Laboratoire de Physiologie Cellulaire, Institut National de la Santé et de la Recherche Médicale EPI 9938, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France
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ABSTRACT |
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Patch-clamp recordings were used to study ion
currents induced by cell swelling caused by hypotonicity in human
prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl was the primary
charge carrier (termed ICl,swell). The
selectivity sequence of the underlying volume-regulated anion channels
(VRACs) for different anions was
Br
I
> Cl
> F
> methanesulfonate
glutamate, with relative
permeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currents
as well as single-channel currents showed moderate outward
rectification. Unitary VRAC conductance was determined at 9.6 ± 1.8 pS. Conventional Cl
channel blockers
5-nitro-2-(3-phenylpropylamino)benzoic acid (100 µM) and DIDS (100 µM) inhibited whole cell ICl,swell in a voltage-dependent manner, with the block decreasing from 39.6 ± 9.7% and 71.0 ± 11.0% at +50 mV to 26.2 ± 7.2% and
14.5 ± 6.6% at
100 mV, respectively. Verapamil (50 µM), a
standard Ca2+ antagonist and P-glycoprotein function
inhibitor, depressed the current by a maximum of 15%. Protein tyrosine
kinase inhibitors downregulated ICl,swell
(genistein with an IC50 of 2.6 µM and lavendustin A by
60 ± 14% at 1 µM). The protein tyrosine phosphatase inhibitor
sodium orthovanadate (500 µM) stimulated
ICl,swell by 54 ± 11%. We conclude that
VRACs in human prostate cancer epithelial cells are modulated via
protein tyrosine phosphorylation.
volume-regulated chloride channels; tyrosine kinase; cell volume
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INTRODUCTION |
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NUMEROUS
CELLULAR PHYSIOLOGICAL processes such as active solute uptake
(24), fluid and electrolyte secretion (27),
hormonal action (15), proliferation (10),
differentiation (51, 52), and apoptosis (3)
are associated with osmotic perturbation and regulation of cell volume.
It is now clear that multiple mechanisms participate in the maintenance
of cell volume (for reviews, see Refs. 16 and 22). The primary event
during regulatory volume decrease seems to be the activation of
volume-regulated anion channels (VRACs). Despite the vast amount of
experimental data about volume-regulated Cl channels
accumulated in recent years, it is still not clear whether expression
of these channels is a common feature of all cells or whether changes
in their expression patterns reflect a special developmental and/or
functional state. Furthermore, the VRAC activation and regulation
mechanisms are not clearly understood, and findings have been
contradictory (for reviews, see Refs. 28 and 34). A wide variety of
factors have been proposed as modulators for these channels: G proteins
(40, 50), arachidonic acid and its metabolites (9,
12), phosphorylation reactions involving Ca2+/calmodulin-dependent protein kinase II
(17), protein kinase C (PKC; Ref. 38), protein kinase A
(14), tyrosine kinase (47), cytoskeleton
(4), and gene expression (55). However, no
ubiquitous modulating factor for volume-sensitive Cl
channels themselves, or their volume sensors, has yet been demonstrated.
Volume-regulated Cl channels have been observed in
several cell types: epithelial cells, fibroblasts, chromaffin cells,
endothelial cells, melanoma cells, and so forth (for reviews, see Refs.
28 and 34). The study of these channels in cancer cells has generated particular interest because of their possible involvement in the process of tumorigenesis and transformation. This is shown, for instance, by their strong upregulation in cervical cancer cells compared with normal cells (7) and by their downregulation after the cells switch from a proliferating to a nonproliferating differentiated state (for reviews, see Refs. 28 and 29).
Prostate cancer is the second leading cause of cancer-related deaths in men (53). However, despite the magnitude of the problem, relatively little is known about the basic biology and growth regulation of prostate epithelial cells. Many of the basic properties of prostate carcinogenesis, including the role of membrane ion channels, can be investigated with the use of simple in vitro models of transformed human prostate cells. One of these models is the androgen-sensitive human prostate cancer cell line LNCaP (lymph node carcinoma of the prostate), which was derived from a lymph node of a subject with metastatic carcinoma of the prostate (18). This cell line retains many of the characteristic properties of human prostate carcinoma such as androgen-dependent growth, the presence of androgen receptors, the production of acid phosphatase, and so forth (11).
