From the Laboratory of Molecular Genetics and
§ Laboratory of Oncology, Gaslini Institute,
16148 Genova, Italy
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ABSTRACT |
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The activity of volume-sensitive
Cl Virtually all cells in a multicellular organism undergo swelling
or shrinking following changes of intracellular or extracellular osmotic pressure. Although the osmolarity of body fluids, particularly in mammalians, is tightly controlled, significant variations may occur
in physiological or pathological conditions. For example, a dramatic
drop in extracellular osmolarity occurs in the renal medulla during the
passage from antidiuresis to diuresis (1). Cell swelling may also occur
in the brain during hypoxic and toxic conditions or following treatment
of diabetic ketoacidosis (2-5) and in the heart following myocardial
ischemia (6, 7).
Usually cells respond to osmotic stresses by transporting organic and
inorganic osmolytes through the plasma membrane. The lowering of
extracellular osmolarity and the consequent cell swelling activate
potassium and chloride channels (8). The resulting exit of KCl drives
water efflux and therefore restores the original cell volume, a
mechanism termed regulatory volume decrease. A key element in this
process is represented by volume-sensitive Cl Cell Culture--
The 9HTEo Solutions--
The isotonic standard solution used for efflux
experiments contained 130 mM NaCl, 2 mM KCl, 1 mM KH2PO4, 2 mM
CaCl2, 2 mM MgCl2, 10 mM Na-Hepes pH 7.3, 10 glucose, and 20 mannitol
(osmolality = 300 mosmol/kg). Hypotonic solutions (HS) were
prepared by omitting mannitol and by lowering NaCl to 90 mM
(205 mosmol/kg; 68% HS) or to 115 mM (250 mosmol/kg; 83%
HS).
Taurine and ATP Efflux--
For this study, we have taken
advantage of the relatively large taurine permeability of VSOAC (11) to
perform efflux experiments. This approach seems more appropriate than
patch-clamp measurements to study the regulation of volume-sensitive
channels, because taurine efflux experiments leave the cell interior
undisturbed. The procedure for taurine efflux has been previously
described (11). Briefly, cells were incubated for 1 h at 37 °C
with 10 µM taurine and 0.2 µCi/ml
[3H]taurine. At the end of incubation, the loading medium
was discarded, and the cells were washed three times with ice-cold
taurine-free medium. Taurine efflux experiments started with the
addition of 1 ml of standard isotonic solution prewarmed at 37 °C.
Every 5 min, for a total of 35 min, the efflux medium containing the
taurine released by the cells was removed from the Petri dish and
rapidly replaced by another ml of solution. The hypotonic shock was
applied 15 min after the beginning of efflux. At the end of the
experiment, cells were lysed by overnight incubation with 0.25 M NaOH. The radioactivity present in the efflux samples and
in cell lysates was determined by liquid scintillation. Total
incorporated taurine T0 was obtained by adding
the radioactivity of all efflux samples to that remaining in the NaOH
extracts. The taurine remaining in the cells at a given time
t (Tres) was determined by
subtracting from T0 the amount of radioactivity
in the efflux samples up to time t. The time course of
taurine efflux was expressed by plotting fractional efflux (FE)
versus time. The FE at a given time was calculated according
to FE = Tex/Tres, where
Tex is the amount of taurine released in a
single efflux interval, and Tres is the amount
of residual taurine remaining in the cells at the beginning of that interval.
For ATP release experiments, cells were loaded for 1 h with 2 µCi/ml [3H]AMP. After loading, the cells were treated
with the same protocol of taurine efflux experiments except for the
lysis, which was accomplished by three rapid freeze-thaw cycles. The
radioactivity collected in the efflux samples was normalized for the
total radioactivity accumulated into the cells as explained above.
Except where indicated, isotonic and hypotonic solutions during the
efflux always contained 1 µM dipyridamole to block the
uptake of adenosine.
