Kwon2,
Gian C.
Gazzola1, and
Ovidio
Bussolati1
1 Dipartimento di Medicina Sperimentale, Sezione di
Patologia Generale e Clinica, Università degli Studi di Parma,
43100 Parma, Italy; and 2 Division of Nephrology, School of
Medicine, The Johns Hopkins University, Baltimore, Maryland 21205
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ABSTRACT |
The expression of the osmosensitive
sodium/myo-inositol cotransporter (SMIT) is regulated by
multiple tonicity-responsive enhancers (TonEs) in the 5'-flanking
region of the gene. In response to hypertonicity, the nuclear abundance
of the transcription factor TonE-binding protein (TonEBP) is increased,
and the transcription of the SMIT gene is induced. Transport system A
for neutral amino acids, another osmosensitive mechanism, is
progressively stimulated if amino acid substrates are not present in
the extracellular compartment. Under this condition, as in
hypertonicity, cells shrink and mitogen-activated protein kinases are
activated. We demonstrate here that a clear-cut nuclear redistribution
of TonEBP, followed by SMIT expression increase and inositol transport
activation, is observed after incubation of cultured human fibroblasts
in Earle's balanced salts (EBSS), an isotonic, amino acid-free saline. EBSS-induced SMIT stimulation is prevented by substrates of system A,
although these compounds do not compete with inositol for transport through SMIT. We conclude that the incubation in isotonic, amino acid-free saline triggers an osmotic stimulus and elicits
TonEBP-dependent responses like hypertonic treatment.
hypertonic stress; system A; glutamine; stress proteins; cell
volume; tonicity-responsive enhancer-binding protein
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INTRODUCTION |
LONG-TERM ADAPTATION OF
MAMMALIAN CELLS to hypertonic stress involves the accumulation of
nonperturbing, osmoprotective solutes in the intracellular compartment
(30). Increased transport of such osmolytes through
secondary active, sodium-dependent mechanisms constitutes a device
employed to this aim in several cell models. The expression of one of
these mechanisms, the sodium/myo-inositol cotransporter
(SMIT) (Ref. 28 and Dall'Asta, unpublished observations), coded by the gene SLC5A3 (2, 31), is induced by
extracellular hypertonicity (29, 49). Several studies,
mostly performed in renal cells (29, 49, 51), led not only
to the cloning of the transporter but also to a detailed molecular
characterization of its sensitivity to osmotic stress. In particular,
it has been shown that SLC5A3/SMIT expression is controlled by multiple
elements located at the 5'-flanking region of the gene
(38), regulated by binding with an osmosensitive
transcription factor named TonEBP, tonicity-responsive enhancer-binding
protein (33). Upon hypertonic stress, TonEBP translocates
to the nucleus and activates SLC5A3 expression, thus promoting a slow
increase of inositol transport (33, 48). The
osmosensitivity of myo-inositol transport is not restricted
to renal models because it has also been described in endothelial
(46), lens (52), and glial cells (35,
41).
Another osmosensitive mechanism is the sodium-dependent transport
system A (7), a secondary active mechanism strictly
coupled to the transmembrane gradient of sodium electrochemical
potential (8, 18). This transport system mediates the
uptake of neutral amino acids with short polar side chains and
tolerates N-alkylation of the substrates. Because of this
unique feature,
-(methylamino)isobutyric acid (MeAIB), a
nonmetabolizable amino acid analog, is the prototypical substrate of
the system (7). The osmosensitivity of the system has been
investigated in a variety of cell models, such as chicken fibroblasts
(44), rat thymocytes (27), bovine kidney
NBL-1 cells (40), mesangial cells (50),
vascular smooth muscle cells (6), and Madin-Darby canine
kidney (MDCK) cells (5, 22). In cultured human fibroblasts
(12) and endothelial cells (9), the
stimulation of system A activity is required for volume recovery after
hypertonic stress. The molecular mechanisms involved in osmotic
sensitivity of system A, as well as the possible role of TonEBP
therein, have not yet been investigated because cloning of system A
transporter has been obtained only very recently (42). The
activity of system A markedly increases also upon a prolonged incubation in an amino acid-free, isotonic saline solution. This regulatory mechanism, named adaptive increase, was originally described
in mesenchymal cells of avian origin (19), but it has also
been found in many other models (see Ref. 32 for review). The possible relationships between adaptive regulation and hypertonic enhancement of system A activity have been widely debated (10, 32). Recently, we found that incubation in isotonic, amino
acid-free saline, the experimental condition that triggers adaptive
upregulation of system A, is followed by a significant cell shrinkage
and a persistent activation of mitogen-activated protein kinase
pathways in cultured human fibroblasts (14). Both of these
changes also follow hypertonic stress in either the same cell model
(14) or in other cell types (23, 37, 43). On
the basis of those findings, we have proposed that incubation in the
absence of amino acids, although carried on under nominally isotonic
conditions, triggers osmocompensatory mechanisms similar to those
elicited by hypertonic treatment (14).
