Volume-regulated chloride conductance in the LNCaP human prostate cancer cell line

Y. M. Shuba*, N. Prevarskaya*, L. Lemonnier, F. Van Coppenolle, P. G. Kostyuk, B. Mauroy, and R. Skryma

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-approx 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega . 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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Composition of the extracellular solutions

Single-channel recordings were performed in cell-attached and inside-out configurations with specified bath and pipette solutions for each experiment. Electrophysiological experiments and data analyses were performed with the use of a HEKA amplifier and Pulse/PulseFit (HEKA Electronic, Germany) and Origin 5.0 (Microcal, Northampton, MA) software. Results are expressed as means ± SE where appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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."

We have previously shown that the K+ current in LNCaP cells was sensitive to blocking by tetraethylammonium (TEA) (42). Therefore, to isolate and study ICl,swell, we prepared isotonic and hypotonic TEA external solutions (see Table 1) in which all of the Na+ was replaced with TEA. Figure 1A shows representative current traces of LNCaP cells in isotonic and hypotonic solutions after the full effect of hypotonicity had developed. Basically, no current was detected under isotonic conditions in the presence of TEA. This is consistent with the previous finding that K+ current is the sole voltage-dependent current in LNCaP cells. Exposure to the hypotonic solution triggered the development of a leaklike current that activated instantaneously as a result of stepwise shift of the potential but showed signs of rectification in the outward direction (the traces in response to the same voltage increment became more and more sparse as the potential became more positive) (Fig. 1, A and B). Current induction could be partially reversed on return to the isotonic TEA solution (Fig. 1C). Return to the isotonic TEA solution was also accompanied by an immediate increase in the current in the outward direction, suggesting its predominantly Cl- nature, since isotonic TEA had higher Cl- concentrations than hypotonic TEA. The Cl- nature of the current was also apparent from the close proximity of its reversal potential to the theoretical equilibrium potential for Cl- (Fig. 1B).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1.   Hypotonicity-evoked Cl- current (ICl,swell) in LNCaP cells. A: traces of the currents obtained in representative cells in isotonic tetraethylammonium (TEA) (left) and hypotonic TEA (right) external solutions (see Table 1) in response to the indicated voltage-clamp protocol (10-mV voltage increments). B: respective current-voltage (I-V) relationship. C: time course of the changes in holding current (bottom, circles) and current at +20 mV (top, diamonds) in the same cell during the transitions from isotonic TEA (open symbols) to hypotonic TEA (solid symbols) solution and back to isotonic TEA (open symbols); transition from isotonic to hypotonic TEA is taken as time 0; breaks in symbol continuity represent periods when the I-V relationship was acquired for A. D: traces of ICl,swell obtained in another cell exhibiting pronounced current inactivation at potentials above +40 mV; currents were elicited by voltage-clamp pulses of 400-ms duration ranging from -100 mV to +100 mV in 20-mV increments (see inset).

Employing longer voltage-clamp pulses (400 ms) to higher levels of depolarization (up to +100 mV) showed that ICl,swell starts to inactivate at positive potentials up to +40 mV (Fig. 1D). The extent of current inactivation progressively increased with depolarization and was quite variable in different cells. For some of the cells, it could reach up to 80% within 400 ms at +100 mV (Fig. 1D). High-voltage stimulation was poorly tolerated by the cells; therefore, the voltage did not exceed +50 mV in most of our whole cell experiments, and the process of ICl,swell inactivation was not investigated any further.

To determine whether ICl,swell activation could be modulated by intracellular Ca2+, we conducted a series of ICl,swell recordings in LNCaP cells dialyzed with intracellular solution in which EGTA was equimolarly replaced by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid, a much faster Ca2+ buffer. This substitution did not noticeably affect the characteristics of ICl,swell, suggesting that Ca2+ is not involved in its activation.

