(Received for publication, July 5, 1995; and in revised form, September 13, 1995)
From the
To determine whether endogenous P-glycoprotein, the MDR1 gene product that functions as a drug transport pump, is a
volume-sensitive Cl channel molecule or a protein
kinase C-mediated regulator of the Cl
channel,
whole-cell patch-clamp and molecular biological experiments were
carried out in a human small intestinal epithelial cell line.
Endogenous expression of P-glycoprotein was confirmed by Northern blot
analysis, reverse transcription-polymerase chain reaction, Western blot
analysis, and immunostaining. The P-glycoprotein expression was
abolished by the antisense (but not sense) oligonucleotide for the MDR1 gene, whereas the magnitude of the Cl
current activated by osmotic swelling was not distinguishable
between both antisense- and sense-treated cells. The volume-sensitive
Cl
currents were not specifically affected by the
anti-P-glycoprotein monoclonal antibodies, MRK16, C219, and UIC2. An
inhibitor of P-glycoprotein-mediated pump activity, verapamil, was
found to never affect the Cl
current. A substrate for
the P-glycoprotein-mediated drug pump, vincristine or daunomycin, did
not prevent swelling-induced activation of the Cl
current. Furthermore, the Cl
current was not
affected by an activator of protein kinase C
(12-O-tetradecanoylphorbol-13-acetate or
1-oleoyl-2-acetyl-sn-glycerol). Thus, it is concluded that the
endogenous P-glycoprotein molecule is not itself a volume-sensitive
Cl
channel nor a protein kinase C-mediated regulator
of the channel in the human epithelial cells.
Cell volume regulation is one of the most fundamental functions
of living cells. A variety of ion channels and transporters are known
to be activated upon osmotic cell swelling and involved in volume
regulation(1, 2, 3) . Among them,
volume-sensitive Cl channels have recently been
studied in a variety of cell
species(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
However, the volume-sensitive Cl
channel molecule has
not as yet been identified.
P-glycoprotein, the MDR1 gene
product, which is an ATP-dependent transporter responsible for
multidrug resistance to many anti-neoplastic agents(17) , was
recently proposed as a candidate molecule of the volume-sensitive
Cl channel(18, 19) . Higgins and his
collaborators (18) suggested that P-glycoprotein (P-gp) (
)is bifunctional, switching between a drug pump upon
exposure to its substrates and a Cl
channel upon
osmotic swelling, based on the following observations. 1)
Volume-sensitive Cl
conductances correlated with P-gp
expression induced by transfection with MDR1(18) ; 2)
inhibitors of the drug pump inhibited the volume-sensitive
Cl
conductance(18, 20) ; 3) the
volume-sensitive Cl
conductance was abolished by
treatment with antisense oligonucleotides against the 5`-end of the MDR1 gene(18) ; and 4) intracellular administration of
a substrate for the P-gp-mediated drug pump prevented swelling-induced
activation of the Cl
conductance but failed to
inhibit the preactivated volume-sensitive Cl
conductance(19, 20) .
Subsequently, many
laboratories have failed to find any correlation between the size of
volume-sensitive Cl currents and the P-gp expression
level in multidrug-resistant or MDR1-transfected cells and
their control counterparts (21, 22, 23, 24, 25) .
Moreover, volume-sensitive Cl
currents in many cell
types have been found to be insensitive to blockers of P-gp-mediated
drug
transport(12, 22, 23, 24, 26, 27) .
Higgins and his collaborators (28) then proposed an alternative
hypothesis that P-gp regulates volume-sensitive Cl
channels via protein kinase C (PKC)-mediated phosphorylation of
P-gp.