To validate this hypothesis, as well as to elucidate further details of
the mechanisms involved in the regulation of osmolyte transport, the
TonEBP-dependent signaling pathway has now been investigated in cells
incubated in an amino acid-free, isotonic saline solution, employing
inositol transport as an osmosensitivity reporter function. We
demonstrate here that incubation in isotonic saline triggers the
nuclear translocation of TonEBP, the induction of SLC5A3, and,
consequently, a marked increase in inositol transport.
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MATERIALS AND METHODS |
Cell culture and experimental treatment.
Human foreskin fibroblasts were obtained from a healthy 15-yr-old
donor. Cells were routinely grown in 10-cm diameter dishes in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum (FBS). The conditions of culture were as follows: pH
7.4, atmosphere 5% CO2 in air, temperature 37°C.
Cultures were passed weekly and used at confluence. The experiments
were made on fibroblast subcultures resulting from 1 × 105 cells seeded onto 2-cm2 wells of disposable
24-well trays (Nunc) and incubated for 3-4 days in 1 ml of growth medium.
Hypertonic DMEM (398 ± 7 mosmol/kgH2O) was obtained
by adding 100 mM sucrose to complete DMEM (310 ± 12 mosmol/kgH2O), supplemented with 10% FBS. The osmolality
of the solution was checked with a vapor pressure osmometer (Wescor 5500).
For incubation in saline solution, cell monolayers were washed twice in
Earle's balanced salt solution (EBSS) containing (in mM) 123 NaCl, 26 NaHCO3, 5 KCl, 1.8 CaCl2, 1 NaH2PO4, and 0.8 MgSO4. Cells were
then incubated in the same solution, supplemented with 10% dialyzed
FBS for the indicated periods of time. The osmolality of the solution
was 280 ± 13 mosmol/kgH2O (n = 6).
The employment of dialyzed serum was required to establish conditions
of complete amino acid starvation (18).
Transport measurements.
The transport of myo-inositol was evaluated according to a
previously described method for the estimation of solute fluxes into
adherent cells (16) with proper modifications. When not stated otherwise, inositol was employed at 40 µM. After the
experimental treatment, cell monolayers were rapidly washed twice with
EBSS and incubated for indicated periods of time at 37°C in 0.2 ml of
the same solution containing labeled myo-inositol. The
sodium-independent transport was determined in a sodium-free
saline solution in which choline replaced sodium in the EBSS. Transport
assay was terminated by rapidly rinsing the cell monolayer twice with 3 ml of ice-cold 300 mM urea, and cells were extracted in situ by the
addition of 0.2 ml of ethanol. Extracts were added to 0.6 ml of
scintillation fluid and counted for radioactivity in a Wallac Microbeta
Trilux counter. Cell monolayers were then dissolved with 0.5% sodium deoxycholate in 1 N NaOH, and protein content was determined using a
modified Lowry procedure (16). The influx of
myo-inositol was expressed as nanomoles of polyol per
milligram of protein per minute. The kinetic parameters were evaluated
by nonlinear regression analysis of transport data using the following
equations
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(1)
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for a single saturable system and
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(2)
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for a saturable system plus diffusion, where v is the
initial velocity of inositol uptake, Vmax is the
maximal velocity, Km is the Michaelis-Menten
constant, and Kd is the diffusion constant.
Cell volume.
Cell volume, expressed as microliters per milligrams of protein, was
estimated from the distribution space of urea according to a method
previously employed in cultured human fibroblasts (12).
[14C]urea (2 µCi/ml, 0.5 mM final concentration) was
added during the last 10 min of incubations. The experiment was stopped
with two rapid washes in 3 ml of ice-cold 300 mM unlabeled urea in water. Alcohol-soluble pools were extracted with absolute ethanol and
added to scintillation fluid to be counted for radioactivity. Protein
content was determined as described above. Under the experimental conditions adopted, the cell content of urea reached a steady-state level by 5 min of incubation (not shown). A highly significant linear
relationship exists between urea distribution space and the
extracellular osmolality that extrapolates to the origin
(Fig. 1).

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Fig. 1.
Measurement of cell volume with urea distribution space.
Cultured human fibroblasts were incubated for 10 min at 37°C in
isotonic DMEM supplemented with 0, 60, 120, or 200 mM sucrose. Urea
distribution space was determined as described in MATERIALS AND
METHODS. Line represents the best fit linear regression
(y = 7.066 × 0.093; P < 0.01).
