Unité Mixte de Recherche Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, O6108 Nice Cedex 2, France
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ABSTRACT |
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Cl
currents induced by cell swelling were characterized in an immortalized
cell line (DC1) derived from rabbit distal bright convoluted tubule by
the whole cell patch-clamp techniques and by
125I
efflux experiments. Exposure of cells to
a hypotonic shock induced outwardly rectifying Cl
currents that could be blocked by 0.1 mM
5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM DIDS, and by 1 mM
diphenylamine-2-carboxylate. 125I
efflux
experiments showed that exposure of the monolayer to a hypotonic medium
increased 125I
loss. Preincubation of cells
with LaCl3 or GdCl3 prevented the development
of the response. The addition of 10 µM adenosine to the bath medium
activated outwardly rectifying whole cell currents similar to those
recorded after hypotonic shock. This conductance was inhibited by the
A1-receptor antagonist 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), LaCl3, or GdCl3 and was activated by
GTP
S. The selective A1-receptor agonist
N6-cyclopentyladenosine (CPA) mimicked the
effect of hypotonicity on 125I
efflux. The
CPA-induced increase of 125I
efflux was
inhibited by DPCPX and external application of LaCl3 or
GdCl3. Adenosine also enhanced Mn2+ influx
across the apical membrane. Overall, the data show that DC1 cells
possess swelling- and adenosine-activated Cl
conductances
that share identical characteristics. The activation of both
conductances involved Ca2+ entry into the cell, probably
via mechanosensitive Ca2+ channels. The effects of
adenosine are mediated via A1 receptors that could mediate
the purinergic regulation of the volume-sensitive Cl
conductance.
cell volume; kidney; A1 receptor
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INTRODUCTION |
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MOST ANIMAL
CELLS POSSESS volume-regulation mechanisms as a response to
changes in extracellular osmolarity (14). After cell
swelling, volume is restored by the loss of ions and other osmolytes,
whereas the concomitant loss of water induces a regulatory volume
decrease (RVD). Swelling-activated Cl channels have been
observed in a wide variety of cell types (6, 9). We have
previously demonstrated that proximal and distal convoluted tubules in
primary culture exhibit Cl
and K+ currents
when exposed to a hypotonic shock. Moreover, these cells are sensitive
to changes in osmolarity of the bathing medium and are capable of RVD
on exposure to hypotonic conditions (27, 31).
Although most reports in the literature agree that the movement of
Cl across the cell membrane is essential in this
regulation, the precise mechanism underlying the activation of
swelling-induced Cl
conductances is largely unknown. Of
the factors that could modulate these channels, Ca2+ plays
an important role, but conflicting results have been reported concerning the origin of Ca2+ in this mechanism. For
example, some studies have highlighted a positive role for cytoplasmic
Ca2+ due to its release from internal cellular stores
(11, 17), whereas other studies have indicated that
Ca2+ entry across the plasma membrane could be one of
the mediators of the hypotonicity-induced activation of
Cl
channels (18, 24).
In distal bright convoluted tubule cells (DCTb) in primary culture,
external Ca2+ is clearly implicated in the regulation of
volume-sensitive Cl channels (27). With this
in mind, we have undertaken studies to elucidate the mechanisms
governing this influx of Ca2+ from the extracellular medium
and have used the DC1 cell line immortalized from primary cultures of
DCTb for the purpose (28). These cells express both
A1 and A2 receptors, and adenosine can be shown
to activate an apical CFTR conductance via a pathway involving
A2A receptors and adenylate cyclase (28).
Moreover, this nucleoside also enhances an increase in calcium influx
via A1 receptors (25).
In the kidney, several observations indicate that A1
receptors decrease cAMP production but also enhance the inositol
phosphate cascade and the liberation of Ca2+ from internal
Ca2+ stores (1). Moreover, according to
Schwiebert et al. (29) adenosine could activate a
Cl channel implicated in the cell volume regulation of
the cortical collecting ducts. In view of these results, we have
carried out whole cell patch-clamp and 125I
efflux experiments to investigate whether the A1 receptor
could be implicated in the activation of a swelling-induced
Cl
conductance in the DC1 cell line. It can be shown here
that DC1 cells exhibit a swelling-activated Cl
conductance and express A1 receptors. Second, stimulation
of this receptor by adenosine activates a Cl
current
having properties of the swelling-activated Cl
conductance. The mechanism of this stimulation implicates
Ca2+ entry into the cell probably via mechanosensitive
Ca2+ channels.
