Amino acids are compatible osmolytes for volume recovery after
hypertonic shrinkage in vascular endothelial cells
Valeria
Dall'Asta1,
Ovidio
Bussolati1,
Roberto
Sala1,
Alessandro
Parolari2,
Francesco
Alamanni2,
Paolo
Biglioli2, and
Gian C.
Gazzola1
1 Istituto di Patologia
Generale, Università degli Studi di Parma, 43100 Parma; and
2 Dipartimento di Cardiochirurgia,
Università degli Studi di Milano, Centro Cardiologico Fondazione
Monzino Istituto di Ricovero e Cura a Carattere Scientifico, 20122 Milan, Italy
 |
ABSTRACT |
The response to chronic hypertonic stress has been studied in
human endothelial cells derived from saphenous veins. In complete growth medium the full recovery of cell volume requires several hours
and is neither associated with an increase in cell
K+ nor hindered by bumetanide but
depends on an increased intracellular pool of amino acids. The highest
increase is exhibited by neutral amino acid substrates of transport
system A, such as glutamine and proline, and by the anionic amino acid
glutamate. Transport system A is markedly stimulated on hypertonic
stress, with an increase in activity roughly proportional to the extent
and the duration of the osmotic shrinkage. Cycloheximide prevents the increase in transport activity of system A and the recovery of cell
volume. It is concluded that human endothelial cells counteract hypertonic stress through the stimulation of transport system A and the
consequent expansion of the intracellular amino acid pool.
glutamine; system A; amino acid transport; regulatory volume
increase; bumetanide
 |
INTRODUCTION |
ADAPTATION TO EXTRACELLULAR hypertonicity is a property
of most mammalian cells (18, 25). However, the mechanisms employed for
volume restoration after hypertonic shrinkage, indicated as regulatory
volume increase (RVI), are different in the various tissues. A
convenient device for counteracting osmotic stress is the increased
accumulation of compatible organic osmolytes through
Na+-dependent cotransport systems.
One of these mechanisms, system A, accumulates neutral amino acids and
methylamines in the intracellular compartment. Stimulation of system A
activity by hyperosmotic stress, known for a long time (40), is a slow,
protein synthesis-dependent process that may involve the synthesis of
new carriers or, alternatively, of regulatory proteins that would
stimulate transport activity of preexisting transporters (29). In
recent years the stimulation of system A activity has been identified
as an important component of cell responses to hypertonic stress in a
variety of cell models (5-7, 9, 15, 17, 19, 39, 43). At least in
human fibroblasts the competence for a hypertonic RVI is strictly
dependent on the availability of neutral amino acid substrates of
system A. In these cells, volume recovery relies on the expansion of the intracellular pool of amino acids and, in particular, of glutamine (9, 15).
As far as the endothelium is concerned, it is known that many types of
endothelial cells exhibit increased activities of
Na+-K+-2Cl
cotransport and
Na+/H+
antiport after hypertonic treatment (11, 30, 32). However, it is not
clear whether transport activation results in a complete volume
recovery, although even the capability of endothelial cells to exert an
effective RVI is uncertain (28, 32,
38). More recent data indicate that, also in endothelium, transport
systems for organic osmolytes are stimulated by hypertonic stress (23, 41, 42), although no attempt has been made in those studies to
demonstrate that transport stimulation is able to correct cell shrinkage effectively.
Recent results from our group have demonstrated that, under hypertonic
conditions, marked endothelial damage is detected that is significantly
reduced by glutamine supplementation of the extracellular medium (35).
Those data prompted us to evaluate the role of the transport of
glutamine and other neutral amino acids in the endothelial response to
extracellular hypertonicity. Employing an adult human model,
endothelial cells derived from saphenous veins, we demonstrate that, on
hypertonic stress, the human endothelium in vitro is able to complete
cell volume recovery through an increase of the intracellular pool of
amino acids sustained by an enhanced activity of transport system A.
 |
METHODS AND MATERIALS |
Cell Culture
Human saphenous vein endothelial cells (HSVECs) were obtained from
patients undergoing coronary artery bypass grafting, as previously
described (35). Cells were routinely grown in collagen-coated 10-cm-diameter dishes in medium 199 (M199), with glutamine
concentration raised to 2 mM. Culture medium was supplemented with 20%
fetal bovine serum (FBS), 50 µg/ml endothelial cell growth
supplement, and 90 U/ml heparin. The conditions of culture were pH 7.4, atmosphere 5% CO2 in air, and
temperature 37°C. Cultures were characterized by typical
cobblestone morphology and by positiveness to factor VIII and
CD31/PECAM-1 antigens (35). Three strains of HSVECs were isolated,
characterized, and employed for the investigations presented here with
qualitatively similar results.
Experimental Procedures
All the experiments were performed using the cluster-tray method for
rapid measurement of solute fluxes in adherent cells (13) with
appropriate modifications. The experiments were carried out on HSVEC
subcultures resulting from 3.5 × 104 cells seeded into
2-cm2 wells of disposable 24-well
trays (Nunc, Life Technology, Milan, Italy) in 1 ml of growth medium.
Cells were used after 3-5 days, when cultures were almost
confluent (12 ± 2 µg
protein/cm2). Culture medium was
always renewed 24 h before the experiment. Hypertonic media were
obtained by additions of 50 or 100 mM sucrose to complete M199. The
osmolalities of the solutions were routinely checked with a vapor
pressure osmometer (model 5500, Wescor Instruments, Logan, UT). After
supplementation with 20% FBS, values of 295 ± 7, 328 ± 15, and
372 ± 12 mosmol/kg were found for control M199 (hereafter indicated
as isotonic M199), hypertonic M199 supplemented with 50 mM sucrose
(hereafter indicated as hypertonic M199 at 330 mosmol/kg), and
hypertonic M199 supplemented with 100 mM sucrose (hereafter indicated
as hypertonic M199 at 370 mosmol/kg), respectively.