We have previously shown that LNCaP cells express a novel type of
K+ channel that is likely to play an essential role in the
physiology of these cells and, more specifically, in cell proliferation
(41, 42). In this research, whole cell and single-channel
patch-clamp techniques were used to identify and characterize the
volume-regulated Cl channels in human prostate cancer
cells for the first time. We also show that these channels are
regulated through tyrosine phosphorylation. With the consideration that
the proliferation and apoptosis of prostate cancer cells are under
hormonal control and that osmotic changes often accompany these
processes, the study of volume-sensitive anion channels is likely to
provide important information for understanding the growth mechanisms
of prostate tumors.
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MATERIALS AND METHODS |
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Cell cultures. LNCaP from the American Type Culture Collection were grown in RPMI 1640 (Biowhittaker, Fontenay sous Bois, France) supplemented with 5 mM L-glutamine (Sigma Chemical, L'Isle d'Abeau, France) and 10% fetal bovine serum (Seromed, Poly-Labo, Strasbourg, France). The culture medium also contained 50,000 IU/l penicillin and 50 mg/l streptomycin. Cells were routinely grown in 50-ml flasks (Nunc, Poly-labo) and kept at 37°C in a humidified incubator in a 95% air-5% CO2 atmosphere. For electrophysiological experiments, the cells were subcultured in petri dishes (Nunc) coated with polyornithine (Sigma, 5 mg/l) and used after 4-6 days.
Electrophysiology and solutions.
Macroscopic membrane ion currents in LNCaP cells (average whole cell
membrane capacitance of 25.5 ± 1.2 pF; n = 46)
were recorded using the patch-clamp technique in its whole cell
configuration. The compositions of the isotonic (310 mosM) and
hypotonic (190 mosM) extracellular solutions are presented in
Table 1. The basic intracellular pipette
solution (osmolarity of 290 mosM) contained (in mM) 100 K(OH), 40 KCl,
1 MgCl2, 10 HEPES, and 10 EGTA, pH 7.2 (adjusted with
glutamic acid). Unless otherwise specified, this solution was also
supplemented with 5 mM MgATP. The resistance of the pipette varied
between 3 and 5 M. Necessary supplements were added directly to the
respective solutions, in concentrations that could not significantly
change the osmolarity. Changes in the external solutions were carried
out using a multibarrel puffing micropipette with common outflow that
was positioned in close proximity to the cell under investigation.
During the experiment, the cell was continuously superfused with the
solution via puffing pipette to reduce possible artifacts related to
the switch from static to moving solution and vice versa. Complete
external solution exchange was achieved in <1 s.
5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), DIDS, 8-bromo-cAMP
(8-BrcAMP), and verapamil were obtained from Sigma, whereas genistein,
lavendustin A, and sodium orthovanadate were obtained from Calbiochem.
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RESULTS |
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Evidence for the Cl nature of hypotonicity-evoked
current.
In our previous studies on membrane ion conductances in LNCaP cells,
only one type of voltage-dependent conductance, i.e., K+
conductance, was identified (41, 42). However, exposure of the cell to the normal external solution with 40% reduced osmolarity (due to a 2-fold reduction in NaCl) initiated development of an additional leaklike current without affecting the depolarization-evoked K+ current. A novel leaklike current, activated within
1-2 min after application of hypotonic solution, required
~5-10 min to fully develop and was preceded by considerable cell
swelling. The reversal potential of this current (
16 ± 3 mV;
n = 4) was very close to the estimated theoretical
equilibrium potential for Cl
(
17 mV), suggesting that
Cl
was the major charge; this current was therefore
called "ICl,swell."
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Pharmacology.