Values are means of 4 determinations with SD shown when greater than
point size.
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Intracellular ion contents and concentrations.
Intracellular ion contents and concentrations were determined as
described previously (12) with slight modifications.
Briefly, cell monolayers were fixed with ethanol (0.1 ml) that was
allowed to dry. The water-soluble pool was extracted in 2 ml of 10 mM CsCl. Potassium and sodium cell contents were determined with a Varian
AA-275 atomic absorption spectrophotometer, using KCl and NaCl in 10 mM
CsCl as standards. Values of the intracellular concentrations of ions
were calculated from values of ion contents, and cell volumes were
determined in parallel cultures in the same experiment.
Immunocytochemistry.
Cells were seeded in four-well Labtech chamber slides (Nunc) at a
concentration of 3 × 104 cells/well in DMEM
supplemented with 10% FBS. After the experimental treatment, the
slides were rapidly washed three times in PBS, fixed in 3.7%
paraformaldehyde for 15 min at room temperature, and then rinsed twice
with PBS containing 0.1% glycine. Fixed cells were permeabilized with
a 1-min incubation in methanol at
20°C and immediately treated with
acetone for 1 min at
20°C. After two washings in PBS, the slides
were incubated in a blocking solution (PBS containing 3% bovine serum
albumin) for 1 h at 37°C. The incubation with 1:50 polyclonal
TonEBP antiserum (33) was performed for 1 h at 37°C
in the same blocking solution diluted 1:2 in PBS. Cells were then
washed twice with PBS containing 0.1% Tween 20 (PBST) and incubated at
37°C for 30 min in the blocking solution with biotinylated goat
anti-rabbit Ig diluted 1:300 as secondary antibody. After two washings
in PBST, the slides were incubated as above in 1:100 diluted
fluorescein-conjugated streptavidin. Finally, cells were washed four
times with PBST and two times with water.
The slides were observed with a confocal laser scanning microscope
(Multiprobe 2001; Molecular Dynamics) equipped with an argon laser and
based on a Nikon inverted microscope. Images were converted in TIFF
files, digitally composed, and directly printed on photographic paper.
Signal intensity was measured on a pseudocolor scale in which black and
dark blue zones represent no- or low-signal areas, whereas increasingly
lighter blue, yellow, red, and white zones are areas with progressively
higher signals.
Preparation of nuclear extracts.
Nuclear extracts were prepared using a modification of the method of
Han and Brasier (20). Cells, grown in 175-cm2
flasks, were washed twice with ice-cold PBS, scraped in the same solution, and collected by low-speed centrifugation. The pellet was
suspended in buffer A [50 mM Tris · HCl (pH 7.4),
10 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5% Igepal CA-630
(Sigma), and protease inhibitors]. After 30 min on ice, the lysates
were centrifuged at 4,000 g for 5 min at 4°C, the
supernatant constituting the cytoplasmic extract. For the purification
of nuclei, nuclear pellets were resuspended in buffer B (50 mM Tris · Hcl, pH 7.4, 10 mM KCl, 1 mM EDTA, 1.7 M sucrose, 1 mM DTT, and protease inhibitors) and centrifuged at 15,000 g
for 30 min at 4°C. Pelleted nuclei, resuspended in buffer
C (50 mM Tris · HCl, pH 7.4, 400 mM KCl, 1 mM EDTA, 10%
glycerol, 1 mM DTT, and protease inhibitors), were kept on ice for 30 min at 4°C with frequent vortexing. The nuclear suspension was
centrifuged at 15,000 g for 5 min at 4°C, and the supernatant was saved. Both cytoplasmic and nuclear extracts were normalized for protein amounts determined by the Bradford assay using
bovine serum albumin as a standard (Bio-Rad).
Electrophoresis and Western blotting.
Protein samples were suspended in SDS-PAGE sample buffer, separated on
a 6% SDS-polyacrylamide gel, and transferred onto a polyvinylidene
difluoride membrane. The membrane was blocked in Tris-buffered saline
containing 1% casein, 0.33% gelatin, and 1% bovine serum albumin for
2 h at 30°C. TonEBP antiserum (33), diluted 1:100,
was added for 1 h at 30°C. After four washes, the membrane was
incubated with horseradish peroxidase-coupled anti-rabbit IgG antibody
(Bio-Rad), washed extensively, and developed using a colorimetric kit
(Bio-Rad).
Northern blotting.