Determinations and Measurements
Cell volume.
Cell volume was estimated as the urea distribution space, according to
a method employed by our group for cultured human fibroblasts (9, 15)
and validated in cultured endothelium by O'Neill and Klein (32).
[14C]urea (1.5 µCi/ml, 0.5 mM final concentration) was added during the last 10 min
of incubation. The incubations were terminated by two rapid washings
(<5 s) with an ice-cold solution of 300 mM urea in water. Cells were
covered with 0.2 ml of ethanol, and the radioactivity in cell extracts
was counted in a Wallac Microbeta Trilux counter after the addition of
Hisafe III scintillation fluid (Wallac, Turku, Finland). Cell
monolayers were dissolved with 0.5% sodium deoxycholate in 1 N NaOH
for determination of protein content directly in the well with use of a
modified Lowry procedure, as previously described (13). Values of cell
volume are expressed as microliters per milligram of protein. The
volume of control cells was the mean of the values obtained at each
experimental time in cells maintained under isotonic conditions.
Intracellular ion contents and concentrations.
Cell monolayers, fixed in place with 0.1 ml of ethanol, were allowed to
dry. Na+ and
K+ contents in the water-soluble
pool, extracted in 2 ml of 10 mM CsCl, were determined with a Varian
atomic absorption spectrophotometer, with NaCl and KCl used as
standard, and expressed as micromoles per milligram of protein. Values
of intracellular ion concentrations were calculated from ion content
and cell volume values determined in parallel cultures under the same
experimental conditions.
Intracellular amino acid contents and concentrations.
Cell monolayers were washed twice with ice-cold
MgCl2 and extracted in a 5%
solution of acetic acid in ethanol. The intracellular content of the
single amino acid species was determined by HPLC analysis with a
Biochrom 20 amino acid analyzer (Amersham Pharmacia Biotech) employing
a high-resolution column (Bio 20 Peek Lithium) and the physiological
fluid chemical kit (Amersham Pharmacia Biotech) for elution. The column
effluent was mixed with ninhydrin reagent, passed through the
high-temperature reaction coil, and read by the photometer unit. Cell
contents of the single amino acid species are expressed as nanomoles
per milligram of protein. The intracellular concentration of amino
acids was calculated from the amino acid contents and cell volumes
determined in parallel cultures under the same experimental conditions.
Amino acid transport activity.
The activity of amino acid transport systems was evaluated by measuring
the influx of preferential substrates under conditions described.
Unless stated otherwise, the assay was performed in Earle's balanced
salt solution containing (in mM) 123 NaCl, 26 NaHCO3, 5 KCl, 1.8 CaCl2, 1 NaH2PO4,
0.8 MgSO4, and 5.5 glucose. After
the transport assay the incubation was terminated by two rapid rinses
with ice-cold urea (300 mM), monolayers were extracted in 0.2 ml of
ethanol, and radioactivity was counted in a Wallac Microbeta Trilux
counter. Amino acid uptake is expressed as nanomoles or picomoles per
milligram of protein per minute.
Materials
FBS was purchased from Euroclone (Milan, Italy). Culture media (M199
and glutamine-free M199) were purchased from Life Technology. L-[2,3,4-3H]arginine
monohydrochloride (45-70 Ci/mmol),
2-[1-14C]methylaminoisobutyric
acid (56 mCi/mmol),
L-[1-14C]glutamic
acid (52 mCi/mmol), and
L-[U-14C]glutamine (249 mCi/mmol) were obtained from NEN Life Science (Boston, MA).
[14C]urea (4.2 Ci/mol),
L-[2,3-3H]proline
(45 Ci/mmol),
L-[2,3-3H]aspartic
acid (33 Ci/mmol), and
L-[3-3H]threonine (19 Ci/mmol) were from Amersham Pharmacia Biotech (Milan, Italy). Ethanol
was obtained from Carlo Erba (Milan, Italy), 2-methylaminoisobutyric
acid (MeAIB) from Aldrich-Europe (Milan, Italy), and bumetanide from
Inalco (Milan, Italy). Sigma-Aldrich (Milan, Italy) was the source of
all other chemicals.
 |
RESULTS |
RVI of Human Endothelial Cells
The results presented in Fig.
1 demonstrate that HSVECs,
incubated in complete growth medium, effectively regulate cell volume after a hypertonic stress. The substitution of isotonic medium (290 mosmol/kg) with hypertonic media provokes a decrease in cell volume
close to that expected for a perfect osmometer (from 7.42 to 6.62 or
5.78 µl/mg of protein after 1 h at 330 or 370 mosmol/kg, respectively). Under either hypertonic condition an RVI restores the
initial cell volume. At 330 mosmol/kg, RVI is complete after 6 h of
incubation, whereas 10 h of incubation are required for a complete
volume recovery in HSVECs incubated at 370 mosmol/kg.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
Changes in cell volume on incubation of human saphenous vein
endothelial cells (HSVECs) in hypertonic media. Cell monolayers were
incubated in isotonic or hypertonic medium 199 (M199), and cell volumes
were measured. Values are means ± SD of 6 independent
determinations within 1 representative experiment. Dashed line, control
value calculated as mean of values obtained with cells maintained in
isotonic medium for indicated times (7.42 ± 0.085 µl/mg of
protein, n = 30). Experiment was
repeated 4 times with similar results.