Ion Pairing Reverse-phase HPLC Analysis of 3H-labeled
Nucleotides and Their Derivatives--
Two hundred µl of cell
supernatants or 100 µl of cell lysates were analyzed in an LKB liquid
chromatograph (Amersham Pharmacia Biotech) equipped with a Supelcosil
LC-18-T column (15 cm x 4.6-mm inner diameter, 3-mm particle size;
Supelco, Bellefonte, PA) and a Supelguard LC-18-T cartridge precolumn
(Supelco). Buffer A was 60 mM
KH2PO4, 5 mM tetrabutylammonium
hydrogen sulfate, pH 6.0, containing 5% (v/v) methanol. Buffer B was
70% buffer A and 30% (v/v) methanol. The mobile phase was developed
at a constant flow rate of 1.5 ml/min as follows: from 0 to 6 min,
0-60% buffer B; from 6 to 10 min, 60% buffer B; from 10 to 12 min,
60-0% buffer B. The system and column were reequilibrated in buffer A
for an additional 8 min before the subsequent injection. The column
eluate was monitored through a model Flow-one Beta radioactivity
detector (Packard Instruments Co, Meriden, CT). Scintillation liquid
was Ultima-Flo M (Packard) at a flow rate of 3 ml/min, which yielded an
approximately 25% efficiency. Peaks were quantitated by means of the
Flo-one/Beta software (Packard), and substances were identified on the
basis of coelution with their unlabeled counterparts monitored at 254 nm by a LKB multiwavelength UV detector.
Analysis of ATP Degradation--
Cells were plated and cultured
as described for efflux studies. To detect the activity of ectoenzymes,
cells were incubated at 37 °C with the saline solution containing
nonradioactive ATP or AMP-PNP. The cell supernatants were then analyzed
by HPLC as explained above.
Materials--
Culture media and fetal clone II were purchased
from HyClone (Cramlington, UK). [3H]Taurine and
[3H]AMP were from Amersham Pharmacia Biotech. DMPX,
DPCPX, U73122, and CGS21680 were from RBI (Natick, MA). AMP-PNP was
purchased from Roche Molecular Biochemicals. All other chemicals were
from Sigma.
Statistics--
Data are presented as mean ±S.E. The points
shown in each figure represent the mean of at least four experiments.
Statistical significance was assessed by Student's t test
for unpaired data.
According to our hypothesis, the hypotonic shock would cause a
release of endogenous ATP. ATP would then be converted to adenosine by
extracellular local catabolism. Adenosine, by interacting with adenosine receptors, would be responsible for the activation of VSOAC
during the hypotonic shock. To test this model, we applied the
hypotonic shock (68% HS) in combination with known adenosine receptor
antagonists. DPCPX, which is a specific inhibitor of A1
receptors at low nM concentrations (17), significantly
inhibited the peak of swelling-induced taurine release (Fig.
1, A and B). The
effect was partial and dose-dependent with a maximal
inhibition of 34% and a half-effective concentration equal to 16 nM (Fig. 1B). Similarly, DMPX, an antagonist
that acts at concentrations in the low µM range with a
slight selectivity for A2 versus A1 receptors (18), was also able to inhibit taurine efflux (Fig. 1,
A and B). Maximal inhibition and half-effective
concentration were 44% and 560 nM, respectively. When DMPX
and DPCPX were used together at maximal concentrations (10 and 1 µM, respectively), the inhibition of taurine efflux was
not different from that obtained by each antagonist individually. The
inhibition of taurine efflux by adenosine receptor antagonists
suggested that endogenous adenosine really contributes to
swelling-dependent VSOAC activation. The next step was to
assess the source of adenosine. Adenosine could directly originate from
the cells or by hydrolysis of released ATP. Accordingly, we used
channels was studied in human tracheal
epithelial cells (9HTEo
) by taurine efflux experiments. The efflux
elicited by a hypotonic shock was partially inhibited by adenosine
receptor antagonists, by
,
-methyleneadenosine 5'-diphosphate (
MeADP), an inhibitor of the 5'-ectonucleotidase, and by
adenosine deaminase. On the other hand, dipyridamole, a nucleoside
transporter inhibitor, increased the swelling-induced taurine efflux.
Extracellular ATP and adenosine increased taurine efflux by
potentiating the effect of hypotonic shock.
MeADP strongly
inhibited the effect of extracellular ATP but not that of adenosine.
These results suggest that anion channel activation involves the
release of intracellular ATP, which is then degraded to adenosine by
specific ectoenzymes. Adenosine then binds to purinergic receptors,
causing the activation of the channels. To directly demonstrate ATP
efflux, cells were loaded with [3H]AMP, and the release
of radiolabeled molecules was analyzed by high performance liquid
chromatography. During hypotonic shock, cell supernatants showed the
presence of ATP, ADP, and adenosine.