Anion selectivity of the channels underlying ICl,swell has been quantified on the basis of the shifts in reversal potentials caused by the replacement of 80 mM Cl- in the external hypotonic TEA solution with equimolar concentrations of either F-, Br-, I-, methanesulfonate (MS), or glutamate (Glu). The results of these experiments yielded the following relative permeability values: PF/PCl = 0.77 ± 0.02 (n = 5), PBr/PCl = 1.26 ± 0.09 (n = 5), PI/PCl = 1.20 ± 0.01 (n = 5), PMS/PCl = 0.49 ± 0.02 (n = 7), and PGlu/PCl, = 0.036 ± 0.006 (n = 4). These anions can, therefore, be placed in the following order of permeation through the VRACs underlying ICl,swell in LNCaP cells: Br-approx I- > Cl- > F- > Ms Glu.

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).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   Pharmacology of ICl,swell in LNCaP cells. A: representative traces of ICl,swell in response to the voltage ramp pulses before and after application of 100 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). B: I-V relationship of ICl,swell derived from the traces shown in A before (contr) and after exposure to NPPB; inset demonstrates the voltage dependence of the effects of NPPB by plotting the percentage of current inhibition vs. membrane potential. Vertical oscillations of the plot reflect the uncertainty in determining the block in the vicinity of the reversal potential. C and D: same as in A and B, showing the effects of 100 µM DIDS. E and F: same as in A and B, showing the effects of 50 µM verapamil (verap). Data obtained from 3 different cells. All I-V relationships presented were obtained after subtraction of the baseline currents before the development of the effects of hypotonicity.

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.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of intracellular ATP on ICl,swell in LNCaP cells. A and B: bar graphs (means ± SE) showing differences in the maximal amplitudes of ICl,swell (A) and in times required to reach half-maximal amplitude (B) in response to 3 repetitive hypotonic challenges (application of hypotonic TEA solution) in the cells dialyzed with nominally ATP-free (solid bars, n = 6) and 5 mM MgATP-containing (shaded bars, n = 4) intracellular solutions. Responses to hypotonicity (1, 2, and 3) were acquired approximately 2-9, 15-22, and 33-40 min, respectively, after whole cell recording configuration was established. ICl,swell density (pA/pF) was measured at +20 and -50 mV, respectively.

Protein tyrosine phosphorylation has been shown to be involved in osmoregulation of several cell types (for review, see Ref. 28). We therefore investigated whether the ICl,swell in LNCaP cells was sensitive to agents that affect protein tyrosine phosphorylation. Bath application of genistein, a protein tyrosine kinase (PTK) inhibitor, caused a gradual reduction in ICl,swell (n = 19). Figure 4, A-C, shows examples of currents in the presence and absence of 10 µM of genistein and their respective current-voltage relationships. The effect of genistein was dose dependent (Fig. 4D) and developed within 4-6 min after application. At 100 µM (we did not use higher concentrations because of possible nonspecific effects), genistein induced inhibition of ICl,swell by 60%. By fitting experimental data for the concentration dependence of the blocking effect to the Hill equation, we determined the value of IC50 for genistein block at 2.6 µM (Fig. 4D).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of the protein tyrosine kinase inhibitor genistein (GST) on ICl,swell in LNCaP cells. A and B: traces of ICl,swell obtained in representative cells in hypotonic TEA solution in response to the voltage-clamp protocol shown in B before (A) and 6 min after (B) bath application of 10 µM GST. C: respective I-V relationship constructed from current traces in A and B. D: dose-response relationship of the effect of GST on ICl,swell. Symbols indicate mean experimental values for 3-6 cells at each concentration, vertical bars indicate ±SE. Continuous curve indicates best fit by Hill equation Idrug/Icontr = (1 - A)/{1 + ([C]/IC50)p} + A, where Idrug and Icontr are current amplitudes in the presence and absence of the drug, [C] is drug concentration, IC50 is drug concentration for 50% inhibition, p is the Hill coefficient, and A is the proportion of drug-insensitive current component; parameters of the fit are shown on the graph.