|
|
In endothelial cells, hypertonic stress produces a marked activation of
Na+-K+-2Cl
cotransport and
Na+/H+
antiport (11, 30-33). Results obtained in HSVECs demonstrate, however, that endothelial RVI does not involve any increase of the cell
content of inorganic cations. During the first 3 h of hypertonic
incubation, cell Na+ and
K+ do not change significantly,
whereas a modest, albeit significant, decrease in cell
K+ content is detected thereafter
(Fig. 2). Because of the decrease in cell
volume (Fig. 1), the intracellular concentrations of
Na+ and
K+ rise during the first hour of
incubation but, subsequently, fall to the initial values in parallel
with cell volume rescue. These data are not consistent with a
significant contribution of
Na+-K+-2Cl
cotransport or
Na+/H+
antiport to the RVI of HSVECs. Moreover, the inhibition of
Na+-K+-2Cl
cotransport appears to have little effect on RVI. If hypertonic stress
is carried on in the presence of bumetanide (30 µM), an inhibitor of
the cotransport, neither the volume recovery (Fig. 3, top)
nor the cell content of K+ (Fig.
3, bottom) is significantly
modified.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
Changes in cell Na+ and
K+ during regulatory volume
increase of HSVECs. Top: cell contents
of Na+ and
K+ were determined in HSVECs
incubated under isotonic or hypertonic conditions. Values are means ± SD of 6 independent determinations within 1 representative
experiment. Bottom: intracellular
concentrations of Na+ and
K+
([cation]in)
calculated from cell content (top)
and cell volume data determined in parallel cultures. Dashed lines,
mean of values obtained in control cells maintained under isotonic
conditions. Experiment was repeated 3 times with similar results.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of bumetanide on regulatory volume increase of HSVECs. Cell
monolayers were incubated in isotonic or hypertonic M199 in absence or
presence of 30 µM bumetanide. Cell volume and cell content of
K+ were determined after 1 or 10 h
of incubation. Values are means ± SD of 4 independent
determinations within 1 representative experiment. Experiment was
repeated twice with similar results.
|
|
Volume restoration of HSVECs is associated with dramatic changes of the
intracellular amino acid pool. Table 1
presents the chromatographic analysis of free amino acids in cells
incubated for 10 h in isotonic or hypertonic M199. Glutamate and
glutamine are the major amino acids in cells maintained in isotonic
medium representing one-half of the overall pool, with glutamate alone accounting for almost one-third of the total. After 10 h of hypertonic incubation (i.e., in cells that have successfully recovered their volume) the cell content of most amino acids is increased, with a more
pronounced change in cells incubated at 370 than at 330 mosmol/kg. In
hypertonically stressed cells the largest absolute change is shown by
glutamine (>150 nmol/mg of protein), followed by glutamate. The
highest relative change is observed for proline, the content of which
exhibits a sixfold increase in cells incubated for 10 h at 370 mosmol/kg compared with cells maintained under isotonic conditions. The
intracellular concentration of amino acids is calculated to be ~70 mM
in isotonic M199 (290 mosmol/kg), a value substantially comparable to
that obtained in other endothelial models by Manolopoulos et al. (26).
The intracellular concentration of amino acids rises to 105 and 140 mM
in cells incubated at 330 and 370 mosmol/kg, respectively. The
expansion of the intracellular amino acid pool, detected after
long-term hypertonic incubation, appears, therefore, roughly sufficient
to account for the RVI exhibited by human endothelium.
View this table:
[in this window]
[in a new window]
|
Table 1.
Cell content and intracellular concentration of free amino acids in
HSVECs incubated under isotonic or hypertonic conditions
|
|
Role of Glutamine in RVI of HSVECs
The results described above demonstrate that glutamine plays an
important role in endothelial RVI; in particular, the increase in the
cell content of glutamine represents a major portion (~40%) of the
overall expansion of the intracellular amino acid pool detected in
hypertonically stressed cells.
However, glutamine is the amino acid present at the highest
concentration in the culture medium employed for HSVEC culture (see
METHODS AND MATERIALS). Therefore,
to elucidate the role of glutamine in endothelial volume recovery, the
hypertonic treatment has been performed while maintaining HSVECs in a
nominally glutamine-free M199. It is noteworthy that glutamine is
present in this medium at a concentration of ~0.1 mM because of the
contribution of serum supplement. Figure 4
shows that, under these conditions, RVI is significantly delayed and
HSVECs are not able to complete volume recovery even after 10 h of
hypertonic incubation.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Changes in cell volume on incubation of HSVECs in hypertonic media in
absence of glutamine. Cell monolayers were incubated in isotonic or
hypertonic M199 in presence or absence of glutamine. Values are means ± SD of 6 independent determinations within 1 representative
experiment. Dashed line, control value calculated as mean of values
obtained with cells maintained in isotonic medium for indicated times
(7.65 ± 0.062 µl/mg of protein,
n = 30). Experiment was repeated 4 times with similar results.
|
|
When these cells are compared with controls incubated under hypertonic
conditions in glutamine-supplemented medium (Fig.