Confluent fibroblasts were exposed to hypertonic medium containing 10%
FBS or to isotonic EBSS supplemented with 10% dialyzed FBS for the
indicated periods. Total RNA was extracted with TRIzol (Life
Technologies, Italy). For Northern analysis, RNA (10-20 µg/lane)
was separated under denaturating conditions on 1% agarose gel
containing 2.2 M formaldehyde. After being stained with ethidium bromide to document equal sample loading and absence of degradation, total RNA was transferred overnight to a positively charged nylon membrane (Nytran Super Charge; Schleicher and Schüll) and then linked to the membrane at 80°C. Hybridization of the blot was carried
out at 42°C overnight with a canine SMIT cDNA probe (28) that shares a homology of 94% with human SMIT (2). After
being stripped, the membrane was rehybridized with a human
glyceraldehyde-3-phosphate dehydrogenase probe (gift from Dr. R. Allen,
American Red Cross Laboratories). The probes were labeled with
[
-32P]dCTP. The blots were washed according to the
manufacturer's instructions, with the last wash performed at 60°C
for 30 min, and then exposed to Kodak XAR film at
70°C for the
appropriate time. Densitometric analysis of the autoradiograms was
performed with a Molecular Dynamics laser densitometer.
Statistical analysis.
Whenever appropriate, statistical analysis was performed with analysis
of variance, unless otherwise stated.
Materials.
FBS and DMEM were purchased from Life Technologies,
[14C]urea (53 mCi/mmol),
[2-3H]myo-inositol (17 Ci/mmol), and
[
-32P]dCTP (3,000 Ci/mmol) were from Amersham.
Biotinylated goat anti-rabbit Ig and fluorescein-conjugated
streptavidin were obtained from Dako. Ethanol was obtained from Carlo
Erba (Milan, Italy). The source of all other chemicals was Sigma.
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RESULTS |
TonEBP redistribution into the nucleus upon incubation of cultured
human fibroblasts in isotonic saline solution.
The incubation of cultured human fibroblasts in isotonic saline
solution caused a marked and progressive cell shrinkage (Fig. 2A). The decrease in cell
volume was already detectable after 15 min of incubation in EBSS. After
3 h of treatment, cell volume was decreased by 30% with respect
to control; this value was maintained thereafter, with no apparent
regulatory volume increase, for at least 12 h. Throughout this
period, the intracellular concentrations of cations underwent
significant alterations (Fig. 2B). However, while
intracellular sodium rapidly returned to control values after a
transient increase, the intracellular concentration of potassium rose
steadily for the first 3 h of treatment, closely paralleling cell
shrinkage. Thereafter, the intracellular potassium concentration
was maintained at values higher than control by roughly 40%. These
data indicate that the incubation of cultured human fibroblasts under
amino acid-free conditions causes a significant cell shrinkage with an
increase in cell ion strength.

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Fig. 2.
Changes in cell volume and intracellular concentrations
of sodium and potassium during the incubation of cultured human
fibroblasts in Earle's balanced salt solution (EBSS). A:
cell volume was determined at the indicated times of incubation in
EBSS + 10% fetal bovine serum (FBS) as described in
MATERIALS AND METHODS. Data are means of 6 independent
determinations with SD. B: the intracellular concentrations
of sodium (open bars) and potassium (filled bars) were calculated from
the ion contents determined as described in MATERIALS AND
METHODS at the indicated times of incubation in EBSS + 10%
FBS in the same experiment shown in A. Data are means of 6 independent determinations with SD. The experiment was repeated 3 times
with comparable results. *P < 0.05 vs. DMEM;
**P < 0.01 vs. DMEM.
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In other cell models, cell shrinkage and the concurrent increase in
cell ion strength, caused by a hypertonic treatment, are associated
with an increase in TonEBP abundance in the nucleus (33, 47,
48). We compared TonEBP behavior in fibroblasts incubated under
either hypertonic conditions or in amino acid-free solutions (Fig.
3). The immunocytochemical analysis
performed with confocal microscopy indicates that TonEBP positivity was faint and widespread in control cells (Fig. 3A). After 90 min of incubation either under hypertonic conditions (Fig.
3B) or in EBSS (Fig. 3C), a clear-cut increase in
nuclear signal was detected, pointing to a nuclear localization of
TonEBP in treated cells. After 3 and 6 h of incubation under
either condition, an increased nuclear abundance of TonEBP was still
detectable (not shown). Western blot analysis (Fig. 3D)
confirms that a nuclear relocalization of TonEBP was detectable in both
hypertonic medium and EBSS, starting from 4 h of incubation. Under
either condition, an increase of TonEBP in the nuclear extract was
detected together with a concurrent decrease in the cytoplasmic signal.
These results demonstrate that both hypertonic treatment and amino
acid-free incubation cause the nuclear redistribution of TonEBP.

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Fig. 3.
