Received 2001-05-07Accepted 2001-07-28

Corresponding author. Tel: 00-44-29-20876670; Fax: 00-44-29-20874094; E-mail: CHENL1@cardiff.ac.uk Acta Physiologica Sinica

Feb. 2002, 54 (1), 1～6

Research Paper

The role of MDR1 gene in volume-activated chloride currentsin pigmented ciliary epithelial cells

CHEN Li-Xin1, 3,*, WANG Li-Wei2, 3, Tim JACOB3

1Laboratory of Cell Biology, 2Department of Physiology, Guangdong Medical College, Zhanjiang 524023, China; 3Cardiff School of Biosciences, Cardiff University, Cardiff, UK)

Abstract: The role of multidrug resistance (MDR1) gene in the activation of volume-activated chloride currents in bovine pigmented ciliary epithelial (PCE) cells was investigated by the patch-clamp technique, the antisense approach, the immunofluorescent technique and the confocal microscopy. PCE cells express P-glycoprotein (P-gp, the product of MDR1 gene). An MDR1 antisense oligonucleotide suppressed MDR1 expression (93% reduction of P-gp immunofluorescence), delayed the activation of a volume-activated chloride current (latency prolonged by 109%), reduced the activation rate by 62% and decreased the peak value of the current by 56%. The transfection reagent lipofectin and the mismatch control oligonucleotide did not significantly affect the current. The data indicate that the volume-activated chloride current is associated with the endogenous expression of MDR1 gene in PCE cells.

Key words: epithelial cells; chloride channels; oligonucleotides, antisense; gene expression; genes; MDR

MDR1基因在睫状体色素上皮细胞容积激活性氯电流中的作用

陈丽新1,3,*, 王立伟2,3， Tim JACOB3

广东医学院 1细胞生物学研究室、 2生理教研室， 湛江 524023； 3Cardiff School of Biosciences, Cardiff University, Cardiff, UK

摘要: 用膜片钳、反义寡核苷酸、免疫荧光及激光共聚焦显微镜等技术, 研究MDR1基因在牛睫状体色素上皮(pigmented ciliary epithelial, PCE)细胞容积激活性氯电流中的作用。PCE细胞表达MDR1基因产物-P糖蛋白(P-gp)。反义MDR1寡核苷酸抑制MDR1基因的表达(P-gp免疫荧光减少93％)，延缓容积激活性氯电流的出现(潜伏期延长109％)，并导致激活率降低62％及电流峰值减小56%。而核酸转染剂阳离子脂质体和非配对性的寡核苷酸对电流没有显著性影响。上述观察结果表明, 睫状体色素上皮细胞容积激活性氯电流与内源性MDR1 表达有关。

关键词: 上皮细胞; 氯通道; 寡核苷酸; 反义; 基因表达; 基因; MDR

学科分类号: Q25

We previously reported that exposure to a hypotonic solution induced a volume-activated Cl－ current in the pigmented ciliary epithelial (PCE) cell[1]. The volume-activated chloride channels could play a crucial role in the net transfer of ions across the ciliary epithelium[2]. However, the molecular nature of the volume-activated Cl－ channels is still unresolved. The candidate proteins of volume-activated Cl－ channels or channel regulators include PICln[3], ClC-3[4], P-glycoprotein (P-gp, the MDR1 gene product)[5], and ClC-2[6]. The property of the volume-activated Cl－ current in the PCE cell suggests that it may be associated with P-gp[1]. In this study, we investigated the role of P-gp in the activation of volume-activated Cl－ currents in PCE cells by inhibiting the expression of endogenous MDR1 gene using an antisense technique.

1MATERIALS AND METHODS

1.1 Cell preparation Bovine ciliary epithelial cells were prepared by a method described previously[1,7,8]. The ciliary epithelium was dissociated with 0.25% trypsin. The isolated ciliary epithelial cells that were resuspended in culture medium E199 (Medium 199 with Earle′s salts, L-glutamine and HEPES, without sodium bicarbonate, Sigma) with 10% fetal calf serum were plated onto glass coverslips and incubated at 37℃ overnight before oligonucleotide treatments.

1.2 Antisense study The antisense oligonucleotide (5′ ATC CAT CCC GAC CTC 3′), complementary to the region around the initiation codon of MDR1 mRNA[9], and the mismatch control oligonucleotide (5′ CTC CAT CAC CAC CTC 3′) were synthesized by Seven Biotech Ltd (Milton Keynes, UK). Both oligonucleotides were phosphorothioated at the first three bases of both ends. To facilitate the uptake of oligonucleotides by the cells, the transfection reagent lipofectin (Gibco-BRL, UK) was used. The cells that were prepared as above and incubated overnight were then cultured in serum-free E199 with or without 200 μg/ml oligonucleotide and 20 μg/ml lipofectin at 37 ℃ for 48 h before immunofluorescence detections and patch-clamp experiments.