5), the composition of their intracellular
amino acid pool is markedly altered. As expected, glutamine is almost
completely absent, but cell glutamate is also markedly lowered. In
contrast, the cell content of some other amino acids, such as proline,
serine, glycine, and alanine, is higher in cells incubated under
glutamine-free hypertonic medium. As a whole, the intracellular amino
acid pool is 703 and 896 nmol/mg of protein in cells incubated under
hypertonic conditions in the absence and in the presence of glutamine,
respectively.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Cell content of free amino acids in HSVECs incubated under hypertonic
conditions in presence and absence of glutamine. HSVECs were incubated
for 10 h under conditions indicated; then cells were extracted and HPLC
was performed. Experiment was repeated twice with comparable results.
|
|
Changes in Amino Acid Transport During Endothelial RVI
Figure 6 shows the results of an experiment
aimed to ascertain the effect of hypertonic treatment on the activity
of various amino acid transport systems (3). To this purpose the
transport of preferential substrates has been measured under
characterizing conditions. The most dramatic change (a >6-fold
increase) is exhibited by transport system A, assessed through the
uptake of the specific substrate MeAIB. Modest increases are detected
for systems y+ (for cationic amino
acids), x
C (an
Na+-independent agency for anionic
amino acids), ASC, and L (for neutral amino acids). It is noteworthy
that all these transport mechanisms are transstimulated, whereas system
A is transinhibited (14). No significant change is detected for system
X
AG, an
Na+-dependent route for anionic
amino acid uptake.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 6.
Activity of amino acid transport systems in HSVECs. Effect of
hypertonic incubation. After 10 h of incubation in isotonic or
hypertonic M199, activity of amino acid transport systems was
determined in HSVECs by measuring 1-min uptake of the following
selective or preferential amino acids under characterizing conditions
detailed below: for system A, 2-methylaminoisobutyric acid (MeAIB, 100 µM, 1 µCi/ml) in Earle's balanced salt solution (EBSS); for system
ASC, L-threonine (50 µM, 2 µCi/ml) in EBSS containing 10 mM MeAIB; for system L,
L-leucine (10 µM, 2 µCi/ml)
in Na+-free EBSS; for system
y+,
L-arginine (20 µM, 1 µCi/ml)
in Na+-free EBSS; for system
x C,
L-glutamate (30 µM, 1 µCi/ml) in Na+-free EBSS; for
system X AG,
L-aspartate (10 µM, 4 µCi/ml) in EBSS. Na+-free EBSS
was obtained by substitution of
Na+ with choline in
Na+ salts. Bars are means of 3 independent determinations in a representative experiment; error bars,
SD. Experiment was repeated 3 times with similar results.
|
|
In mesenchymal cells, glutamine entry occurs through three distinct
transport systems, with major contributions from the
Na+-dependent systems A and ASC
and a small contribution from the Na+-independent system L (10).
This situation is substantially similar in human umbilical vein
endothelial cells, where no clear-cut evidence has been obtained that
an N-type transport system is present (3). As far as HSVECs are
concerned, a 10-h hypertonic incubation causes a significant, although
small, increase of L-glutamine influx (Fig. 7). However, a discrimination
of the entry routes for glutamine indicates that MeAIB-inhibitable
uptake, roughly corresponding to the contribution of system A (3),
exhibits a threefold increase under hypertonic conditions, whereas
neither the MeAIB-insensitive nor the small
Na+-independent portion is
significantly modified.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 7.
Characterization of glutamine transport in HSVECs incubated under
isotonic and hypertonic conditions. Cell monolayers were incubated for
10 h in isotonic or hypertonic (370 mosmol/kg) M199. After incubation,
cell monolayers were washed and incubated for 1 min in isotonic or
hypertonic EBSS containing
[14C]glutamine (100 µM, 1 µCi/ml) in absence or presence of 10 mM MeAIB.
Na+-independent uptake was
determined in a modified EBSS (isotonic or hypertonic) in which choline
replaced Na+ salts. Bars are means
of 3 independent determinations in a representative experiment; error
bars, SD. Experiment was repeated twice with similar results.
|
|
Changes in the Activity of Transport System A During the Volume
Recovery Process
To follow more closely the transport activity of system A during the
hypertonic incubation, the influx of
L-proline, the best natural
substrate of the system (3, 14), has been measured at different times
during the treatment. To this purpose, a tracer amount of the labeled
amino acid has been added to isotonic or hypertonic medium (Fig.
8). The switch of extracellular osmolality from 290 to 330 mosmol/kg causes a clear-cut increase of
L-proline influx; after a 6-h
hypertonic treatment, when the volume recovery is substantially
complete (cf. Fig. 1), system A transport activity is stimulated
twofold compared with cells maintained in isotonic M199. No further
increase is detectable after this period. In contrast, at 370 mosmol/kg
the increase in proline transport is observed, although at a slower
rate, up to 10 h, when it reaches values fourfold higher than controls
maintained under isotonic conditions. Interestingly, in cells incubated
under hypertonic conditions in the absence of glutamine, where cell
volume is only partially recovered even after 10 h (cf. Fig. 4), system
A transport activity increases massively and steadily for 10 h, when
10-fold-stimulated values are attained.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 8.
Time course of increase in system A transport activity during RVI of
HSVECs. Cell monolayers were incubated for 10 h in hypertonic M199 in
presence or absence of glutamine. At each time,
L-proline uptake was measured by
adding
L-[3H]proline
(2 µCi/ml) to medium; after 10 min, cells were washed 3 times with
ice-cold 300 mM urea, and cell-associated radioactivity was determined.