Changes in tonicity-responsive
enhancer-binding protein (TonEBP) distribution upon incubation of human
fibroblasts in EBSS. Confocal microscopy of the distribution of TonEBP
in cells maintained in DMEM + 10% FBS (A) or incubated
for 90 min in hypertonic DMEM + 10% FBS (B) or in
isotonic EBSS + 10% dialyzed FBS (C). After the
experimental treatment, fibroblasts grown on 4-well chamber slides were
fixed and stained with the TonEBP antiserum. The experiment was
repeated 4 times with comparable results. Bar = 20 µm.
D: Western blot analysis of TonEBP in nuclear and
cytoplasmic extracts prepared from cells cultured in DMEM (control, C)
incubated for 4 h in hypertonic DMEM (H) or in isotonic EBSS (E).
The experiment was repeated twice with comparable
results.
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Characterization of inositol transport in cultured human
fibroblasts.
Because SMIT carrier is a target of TonEBP (33), the
evaluation of inositol transport represents a convenient device to ascertain the functional consequences of TonEBP activation induced by
the incubation in EBSS. Only limited attention has been thus far
devoted to the transport of the polyol in human fibroblasts (3,
15, 34). Moreover, the contradictory kinetic data reported in
those studies may reflect the sensitivity of the transport process to
experimental conditions such as the type and concentration of serum
employed for cell culturing (1). In a series of
experiments, recounted in Fig.
4,
we have, therefore, performed a characterization of inositol transport
in cultured human fibroblasts to define the operational features of the
transporter(s) involved in the experimental conditions employed in this
study.

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Fig. 4.
Influx of myo-inositol in cultured human
fibroblasts. A: cultured human fibroblasts, grown onto
24-well culture multidish in DMEM, were washed with EBSS. The
intracellular accumulation of 100 µM
[3H]myo-inositol was then measured in EBSS
([Na+]out = 142 mM; ,
sodium present) or in a modified sodium-free EBSS ( ,
sodium absent). B: kinetic analysis of 10-min
myo-inositol uptake in a range of polyol concentrations from
0.01 to 1.28 mM ( , sodium-dependent uptake;
, sodium-independent uptake). The sodium-dependent
uptake was obtained by subtracting the uptake measured in the absence
of sodium from the total uptake of the polyol. Lines represent the
linear regression ( ) or the best fit nonlinear
regression calculated from Eq. 1 ( ).
v, Initial velocity of inositol uptake. C:
Eadie-Hofstee plot of the sodium-dependent component shown in
B. Line represents the best fit linear regression.
D: effects of chronic treatment (24 h) with 1 µM phorbol
12,13-dibutyrate (PDBu) or acute treatment (1 h) with 400 µM
8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) on 10-min
myo-inositol uptake. Experimental treatments were performed
in complete growth medium, whereas transport assay was performed in
EBSS. In all cases, data are means of 3 independent determinations with
SD shown when greater than the size of the point. *P < 0.05 vs. control; **P < 0.01 vs. control.
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Figure 4A reports the time course of myo-inositol
accumulation either in the presence or in the absence of sodium. The
polyol influx was linear up to 60 min under both conditions. It was
concentrative only in the presence of sodium, whereas it was
exceedingly low in the absence of the cation. The kinetic analysis of
inositol transport was performed in a wide range of concentrations from 0.01 to 1.28 mM (Fig. 4B). Sodium-independent influx was not
saturable in the range of concentrations adopted and was thus formally
undistinguishable from diffusion. Once subtracted, the
sodium-independent component, sodium-dependent inositol, transport
was satisfactorily fitted by an equation (Eq. 1, see
MATERIALS AND METHODS) that describes the influx as the
operation of a single saturable mechanism endowed with a moderately
high affinity (Km = 50 µM), a conclusion
shown through Eadie-Hofstee transformation (Fig. 4C). The
value of Km is comparable to values found by
other investigators for inositol transport in human fibroblasts
(3) and in the basolateral membrane of MDCK cells
(28). Consistent with the results obtained in MDCK
cells (36) and at variance with those obtained in retinal pigment epithelial cells (26), inositol transport was
markedly stimulated by protein kinase C downregulation, obtained with a prolonged incubation in the presence of phorbol esters, and
significantly decreased by acute protein kinase A activation (Fig.
4D). These results indicate that the transporter
expressed in cultured human fibroblasts has kinetic, operational, and
regulatory features similar to those described for the SMIT transporter
of MDCK cells.
Stimulation of inositol transport by amino acid-free incubation.