1.3 Immunocytochemistry The expression of MDR1 gene was detected by an immunofluorescence technique[7,8]. The cells were fixed in 4% paraformaldehyde, permeabilized in 0.3% Triton X-100, blocked in 10% sheep serum (Sigma) and incubated at 4 ℃ in the presence or absence of the anti-P-gp antibody JSB-1 (diluted 1∶10; Monosan, Sanbio, Netherlands) overnight. The cells were then incubated at room temperature (20~22 ℃) for 1 h in sheep anti-mouse IgG conjugated with fluorescein isothiocyanate (diluted 1∶100; Sigma) and examined by a confocal microscope (Noran Instruments, USA). The fluorescence intensity (grey level) of the fluorescence image of individual cell was measured using the MetaMorph image analysis system (Universal Imaging Corporation, USA) and expressed on a scale of 0~255 U (min-max).

1.4 Patch-clamp experiments Membrane currents of the cells were recorded with the whole-cell patch-clamp technique described by Hamill et al.[10], and the volume-activated chloride currents were measured during exposure to hypotonic conditions using the methods described previously[1,7,8] with a List EPC-7 patch-clamp amplifier (List Electronic, Germany). Throughout the experiments the cells were held at the Cl－ equilibrium potential (0 mV) and then stepped to ±40, 0 and ±80 mV for 200 ms and with 4 s interval between pulses. All current measurements were made 10 ms after the onset of each voltage pulse. The pulse generation and current analysis were carried out with the EPC software package (CED, Cambridge, UK). All patch-clamp recordings were made at room temperature (20～22 ℃).

1.5 Solutions and chemicals Bath and pipette solutions were chosen to enable Cl－ current measurements[7]. The isotonic bath solution contained (mmol/L): 105 NaCl, 2 CaCl2, 0.5 MgCl2, 10 HEPES and 70 D-manitol. The osmolarity was adjusted to 300 mOsmol/L. The hypotonic bath solution was obtained by omitting the D-manitol from the solution, giving an osmolarity of 230 mOsmol/L. The pipette solution was composed of (mmol/L): 105 N-methyl-D-glucamine chloride (NMDG-Cl), 1.2 MgCl2, 10 HEPES, 1 EGTA, 2 ATP and 70 D-mannitol with osmolarity of 300 mOsmol/L. The pH of the solutions was adjusted to 7.4 (bath) and 7.25 (pipette) with Tris base. The osmolarity was measured with a freezing-point osmometer (OSMOMAT 030; Gonotec, Germany).

1.6 Statistics Values were expressed as mean ( standard error (number of observations). Student′s t test was used to test for significant differences and P<0.05 was taken to be significant.

2RESULTS

2.1 P-gp expression in pigmented ciliary epithelial cells

The immunocytochemical experiments verified that PCE cells expressed P-gp. Under the control condition (no additives), in the presence of anti-P-gp antibody JSB-1, the cells exhibited fluorescence. The mean value of P-gp immunofluorescence in pigmented cells was 28.8±2.2 U (n=33). To eliminate the possibility of cell autofluorescence, control experiments were carried out. In the absence of JSB-1, there was no background fluorescence in pigmented cells (n=17).

2.2 Antisense suppression of P-gp expression

After showing that P-gp was expressed in PCE cells, MDR1 antisense oligonucleotide was introduced and the P-gp expression was suppressed (Fig.1A). Incubation of PCE cells in 200 μg/ml MDR1 antisense oligonucleotide plus 20 μg/ml lipofectin for 48 h decreased significantly P-gp immunofluorescence by 93%, from th- value of 28.8±2.2 U (ctrl; n=33) to 2.1±0.5 units (anti-MDR1; n=22, P<0.01). However, the control mismatch oligonucleotide (200 μg/ml) with 20 μg/ml lipofectin had no significant effect on P-gp expression (mismatch oligo; 26.7±3.2 U, n=23, P>0.05) (Fig.1B).

Fig.1.A: P-gp immunofluorescence in PCE cells. White and red represent strong, and purple and black indicate weak fluorescence (shown in the colour bar to the right of the images). Cells were cultured in control solution (ctrl) or treated with lipofectin plus MDR1 antisense oligonucleotide (anti-MDR1) or plus mismatch oligonucleotide (mismatch oligo). B: Quantification of P-gp immunofluorescence (Im-P-gp) under different conditions. The fluorescence intensity is expressed on a scale of 0~255 U (min-max).