The purpose of the present study is to directly examine
whether endogenous P-gp is a volume-sensitive Cl
channel molecule or a PKC-mediated regulator of the Cl
channel. We employed a human small intestinal epithelial cell
line (Intestine 407) as a model system for the following reasons. (a) Small intestinal epithelial cells endogenously express
sizable P-gp(29, 30) ; (b) cell volume
regulation is a prerequisite for the normal function of enterocytes in
which accumulation of osmotically active solutes is inevitably produced
by their vigorous active transport(31, 32) ; (c) in Intestine 407 cells prominent activation of
volume-sensitive Cl
currents is actually induced by
osmotic swelling(4) , and the channel properties have been well
characterized (4, 33) ; and (d) in this cell
line the PKC activity has been known to play a significant role in
receptor-mediated cell responses (34) . The present results
clearly indicated that the endogenous activity of volume-sensitive
Cl
channel is totally independent of endogenously
expressed P-gp in this cell line.
Amplification and detection of human MDR1 and MDR2 mRNA in Intestine 407 and Hep G2 cells were achieved by reverse transcription-polymerase chain reaction (RT-PCR), as described previously(35) . MDR1-specific sequences were amplified by using the sense strand primer CTTCAGGGTTTCACATTTGGC (residues 2314-2334) and the antisense strand primer CTCATGAGTTTATGTGCCACC (residues 2889-2909), which yield a 596-base pair (bp) PCR product. MDR2-specific sequences were CTTTCAAGCAGTAGCCACG (residues 585-603; sense strand) and TGTGCCCTCATCAGGGT (residues 1340-1356; antisense strand). The resulting PCR product using these amplimers was 772 bp.
For the immunofluorescence detection of P-gp Intestine 407 cells were cultured on glass coverslips or tissue culture chambers (16-well Chamber Slide; Nunc, Naperville). The cells were fixed in phosphate-buffered saline (PBS) containing 1% paraformaldehyde for 30 min at 4 °C. They were washed with PBS, and blocking was made with 10% normal goat serum in PBS for 30 min at room temperature. They were then incubated overnight at 4 °C with 0.3 ml of PBS containing 2.7 µg of monoclonal antibody (MRK16) and then washed and incubated for 2 h at room temperature with 0.3 ml of biotinylated sheep antimouse Ig (Amersham) diluted 1:200 with PBS. They were then washed and incubated for 2 h at room temperature with 0.3 ml of streptavidin Texas Red (diluted 1:300 with PBS, Amersham). After a third wash with PBS, the cells were mounted on slides and examined with a fluorescence microscope (model BH-2; Olympus, Tokyo).
The pipettes were fabricated from borosilicate
glass capillaries (Hilgenberg, Malsfeld, Germany) using a puller (P-97;
Sutter Instrument, Novato). Wide-tipped patch pipettes with a tip
resistance of around 1.5 M when filled with the pipette solution
were employed in order to reduce the voltage drop across the residual
series resistance and to efficiently introduce a monoclonal antibody
into the cytosol.
Whole-cell recordings were carried out at room temperature (22-26 °C), as described previously(4) .
The following agents were
added to the bath solution: verapamil, nifedipine, cyclosporin A,
vincristine, 1,9-dideoxyforskolin (DDFSK), tamoxifen,
12-O-tetradecanoylphorbol-13-acetate (TPA), and
1-oleoyl-2-acetyl-sn-glycerol (OAG). When necessary,
vincristine or daunomycin was added to the pipette solution. All the
agents except for cyclosporin A (a gift from Dr. T. Tsuruo, University
of Tokyo) and TPA (Wako, Osaka) were from Sigma. Stock solutions
(10-250 mM) of tamoxifen (in methanol), TPA (in
ethanol), and others (in dimethyl sulfoxide) were diluted to the
desired final concentrations immediately before use. The vehicle alone
at the concentrations employed (0.1%) did not affect the
volume-sensitive Cl
currents in Intestine 407 cells.
In some experiments, an anti-P-gp monoclonal antibody, MRK16 (a gift
from Dr. T. Tsuruo)(40) , MRK17 (a gift from Dr. T. Tsuruo) (40) or UIC2 (Ingenex, Menlo Park)(41) , or the
purified mouse IgG (ICN, Costa Mesa) was applied to the
bath solution. Another anti-P-glycoprotein antibody, C219 (Centocor,
Malvern)(42) , was added to the pipette solution.