Pharmacological sensitivity of
ICl,swell was examined with the use of
NPPB, DIDS, and verapamil. The first two agents are conventional anion
transport inhibitors, whereas verapamil is not only a standard
Ca2+ channel antagonist but is also known as an inhibitor
of P-glycoprotein-mediated drug transport (34, 49). Figure
2, A and B, shows
fully developed raw currents as well as respective ramp-derived
current-voltages from a typical cell exposed to hypotonic solution
before and during application of 100 µM NPPB. At 100 µM, NPPB
blocked 39.6 ± 9.7% (n = 13) of the +50-mV
current. Plotting the percentage of current block against membrane
potential (Fig. 2B, inset) showed that inhibition
by NPPB is slightly voltage dependent, decreasing to 26.2 ± 7.2%
(n = 13) at 100 mV. In contrast, the blocking of ICl,swell by 100 µM DIDS was strongly voltage
dependent (Fig. 2, C and D). At +50 mV, DIDS
produced 71.0 ± 11.0% (n = 5) inhibition, which
stayed almost constant over the entire range of positive membrane
potentials, whereas inhibition sharply decreased at membrane potentials
below 0 mV, constituting only 14.5 ± 6.6% (n = 5) at
100 mV (Fig. 2D, inset). Verapamil (50 µM) produced only a minor (under 15%) voltage-independent reduction
in ICl,swell (Fig. 2, E and
F).
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Modulation.
Given that cytosolic ATP dependency is one of the common features of
VRACs (34), a nominally ATP-free pipette solution was used
to test the possible effects of ATP on ICl,swell
activation in LNCaP cells. Despite significant variability in responses
from individual cells, the following most obvious differences in the time courses of ICl,swell development and
deactivation in cells dialyzed with nominally ATP-free and 5 mM MgATP
intracellular solutions were noted: 1) ATP withdrawal
resulted in ICl,swell rundown after repetitive
challenges with hypotonicity, as opposed to run-up in the presence of
ATP (Fig. 3A); and
2) without ATP supplementation, deactivation of
ICl,swell occurred much faster and more
completely on return to normal osmolarity. However, in the presence of
ATP, it was slowed down and less complete, resulting in the
"accumulation" of significant residual current after each return to
isotonicity. The time it took to reach half of maximum ICl,swell was taken as the measure of the rate
of current activation and did not show significant differences.
However, in the absence of ATP, this rate tended to increase with each
hypotonic challenge, whereas, in the presence of ATP, it was nearly
constant (Fig. 3B). These findings suggest that ATP plays a
role in ICl,swell activation in LNCaP cells.
Nevertheless, because of varying initial levels and speeds of reduction
during dialysis of endogenous ATP in different cells, simple removal of
ATP from the pipette solution is not sufficient to uncover all aspects
of its action. Additional studies with the use of metabolic and
phosphorylation inhibitors are required to have a better understanding
of the mechanisms and modes of ICl,swell
modulation by ATP.
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Single-channel recordings.
Having demonstrated the presence of ICl,swell in
LNCaP cells on the whole cell level, we were interested in examining
the properties of the single channels underlying this current.
Single-channel recordings were performed in cell-attached mode with
hypotonic TEA solution in both the bath and the patch pipette. In most
cases, no single-channel activity was observed with isotonic TEA in the bath. However, in four of seven patches, the replacement of the isotonic TEA bath solution with hypotonic TEA initiated the development of the type of single-channel activity presented in Fig.
6A. This activity was
characterized by a unitary amplitude of <1 pA, very long openings
without high-frequency flickering between open and closed states (Fig.
6B), and apparent strong rectification in the outward
direction. The ramp voltage-clamp protocol was used to cover a broad
range of membrane potentials with each pulse and, at the same time, to
acquire the current-voltage relationship that yielded single-channel
traces showing a very small inward current at potentials below the cell
resting potential (Fig. 6C, inset). Linear fit of
the current-voltage relationship constructed from the ramp portions of
the traces produced a channel slope conductance in the outward
direction of 9.6 ± 1.8 pS (n = 4) (Fig. 6C). This activity was never observed in the presence of 100 µM DIDS in the pipette (n = 6) and was apparently
dependent on the tonicity of the extracellular solution. These
observations, combined with the fact that neither the K+
channels known to be present in LNCaP cells (42) nor any
other hypothetical cation-selective channel could produce unitary
activity with similar features under these experimental conditions,
strongly suggest that this activity is associated with the operation of single VRACs.