2.3 Volume-activated Cl－ current in PCE cells

Whole-cell membrane currents in PCE cells were recorded by the patch-clamp technique. Fig.2. Volume-activated Cl－ currents in the PCE cell. The cell was held at 0 mV and stepped to ±40, 0 and ±80 mV. Iso, isotonic condition. Hypo, 10 min after exposure to hypotonic solution.Volume-activated chloride currents were induced by perfusing the cells with the hypotonic solution (230 mOsmol/L). In control cells (no additives), the chloride currents were activated after a certain latency (taken as the time between the exposure to the hypotonic solution and the activation of the current), and then reached the peak in 3～5 min after activation. The currents were weakly outward rectified and lacked time dependent inactivation at the voltages tested (Fig.2). Similar results were observed in 25 cells. We previously demonstrated that the currents were ATP dependent and were carried by chloride[１]

2.4 Inhibition of volume-activated Cl－ current by MDR1 antisense oligonucleotide

The above-mentioned results demonstrated that an MDR1 antisense oligonucleotide inhibited P-gp expression. This antisense technique was then employed to investigate the role of endogenous P-gp in the activation of volume-activated Cl－ current in PCE cells. The results showed that 48 h incubation in 200 μg/ml MDR1 antisense oligonucleotide and 20 μg/ml lipofectin inhibited the volume-activated Cl－ currents (Fig.3). The activation of the volume-activated chloride currents was delayed. The latency increased by 109%, from 119±21 s (control, n=11) to 249±29 s (n=10, P<0.01). The volume-activated chloride currents in response to all the voltage steps (±40, 0 and ±80 mV) were also decreased. The peak current at +80 mV was reduced by 56%, from 1517±192 pA (control, n=11) to 671±78 pA (n=10, P<0.01).

Fig.3.Effects of MDR1 antisense oligonucleotide on volume-activated Cl－ currents. Hypo: hypotonic condition. The cell was held at 0 mV and cycled through ±40, 0 and ±80 mV.

Fig.4.Effects of MDR1 antisense oligonucleotide on the activation rate of the volume-activated Cl－ current. Time 0 represents the onset of the activation of the volume-activated Cl－ current.

The antisense oligonucleotide not only prolonged the latency and inhibited the peak currents but also inhibit-ed the rate of the volume-activated chloride currents to reach the peak by 62% (Fig. 4). The lines in the figure represent the curve fitting to the data using the equation y=ae－bx+ c. The current rises from c (current at time 0, the onset of the activation of the volume-activated Cl－ current) to a+c (peak current) with a time constant of 1/b. In the control cells, once activated, the current increased gradually and reached the peak with the time constant of 141±18 s (n=6). However, the time constant in the cells treated with the antisense oligonucleotide increased to 228±24 s (n=5, P<0.01).

2.5 Effects of lipofectin on the volume-activated Cl－ current

The effect of transfection reagent lipofectin on the volume-activated Cl－ current was tested. PCE cells treated with 20 μg/ml lipofectin alone exhibited a volume-activated Cl－ current (1471±112 pA at +80 mV step, n=9) that was not significantly different from that of the control cells (1517±192 pA, n=11, P>0.05).

2.6 Effects of mismatch control oligonucleotide on the volume-activated Cl－ current

The cells were treated with 200 μg/ml mismatch oligonucleotide and 20 μg/ml lipofectin in the same condition as for antisense treatment.

Fig.5.Peak volume-activated Cl－ currents under different treatments. The mean currents at +80 mV (positive currents) and －80 mV (negative currents) under different conditions were given．

The data showed that the mismatch oligonucleotide did not significantly affect the volume-activated Cl－ currents (Fig. 5). The peak current (1420±123 pA at +80 mV step, n=6) was not significantly different from that of the control cells (no additives; 1517±192 pA, n=11, P>0.05), but was much larger than that of the cells incubated in the antisense oligonucleotide (671±78 pA, n=10, P<0.01).

3 DISCUSSION

The secretion of aqueous humour involves ionic transport through the ciliary epithelium, which consists of a non-pigmented (NPCE) layer and a pigmented (PCE) layer. It has been hypothesized that the volume-activated Cl－ current in ciliary epithelial cells is the key factor in the regulation of the formation of aqueous humour. In this study, a volume-activated Cl－ current was recorded in the PCE cell. We have previously demonstrated that this current is intracellular ATP dependent, outward rectified and is blockable by extracellular application of ATP and tamoxifen[1]. The property of the current suggests its association with the MDR1 gene product P-gp, one of the candidates of volume-activated Cl－ channels or channel regulators. This has been verified by the antisense inhibition study in this report.

Antisense technique is a versatile approach for shutting off endogenous cellular genes. It targets the gene′s mRNA rather than the gene itself[11]. Antisense oligonucleotides are designed to hybridize to a specific mRNA. This hybrid formation causes a steric or conformational obstacle for protein translation. Also, double stranded mRNA or oligonucleotide-mRNA hybrids are substrates for RNAse-H. As a result the production of specific protein is inhibited. In this study, the antisense technique was used to knock down P-gp expression. Our results showed that PCE cells expressed P-gp and possessed a Cl－ current activated by hypotonic swelling. A MDR1 antisense oligonucleotide inhibited P-gp expression and the volume-activated Cl－ current. The evidence strongly indicates that P-gp is indeed involved in the activation of the volume-activated Cl－ current.