Values are means ± SD of 4 independent determinations in a
representative experiment. Dashed line, control value calculated as
mean of values obtained with cells maintained in isotonic medium for
indicated times. Experiment was repeated 5 times with similar
results.
|
|
To investigate the role of protein synthesis in the upregulation of
system A, the hypertonic stress has been performed in the presence of
3.5 µM cycloheximide. At this concentration, protein synthesis is
inhibited by 75%, as demonstrated by determinations of leucine
incorporation in the acid-insoluble cell fraction (not shown). Figure
9 shows that cycloheximide completely
prevents the increase in
L-proline uptake, thus
demonstrating that the upregulation of system A requires an active
protein synthesis (Fig. 9, top).
Interestingly, volume recovery is also severely hindered in the
presence of cycloheximide (Fig. 9,
bottom).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of cycloheximide on increase of system A transport activity
(top) and RVI
(bottom) in HSVECs. Cell monolayers
were incubated for 10 h in hypertonic M199 in absence or presence of
cycloheximide (3.5 µM). At times indicated,
L-proline uptake (by 10-min
addition of
L-[3H]proline
to M199, see Fig. 8) and cell volume (by addition of
[14C]urea, see Fig. 1)
were measured. Values are means ± SD of 4 independent
determinations in a representative experiment. Dashed lines, control
values calculated as mean of values obtained with cells maintained in
isotonic medium for indicated times. Experiment was repeated twice with
similar results.
|
|
 |
DISCUSSION |
The main conclusion reached in this report is the demonstration that
amino acids are the organic osmolytes employed by endothelial cells for
a successful long-term RVI on chronic hypertonic stress. The
osmoregulatory accumulation of amino acids is dependent on a slowly
ensuing, protein synthesis-dependent stimulation of the activity of
transport system A for neutral amino acids. Transport enhancement is
proportional to the degree and the duration of the osmotic shrinkage.
If the transport increase is suppressed with cycloheximide, volume
restoration is also completely blocked. L-Glutamine is the amino acid
that exhibits the highest absolute increase at the intracellular level,
whereas L-proline exhibits the
highest relative increase. Both these amino acids are good substrates
of system A in cultured human endothelium (3, 23). However, the
expansion of the intracellular pool is not restricted to these amino
acids or to other substrates of system A, such as alanine, serine, or
glycine. Hypertonically stressed cells, indeed, exhibit enhanced
intracellular accumulation of typical substrates of systems L (e.g.,
phenylalanine and leucine) and ASC (e.g., threonine). Because glutamine
is also a substrate of these systems, it is likely that these amino
acids derive from a heteroexchange of intracellular glutamine with
extracellular amino acids (8). This interpretation is consistent with
the moderate stimulation of transport systems L and ASC observed in endothelial cells after RVI (cf. Fig. 6). Glutamate is also accumulated during RVI, but at variance with the results obtained in nonendothelial models (12), the activity of system
X
AG for anionic amino acid
transport does not increase on hypertonic stress in HSVECs (cf. Fig.
6). The excess intracellular content of glutamate, therefore, likely
derives from glutamine hydrolysis. Consistently, in hypertonically
shrunken HSVECs, glutamine is only metabolized to glutamate
(unpublished results).
Most of the available literature points to a major role of
Na+-K+-2Cl
cotransport as a mechanism for endothelial volume regulation (30-33). However, these studies reach divergent conclusions as far
as the response to hypertonic stress is concerned. Employing endothelial cells from bovine aorta, pulmonary artery, and cerebral microvessels, O'Donnell and co-workers (30, 31) demonstrate a rapid,
although incomplete, hypertonic RVI. The adaptive response is severely
hindered by bumetanide and is associated with a brisk stimulation of
Na+-K+-2Cl
cotransport. On the contrary, O'Neill and co-workers (32, 33) observe
neither an increase of cell K+ nor
cell volume recovery in hypertonically shrunken bovine aortic endothelial cells, although they also detect a clear-cut stimulation of
Na+-K+-2Cl
cotransport. These authors, however, describe an effective RVI, associated with a bumetanide-inhibitable net influx of
K+, provided that isotonic
shrinkage is caused by the replacement of extracellular
Na+ and
K+ or a preincubation in hypotonic
medium (32).
The results presented here, obtained in human endothelial cells from
saphenous veins, indicate that the role of
Na+-K+-2Cl
cotransport in long-term hypertonic RVI is, at best, marginal. This
conclusion is based on the absence of significant changes in
K+ content during the volume
recovery process. Moreover, bumetanide does not affect significantly
the cell volume and the cell content of
K+ in hypertonically shrunken
cells (cf. Fig. 3). Rather, as discussed above, volume recovery relies
on the increased accumulation of amino acids due to the activity of
transport system A. Preliminary data obtained in human umbilical vein
endothelial cells indicate that also in this endothelial model RVI is
strictly dependent on neutral amino acid accumulation (V. Dall'Asta,
unpublished results). Moreover, Kempson et al. (23) reported a
hypertonic upregulation of transport system A in vascular endothelial
cells from calf pulmonary artery, although no assessment of the
functional consequences of the transport change has been performed in
that study. Osmosensitivity of system A appears, therefore, to be a consistent feature of endothelial cells as well as of many other cell
models (5-7, 9, 15, 17, 19, 39, 43). It should be stressed,
however, that the results presented here do not exclude the possibility
that stimulation of the activity of
Na+-K+-2Cl
cotransport does indeed occur during the first phases of the hypertonic
treatment. Rather, they indicate that cotransport activation is not
really important for long-term volume restoration. On the other hand,
where
Na+-K+-2Cl
cotransport activation is associated with an effective restoration of
cell volume, hypertonic treatment occurs in complete growth medium (30,
31), so that these studies cannot exclude a role for amino acid
accumulation in the observed volume recovery. Interestingly, where no
significant RVI is detected, amino acid-free hypertonic solutions are
usually employed (32).