Lavendustin A, another more specific PTK inhibitor (35), produced a downregulating effect similar to genistein. At 1 µM, lavendustin A reduced ICl,swell by 60 ± 14% (n = 4) at +30 mV. Figure 5A shows the time course of the current inhibition by 1 µM lavendustin A in the representative cell as well as the traces of the currents before and after application of the inhibitor that were used to construct the current-voltage relationships presented in Fig. 5B.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of the protein tyrosine kinase inhibitor lavendustin A (LAV) and tyrosine phosphatase inhibitor sodium orthovanadate (VO4) on ICl,swell in LNCaP cells. A: time course of the development of ICl,swell measured at +30 mV in the representative cell, after exposure to hypotonic TEA solution and its inhibition by 1 µM lavendustin A. Applications of the solutions are marked by horizontal bars; start of exposure to hypotonic TEA is taken as time 0; the breaks a and b in symbol continuity correspond to the periods when the currents presented in insets a and b were acquired (the voltage-clamp protocol used to acquire currents is shown in inset b). B: I-V relationship of the control ICl,swell and ICl,swell in the presence of 1 µM lavendustin A constructed from current traces presented in insets a and b of A. C and D: same as in A and B, showing the effect of 0.5 mM sodium orthovanadate on another cell.

In contrast to genistein and lavendustin A, but consistent with the involvement of protein tyrosine phosphorylation in ICl,swell regulation in LNCaP cells, the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate (500 µM) potentiated this current by 54 ± 11% (n = 3) (Fig. 5, C and D). Application of sodium orthovanadate was often accompanied by small (5-10 mV) negative shifts in ICl,swell reversal potential. This suggests that, in addition to its effect on ICl,swell via PTP inhibition, sodium orthovanadate may also slightly change the selectivity of the channel and/or activate another membrane conductance. This, however, was not further investigated.

We also investigated whether ICl,swell could be modulated by cAMP, by applying the membrane-permeable cAMP analog 8-BrcAMP to the cell at 250 µM once the current had fully developed. 8-BrcAMP caused a further 15% increase in the current (in 5 of 7 cells, data not shown), suggesting that cAMP-dependent phosphorylation may also be involved in its modulation.

The PKC activator phorbol myristate acetate (PMA, 10 nM) had no significant effect on any of the cells studied (n = 5).

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.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6.   Activity of single, volume-regulated anion channels in LNCaP cells. A: development of single-channel activity in multichannel cell-attached patch following LNCaP cell exposure to hypotonic TEA solution (marked by vertical bar); recordings (left) were obtained in response to voltage-clamp pulses to reversal potential of +150 mV applied every 30 s. Superimposed solid lines indicate the levels of zero current, whereas horizontal dotted lines are multiples of 1.1 pA. Open probability (Po) values vs. time calculated, assuming 5 functional channels in the patch, are presented opposite each record as the height of the bars (right). B: magnified view of single-channel activity obtained in another cell-attached patch at reversal potentials of +100 mV (left) and +150 mV (right) after LNCaP cell exposure to hypotonic TEA solution. C: I-V relation of a single, volume-sensitive Cl- channel. I-V was constructed from the ramp portions of single-channel recordings in cell-attached configuration (see inset) by selecting the parts corresponding to the channel opening with subsequent averaging and digital smoothing of the resulting curve to reduce noise. Superimposed linear fit produces the slope conductance value of 9.94 pS. D: I-V relationship of single volume-sensitive Cl- channel obtained in symmetrical Cl- (see text for details). Superimposed linear fits provide the values of outward and inward slope conductances of 7.5 and 4.2 pS, respectively; inset presents pulse protocol and 6 superimposed single-channels recordings acquired in 2 different patches in cell-attached and inside-out configurations that were used to construct I-V.