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DISCUSSION |
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Volume-regulated ion channels permeable to K+,
Cl, or nonselective cation channels have been described
in various nonexcitable cells in which they play a major role in volume
regulation (for reviews, see Refs. 28 and 34). However, very little is
known about these channels and their modulation in prostate cells where they may be critically involved in the process of malignant
transformation and generation of the response to mitogenic and hormonal stimulus.
The results described in this report provide evidence for the
activation of a Cl current,
ICl,swell, activated by cell swelling, in LNCaP
cells originating from human carcinoma of the prostate. This
current, which reversed close to the expected Cl
equilibrium potential, was almost completely abolished after substitution of Cl
by impermeable anions, had a
characteristic anion selectivity profile of
Br
I
> Cl
> F
> MS
Glu (e.g., Ref. 28), and was sensitive
to the blocking action of the conventional Cl
channel
inhibitors NPPB and DIDS.
Voltage dependence and conductance.
ICl,swell in LNCaP cells showed
moderate outward rectification similar to that observed in other cell
types (34). It is suggested that outward rectification of
the macroscopic current is mainly due to a property inherent in the
single VRAC per se, since this outward rectification was also detected
on a single-channel level in excised patches under symmetrical
Cl conditions (26).
Pharmacology.
The pharmacological properties of VRACs in LNCaP cells are similar to
those previously reported in other cell types (28, 34).
Specifically, the stilbene derivative DIDS inhibited VRAC-mediated Cl current in a voltage-dependent manner and was almost
ineffective at negative membrane potentials. NPPB, a carboxylate analog
Cl
channel blocker, also inhibited this current, but
blocking was not significantly voltage dependent: the outward and
inward currents were almost equally affected. Although reports showed
that verapamil, more commonly known to inhibit some types of
Ca2+ and K+ channels as well as the
P-glycoprotein function, was also capable of inhibiting VRAC in some
cells (30, 54), it appeared to be a very weak VRAC blocker
in LNCaP cells. This finding is similar to observations in intestine
407 cells (48) and some other cell types (34)
in which verapamil did not show strong VRAC blocking potency. Thus the
pharmacological properties of VRACs differ significantly from cell to
cell. This variability may indicate the existence of several types of
VRACs as well as different, cell-specific volume-sensing mechanisms
involved in VRAC regulation (for review, see Ref. 28).
Regulation.
Although it is generally considered that VRAC activity is ATP
dependent, varying effects of intracellular ATP have been reported (for
review, see Ref. 34). When cells were dialyzed with ATP-free intracellular solution in the absence of extracellular glucose, the
following effects on volume-activated whole cell Cl
currents were observed: almost complete inhibition (1),
partial inhibition (21), induction of gradual rundown
(23, 54), and almost no effect (13, 31). Some
findings indicate that the use of nominally ATP-free intracellular
solutions may not be sufficient to reveal the ATP dependency of
ICl,swell and that the active depletion of
intracellular ATP with metabolic inhibitors is required
(33). Moreover, recent studies suggest that activation of
the channels underlying ICl,swell may occur via
two mechanisms, one ATP dependent and the other ATP independent, and
that the preference between the two mechanisms depends on the rate of
the cell swelling, with a shift to the ATP-independent mechanism at higher rates (2). These studies also demonstrate that the
ATP dependence of ICl,swell is due to ATP
binding rather than hydrolysis and/or phosphorylation reactions
(2).
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ACKNOWLEDGEMENTS |
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This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (INSERM), La Ligue Nationale Contre le Cancer and l'ARC (France), and by the International Association for the promotion of the cooperation with scientists from the New Independent States of the former Soviet Union. Y. M. Shuba was supported by INSERM and University of Science and Technology of Lille International Cooperation Programs.
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FOOTNOTES |
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* Y. M. Shuba and N. Prevarskaya contributed equally to this work.
Present address of Y. M. Shuba and P. G. Kostyuk: Bogomoletz Institute of Physiology, Bogomoletz Str., 4, Kiev, Ukraine.
Address for reprint requests and other correspondence: R. Skryma, Laboratoire de Physiologie Cellulaire, INSERM EPI 9807, Bâtiment SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France (E-mail: phycel{at}pop.univ-lille1.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 22 November 1999; accepted in final form 8 May 2000.
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