On the basis of the present and previous studies (23, 30-33), it
is possible to compare hypertonic stimulation of transport system A and
of
Na+-K+-2Cl
cotransport. The activation of the latter mechanism is rapid (<1 min)
and depends on phosphorylation of the cotransport (24, 31). As judged
from K+ influx measurements,
maximal activation is reached after 20 min of hypertonic incubation,
with a slow decrease thereafter (30). Maximal sensitivity of
cotransport to extracellular tonicity is detected between 280 and 320 mosmol/kg (30). In contrast, the stimulation of system A requires at
least 3 h of hypertonic incubation and an active protein synthesis.
Transport activity rises steadily for several hours and remains
elevated until volume is completely restored. The stimulatory effect is
significantly higher at 370 than at 330 mosmol/kg (present study),
reaching a maximum at 400 mosmol/kg (23). Stimulation of
Na+-K+-2Cl
cotransport and the increase in system A activity may represent, therefore, two complementary and, possibly, sequential aspects of the
same regulatory response to hypertonic stress. Although cotransport may
be employed for the rapid correction of small osmotic fluctuations,
long-term restoration of cell volume in response to larger and chronic
hypertonic challenges relies on a slow stimulation of neutral amino
acid transport and the consequent expansion of the intracellular amino
acid pool characterized here. Interestingly, the progressive and slow
accumulation of amino acids parallels the fall of intracellular
K+ concentration and the small
decrease in K+ content. At late
times of hypertonic treatment, cotransport stimulation could thus
warrant a rapid efflux of K+
rather than a net influx of the cation.
Although
Na+-K+-2Cl
cotransport does not account for RVI in HSVECs or in cultured human
fibroblasts (9) under hypertonic conditions, it could work as a
volume-active mechanism in other situations of physiological relevance.
For instance, a cooperation between amino acid transport system A and
Na+-K+-2Cl
cotransport has been described during the isotonic volume increase associated with cell cycle progression in human fibroblasts (4). It is
possible that a similar mechanism works also in endothelial models,
since bumetanide hinders cell cycle progression in bovine endothelial
cells derived from adult aortic arch (34).
The control of cell shape and volume appears to be of pivotal
importance for a variety of functions of endothelial cells (1, 2, 16,
20), particularly for their barrier activity (21, 22, 27, 36-38).
Thus, if the behavior exhibited by HSVECs in vitro reflects that of
endothelium in vivo, the prolonged period required by endothelial cells
to recover the original volume after hypertonic stress may have
important consequences. Whether manipulations of extracellular neutral
amino acid concentration affect endothelial functions deserves further
investigation. However, the results presented here demonstrate that
glutamine is employed as an osmolyte by endothelial cells, thus
explaining the protective effect exerted by the amino acid on
endothelium challenged by hypertonic stress in vitro (35). On the basis
of this conclusion, potential benefits of glutamine supplementation of
hypertonic solutions employed for the preservation of organs and
vascular grafts should be assessed.
 |
ACKNOWLEDGEMENTS |
This study was partially supported by Centro Cardiologico
Fondazione Monzino Istituto di Ricovero e Cura a Carattere Scientifico (Milan, Italy), Fondazione Cassa di Risparmio di Parma (Parma, Italy),
and Consiglio Nazionale delle Ricerche, Target Project Biotechnology
(Rome, Italy).
 |
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 and other correspondence: G. C. Gazzola,
Istituto di Patologia Generale, Plesso Biotecnologico Integrato,
Università degli Studi di Parma, Via Volturno, 39, 43100 Parma,
Italy (E-mail: gazzola{at}ipruniv.cce.unipr.it).
Received 2 November 1998; accepted in final form 4 January 1999.
 |
REFERENCES |
1.
Asakawa, H.,
J. Miyagawa,
T. Hanafusa,
M. Kuwajima,
and
Y. Matsuzawa.
High glucose and hyperosmolarity increase secretion of interleukin-1
in cultured human aortic endothelial cells.
J. Diabetes Complications
11:
176-179,
1997[Medline].
2.
Bohlen, H. G.
Mechanism of increased vessel wall nitric oxide concentrations during intestinal absorption.
Am. J. Physiol.
275 (Heart Circ. Physiol. 44):
H542-H550,
1998[Abstract/Free Full Text].
3.
Bussolati, O.,
R. Sala,
A. Astorri,
B. M. Rotoli,
V. Dall'Asta,
and
G. C. Gazzola.
Characterization of amino acid transport in human endothelial cells.
Am. J. Physiol.
265 (Cell Physiol. 34):
C1006-C1014,
1993[Abstract/Free Full Text].
4.
Bussolati, O.,
J. Uggeri,
S. Belletti,
V. Dall'Asta,
and
G. C. Gazzola.
The stimulation of Na,K,Cl cotransport and of system A for neutral amino acid transport is a mechanism for cell volume increase during the cell cycle.
FASEB J.
10:
920-926,
1996[Abstract/Free Full Text].
5.
Chen, J. G.,
M. Coe,
J. A. McAteer,
and
S. A. Kempson.
Hypertonic activation and recovery of system A amino acid transport in renal MDCK cells.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F419-F424,
1996[Abstract/Free Full Text].
6.
Chen, J. G.,
and
S. A. Kempson.
Osmoregulation of neutral amino transport.