The much stronger outward rectification of single-channel currents compared with the whole cell ICl,swell suggests that intracellular Cl- concentrations in the intact LNCaP cells are quite low. To further examine the rectification properties and Cl- dependency of single VRACs, experiments were carried out on cell-attached patches with cell resting potential of zero and inside-out patches with the intrapipette Cl- concentration reduced to 20 mM (IP solution in Table 1). The cell's resting potential was moved toward zero with either isotonic (140 mM K+) or hypotonic (100 mM K+) K+-rich bath solutions containing 20 mM Cl- (with Glu for the remainder; K-rich solution in Table 1). Single-channel activity was preserved for some time (up to 3-5 min) after excision of the patch into inside-out mode. In both cell-attached and inside-out modes, single-channel currents elicited by ramp pulses spanning from -100 to +100 mV were both outward and inward and their amplitudes did not change after patch excision. This is demonstrated by the inset in Fig. 6D, which presents six superimposed single-channel recordings acquired in two different patches in response to the depicted pulse protocol before and after patch excision. The ramp portions of the recordings between the levels of membrane potentials ±100 mV were used to construct the current-voltage relationship of Fig. 6D, characterized by moderate outward rectification and a reversal potential close to 0 mV. The fit of the current-voltage relationship with two linear functions showed that the channel does rectify, and the slope conductance for the outward current is ~1.8 times higher than that of the inward one, i.e., 7.5 pS vs. 4.2 pS (Fig. 6D); the decrease in the outward conductance compared with the one presented in Fig. 6C reflects the reduction of the extracellular Cl- concentration from 80 to 20 mM. The reversal potential of 0 mV is consistent with Cl- as a charge carrier because the bath solution had a resting potential of zero and the pipette solution contained equal concentrations of Cl- (20 mM). Our data on the rectification properties of single VRACs under symmetric Cl- conditions (the data of Fig. 6D) and in intact cells (the data in Fig. 6C) are also consistent with the hypothesis that most of the rectification observed in intact cells is due to low intracellular Cl-.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-approx 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).

Our single-channel data, obtained in cell-attached and inside-out patches, show that the ICl,swell in LNCaP cells is based on the activation of a rather low-conductance channel (4-10 pS). This channel exhibits an intrinsic property of outward rectification, as, under symmetric Cl- conditions, its conductance is ~1.8 times higher in the outward direction than in the inward direction. It is still unclear whether channel operation is characterized by different open probabilities in the two directions, which could also contribute to the observed outward rectification of the whole cell ICl,swell. Our data on the differences in the rectification properties of single VRACs under symmetric Cl- conditions and in intact cells are also consistent with the relatively low intracellular Cl- concentration in LNCaP cells.

Estimates of the unitary conductances of VRACs largely depend on the method used to obtain them, i.e., stationary and nonstationary noise analysis or direct single-channel measurements (19), and values for different cell models can vary from very low (0.1 pS) to extremely high (400 pS). For instance, single-channel conductance, derived from the stationary noise analysis of macroscopic current, was estimated at <2 pS in chromaffin cells (9) and T lymphocytes (23) and was ~0.2-5.8 pS in a study of 15 different cell species (32). Intermediate single-channel conductances between 20 and 90 pS have been reported in epithelial cells, glia cells, osteoblasts, osteoclasts, epididymal cells, and muscle cells (28, 44). Large-conductance VRACs between 200 and 400 pS have been described in cardiac myocytes, neuroblastoma cells, astrocytes, and renal epithelial cells (8, 20, 39). These differences in unitary conductance may point to a broad population of VRACs in different cell models. Furthermore, the existence of several types of VRACs may be suggested on the basis of their different gating behavior. "Intermediate conductance" VRACs inactivate rapidly, with time constants in the range of 100 ms at +80 mV, whereas nonactivating or slowly inactivating VRACs seem to have a small single-channel conductance (28). Our experiments showed that ICl,swell in LNCaP cells based on the activation of rather small-conductance channels exhibits little or no inactivation between -100 mV and +50 mV but that inactivation is enhanced above +80 mV.

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).

In our experiments on LNCaP cells, the simple omission of ATP from the intracellular solution did not prevent activation of ICl,swell, although the rate and magnitude of current development after exposure to the hypotonic solution showed significant variability. This probably reflects variations in the rate of reduction of endogenous ATP levels during dialysis. Despite this variability, ICl,swell in LNCaP cells dialyzed with ATP-free solutions was characterized by such common features as gradual rundown on repetitive challenges with hypotonicity, as well as rapid and virtually complete deactivation on each return to isotonicity. This behavior contrasted with that of ATP-dialyzed cells, in which ICl,swell exhibited run-up and slower, incomplete deactivation in response to the same operations, consistent with the involvement of ATP in its activation.