In the experiment reported in Fig. 5,
inositol transport was measured during a prolonged incubation of human
fibroblasts in EBSS. The experimental treatment led to a marked,
slowly ensuing stimulation of the polyol uptake. The time course of the
effect, shown in Fig. 5A, indicates that the transport
stimulation became detectable after 6 h of incubation in EBSS. The
incubation in hypertonic DMEM also stimulated inositol uptake in human
fibroblasts. The time course of the effect was comparable in the two
conditions for up to 18 h of treatment, although hypertonic
treatment caused a larger stimulation of inositol transport. In cells
incubated in EBSS, inositol influx rose further, up to 24 h, while
no further increase in polyol uptake was detected under hypertonic
conditions after 18 h of treatment. In both cases, however,
cycloheximide (18 µM) completely suppressed transport stimulation
(not shown). Figure 5, B and C, shows the results
of the kinetic analysis of inositol transport performed after 15 h
of either hypertonic or EBSS incubation. The kinetic parameters,
reported in Table 1, indicate
that both treatments enhanced inositol transport through a marked
increase of transport of Vmax, while the
Km was not significantly affected under either
condition. At this time, the Vmax change induced
by hypertonic treatment was greater than that caused by amino acid
deprivation. Northern analysis, performed with a SMIT full-length
probe, consistently indicated that a markedly enhanced abundance of
SMIT mRNA was detectable in both hypertonically, and, although to a
lesser degree, EBSS-treated fibroblasts (Fig. 5D).

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Fig. 5.
Changes of inositol transport and
sodium/myo-inositol cotransporter (SMIT) expression induced
by hypertonic treatment or incubation in EBSS. A: cultured
human fibroblasts were transferred from DMEM ( ) into
EBSS ( ) or hypertonic DMEM ( ). At the
indicated times, cells were washed twice in EBSS, and 10-min inositol
uptake was assayed as described in MATERIALS AND METHODS.
B: kinetic analysis of 10-min myo-inositol uptake
measured in a range of concentrations from 0.01 to 1.28 mM in cells
maintained in DMEM ( ), incubated for 15 h in
hypertonic DMEM ( ), or incubated for 15 h in EBSS
( ). Data are means of 3 independent determinations with
SD shown when greater than the size of the point. Lines represent the
best fit of data to Eq. 2 (see MATERIALS AND
METHODS). C: Eadie-Hofstee transformation of the data
presented in B after subtraction of the sodium-independent
component. Lines represent best fit linear regressions. D:
analysis of SMIT mRNA levels by Northern blot. Total RNA (15 µg) was
isolated from human fibroblasts maintained in DMEM (control, C) or
exposed for 9 h to hypertonic DMEM (H) or to isotonic EBSS (E).
The membrane was sequentially hybridized with SMIT or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes. The increase
of expression of SMIT, normalized for GAPDH signal, was +980% in
hypertonically treated cells and +250% in cells incubated in EBSS. The
experiment was repeated 3 times with comparable results.
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These results indicate that the nuclear translocation of TonEBP,
promoted by incubation in isotonic EBSS, is followed by the induction
of SMIT and by the consequent increase of inositol uptake.
Characteristics of EBSS-induced stimulation of inositol transport.
Upregulation of membrane transport caused by substrate deprivation is a
very well-known phenomenon, described for several kinds of solutes,
such as neutral amino acids (17, 19), and, in lower
organisms, inositol itself (39). These regulatory
mechanisms are usually called "adaptive changes." Because inositol
is present in DMEM formula, the possibility exists that the stimulation
of polyol transport observed after a prolonged incubation in EBSS is
referable to an adaptive response. Figure
6A shows the results of an
experiment in which the incubation of cultured human fibroblasts in
EBSS was performed in the presence of different concentrations of
inositol. Although transport increase was only partially blocked by 100 µM inositol, i.e., at the concentration of the polyol present in
FBS-supplemented DMEM, an almost complete suppression was reached at a
very high concentration of inositol (5 mM). Because, at this huge,
supraphysiological concentration, inositol could have little nutritional role, but, rather, an osmoprotective effect, we
hypothesized that other organic osmolytes could interfere with
EBSS-induced SMIT expression.

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Fig. 6.
Modulation of EBSS-induced increase in inositol transport
by extracellular organic osmolytes. A: the uptake of
myo-inositol (10 min) was measured in human fibroblasts
incubated for 15 h in DMEM (open bar), in EBSS (solid bar), or in
EBSS supplemented with the indicated concentrations of the polyol
(hatched bars). B: inositol uptake was measured after a 15-h
incubation in DMEM (open bar), in EBSS (solid bar), or in EBSS
supplemented with the indicated amino acids employed at 0.5 mM (hatched
bars). In all cases, data are means of 3 independent determinations
with SD. **P < 0.01 vs. values of inositol transport
obtained in cells incubated in EBSS. MeAIB, -(methylamino)isobutyric
acid; Pro, proline; Gln, glutamine; Leu, leucine; Val, valine; Glu,
glutamic acid; Arg, arginine.