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MATERIALS AND METHODS |
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Cultures
The DC1 renal cell line was obtained from primary cultures of rabbit distal convoluted tubules after transfection of cells with the PSV3 neoplasmid and G418 selection. The technique of transformation of primary cultures is described in a previous paper (28). Cultures were seeded on collagen-coated 35-mm petri dishes or on collagen-coated permeable Millipore filters filled with a culture medium composed of equal quantities of DMEM and Ham's F-12 (GIBCO BRL, Grand Island, NY). The medium was supplemented with 15 mM NaHCO3, 20 mM HEPES at pH 7.4, 2 mM glutamine, 5 mg/l insulin, 50 nM dexamethasone, 10 µg/l epidermal growth factor, 5 mg/l transferrin, 30 nM sodium selenite, and 10 nM triiodothyronine. Cultures were maintained at 37°C in a 5% CO2-95% air-water-saturated atmosphere. DC1 cells were used between passages 12 and 25.Whole Cell Patch-Clamp Experiments
Whole cell currents were recorded from DC1 cells (3-4 days of age) grown on collagen-coated supports and maintained at 33°C for the duration of the experiments. The ruptured-patch whole cell configuration of the patch-clamp technique was used. Patch pipettes (resistance 2-3 MVoltage-clamp commands, data acquisition, and data analysis were
controlled via a computer equipped with a Digidata 1200 interface (Axon
Instruments). pCLAMP software (versions 5.51 and 6.0; Axon Instruments)
was used to generate whole cell current-voltage
(I-V) relationships, with the membrane currents
resulting from voltage pulse stimuli filtered at 1 kHz, sampled at 2.5 kHz, and stored directly onto the computer's hard disk. Cells were
held at a holding potential of 50 mV, and 400-ms pulses from
100 to
+120 mV were applied in increments of 20 mV every 2 s.
125I Efflux from DC1 Monolayers
Calculation.
From back addition of the radioactivity in the efflux samples to the
radioactivity remaining in the cells, the apical and basolateral efflux
rate constants were calculated according to the following equations,
which provide the fraction of total radioactivity lost per unit time
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Fluorescence Experiments
Image analysis was performed by using an optical system composed of a Zeiss ICM405 inverted microscope and a Zeiss ×40 objective for epifluorescent measurements with a 75-W xenon lamp. The excitation beam was filtered through narrow-band filters (350, 360, and 380 nm, Oriel) mounted in a motorized wheel (Lambda 10-2, Sutter Instrument) equipped with a shutter that controlled exposure times. The incident and emitted fluorescence radiation beams were separated by using a Zeiss chromatic beam splitter. Fluorescence emission was detected via a 510-nm narrow-band filter (Oriel). The transmitted light images were viewed by an 8-bit extended ISIS camera (Extended ISIS, Photonic Science, Sussex, UK) equipped with an integration module to maximize the signal-to-noise ratio. The video signal from the camera was fed into an image processor integrated with a DT2867 image card (Data Translation) installed in a Pentium 100-MHz PC. The processor converted the video signal into 512 lines of 768 square pixels/line and 8 bits/pixel. The 8-bit information obtained for each pixel represented one of a possible 256 gray levels, ranging from 0 (for black) to 255 (for white). Image acquisition and analysis were performed by using AIW software (version 2.1, Axon Instruments). Final calculations were made by using Excel software (Microsoft).Intracellular Ca2+ measurements were carried out on 4- to 8-day-old DC1 cell monolayers grown in petri dishes and loaded for 45 min at room temperature with a solution of 5 µM fura 2-acetoxymethyl ester (AM) containing 0.01% pluronic acid. The cells were then washed with a NaCl solution containing (in mM) 140 NaCl, 5 KCl, 1 MgSO4, 5 glucose, and 20 HEPES, pH 7.40, with 1 Tris. Cells were successively excited at 350 and 380 nm, with images digitized and stored on the computer hard disk for later analysis. Each raw image was the result of an integration of four to five frames averaged four times. The acquisition rate used was one image every 10 s. For each monolayer, intracellular Ca2+ was monitored in 18-20 random cells. Ca2+ concentrations ([Ca2+]i) were calculated from the dual wavelength-to-fluorescence ratio by using the Grynkiewicz equation (13).