It has been suggested that some protein kinases may modulate VRACs via phosphorylation of the channel, its accessory proteins, or its volume sensor (34). In our experiments, the membrane-permeable cAMP analog 8-BrcAMP only slightly stimulated ICl,swell, suggesting that cAMP-dependent protein kinase A plays only a minor role in VRAC regulation in LNCaP cells. A similar virtual cAMP independence of volume-activated Cl- current has been found in many other cell types (5, 12, 30).

The PKC activator PMA also had no effect on VRAC in LNCaP cells, suggesting that the PKC pathway is not involved in its modulation either. The ineffectiveness of PKC activators (12-O-tetradecanoylphorbol-13-acetate and indolactan) on VRAC current has also been demonstrated in bovine endothelial cells (45) and osteoblast-like cells (13).

It is known that simple hypotonic shock is capable of triggering tyrosine kinase activity, resulting in tyrosine phosphorylation of mitogen-activated protein (MAP) kinases and possibly of the focal adhesion protein p125FAK (46, 47). On the other hand, tyrosine kinases are also known to be involved in the signal transduction of such hormones as prolactin (36, 37) and growth hormone (7), as well as growth factors (25), which play an important role in prostate cell proliferation. We therefore investigated whether tyrosine kinase phosphorylation was in any way involved in modulating VRACs in LNCaP cells. The tyrosine kinase inhibitors genistein and lavendustin A were found to downregulate VRAC current, whereas the tyrosine phosphatase inhibitor sodium orthovanadate had an opposite, potentiating effect.

The involvement of PTK in VRAC activation is still not firmly established. To summarize, no proof of PTK involvement has been shown in rat osteoblast-like cells (13) and in the CPAE calf endothelial cell line (45). On the other hand, tyrosine protein kinase inhibitors have been shown to prevent the activation of cardiac ICl,swell (43). The inhibitory effects of tyrphostin and genistein on ICl,swell were also documented in CPAE endothelial cells (50); however, Tilly et al. (47) demonstrated the reduction of the osmoregulated shock-induced ion efflux by the PTK inhibitors herbimycin A and genistein in 125I-- and 86Rb+-loaded intestinal human epithelial cells.

The mechanism by which PTK stimulates volume-regulated Cl- channels is unknown. Because MAP kinase activity is upregulated not only through tyrosine-linked receptors but also through G protein-coupled receptors and integrins (6) and VRAC currents are stimulated by the G protein agonist lysophosphatidic acid (for review, see Ref. 28), it has been suggested that tyrosine phosphorylation of MAP kinases may be responsible for VRAC regulation. However, a study by Szücs et al. (45) showed that VRAC currents were not affected by lysophosphatidic acid, an activator of p42MAP kinase and p125FAK or by wortmannin, a MAP kinase inhibitor. It would, therefore, appear that activation of VRACs involves a mechanism distinct from that of the MAP kinases. Okada (34) suggested that the tyrosine kinase cascades may modulate VRAC by affecting cytoskeletal rearrangements. Direct constitutive association of tyrosine kinase with VRAC is also possible. This type of constitutive tyrosine phosphorylation has previously been demonstrated in K+ channels stimulated by prolactin (36).

From all our data, we conclude that changes in cell volume activate a Cl- current in human cancer prostate cells and that tyrosine phosphorylation is necessary to sustain the activity of the respective underlying anion channels.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Best, L, Sheader EA, and Brown PD. A volume-activated anion conductance in insulin-secreting cells. Pflügers Arch 413: 363-370, 1996.

2.   Bond, T, Basavappa S, Christensen M, and Strange K. ATP dependence of the ICl,swell channel varies with rate of cell swelling. Evidence for two modes of channel activation. J Gen Physiol 113: 441-456, 1999[Abstract/Free Full Text].