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To test this hypothesis, we performed an experiment in which EBSS
incubation was carried out in a saline solution supplemented with
single amino acids employed at a concentration of 0.5 mM (Fig.
6B). Amino acids that are not transported through system A
in cultured human fibroblasts, such as L-glutamate
(11) and L-arginine (45), did not
affect EBSS-induced stimulation of inositol transport significantly.
L-Leucine and L-valine, relatively poor
substrates of system A (18), produced a significant
repression of the induction of inositol transport. However, cells
incubated in EBSS supplemented with these amino acids still exhibited
inositol transport values significantly higher (P < 0.05) than control cells maintained in DMEM. On the contrary, other
amino acids, such as L-proline and L-glutamine,
which are good substrates of system A, and, hence, actively accumulated
in the intracellular compartment (13, 18), completely
suppressed the stimulation of inositol transport. Interestingly, the
nonmetabolizable amino acid analog MeAIB, the prototypical substrate of
system A, also abolished SMIT induction.
However, the amino acids that repress SMIT induction do not interact
with the transporter as substrates. Indeed, inositol uptake was not
inhibited by MeAIB, proline, glutamine, valine, or leucine if these
compounds were present only during the uptake of the polyol (Fig.
7). Therefore, the capability of specific amino acids to suppress SMIT induction cannot be referred to a direct
competition between these compounds and myo-inositol for the
transporter.

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Fig. 7.
Lack of inhibition of inositol transport by neutral amino
acids. Ten-minute uptake of myo-inositol was measured under
control conditions (no preincubation in EBSS) in the absence (control,
empty bar) or in the presence (hatched bars) of the
indicated compounds employed at 2 mM. Data are means of 3 independent
determinations with SD.
|
|
Effect of inositol on cell volume recovery.
In cells shrunken through an amino acid-free incubation, neutral
amino acids, which are substrates of system A, exert a rapid osmocompensatory effect leading to a rapid recovery of cell volume (14). The effect of inositol supplementation on the
recovery of cell volume was tested under the same conditions (Fig.
8). The addition of 5 mM
myo-inositol to EBSS after a 12-h incubation in the absence
of organic osmolytes led to a gradual recovery of cell volume that
required more than 12 h. Cell volume restoration paralleled the
accumulation of the polyol. After 21 h of inositol supplementation, both cell volume and cell content of the polyol reached a steady state.

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Fig. 8.
Osmoprotective effect of inositol in human fibroblasts
incubated in EBSS. Cultured human fibroblasts, grown in complete DMEM
( ), were transferred to EBSS ( ). After
12 h, the incubation was prolonged for a further 26 h in the
same solution or in EBSS supplemented with 5 mM myo-inositol
( ). Cell volume was measured at the indicated times. In
parallel cultures, EBSS was supplemented with 5 mM
[3H]myo-inositol, and the intracellular levels
of the polyol measured at the indicated times of incubation are shown
( ). Data are means of 4 independent determinations with
SD shown when greater than the size of the point.
|
|
 |
DISCUSSION |
The volume-restoring accumulation of most compatible osmolytes is
due to the stimulation of several sodium-coupled transporters, such as
the sodium-chloride-betaine cotransporter BGT1 (21) and
the SMIT transporter (51). The regulatory mechanism occurs at the transcription level and involves (a) tonicity-responsive element(s) that present(s) a binding site for an osmosensitive transcription factor, TonEBP. The activity of this Rel-like DNA binding
protein is significantly stimulated through both an increase in its
abundance and a nuclear redistribution when cells are incubated under
hypertonic conditions (33). Here we show that the
incubation of human fibroblasts in isotonic EBSS (280 ± 13 mosmol/kgH2O), an amino acid-free saline solution, produces
an osmotic stimulus, triggering TonEBP redistribution and activation.
When these cells are transferred into the amino acid-free saline, they
progressively shrink as a consequence of the depletion of the
intracellular amino acid pool. Under normal conditions, the overall
concentration of intracellular amino acids reaches a value of 140 mM
that is nearly reduced by half after 6 h of incubation under amino
acid-free conditions (14). In amino acid-starved cells,
the osmotic equilibrium relies only on the inorganic ions, thus leading
to a significant increase of intracellular ionic strength, as
demonstrated by the 40% increase of intracellular potassium
concentration after 3 h of EBSS incubation (Fig. 2A).
Therefore, the availability of extracellular amino acids is required
not only for the recovery of hypertonically stressed cells
(9, 12, 14) but also for the maintenance of cell volume
and the control of cell ionic strength under isotonic conditions.