Proc. Soc. Exp. Biol. Med.
210:
1-6,
1995[Abstract].
7.
Chen, J. G.,
L. R. Klus,
D. K. Steenbergen,
and
S. A. Kempson.
Hypertonic upregulation of amino acid transport system A in vascular smooth muscle cells.
Am. J. Physiol.
267 (Cell Physiol. 36):
C529-C536,
1994[Abstract/Free Full Text].
8.
Dall'Asta, V.,
R. Franchi-Gazzola,
O. Bussolati,
R. Sala,
B. M. Rotoli,
P. A. Rossi,
J. Uggeri,
S. Belletti,
R. Visigalli,
and
G. C. Gazzola.
Emerging roles for sodium dependent amino acid transport in mesenchymal cells.
Amino Acids
11:
117-133,
1996.
9.
Dall'Asta, V.,
P. A. Rossi,
O. Bussolati,
and
G. C. Gazzola.
Response of human fibroblasts to hypertonic stress. Cell shrinkage is counteracted by an enhanced active transport of neutral amino acids.
J. Biol. Chem.
269:
10485-10491,
1994[Abstract/Free Full Text].
10.
Dall'Asta, V.,
P. A. Rossi,
O. Bussolati,
G. G. Guidotti,
and
G. C. Gazzola.
The transport of L-glutamine into cultured human fibroblasts.
Biochim. Biophys. Acta
1052:
106-112,
1990[Medline].
11.
Escobales, N.,
E. Longo,
E. J. Cragoe, Jr.,
N. R. Danthuluri,
and
T. A. Brock.
Osmotic activation of Na+-H+ exchange in human endothelial cells.
Am. J. Physiol.
259 (Cell Physiol. 28):
C640-C646,
1990[Abstract/Free Full Text].
12.
Ferrer-Martinez, A.,
A. Felipe,
B. Nicholson,
F. J. Casado,
M. Pastor-Anglada,
and
J. McGivan.
Induction of the high affinity Na+-dependent glutamate transport system X
AG by hypertonic stress in the renal epithelial cell line NBL-1.
Biochem. J.
316:
689-692,
1995.
13.
Gazzola, G. C.,
V. Dall'Asta,
R. Franchi-Gazzola,
and
M. F. White.
The cluster-tray method for rapid measurement of solute fluxes in adherent cultured cells.
Anal. Biochem.
115:
368-374,
1981[Medline].
14.
Gazzola, G. C.,
V. Dall'Asta,
and
G. G. Guidotti.
The transport of neutral amino acids in cultured human fibroblasts.
J. Biol. Chem.
255:
929-936,
1980[Abstract/Free Full Text].
15.
Gazzola, G. C.,
V. Dall'Asta,
F. A. Nucci,
P. A. Rossi,
O. Bussolati,
E. K. Hoffmann,
and
G. G. Guidotti.
Role of amino acid transport system A in the control of cell volume in cultured human fibroblasts.
Cell. Physiol. Biochem.
1:
131-142,
1991.
16.
Gilcrease, M. Z.,
and
R. L. Hoover.
Neutrophil adhesion to endothelium following hyperosmolar insult.
Diabetes Res.
16:
149-157,
1991[Medline].
17.
Gomez-Angelats, M.,
M. Lopez-Fontanals,
A. Felipe,
F. J. Casado,
and
M. Pastor-Anglada.
Cytoskeletal-dependent activation of system A for neutral amino acid transport in osmotically stressed mammalian cells: a role for system A in the intracellular accumulation of osmolytes.
J. Cell. Physiol.
173:
343-350,
1997[Medline].
18.
Hoffmann, E. K.,
and
P. B. Dunham.
Membrane mechanisms and intracellular signalling in cell volume regulation.
Int. Rev. Cytol.
161:
173-262,
1995[Medline].
19.
Horio, M.,
A. Yamauchi,
T. Moriyama,
E. Imai,
and
Y. Orita.
Osmotic regulation of amino acids and system A transport in Madin-Darby canine kidney cells.
Am. J. Physiol.
272 (Cell Physiol. 41):
C804-C809,
1997[Abstract/Free Full Text].
20.
Ishizaka, H.,
and
L. Kuo.
Endothelial ATP-sensitive potassium channels mediate coronary microvascular dilation to hyperosmolarity.
Am. J. Physiol.
273 (Heart Circ. Physiol. 42):
H104-H112,
1997[Abstract/Free Full Text].
21.
Kajimura, M.,
M. E. O'Donnell,
and
F. E. Curry.
Effect of cell shrinkage on permeability of cultured bovine aortic endothelia and frog mesenteric capillaries.
J. Physiol. (Lond.)
503:
413-425,
1997[Abstract].
22.
Kempski, O.,
M. Spatz,
G. Valet,
and
A. Baethman.
Cell volume regulation of cerebrovascular endothelium in vitro.
J. Cell. Physiol.
123:
51-54,
1985[Medline].
23.
Kempson, S. A.,
M. J. Hoshaw,
R. S. Hinseley,
and
J. A. McAteer.
Hyperosmotic stress up-regulates amino acid transport in vascular endothelial cells.
Kidney Int.
52:
1332-1339,
1997[Medline].
24.
Klein, J. D.,
P. B. Perry,
and
W. C. O'Neill.
Regulation by cell volume of Na+-K+-2Cl
cotransport in vascular endothelial cells: role of protein phosphorylation.
J. Membr. Biol.
132:
243-252,
1993[Medline].
25.