3.   Bortner, CD, and Cidlowski JA. Absence of volume regulatory mechanisms contributes to the rapid activation of apoptosis in thymocytes. Am J Physiol Cell Physiol 271: C950-C961, 1996[Abstract/Free Full Text].

4.   Cantiello, HF. Role of actin filament organization in cell volume and ion channel regulation. J Exp Zool 279: 425-435, 1997[ISI][Medline].

5.   Chan, HC, Fu WO, Chung YW, Huang SJ, Chan PSF, and Wong PYD Swelling-induced anion and cation conductances in human epididymal cells. J Physiol (Lond) 478: 449-460, 1994[Abstract].

6.   Chen, Q, Kinch MS, Lin TH, Burridge K, and Juliano RL. Integrin-mediated cell adhesion activates mitogen-activated protein kinases. J Biol Chem 269: 26602-26605, 1994[Abstract/Free Full Text].

7.   Chou, CY, Shen MR, and Wu SN. Volume-sensitive chloride channels associated with human cervical carcinogenesis. Cancer Res 55: 6077-6083, 1995[Abstract].

8.   Coulombe, A, and Coraboeuf F. Large conductance chloride channels of newborn rat cardiac myocytes are activated by hypotonic media. Pflügers Arch 422: 143-150, 1992[ISI][Medline].

9.   Doroshenko, P, and Neher E. Volume-sensitive chloride conductance in bovine chromaffin cell membrane. J Physiol (Lond) 449: 197-218, 1992[Abstract].

10.   Dubois, JM, and Rouzaire-Dubois B. Role of potassium channels in mitogenesis. Prog Biophys Mol Biol 59: 1-21, 1993[ISI][Medline].

11.   Furuya, Y, Lin XS, Walsh JC, Nelson WG, and Isaacs JT. Androgen ablation-induced programmed death of prostate glandular cells does not involve recruitment into a defective cell cycle or p53 induction. Endocrinology 136: 1898-1906, 1995[Abstract].

12.   Gosling, M, Poyner DR, and Smith JW. Effects of arachidonic acid upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells. J Physiol (Lond) 493: 613-623, 1996[Abstract].

13.   Gosling, M, Smith JW, and Poyner DR. Characterization of a volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells. J Physiol (Lond) 485: 671-682, 1995[Abstract].

14.   Hall, SK, Zhang J, and Lieberman M. Cyclic AMP prevents activation of a swelling-induced chloride-sensitive conductance in chick heart cells. J Physiol (Lond) 488: 359-369, 1995[Abstract].

15.   Haussler, U, Rivet-Bastide M, Fahlke C, Müller D, Zachar E, and Rüdel R. Role of the cytoskeleton in the regulation of Cl- channels in human embryonic skeletal muscle cells. Pflügers Arch 428: 323-330, 1994[ISI][Medline].

16.   Hoffmann, EK, and Dunham PB. Membrane mechanisms and intracellular signalling in cell volume regulation. Int Rev Cytol 161: 173-262, 1995[ISI][Medline].

17.   Hoffmann, EK, Lambert IH, and Simonsen LO. Separate, Ca2+-activated K+ and Cl- transport pathways in Ehrlich ascites tumor cells. J Membr Biol 91: 227-244, 1986[ISI][Medline].

18.   Horoszewicz, JS, Leong SS, Kawinski E, Karr J, Rosenthal H, Chu MT, Mirand EA, and Murphy GP. LNCaP model of human prostate carcinoma. Cancer Res 43: 1908-1918, 1983.

19.   Jackson, PS, and Strange K. Single-channel properties of a volume-sensitive anion conductance. Current activation occurs by abrupt switching of closed channels to an open state. J Gen Physiol 105: 643-660, 1995[Abstract].

20.   Jalonen, T. Single-channel characteristics of the large-conductance anion channel in rat cortical astrocytes primary culture. Glia 9: 227-237, 1993[ISI][Medline].