MeADP inhibited adenosine
formation and caused the appearance of AMP. Under hypotonic conditions,
elevation of intracellular Ca2+ by ionomycin caused an
increase of ATP and adenosine in the extracellular solution. Our
results demonstrate that volume-sensitive anion channels are regulated
with an autocrine mechanism involving swelling-induced ATP release and
then hydrolysis to adenosine.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channels,
called volume-sensitive organic anion channel
(VSOAC),1 which are also
permeable to organic osmolytes such as taurine (9-11). Because taurine
in many cells is accumulated at high concentrations through
Na+-dependent transporters, the opening of
VSOAC causes a significant taurine efflux, thus contributing to
regulatory volume decrease. The mechanism underlying VSOAC activation
is unknown. In a previous work performed on a tracheal epithelial cell
line, we found that extracellular ATP is a potent modulator of
swelling-induced VSOAC activation (11). This finding led us to test the
hypothesis that release of endogenous ATP by the cells could be
responsible for VSOAC activation during the hypotonic shock. Our
experiments did not support this hypothesis because ATP was not
revealed by the luciferin-luciferase method, and exogenous hexokinase
did not inhibit VSOAC activity (11). In contrast with our results, other investigators have found that there is a release of ATP induced
by cell swelling (12, 13). To reconcile these conflicting results, we
have considered the possibility that ATP efflux in our cells could be
masked by the rapid catabolism caused at the extracellular side of the
membrane by specific ectoenzymes (14, 15). The present article deals
with the hypothesis that ATP is indeed released upon cell swelling and
rapidly degraded to adenosine and that this nucleoside is the modulator
of volume-sensitive anion channels, at least in our cell model.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cell line was obtained by
immortalization of human tracheal epithelial cells (16). Cells were
grown at 37 °C in an atmosphere of 5% CO2 using a
medium containing 45% Dulbecco's modified Eagle's medium, 45%
Ham's F-12, and 10% fetal clone II serum plus 100 units/ml
penicillin, 100 µg/ml streptomycin, and 2 mM
L-glutamine. Cells were plated on 35-mm Petri dishes at a density of 40,000/cm2 and cultured for 4 days before
performing taurine and ATP transport studies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MeADP, a potent blocker of 5'-ectonucleotidase, the ectoenzyme
that hydrolyses AMP to adenosine (15, 19).
MeADP inhibited in a
dose-dependent fashion the taurine efflux elicited by the
hypotonic shock (Fig. 2). Maximal effect
was 37%, and half-effective concentration was 510 nM.
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Fig. 1.
Inhibition of swelling-induced taurine
release by adenosine receptor antagonists. A, time
course of taurine FE. The arrow marks the beginning of
hypotonic shock (68% HS). Experiments were carried out in the absence
or in the presence of 50 µM DPCPX or 50 µM
DMPX. The FE was significantly inhibited by DMPX (p < 0.001 and p < 0.05 at times 20 and 25, respectively)
and by DPCPX (p < 0.05 at time 20). B, the
percent of fractional efflux inhibited by DMPX (open
squares) and by DPCPX (filled circles) at time 20 is
plotted versus the antagonist concentration.
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[in a new window]
Fig. 2.
Inhibition of taurine release by
MeADP. A, time course
of taurine efflux in the absence and in presence of 100 µM
MeADP. The arrow marks the beginning
of hypotonic shock (68% HS). FE was significantly inhibited at time 20 (p < 0.001) and time 25 (p < 0.05).
B, the inhibition of taurine efflux measured at time 20 is
plotted versus the
MeADP concentration.
We previously showed that extracellular ATP application results in a
strong potentiation of hypotonic shock effect (11). We asked whether
exogenous ATP also acts through the conversion to adenosine. Therefore,
we applied 100 µM ATP in the 68% HS with or without
MeADP. Interestingly,
MeADP strongly reduced the taurine
efflux elicited by the combined stimulation with ATP and hypotonic
shock (Fig. 3). Actually,
MeADP
inhibition seems to result from complete block of ATP effect in
addition to the expected partial inhibition of the efflux induced by
the hypotonic shock alone. Extracellular application of adenosine also
potentiated swelling-induced taurine release as ATP (Fig. 3).
Nevertheless, under these conditions,
MeADP was completely
ineffective.
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Previous studies suggested that extracellular adenosine and ATP act in
9HTEo and other airway cell lines by increasing intracellular Ca2+ through a phospholipase C (PLC)-based mechanism (20).