Mn2+ influx measurements were made on 4- to 8-day-old DC1 cell monolayers grown in petri dishes and loaded with fura 2-AM as described above. Fluorescence quenching experiments were carried out by the addition of 50 µM MnCl2 to the NaCl medium. Fluorescent measurements were performed at 360 nm (the isosbestic point for fura 2, where the fluorescence signal is independent of the free calcium concentration). The level of Mg2+ influx was quantified by the slope of the calibration line describing quenching kinetics. Each raw image was the result of an integration of six frames averaged four times. The acquisition rate was one image every 5 s.
Chemicals
Adenosine was prepared as a 10 mM stock solution in NaCl buffer. N6-cyclopentyladenosine (CPA) and 8-cyclopentyl-1,3-diproxylxanthine (DPCPX; Sigma) were prepared as 10 mM stock solutions in DMSO. A stock solution of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; Calbiochem) was prepared at 100 mM in DMSO and used at a concentration of 0.1 mM in final solutions. Diphenylamine-2-carboxylate (DPC; Aldrich) was prepared as 1 M stock solution in DMSO and dissolved at 1 mM in the incubation medium. DIDS (Sigma) was dissolved in the final solutions at a concentration of 1 mM. Fura 2-AM (Molecular Probes) was dissolved at 3 mM in DMSO and added to the loading solution at a final concentration of 5 µM plus 0.01% pluronic acid.Solutions
The compositions of the different solutions used in these experiments are given in Table 1.
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RESULTS |
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Effects of Hypotonic Shock on Cl Permeability of
Cultured DC1 Cells
Whole cell experiments.
Nature of the Cl current induced by hypotonicity.
Whole cell currents were recorded with Ca2+-free pipette
solutions containing 140 mM NMDGCl, whereas hyperosmotic extracellular solutions contained 140 mM NMDGCl and 50 mM mannitol. After successful gigaseal formation, the whole cell configuration was obtained in 25%
of cases. Voltage-clamp experiments were performed by holding the
membrane potential at
50 mV and then applying voltage steps of 400-ms
duration every 2 s from
100 to 120 mV in 20-mV increments. When
the osmolarity of the extracellular solution was 350 mosmol/kgH2O, the voltage-step protocol elicited small
currents (Fig 1A) that changed
linearly with the membrane voltage and had a slope conductance of
0.57 ± 0.02 nS and a reversal potential of
1.5 ± 0.9 mV
(Fig 1D). The amplitude of the currents was 69 ± 11 pA
(n = 15) at +100 mV. Because of its small amplitude,
the nature of the current was not analyzed further, although its
reversal potential suggests that Cl
may well have been
the charge carrier.
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125I efflux experiments.
Effect of DC1 cell exposure to a hypotonic medium.
The presence of a Cl
conductance activated by hypotonic
shock was also assayed by using I
efflux measurements. In
one set of experiments, DC1 cells were grown on collagen-coated petri
dishes, and apical effluxes were measured after the cells were loaded
with 125I
. Figure
5 presents the
125I
efflux rate constant (expressed as a
percentage of the initial value at time t = 1 min) as a
function of time. Initial effluxes, measured in the isotonic control
medium, were independent of time, the efflux rate remaining constant at
5.03 ± 0.12 · 10
2/min (mean ± SE,
n = 70). 125I
efflux
variations were then examined when cultured monolayers were exposed to
a hypotonic medium (290 mosmol/kgH2O), which was obtained
by diluting the control solution. Figure 5A shows that exposure of the monolayers to a hypotonic medium led to an increase in
125I
efflux, which reached a maximum level
3-4 min after onset of the exposure. The mean increase in
the 125I
efflux was 246.9 ± 37.7%
(n = 9).