21.   Kinard, TA, and Satin LS. An ATP-sensitive Cl- channel current that is activated by cell swelling, cAMP, and glyburide in insulin-secreting cells. Diabetes 44: 1461-1466, 1995[Abstract].

22.   Lang, F, Ritter M, Völkl H, and Häussinger D. The biological significance of cell volume. Renal Physiol Biochem 16: 48-65, 1993[Medline].

23.   Lewis, RS, Ross PE, and Cahalan MD. Chloride channels activated by osmotic stress in T lymphocytes. J Gen Physiol 101: 801-826, 1993[Abstract].

24.   MacLeod, RJ, and Hamilton JR. Volume regulation initiated by Na+-nutrient cotransport in isolated mammalian villus enterocytes. Am J Physiol Gastrointest Liver Physiol 260: G26-G33, 1991[Abstract/Free Full Text].

25.   Magni, M, Pandiella A, Helin K, Meldolesi J, and Beguinot L. Transmembrane signalling at the epidermal growth factor receptor. Biochem J 277: 305-311, 1991[ISI][Medline].

26.   Meyer, K, and Korbmacher C. Cell swelling activates ATP-dependent voltage-gated chloride channels in M-1 mouse cortical collecting duct cells. J Gen Physiol 108: 177-193, 1996[Abstract].

27.   Nakahari, T, Murakami M, and Kataoka T. Shrinkage of rat mandibular acinar cell with acetylcholine detected by video-enhanced contrast microscopy. Jpn J Physiol 39: 609-615, 1989[ISI][Medline].

28.   Nilius, B, Eggermont J, Voets T, and Droogmans G. Volume-activated Cl- channels. Gen Pharmacol 27: 1131-1140, 1996[Medline].

29.   Nilius, B, Eggermont J, Voets T, Buyse G, Manolopoulos V, and Droogmans G. Properties of volume-regulated anion channels in mammalian cells. Prog Biophys Mol Biol 68: 69-119, 1997[ISI][Medline].

30.   Nilius, B, Oike M, Zahradnik I, and Droogmans G. Activation of a Cl- current by hypotonic volume increase in human endothelial cells. J Gen Physiol 103: 787-805, 1994[Abstract].

31.   Nilius, B, Sehrer J, De Smet P, Van Driessche W, and Droogmans G. Volume regulation in a toad epithelial cell line: role of coactivation of K+ and Cl- channels. J Physiol (Lond) 487: 367-378, 1995[Abstract].

32.   Nilius, B, Sehrer J, Viana F, De Greef C, Raeymaekers L, Eggermont J, and Droogmans G. Volume-activated Cl- currents in different mammalian non-excitable cell types. Pflügers Arch 428: 364-371, 1994[ISI][Medline].

33.   Oiki, S, Kubo M, and Okada Y. Mg2+ and ATP-dependence of volume-sensitive Cl- channels in human epithelial cells. Jpn J Physiol 44: S77-S79, 1994[ISI][Medline].

34.   Okada, Y. Volume expansion-sensing outward-rectifier Cl- channel: fresh start to the molecular identity and volume sensor. Am J Physiol Cell Physiol 273: C755-C789, 1997[Abstract/Free Full Text].

35.   Onoda, T, Iinuma H, Sasaki Y, Hamada M, Isshiki K, Naganawa H, Takeuchi T, Tatsuta K, and Umezawa K. Isolation of a novel tyrosine kinase inhibitor, lavendustin A, from Streptomyces griseolavendus. J Nat Prod 52: 1252-1257, 1989[ISI][Medline].

36.   Prevarskaya, NB, Skryma RN, Vacher P, Daniel N, Djiane J, and Dufy B. Role of tyrosine phosphorylation in potassium channel activation. J Biol Chem 270: 24292-24299, 1995[Abstract/Free Full Text].

37.   Prevarskaya, NB, Skryma RN, Vacher P, Daniel N, Bignon C, Djiane J, and Dufy B. Early effects of PRL on ion conductances in CHO cells expressing PRL receptor. Am J Physiol Cell Physiol 267: C554-C562, 1994[Abstract/Free Full Text].