If the hypotonic shock induced ATP and adenosine release, a PLC
inhibitor should reduce the swelling-induced taurine efflux. We used
U73122, an amino steroid that is able to potently block PLC when used at concentrations in the low µM range (21). U73122 (2 µM) reduced the peak of taurine efflux by 40% (Fig.
4A).
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We reasoned that if adenosine were the endogenous agonist that mediates
the effect of hypotonic shock on taurine channels, adenosine deaminase
(ADA), which converts adenosine to inosine, should have an inhibitory
effect. ADA caused a significant decrease of the swelling-induced
taurine efflux, although smaller than that of MeADP, DPCPX, and
DMPX (Fig. 4B).
Nucleosides are taken up by the cells through specific transporters,
some of which are sensitive to inhibitors like nitrobenzylthioinosine (NBMPR) and dipyridamole (22, 23). We applied these compounds during
the hypotonic shock to assess if they had an effect. Dipyridamole significantly increased taurine efflux under hyposmotic conditions (Fig. 5A). NBMPR was instead
almost ineffective (Fig. 5B).
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Previous experiments revealed that a mild hypotonic shock (83% HS)
causes only a slight activation of the Cl channels.
However, this effect can be strongly potentiated by Ca2+-elevating agents such as ATP and ionomycin to obtain a
channel activation comparable with that achieved with a stronger
hypotonic shock (11). Fig. 6 shows that
adenosine also acts by synergistically increasing the volume-sensitive
taurine efflux. CGS21680, an agonist selective for A2A
adenosine receptors (24), also increased taurine efflux
(n = 3, not shown). Interestingly,
MeADP did not
inhibit adenosine effect but strongly antagonized that of ATP (Fig. 6). We also evaluated other nucleotides to clarify the mechanisms through
which ATP affects channel activity. UTP and 2MeSATP were slightly
active. In both cases
MeADP was ineffective. We tested AMP-PNP
because it has been often used as a nonhydrolyzable ATP analog.
Accordingly, this compound had to be ineffective if ATP acts through
the conversion to adenosine. Unexpectedly, AMP-PNP was active, although
less than ATP, and its activity was strongly reduced by
MeADP
(Fig. 6).
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Previous attempts to measure ATP release from 9HTEo cells by the
luciferin-luciferase assay were unsuccessful (11). In the present work,
we have therefore utilized an HPLC-based approach. We reasoned that
this method, by revealing the presence of ATP catabolites, would have
allowed detection of ATP release even in the presence of a rapid
extracellular hydrolysis. Accordingly, 9HTEo
cells were loaded with
[3H]AMP for 1 h. After this period, analysis of cell
lysates revealed that 53.7 ± 2.3% (n = 5) of the
radioactivity was due to labeled ATP. ADP, AMP, adenosine, and inosine
accounted for 22.1 ± 1.1%, 8.6 ± 0.9%, 3.8 ± 0.4%,
and 7.8 ± 0.5% total accumulated radioactivity, respectively. It
is probable that [3H]AMP enter the cells as adenosine by
the action of ectonucleotidase and adenosine transporters. Indeed, cell
loading was strongly inhibited by dipyridamole or
MeADP (not
shown). Under isotonic conditions, cells loaded with
[3H]AMP released small amounts of radioactivity (Fig.
7A). Upon applying the
hypotonic solution (68% HS), a small but significant increase of
released radioactivity was observed in the first 10 min (Fig. 7,
A and B). A milder hypotonic shock (83% HS) was
instead without effect (not shown). We also wanted to assess the effect of intracellular Ca2+ elevation alone or in combination
with the hypotonic shock. We found that ionomycin elicited per
se an increased radioactivity release from
[3H]AMP-loaded cells (Fig. 7B). Stimulation
with ionomycin plus hypotonic shock (68% HS) induced a much higher
efflux (Fig. 7B). Indeed, the effect was larger than the sum
of the single responses to ionomycin and hypotonic shock.
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HPLC analysis revealed that hypotonic shock, in the first 10 min of
stimulation, caused the release of radioactive species whose elution
times corresponded to those of adenosine, ADP, and ATP (Fig.