However the inhibition of P-gp expression and the volume-activated Cl－ current may be the non-specific effects of the oligonucleotide synthesized or of the transfection reagent lipofectin. To eliminate this possibility, the effect of a mismatch control oligonucleotide (three bases mismatched) and lipofectin on P-gp expression and the volume-activated Cl－ current was investigated. The results demonstrated that neither the mismatch control oligonucleotide nor lipofectin had any significant effect on P-gp expression and the current.

Is P-gp a chloride channel or a channel regulator in PCE cells? P-gp was first proposed to be both a drug transporter and a volume-activated chloride channel[5,12], it was then assumed to be a channel regulator[13,14]. In our experiments, inhibition of P-gp expression by an anti-MDR1 antisense oligonucleotide delayed the activation of the volume-activated chloride current and decreased the activation rate of the current. This suggests that endogenous P-gp may work as a channel regulator in PCE cells. However, inhibition of P-gp expression also reduced the peak current. This can be interpreted in two ways. First, the P-gp is the channel itself. Second, P-gp works as a channel regulator. Knocking down the channel regulator P-gp reduced the activation rate of the current. This led to a decrease in the amplitude of the whole-cell current in a certain time. However, mean peak current similar to that of the control cells should be obtained if enough time of exposure to hypotonic solution was allowed. To study this further, we extended the exposure time from 7 min to 15 min after the activation of the chloride current, but the current could not be significantly increased further. Thus, we cannot exclude the possibility that P-gp functions additionally as a chloride channel.

Our immunofluorescence data demonstrated that P-gp immunofluorescence was knocked down by 93% by an anti-MDR1 antisense oligonucleotide. However, the volume-activated chloride current at a +80 mV step was only reduced by 56% by the antisense oligonucleotide. This implies that there is more than one protein that is associated with the activation of the volume-activated Cl－ current in the pigmented ciliary epithelial cell. P-gp is just only one of the proteins. PCE cells may possess multiple Cl－ channels or channel regulators.

In summary, our work has revealed that PCE cells express P-gp. The endogenous P-gp is involved in the activation of the volume-activated chloride current. The evidence suggests that P-gp functions as both a volume-activated chloride channel and a channel regulator.

REFERENCES

[1]Chen LX (陈立新), Wang LW (王立伟). Volume-activated chloride current in pigmented ciliary epithelial cells. Acta Physiol Sin (生理学报), 2000,52:421～426.

[2]Jacob TJC, Civan MM. Role of ion channels in aqueous humour formation. Am J Physiol, 1996,271:C703～C720.

[3]Paulmichl M, Li Y, Wickman K, Ackerman M, Peralta E, Clapham D. New mammalian chloride channel identified by expression cloning. Nature, 1992,356:238～241.

[4]Duan D, Winter C, Cowley S, Hume JR, Horowitz B. Molecular identification of a volume-regulated chloride channel. Nature, 1997,390:417～421.

[5]Valverde MA, Diaz M, Sepulveda M, Gill DG. Hyde SC, Higgins CF. Volume regulated chloride channels are associated with the human multidrug-resistance P-glycoprotein. Nature, 1992,355:830～833.

[6]Gründer S, Thiemann A, Pusch M, Jentsch TJ. Regions involved in the opening of ClC-2 chloride channel by voltage and cell volume. Nature, 1992,360:759～762.

[7]Chen L, Wang L, Jacob TJC. Association of intrinsic PICln with volume-activated Cl－ current and volume regulation in a native epithelial cell. Am J Physiol, 1999,276:C182～C192.

[8]Wang L, Chen L, Jacob TJC. The role of ClC-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition. J Physiol, 2000,524:63～75.

[9]Chen C-J, Chin JE, Ueda K, Clark DP, Pastan I, Gottesman MM, Roninson IB. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug resistant human cells. Cell, 1986,47:381～３８9.

[10]Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch, 1981,391:85～100.

[11]Schlingensiepen R, Brysch, W, Schlingensiepen K-H. Antisense－From Technology to Therapy: Lab Manual and Textbook. Berlin: Blackwell Science, 1997.

[12]Gill DR, Hyde SC, Higgins CF, Valverde MA, Mintenig GM, Sepulveda FV. Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein. Cell, 1992,71:23～32.

[13]Higgins CF. Volume-activated chloride currents associated with the multidrug resistance P-glycoprotein. J Physiol, 1995,482P:31S～36S.

[14]Bond TD, Higgins CF, Valverde MA. P-glycoprotein and swelling-activated chloride channels. Methods Enzymol, 1998,292:359～370.