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Measurement of [Ca2+]i During Hypotonic Shock
We reasoned that the presence of EGTA in the pipette solution did not prevent localized increases in Ca2+ in the vicinity of the plasma membrane secondary to a Ca2+ influx. Fluorescence experiments using fura 2-loaded DC1 cells were therefore carried out to follow cytosolic Ca2+ variations during a hypotonic shock. Figure 6 shows the effect of hypotonic solution on intracellular calcium (i.e., [Ca2+]i) under different experimental conditions. When the cells were bathed with an isotonic NaCl solution (300 mosmol/kgH20) containing 1 mM CaCl2, the resting [Ca2+]i averaged 30.8 ± 5.2 nM (n = 288 cells from 16 monolayers). Swelling the DC1 cells with hypotonic NaCl solution (200 mosmol/kgH2O) induced a transient increase in [Ca2+]i, which reached a maximum value of 120.1 ± 25.1 nM and returned close to the control value within 5 min (Fig. 6A). After the cells were rinsed with isotonic NaCl solution, a second hypotonic shock could be reinduced. However, the amplitude of the [Ca2+]i increase was systematically reduced with [Ca2+]i reaching 71.4 ± 10.5 nM (Fig. 6A). Overall, the results in Fig. 6 show that hypotonic shock induced a significant increase in [Ca2+]i in 75 ± 4% of the 230 cells analyzed in 13 different monolayers. In the experiments in Fig. 6, B and C, the second hypotonic shock was performed in the presence of La3+(50 µM) or Gd3+ (400 µM) respectively. Under these conditions, the second shock did not evoke [Ca2+]i changes in all the cells of the three monolayers studied. These observations strongly suggest that the increase in [Ca2+]i induced by hypotonicity required a Ca2+ influx from the extracellular medium. To eliminate the participation of intracellular Ca2+ store release in the swelling-induced [Ca2+]i increase, experiments were then performed in the presence of thapsigargin. As illustrated in Fig. 6D, emptying intracellular Ca2+ pools did not prevent the increase in [Ca2+]i during the hypotonic shock.
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Effects of Adenosine and P1 Purinoceptor Agonists on
Cl Permeability of Cultured DC1 Cells
Whole cell experiments.
Nature of the Cl current induced by adenosine in
cultured DC1 cells.
In 40% of the trials carried out, adenosine activated a linear cystic
fibrosis transmembrane conductance regulator (CFTR)-like Cl
conductance (28). In 30% of the trials,
adenosine (10 µM) activated an outwardly rectifying Cl
conductance with a time-dependent inactivation at depolarizing potentials and with a maximal effect at 3-4 min (Fig.
7B). At this time, the initial
current recorded at 100 mV was 1.9 times the current measured at
100
mV (at 100 mV, ICl
= 479.3 ± 27.4 pA; at
100 mV, ICl
=
256.5 ± 16.2 pA, n = 18). The reversal
potential of the stimulated current was
1.16 ± 3.4 mV
(n = 18). The I-V relationships
for initial currents measured 11 ms after the onset of the voltage
pulse are illustrated in Fig. 7E.
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125I efflux experiments.
Because we have previously shown that adenosine causes a biphasic
increase in 125I
efflux in DC1 cells,
probably due to the stimulation of both A1 and
A2 receptors (27), it was decided to study the
effect of CPA on 125I
efflux. CPA is a
metabolically stable adenosine analog that is a selective
A1-receptor agonist. The increase in the rate of
125I
efflux induced by CPA was slow, reached
a maximum level equal to 166 ± 12% of control (n = 9) after 3-min exposure to the agonist, then slowly returned to the
control level within 5 min. (Fig. 11A). Overall, the kinetics
of this increase were very similar to those evoked by the exposure of
cells to hypotonic medium. The CPA-induced
125I
efflux was inhibited effectively by DIDS
(1 mM) and DPC (1 mM) (Fig. 11A).
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Measurement of [Ca2+]i During Adenosine Application
Experiments were then undertaken to describe the effect of exogenous adenosine on [Ca2+]i. In these experimental series, the [Ca2+]i of cultured DC1 cells maintained in a bathing solution with 1 mM Ca2+ was 51.9 ± 2.4 nM (n = 720 cells from 37 monolayers). As shown in Fig. 12A, the addition of 10 µM adenosine in NaCl buffer evoked a transient increase in [Ca2+]i, which took place within 20 s (maximum [Ca2+]i = 223.3 ± 6.4 nM, n = 171 cells from 12 monolayers). This response was observed in 80% of the analyzed cells (171 of 214 cells). Ten minutes after adenosine removal, a second application of 10 µM adenosine was unable to induce a second [Ca2+]i increase (Fig 12A). The experiments in Fig. 12, B and C, show that in the presence of La3+(50 µM) or Gd3+ (400 µM), adenosine did not increase [Ca2+]i. As already concluded for the swelling increase in [Ca2+]i, the present results indicate that the adenosine-induced increase in [Ca2+]i was dependent on a Ca2+ influx from the extracellular medium.