8, B and E). These
peaks were absent in isotonic conditions (Fig. 8, A and
C). On the other hand, supernatants of cells stimulated with
ionomycin did not show a significant ATP, ADP, and adenosine release
but a marked peak corresponding to inosine (Fig. 8D). When
cells were treated with ionomycin plus the hypotonic shock (68% HS),
supernatants had a higher content of ATP with respect to that observed
with only the hypotonic shock (Fig. 8F; p < 0.05). Adenosine was also significantly increased (p < 0.01). Interestingly, when hypotonic shock (with or without ionomycin)
was applied in the presence of MeADP, the adenosine peak was
strongly reduced, whereas AMP, which was always undetectable in the
other experimental conditions, was clearly visible (Fig. 8,
E and F).
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Experiments performed using dipyridamole and ADA (Figs. 4 and 5) showed changes in VSOAC activity that we interpreted as due to changes in extracellular adenosine concentration. To validate this interpretation we removed dipyridamole from the efflux medium or, alternatively, we added exogenous ADA (0.2 units/ml). As expected, both procedures decreased the radioactive adenosine in the extracellular medium. Removal of dipyridamole reduced the adenosine peak from 1061 ± 133 to 585 ± 20 cpm (p < 0.05; not shown). On the other hand, application of ADA completely abolished the adenosine peak and caused an increase of inosine from 128 ± 68 to 1401 ± 144 cpm (p < 0.01).
Our results suggest that 9HTEo cells possess ectoenzymes able to
degrade ATP. To directly demonstrate this process, cells were incubated
for variable times with ATP (100 µM). The supernatants were then analyzed by HPLC. After 1 h (not shown) and, more
markedly, after 2 h (Fig.
9B), the peak corresponding to
ATP decreased, and additional peaks appeared. These peaks corresponded
to ADP, AMP, adenosine, and inosine. The process of ATP hydrolysis did not occur when ATP was incubated in the absence of cells. When
MeADP was included in the extracellular solution, the adenosine peak was abolished, whereas that of AMP was significantly increased (not shown). AMP-PNP was able to activate VSOAC in a
MeADP-sensitive way (Fig. 6). This result suggested that this
compound can be converted to adenosine. Accordingly, we incubated the
cells with this compound. HPLC analysis revealed the presence of a
contaminant in the AMP-PNP stock solution (see Fig. 9C).
According to the manufacturer instructions, this compound should be
AMP-PNP after the loss of a phosphate moiety (i.e.
ADP-NH2). After incubation with 9HTEo
cells, the main and
the secondary peaks were decreased, whereas a peak corresponding to
adenosine was clearly evident (Fig. 9D). Incubation without
the cells did not change the pattern of AMP-PNP-related peaks. Also in
this case,
MeADP decreased the adenosine peak and increased the
AMP signal (not shown).
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DISCUSSION |
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Cells are able to face a hypotonic shock by activating ion
channels (VSOAC) permeable to Cl and organic osmolytes.
The resulting efflux of osmolytes and water restores the original cell
volume. The mechanisms responsible for VSOAC activation are unknown,
although protein phosphorylation, membrane stretch, and changes in
cytoskeletal organization have been proposed (25). Recently, it has
been postulated that hypotonically induced ATP release represents an
autocrine stimulus for VSOAC activation (12, 13). Our results indicate
that the hypotonic shock elicits an ATP release also in 9HTEo
tracheal epithelial cells. ATP is then hydrolyzed to adenosine by
specific ectoenzymes. However, in our cells, adenosine and not ATP is
the agonist responsible for VSOAC activation. Several evidences support
this scheme. First, taurine efflux is significantly reduced by
adenosine receptor antagonists such as DPCPX and DMPX. These compounds
act with half-effective concentrations, consistent with an inhibition
at the level of adenosine receptors (17, 18). It is not clear which
adenosine receptor is involved in the regulation of volume-sensitive
Cl
channels in 9HTEo
cells. The inhibition by DMPX and
the activation by CGS21680 suggests the involvement of the
A2A type (24). However, DPCPX, a selective A1
antagonist, has an unusually low IC50 value for an
A2A receptor (24). Future studies with a series of agonists and antagonists could elucidate this point. The 5'-ectonucleotidase inhibitor
MeADP, which blocks the conversion of AMP to adenosine (14, 15, 19), reduces the swelling-induced taurine release to an extent
similar to adenosine receptor antagonists. A comparable reduction is
also obtained by using the PLC inhibitor U73122. This result is
consistent with the finding that exogenous adenosine stimulates PLC
with consequent intracellular Ca2+ increase in airway
epithelial cell lines (20). Our results are also consistent with
previous patch-clamp studies in which we found that stimulation of
adenosine receptors results in VSOAC activation (26).