38.   Robson, L, and Hunter M. Role of cell volume and protein kinase C in regulation of Cl- conductance in single proximal tubule cells of Rana temporaria. J Physiol (Lond) 480: 1-7, 1994[Abstract].

39.   Schwiebert, EM, Mills JW, and Stanton BA. Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line. J Biol Chem 269: 7081-7089, 1994[Abstract/Free Full Text].

40.   Shen, MR, Wu SN, and Chou CY. Volume-sensitive chloride channels in the primary culture cells of human cervical carcinoma. Biochim Biophys Acta 1315: 138-144, 1996[ISI][Medline].

41.   Skryma, RN, Prevarskaya NB, Dufy-Barbe L, Odessa MF, Audin J, and Dufy B. Potassium conductance in the androgen-sensitive prostate cancer cell line, LNCaP: involvement in cell proliferation. Prostate 32: 112-122, 1997.

42.   Skryma, RN, Van Coppenolle F, Dufy-Barbe L, Dufy B, and Prevarskaya NB. Characterization of Ca2+-inhibited potassium channels in the LNCaP human prostate cancer cell line. Receptors Channels 6: 241-253, 1999[ISI][Medline].

43.   Sorota, S. Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current. Pflügers Arch 431: 178-185, 1995[ISI][Medline].

44.   Strange, K, and Jackson PS. Swelling-activated organic osmolyte efflux: a new role for anion channels. Kidney Int 48: 994-1003, 1995[ISI][Medline].

45.   Szücs, G, Heinke S, De Greef C, Raeymaekers L, Eggermont J, Droogmans G, and Nilius B. The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation. Pflügers Arch 431: 540-548, 1996[ISI][Medline].

46.   Tilly, BC, Gaestel M, Engel K, Edixhoven MJ, and de Jonge HR. Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade. FEBS Lett 395: 133-136, 1996[ISI][Medline].

47.   Tilly, BC, Van Den Berghe N, Tertoolen LGJ, Edixhoven MJ, and De Jonge HR. Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances. J Biol Chem 268: 19919-19922, 1993[Abstract/Free Full Text].

48.   Tominaga, M, Tominaga T, Miwa A, and Okada Y. Volume-sensitive chloride channel activity does not depend on endogenous P-glycoprotein. J Biol Chem 270: 27887-27893, 1995[Abstract/Free Full Text].

49.   Valverde, MA, Diaz M, Sepulveda FV, Gill DR, Hyde SC, and Higgins CF. Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein. Nature 355: 830-833, 1992[ISI][Medline].

50.   Voets, T, Manolopoulos V, Eggermont J, Ellory C, Droogmans G, and Nilius B. Regulation of a swelling-activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins. J Physiol (Lond) 506: 341-352, 1998[Abstract/Free Full Text].

51.   Voets, T, Szucs G, Droogmans G, and Nilius B. Blockers of volume-activated Cl- currents inhibit endothelial cell proliferation. Pflügers Arch 431: 132-134, 1995[ISI][Medline].

52.   Voets, T, Wei L, De Smet P, Van Driessche W, Eggermont J, Droogmans G, and Nilius B. Downregulation of volume-activated Cl- currents during muscle differentiation. Am J Physiol Cell Physiol 272: C667-C674, 1997[Abstract/Free Full Text].

53.   Woolf, SH. Screening for prostate cancer with prostate specific antigen. An examination of the evidence. N Engl J Med 333: 1401-1405, 1995[Free Full Text].

54.   Wu, J, Zhang JJ, Koppel H, and Jacob TJC P-glycoprotein regulates a volume-activated chloride current in bovine non-pigmented ciliary epithelial cells. J Physiol (Lond) 491: 743-755, 1996[Abstract].

55.   Yamamoto, T, Kashihara Y, Hazama A, Miwa A, and Okada Y. Expression of c-Jun by hypotonic stimulation in cultured human epithelial cells (Abstract). Jpn J Physiol 45: S27, 1995.


Am J Physiol Cell Physiol 279(4):C1144-C1154
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society