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To confirm that the stimulatory action of adenosine on [Ca2+]i was mediated by the P1 receptor, additional experiments were performed in the presence of the P1-selective receptor antagonist DPCPX. As shown in Fig. 12D, treatment of the monolayer with DPCPX (10 µM) completely inhibited the action of 10 µM adenosine.
In another series, monolayers were stimulated with 10 µM adenosine then, 7-8 min after removal of the nucleoside from the bath solution, the cells were submitted to an hypotonic shock. Under these conditions, the hypotonic shock did not evoke an increase in [Ca2+]i (Fig. 12E).
To verify that after addition of adenosine cells were still responsive, the effects of 10 µM ATP were examined 30 s after the adenosine was added to the cells. The results in Fig. 12F clearly show that this subsequent addition of ATP induced a rapid and transient increase in [Ca2+]i. This increase was significantly higher than the one measured during adenosine stimulation (adenosine: [Ca2+]i = 193.0 ± 23 nM; ATP: [Ca2+]i = 291.2 ± 29 nM, P < 0.001, n = 54 cells from 3 monolayers).
In a last group of experiments, the stimulatory responses of a
lower dose of adenosine were studied. The recordings in Fig. 13A illustrate the
[Ca2+]i variations induced by 1 µM
adenosine. As observed with higher concentration, the addition of 1 µM adenosine induced a transient increase in
[Ca2+]i (maximum
[Ca2+]i = 110.4 ± 10.3 nM,
n = 102 cells from 6 monolayers). However, a subsequent
addition of 1 µM adenosine still induced an increase in
[Ca2+]i (maximum
[Ca2+]i = 114.0 ± 10.8 nM,
n = 102 cells from 6 monolayers). These successive
increases were completely abolished by the application of
Gd3+ (Fig. 13B) or nifedipine (Fig.
13C). Moreover, as illustrated in Fig. 13D, a
first stimulation of [Ca2+]i with 1 µM
adenosine could be followed by a stimulation induced by a hypotonic
shock and then by a new stimulation with 1 µM adenosine.
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Mn2+ Influx Induced by Adenosine
The experiments described above indicated that extracellular Ca2+ was required to activate adenosine Cl
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Effects of ATP on Cl Currents in Cultured DC1
Cells
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To analyze the nature of the receptor implicated in the ATP-induced
Cl currents, experiments were performed in the presence
of purinergic receptor antagonists. The results are reported in Fig.
16. When the Cl
currents
were measured in the absence of EGTA in the pipette solution, the
effect of ATP was completely blocked by 100 µM of the P2-antagonist
suramin whereas the action of ATP was not modified by 10 µM of the
P1-antagonist DPCPX (Fig. 16A). By contrast, when the
Cl
currents were recorded in the presence of 5 mM EGTA in
the pipette solution, the effect of ATP was inhibited by DPCPX and not
modified by suramin (Fig. 16B). Finally, two types of
Cl
currents could be activated by ATP: Ca2+
dependent-like Cl
currents and swelling-sensitive-like
Cl
currents.
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DISCUSSION |
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An increase in cell volume activates a Cl current in
most mammalian cells. Cell swelling can be induced by challenging cells with either a hypotonic extracellular solution or a hypertonic intracellular solution. The present work describes a
hypotonicity-induced Cl
conductance that shares similar
biophysical and pharmacological features with that described in primary
cultures of DCTb cells (27) and in other epithelial cells
(12). Notably, the I-V relationship
is outwardly rectifying, and a time-dependent inactivation is observed
at the most positive membrane potentials (i.e., +80 mV).
The halide selectivity sequence of this current permits the different
types of Cl channels to be identified. In DC1 cells, the
sequence for the hypotonicity-induced Cl
current was
I
= Br
> Cl
, which
is similar to that found in DCTb cells. The sensitivity to various
anion channel blockers is also an indication of the nature of the
channels. DIDS, NPPB, and DPC were found to inhibit the
hypotonicity-induced Cl
current
In contrast to primary cultures of microdissected DCTb cells, the DC1
cell line has the advantage of generating a high yield of cells, thus
making it possible to obtain sufficiently large surface areas of
epithelial cell layers for the effective measurement of tracer
effluxes. In epithelial cells, 125I fluxes
are believed to be a good indicator of Cl
ion movements
across the cell membrane. When cultured monolayers were exposed to a
hypotonic medium (290 mosmol/kgH2O), an increase in
125I
efflux, which reached a maximum level
3-4 min after exposure to the solution, was elicited. This
increase was inhibited by Cl
channel blockers such as
DIDS and DPC. Finally, both experimental approaches lead to clearly
demonstrate the existence of swelling-activated Cl
permeability in cultured DC1 cells.