Another indirect indication of adenosine involvement is the finding
that nucleoside transporter inhibitors increase and that ADA,
conversely, decreases taurine efflux. Such results can be explained by
hypothesizing local variations of adenosine concentrations sensed by
adenosine receptors. Indeed, dipyridamole would increase the
availability of extracellular adenosine by blocking the reuptake. The
stronger effect of dipyridamole compared with NBMPR can be explained by
its ability to also inhibit NBMPR-insensitive transporters (22, 23). On
the other hand, ADA probably decreases taurine efflux by degrading
adenosine to inosine, thus lowering the adenosine concentration, which
is sensed by adenosine receptors. ADA-dependent inhibition
is smaller than that elicited by other treatments (DPCPX, DMPX,
MeADP, U73122). This could be because of the difficulty encountered by a large enzyme to reach the sites on the plasma membrane
where ATP release and hydrolysis occur or because the rate of adenosine
production from ATP is faster than the rate of adenosine deamination to inosine.
To further verify our hypothesis, we applied extracellular ATP to
assess whether it mimics the effect of the endogenous ATP. We found
that ATP potentiates the effect of mild and strong hypotonic shocks
(83% HS and 68% HS, respectively) and that this effect is completely
blocked by MeADP. Such a result suggests that exogenous ATP is
degraded to AMP by ectoATPase and ectoADPase and then to adenosine by
5'-ectonucleotidase. Adenosine then activates VSOAC by interacting with
adenosine receptors. Indeed, the inhibition by
MeADP can be
bypassed by application of exogenous adenosine. In support of this
model is the finding that extracellular UTP, which is not able to
activate adenosine receptors, is a poor agonist of VSOAC. Surprisingly,
AMP-PNP, which is often used as a hydrolysis-resistant ATP analog,
activates taurine efflux. Furthermore, its effect can be blocked by
MeADP. Our experiments, however, demonstrate that incubation of
9HTEo
with AMP-PNP results in the appearance of adenosine in the cell
supernatant. This process is probably due to hydrolysis because it is
abolished by
MeADP. Our observation is consistent with recent
findings that AMP-PNP can be actually hydrolyzed by extracellular
ATPases (27).
Interestingly, other investigators have recently found that
extracellular ATP may act in hypocampal slices by rapid conversion to
adenosine and stimulation of adenosine receptors (27). These authors
have proposed a "channeling" model to explain the process. According to this model, the enzymes responsible for ATP degradation, and adenosine receptors are closely clustered. This organization ensures that the product of each reaction goes directly to the next
enzyme in the cascade. This model, termed preferential substrate delivery, has been also proposed to explain the kinetics of
extracellular ATP hydrolysis in endothelial cells (15). The close
organization of ectoenzymes and receptors and the consequent channeling
of the compounds through the degrading cascade would account for the
poor effect of large molecules like ADA. It has been reported that
ectonucleotidases are clustered in caveolae, i.e. small
invaginations of plasma membrane (28). This organization could also
contribute to confine ATP and its catabolites so that ectoenzymes and
purinergic receptors would sense local and not bulk concentrations. It
is interesting to observe that our results clarify previously
unresolved observations done with patch-clamp experiments on 9HTEo
cells (26). Actually, we found that the activation of volume-sensitive Cl
channels by extracellular ATP could be inhibited by an
adenosine receptor antagonist (DMPX). We may now conclude that this
surprising behavior was because of the hydrolysis of ATP to adenosine,
which is the actual agonist. Such a conclusion reveals that the
mechanism of ATP hydrolysis by ectoenzymes might be relevant even
during patch-clamp experiments in which cells are continuously perfused with fresh solution.
We have demonstrated by an HPLC-based approach that ATP is indeed
released during the hypotonic shock (68% HS) in 9HTEo tracheal epithelial cells. Previous attempts to detect ATP by the luciferase assay were unsuccessful (11). We have no explanation for these previous
negative results. It is possible that rapid nucleotide hydrolysis and
the mechanism of preferential substrate delivery might reduce the
amount of ATP that diffuses from the cells into the bulk solution.