We have also demonstrated that the TonEBP redistribution induced by
isotonic amino acid-free incubation is functionally effective. For this
purpose, we have employed SMIT transporter as a TonEBP-dependent reporter gene, although no data were available on the osmosensitivity of inositol transport in cultured human fibroblasts before those reported here. However, in cultured human fibroblasts, SMIT is induced
not only after hypertonic stress, as expected from the results obtained
in other models (29, 35, 41, 46, 49, 51, 52) but also
after an isotonic, amino acid-free incubation. As a result of SMIT
induction, the transport of inositol in EBSS-incubated human
fibroblasts undergoes a marked increase. The osmoprotective role of the
transport stimulation appears evident from the results reported in Fig.
8. Inositol works as an effective osmoprotective agent, provided that
it is added to the extracellular medium at a high, supraphysiological
concentration for at least 21 h. It should be recalled that in
cells shrunken through an incubation in EBSS, volume is rapidly
restored to control values when amino acid substrates of transport
system A are added to the extracellular medium (14).
However, the transport capacity of transport system A, measured in
human fibroblasts after a 6-h incubation in EBSS (>100
nmol · mg protein
1 · min
1),
is impressively higher than that exhibited by SMIT transporter after a
15-h incubation in the same saline solution (369 pmol · mg
protein
1 · min
1, this report). As
expected, system A substrates induce a faster volume recovery than inositol.
The incubation in isotonic, osmolyte-free saline stimulates both SMIT
and transport system A for neutral amino acids. The activation of
TonEBP, detected under the same condition, suggests that hypertonic
stress is fully mimicked and strengthens the hypothesis that
the adaptive regulation of system A (i.e., the increase in the activity
of the system observed after amino acid starvation) (19)
is actually a consequence of the well-known osmotic sensitivity of this
transport system. These considerations would also suggest that TonEBP
may be involved in the upregulation of transport system A, an issue
that should now be directly addressed, given that the system has been
recently cloned (42). However, it should be recalled that
amino acid deprivation can activate several signaling pathways, some of
which appear to be specific for single amino acids (see Ref.
25 for review).
Although the substrates of transport system A exert no inhibition of
inositol transport (Fig. 7), they effectively suppress the EBSS-induced
stimulation of SMIT if added to the saline solution (Fig.
6B). Under this condition, cell shrinkage and the
upregulation of system A transport activity are also prevented
(14). Amino acids that are not transported by system A,
such as glutamic acid or arginine (11, 45), do not
interfere with the stimulation of inositol transport caused by
incubation in EBSS. These data suggest that only amino acids that can
be effectively accumulated into the intracellular compartment through
system A can block SMIT induction. This result points, therefore, to a
regulatory interaction of SMIT and system A transporters and suggests
that both transport systems respond to a common stimulus, i.e., cell shrinkage and/or the increase in intracellular ionic strength. The
transport activity of system A (14) exhibits markedly
faster changes than inositol uptake (this contribution) after either hypertonic stress or amino acid-free incubation. Consistently, the
hypertonic activation of betaine (5) and
myo-inositol carriers (21) is slower than the
stimulation of system A in MDCK cells. System A stimulation may
thus be the most rapid transport mechanism employed to counteract the
increase in intracellular ionic strength, immediately following the
transient protection yielded by the induction of heat shock proteins.
Interestingly, in mutant Chinese hamster ovary cells that overexpress
system A, changes of heat shock protein levels have also been detected
(24). These considerations indicate that coordinated
regulation of organic osmolytes (4) may be not restricted
to renal cells. Moreover, they suggest that an ordered sequence of
regulatory phenomena and changes counteracts the increase in
intracellular ionic strength induced by hypertonic stress or
deprivation of organic osmolytes. However, the components of this
compensatory sequence, as well as the underlying mechanisms and the
possible pathophysiological implications, are far from being fully elucidated.
 |
ACKNOWLEDGEMENTS |
This work is funded by Ministero dell'Università e della
Ricerca Scientifica e Tecnologica, Project Molecular Bases of Genetic Diseases, Rome Italy (to V. Dall'Asta); Consiglio Nazionale delle Ricerche, Target Project Biotechnology, Rome, Italy (to V. Dall'Asta); and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-42479 and DK-44484 (to H. Moo Kwon).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: O. Bussolati, Dipartimento di Medicina Sperimentale, Sezione di Patologia Generale e Clinica, Plesso Biotecnologico Integrato, Università degli Studi di Parma, Via Volturno, 39, 43100 Parma, Italy (E-mail: ovidio.bussolati{at}unipr.it).
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.
Received 23 August 2000; accepted in final form 21 December 2000.
 |
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