Despite abundant literature, the precise mechanisms underlying
the activation of swelling-activated Cl currents remain
unclear. In the present study the finding that nifedipine,
La3+, and Gd3+ strongly blocked both the
125I
efflux and the Cl
currents
induced by the exposure of cells to a hypotonic solution indicated the
involvement of external Ca2+ in the response. Thus it was
interesting to check the effect of nucleotides in the development of
Cl
currents because these molecules are known to modulate
[Ca2+]i by controlling both Ca2+
entry and Ca2+ release from intracellular pools. For
example, adenosine is an effector molecule that modulates cAMP
production and intracellular Ca2+ in various tissues
(25). Moreover, it has been proposed that this nucleoside
could participate in the regulation of Cl
secretion in
several types of epithelia, including kidney epithelia (5, 21,
29). Notably, we have previously shown the existence of
A1 and A2 receptors in the basolateral membrane
of DC1 cells, whereas adenosine has also been shown to activate an
apical CFTR Cl
conductance in these cells via a pathway
involving A2A receptors, G proteins, adenylate cyclase, and
protein kinase (28). In this previous work, preliminary
experiments clearly indicated that, besides the activation of CFTR
Cl
currents, adenosine increased a second type of
Cl
conductance, the nature of which remained to be
determined. We therefore examined in the present study, the effect of
adenosine via A1 receptors on Cl
conductance
in DC1 cells. As can be seen from the results presented, the exposure
of DC1 cells to adenosine was followed within 1-2 min by an
increase in whole cell currents. In most cells, the adenosine-stimulated currents reversed near 0 mV, were outwardly rectifying, and exhibited time-dependent inactivation at depolarized membrane potentials. It could be concluded that the activation of an
outwardly rectifying Cl
current by adenosine was mainly
due to activation of the adenosine A1 receptor because the
antagonist DPCPX blocked the activation of adenosine-evoked
Cl
currents. Furthermore, these currents were also
dependent on Ca2+ influx because they were totally
inhibited in the presence of nifedipine, La3+, or
Gd3+. I
efflux experiments confirmed the data
obtained by using the whole cell patch-clamp technique.
Overall, the present experiments demonstrated that the A1
adenosine receptor could mediate the purinergic regulation of the volume-sensitive Cl conductance. This hypothesis is
strengthened by the following observations: first, the biophysical
properties of Cl
permeability induced by adenosine were
very similar to those of hypotonicity-activated Cl
currents; second, the A1-receptor antagonist DPCPX blocked
the activation of Cl
currents by adenosine and the
activation of 125I
efflux by CPA but also the
swelling-induced Cl
currents; third, the selective
A1-receptor agonist CPA (25) mimicked the
effect of hypotonic shock on 125I
efflux; and
fourth, adenosine and swelling-activated Cl
currents were
mediated by Ca2+ influx through the cell membrane. The
presence of A1 and A2 adenosine receptors in
renal tissue has been demonstrated in the perfused thick ascending limb
(3) and extensively studied in several cell lines or
primary cultures (2, 5, 21, 29, 30). However, the
physiological role of these receptors in the kidney is not fully
understood. In rabbit renal cortical collecting duct cells
(29), as well as in A6 cells (5) in culture,
adenosine was found to activate an apical Cl
channel via
a pathway involving A1 receptors, whereas in the rat
medullary thick ascending limb adenosine was described as a potent
inhibitor of Cl
reabsorption (3).
A1 receptors also play an important role in the regulation
of distal nephron Na+ reabsorption, although contrasting
effects have been reported in the literature. For example,
A1 stimulation by adenosine increased (16) or
did not modify (5) Na+ reabsorption in A6
cells, whereas it was reported to inhibit Na+ reabsorption
in primary cultures of rat inner medullary connecting tubule
(3).