Interestingly, the ATP release does not occur when the milder hypotonic shock (83% HS) is applied. This behavior explains, at least in part, the quite different channel activity observed with the two hypotonic solutions. Indeed, with 68% HS, the taurine efflux is so fast that more than 50% of intracellular taurine is released in the first 5 min of stimulation (Fig. 1). On the contrary, with 83% HS, only 3% of taurine is released (Fig. 6). In other words, the relationship between the efflux and the extracellular osmolality is not linear, but a threshold for activation exists below 250 mosmol/kg. This threshold is probably because of the activation of ATP release only with strong hypotonic shocks. Actually, when exogenous adenosine is provided under mild hypotonic conditions (Fig. 6), the taurine efflux increases to levels comparable with those attained with 68% HS.
Intracellular Ca2+ increase by application of ionomycin in
isotonic conditions did not result in ATP efflux but caused a large release of inosine. However, combined stimulation with hypotonic solution (68% HS) and ionomycin elicited a stronger release of ATP if
compared with hypotonic shock alone. In parallel, extracellular adenosine was also increased. MeADP markedly decreased adenosine and caused the appearance of AMP in hypotonic conditions. This behavior
suggests that adenosine is not directly released by the cells but
probably arises from ATP hydrolysis. Inosine was not lowered by
MeADP, thus indicating that it does not derive from adenosine.
Actually, the effect of ionomycin in isotonic conditions suggests that
there is a separate Ca2+-dependent mechanism
for inosine release.
Our results do not match exactly those of other investigators (12).
First of all, we find that adenosine and not ATP is the autocrine
agonist of VSOAC during the hypotonic shock. Another difference lies in
the degree of channel activation, which can be accounted for by the
autocrine signaling. Actually, Wang et al. (12) reported
that treating hepatoma cells with ATP-degrading enzymes like apyrase
completely blocks the swelling-induced chloride channel activation. In
our experiments, the various treatments only partially inhibited the
effect of the stronger hypotonic shock. It appears therefore that in
9HTEo cells, adenosine and ATP release are not essential for
swelling-induced activation of VSOAC. However, the release of these
substances appears to play an important modulatory role. Our findings
suggest that important tissue-specific differences might exist in the
mechanism of VSOAC activation.
In conclusion, our experiments reveal that hypotonic shock induces a
release of ATP from airway epithelial cells. ATP is then converted on
the extracellular side of the membrane to adenosine, which binds to
adenosine receptors to increase the activity of volume-sensitive
chloride channels. This autocrine mechanism, which probably affects the
speed and extent of regulatory volume decrease (29), is likely
modulated by intracellular Ca2+. The regulation of ATP
release by cell volume and Ca2+ is probably important in
the process of transepithelial chloride transport. Indeed,
extracellular ATP represents a strong stimulus for chloride secretion
in airway epithelia (30). Several agonists, including ATP, induce an
intracellular Ca2+ increase in airway epithelial cells
(30-32). This response could elicit intracellular ATP release, which
would further stimulate the cells in an autocrine/paracrine fashion.
The sensitivity of the ATP release process to cell volume could help to
control the activation of chloride channels. Indeed, excessive
Cl channel activity and Cl
secretion would
cause cell shrinkage. This should stop the release of ATP, which
otherwise would continue to stimulate Cl
channels.
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ACKNOWLEDGEMENT |
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We thank Dr. Nora Marchese for technical advice.
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FOOTNOTES |
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* This work was supported by Telethon-Italy Grant E.593.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.
¶ To whom correspondence should be addressed: Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, Largo Gerolamo Gaslini 5, 16148 Genova, Italy. Tel.: 39-010-5636532; Fax: +39-010-3779797; E-mail: galietta{at}unige.it.
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ABBREVIATIONS |
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The abbreviations used are:
VSOAC, volume-sensitive organic anion channel;
HS, hypotonic solution;
FE, fractional efflux;
DPCPX, 8-cyclopentyl-1,3-dipropylxanthine;
DMPX, 3,7-dimethyl-1-propargylxanthine;
MeADP,
,
-methyleneadenosine 5'-diphosphate;
2MeSATP, 2-methylthioadenosine triphosphate;
ADA, adenosine deaminase;
NBMPR, S-(4-nitrobenzyl)-6-thioinosine;
AMP-PNP, 5'-adenylylimidodiphosphate;
HPLC, high performance liquid
chromatography;
PLC, phospholipase C.
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