Lang, F.,
G. L. Busch,
M. Ritter,
H. Völkl,
S. Waldegger,
E. Gulbins,
and
D. Häussinger.
Functional significance of cell volume regulatory mechanisms.
Physiol. Rev.
78:
247-306,
1998[Abstract/Free Full Text].
26.
Manolopoulos, V. G.,
T. Voets,
P. E. Declercq,
G. Droogmans,
and
B. Nilius.
Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells.
Am. J. Physiol.
273 (Cell Physiol. 42):
C214-C222,
1997[Abstract/Free Full Text].
27.
Mazzoni, M. C.,
P. Borgstrom,
M. Intaglietta,
and
K. E. Arfors.
Capillary narrowing in hemorrhagic shock is rectified by hyperosmotic saline-dextran reinfusion.
Circ. Shock
31:
407-418,
1990[Medline].
28.
Mazzoni, M. C.,
E. Lundgren,
K.-E. Arfors,
and
M. Intaglietta.
Volume changes of an endothelial cell monolayer on exposure to anisotonic media.
J. Cell. Physiol.
140:
272-280,
1989[Medline].
29.
McGivan, J. D.,
and
M. Pastor-Anglada.
Regulatory and molecular aspects of mammalian amino acid transport.
Biochem. J.
299:
321-334,
1994[Medline].
30.
O'Donnell, M. E.
Role of Na-K-Cl cotransport in vascular endothelial cell volume regulation.
Am. J. Physiol.
264 (Cell Physiol. 33):
C1316-C1326,
1993[Abstract/Free Full Text].
31.
O'Donnell, M. E.,
A. Martinez,
and
D. Sun.
Endothelial Na-K-Cl cotransport regulation by tonicity and hormones: phosphorylation of cotransport protein.
Am. J. Physiol.
269 (Cell Physiol. 38):
C1513-C1523,
1995[Abstract/Free Full Text].
32.
O'Neill, W. C.,
and
J. D. Klein.
Regulation of vascular endothelial cell volume by Na-K-2Cl cotransport.
Am. J. Physiol.
262 (Cell Physiol. 31):
C436-C444,
1992[Abstract/Free Full Text].
33.
O'Neill, W. C.,
and
D. F. Steinberg.
Functional coupling of Na+-K+-2Cl
cotransport and Ca2+-dependent K+ channels in vascular endothelial cells.
Am. J. Physiol.
269 (Cell Physiol. 38):
C267-C274,
1995[Abstract/Free Full Text].
34.
Panet, R.,
M. Markus,
and
H. Atlan.
Bumetanide and furosemide inhibited vascular endothelial cell proliferation.
J. Cell. Physiol.
158:
121-127,
1994[Medline].
35.
Parolari, A.,
R. Sala,
C. Antona,
O. Bussolati,
F. Alamanni,
P. Mezzadri,
V. Dall'Asta,
G. C. Gazzola,
and
P. Biglioli.
Hypertonicity induces injury to cultured human endothelium: attenuation by glutamine.
Ann. Thorac. Surg.
64:
1770-1775,
1997[Abstract/Free Full Text].
36.
Ragette, R.,
C. Fu,
and
J. Bhattacharya.
Barrier effects of hyperosmolar signaling in microvascular endothelium of rat lung.
J. Clin. Invest.
100:
685-692,
1997[Abstract/Free Full Text].
37.
Rapoport, S. I.,
W. R. Fredericks,
K. Ohno,
and
K. D. Pettigrew.
Quantitative aspects of reversible osmotic opening of the blood-brain barrier.
Am. J. Physiol.
238 (Regulatory Integrative Comp. Physiol. 7):
R421-R431,
1980[Medline].
38.
Shepard, J. M.,
S. K. Goderie,
N. Brzyski,
P. J. Del Vecchio,
A. B. Malik,
and
H. K. Kimelberg.
Effects of alterations in endothelial cell volume on transendothelial albumin permeability.
J. Cell. Physiol.
133:
389-394,
1987[Medline].
39.
Soler, C.,
A. Felipe,
F. J. Casado,
J. D. McGivan,
and
M. Pastor-Anglada.
Hyperosmolarity leads to an increase in derepressed system A activity in the renal epithelial cell line NBL-1.
Biochem. J.
289:
653-658,
1993[Medline].
40.
Tramacere, M.,
P. G. Petronini,
A. Severini,
and
A. F. Borghetti.
Osmoregulation of amino acid transport activity in cultured fibroblasts.
Exp. Cell. Res.
151:
70-79,
1984[Medline].
41.
Weik, C.,
U. Warskulat,
J. Bode,
T. Peters-Regher,
and
D. Haussinger.
Compatible organic osmolytes in rat liver sinusoidal endothelial cells.
Hepatology
27:
569-575,
1998[Medline].
42.
Wiese, T. J.,
J. A. Dunlap,
C. E. Conner,
J. A. Grzybowski,
W. L. Lowe, Jr.,
and
M. Y. Yorek.
Osmotic regulation of Na-myo-inositol cotransporter mRNA level and activity in endothelial and neural cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C990-C997,
1996[Abstract/Free Full Text].
43.
Yamauchi, A.,
A. Miyai,
K. Yokoyama,
T. Itoh,
T. Kamada,
N. Ueda,
and
Y. Fujiwara.
Response to osmotic stimuli in mesangial cells: role of system A transporter.
Am. J. Physiol.
267 (Cell Physiol. 36):
C1493-C1500,
1994[Abstract/Free Full Text].
Am J Physiol Cell Physiol 276(4):C865-C872
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society