In several studies, an increase in cytosolic Ca2+ has been
reported during hypotonic stress or during A1 receptor
stimulation by adenosine (17, 18, 25). In the present
study, adenosine and hypotonic shock increased
[Ca2+]i. This increase was suppressed by
Ca2+ channel blockers and by DPCPX, indicating that the
action of both stimuli is mainly due to Ca2+ entry into the
cell and depends on the integrity of the A1 receptor. On
the basis of these results, it could be concluded that the increase in
Cl conductance observed during hypotonicity could be
mediated by Ca2+ influx via A1 receptor
stimulation. Nevertheless, it may be recalled that in the present
study, the activation of a Cl
conductance by hypotonicity
or by adenosine took place in the presence of a high concentration of
EGTA in the pipette solution. The simplest interpretation of this
finding could be that an increase in [Ca2+]i
is not necessarily required to activate Cl
currents and
that Ca2+ influx is sufficient per se to increase the
Cl
conductance during hypotonicity or adenosine
stimulation. However, it is also possible that the cells still
responded by increasing their [Ca2+]i when
the pipette solution contained 5 mM EGTA. The observations of Evans and
Marty (8) shed light on this problem by indicating that
with EGTA as a buffer a whole region of the cell could escape control
by the Ca2+ buffer. Because this region could extend to a
large part of the plasma membrane (8), a local transient
increase in Ca2+ could arise in the presence of EGTA.
It remains that Ca2+ influx is an essential step in
activating swelling- and adenosine-sensitive Cl channels.
To further support this hypothesis, we studied the effects of ionomycin
and ATP in the activation of Cl
channels in hypertonic
bathing medium. Both molecules are known to induce a strong increase in
[Ca2+]i. When applied on DC1 cells, they
induced two types of Cl
conductances depending on the
presence of high EGTA concentrations in the pipette solution. In the
absence of a chelator, the Cl
currents induced by
ionomycin or ATP were roughly identical to the Ca2+
dependent Cl
currents previously found in cultured DCTb
(3). These currents were likely stimulated by an increase
in cytoplasmic Ca2+ due to Ca2+ release from
intracellular stores. By contrast, in the presence of EGTA, the
application of ionomycin or ATP increased the uptake of
Mn2+. This increase is evidence of Ca2+ uptake
across the membrane (5, 26). This maneuver induced large
Cl
currents, which inactivated during depolarizing
voltage steps. Although they are not fully characterized, these
currents closely resemble the swelling-activated Cl
currents described in the present study. Finally, these data bring
further evidence that Ca2+ entry across the plasma membrane
could be one of the mediators of the activation of Cl
currents by hypotonicity. Moreover the fact that Gd3+
completely inhibited Cl
conductance activation and
[Ca2+]i increase by exposure to hypotonic
solution or adenosine suggests that Ca2+ influx occurs
through mechanosensitive channels.
Stretch-activated Ca2+-permeable channels have been
described in many epithelial cells (7), including those of
the proximal tubule (10). The role of external
Ca2+ has already been postulated by McCarty and O'Neil
(18), who showed that RVD in proximal straight tubules was
highly dependent on the extracellular Ca2+ concentration.
Moreover, they concluded that Ca2+ channels could be
responsible for a swelling-activated Ca2+ entry.
Interestingly, a relationship among mechanical stress, Ca2+
entry, and Cl currents has been recently demonstrated in
cultured endothelial cells (23).
The experiments using GTPS showed that G proteins were involved in
the control of the outwardly rectifying Cl
conductance in
DC1 cells. This result is in accordance with that of Schwiebert et al.
(29), who reported that the stimulation of A1
receptors in a rabbit cortical collecting duct cell line activates a
volume-sensitive Cl
channel, via a pathway involving
phospholipase C, protein kinase C, and a G protein (29).
Finally, in addition to the mechanism involving adenylate cyclase and
cAMP, adenosine might act via an alternative transduction mechanism in
DC1 cells to activate a volume-sensitive Cl conductance.
It is possible that a mechanosensitive Ca2+ entry is the
pathway for Cl
conductance activation in response to
hypotonic shock or A1 purinoceptor stimulation.
If A1 purinergic receptors are linked to swelling-activated
Cl conductance, it remains to be understood how these
receptors are stimulated during hypotonic cell swelling. It has been
recently postulated that hypotonically induced ATP release could induce an autocrine stimulus for activating ion channels permeable to Cl
and organic osmolytes (19, 22). In the
present study, ATP also induced swelling-induced-like Cl
current in the presence of 5 mM EGTA in the pipette solution. Interestingly, these currents were blocked by DPCPX but were
insensitive to suramin, indicating that under these conditions the
action of ATP was not through P2 receptors. The best explanation is
that ATP could be degraded to adenosine, which then acts on the
A1 receptors to activate Cl
conductance. This
hypothesis matches with that of Musante et al. (22), who
have demonstrated that hypotonic shock induces an autocrine release of
ATP from airway cells. According to these authors, ATP is then
hydrolyzed to adenosine on the extracellular side of the membrane,
increasing the activity of volume-sensitive Cl
channels.
Such a mechanism could well serve in DC1 cells because renal cells
exhibit a high level of ectonucleotidases (15).
In our experiment, external ATP also induced Ca2+-sensitive
Cl currents via P2 receptors. However, although DC1 cells
exhibited both A1 and P2 receptors (4, 26),
hypotonic shock or adenosine application induced swelling
Cl
currents only (data not shown), irrespective of the
EGTA concentration in the pipette solution. It is therefore reasonable
to postulate that ATP released during shock does not activate
Ca2+-sensitive Cl
currents. Finally, in DC1
cells the activation of Ca2+-induced Cl
currents by ATP could be due to the release of Ca2+ from
internal stores (4, 26) whereas activation of adenosine or
swelling-sensitive Cl
conductance would be mainly due to
Ca2+ influx.
In our study, two successive hypotonic shocks induced a
[Ca2+]i increase as well as an activation of
Cl conductance. By contrast, a double application of 10 µM adenosine did not evoke a second rise in
[Ca2+]i a second Cl
conductance
increase. This inability of adenosine (10 µM) to trigger two
successive responses could be the consequence of a desensitization of
A1 receptors at micromolar nucleotide concentrations (16). However, the possibility of obtaining a second
response to a hypotonic shock indicates that a part of the
A1 receptor population remained potentially active. We
hypothesized therefore that the quantity of adenosine produced during
the first hypotonic shock was too weak to saturate the receptors. This
idea is further supported by the fact that a second Ca2+
response was restored with a low adenosine concentration (1 µM).
At this stage, the question arises as to whether the data obtained with
DC1 cell line could be extrapolated to the original DCTb in primary
culture. Concerning volume-sensitive Cl conductance,
cultured DC1 and DCTb cells exhibited a hypotonicity-sensitive increase
in whole cell Cl
conductance. The biophysical features of
this Cl
conductance were closely identical in both cell
types. Concerning the role of adenosine, experiments performed on
cultured DCTb cells show that extracellular application of adenosine
activated an outwardly rectifying Cl
current in 12% of
the trial, the characteristics of which resembled those observed in DC1
cells. Taken together, these observations indicated that the DC1 cell
line is representative of a primary culture of DCTb cells.
The physiological role of A1 receptors in the distal tubule
has not yet been elucidated. However, adenosine might regulate cell
volume by controlling the swelling-activated Cl
conductance. Schwiebert et al. (29) have proposed that,
during ischemia, the release of adenosine could play an important role in cell volume regulation in the collecting tubule. This proposal could
also be available in the distal tubule because we have demonstrated that distal cells exhibited swelling-activated Cl
and
K+ channels implicated in the RVD phenomenon
(27).
In conclusion, our study provides evidence that A1
receptors play an important role in the regulation of swelling-induced Cl currents. It is suggested that hypotonic shock
stimulates ATP release from the DC1 cells. A1 receptors are
then activated by adenosine generated by the degradation of ATP by
membrane ectoenzymes. This stimulation of A1 receptors
induces an influx of extracellular Ca2+ through
Gd3+-sensitive Ca2+ channels. Finally, the
Ca2+ influx activates the Cl
channels. The
mechanism of this activation is not fully elucidated. However,
according to Verdon et al. (32), the Ca2+
influx during cell swelling would increase the
[Ca2+]i in the vicinity of the cell membrane
[this region of the cell escapes control by the Ca2+
buffer (8)]. This increase will be sufficient to activate protein kinase C. The protein kinase C, in turn, phosphorylates a
regulatory protein. This protein could be the P-glycoprotein which is
implicated in the control of swelling-sensitive Cl
conductance. Such a mechanism has been also proposed by Rubera et al.
(27) in DCTb cells in primary culture.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Poujeol, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6548, Bâtiment Sciences Naturelles Université de Nice-Sophia Antipolis, Parc Valrose, O6108 Nice Cedex 2, France (E-mail: poujeol{at}unice.fr).
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 10 February 2000; accepted in final form 18 September 2000.
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