Figure 1: Expression of MDR1 mRNA and P-glycoprotein in Intestine 407 cells. A, Northern blot analysis of total RNA of Intestine 407 cells. An intense band is detected at around 4.4 kilobase pairs (kb). B, RT-PCR analysis of MDR1 mRNA (lanes 1 and 2) and MDR2 mRNAs (lanes 3 and 4) from Hep G2 cells (lanes 1 and 3) and Intestine 407 cells (lanes 2 and 4). M, DNA size markers. Glyceraldehyde-3-phosphate dehydrogenase-specific PCR product, which was used as an internal cDNA control, was equally observed throughout the experiments (data not shown). C, Western blot analysis of crude membrane fractions from Hep G2 (lane 1) and Intestine 407 (lane 2) cells reacted with the anti-P-glycoprotein monoclonal antibody, C219.
For further confirmation of the P-gp expression indirect immunofluorescence was carried out using MDR1-specific monoclonal antibody, MRK16. As shown in Fig. 2A, Intestine 407 cells were found to highly express P-gp.
Figure 2: Indirect immunofluorescence assay using the anti-P-glycoprotein monoclonal antibody, MRK16. A, immunofluorescence on the control Intestine 407 cells. B, background fluorescence on the control cells (without antibody). C, immunofluorescence on the cells after 4-day incubation with sense oligonucleotides for MDR1 gene. All the cells treated with sense oligonucleotide showed a staining pattern somewhat different from the control cells. D, immunofluorescence on the cells after 4-day incubation with antisense oligonucleotides for MDR1 gene. Scale bar, 20 µm.
In both cells treated with the sense
and antisense oligonucleotides, as shown in Fig. 3(A and B, left traces), activation of whole-cell
Cl currents, essentially to the same extent, was
consistently observed in association with osmotic swelling. These
results are in contrast to a previous antisense study in NIH-3T3/MDR
cells(18) . Voltage dependence of the current profile (Fig. 3, A and B, right traces) and
the mean amplitudes of peak current density (Fig. 3C)
were indistinguishable between the cells treated with the sense and
antisense oligonucleotides. These results clearly indicate that the
activity of the volume-sensitive Cl
channel does not
require the P-gp expression.
Figure 3:
Effects of sense and antisense
oligonucleotides on the volume-sensitive Cl current. A and B, representative traces of whole-cell
Cl
currents recorded before and after hypotonic
challenge (at horizontal lines) during application of
alternative 2-s pulses from 0 to ±40 mV (left) and from
0 to ±20, ±40, ±60, and ±80 mV (left at asterisk; right for the expanded traces) in
the Intestine 407 cells treated with sense (A) and antisense
oligonucleotides (B). (The gain of the chart recorder was
changed by half at the second asterisk in B.) Arrowheads indicate the zero current level. Horizontal
lines above the current traces indicate the time of hypotonic
challenge. Virtually no differences were observed between capacitances
in sense- and antisense-treated cells. C, the mean current
densities of peak Cl
currents recorded at +80 mV
in sense-treated cells (n = 9) and antisense-treated
cells (n = 12). These were not statistically different
from each other. Vertical bars represent
S.D.
Figure 4:
Effects
of anti-P-glycoprotein monoclonal antibody, C219, on the
volume-sensitive Cl currents. A and B, representative traces of whole-cell Cl
current responses to alternating 2-s pulses from 0 to ±40
mV (A) and from 0 to ±100 mV in 20-mV steps (A at asterisk; B for the expanded traces) before
and after hypotonic challenge (at horizontal bar) in Intestine
407 cells dialyzed with intrapipette C219 (10 µg/ml) for over 15
min. The arrowhead indicates the zero current level. C, the mean current density of the peak Cl
current at +80 mV in the presence of C219 (n = 8) and that in the absence of the antibody (n = 5). These were not statistically different from each
other. Vertical bars represent
S.D.
A
monoclonal antibody specific to the MDR1 product, MRK16 (40) , the epitope of which is the extracellular domain of
P-gp(45) , is known to partially inhibit the P-gp-mediated pump
function(40, 45, 46) . Bath application of
MRK16 (27 µg/ml) partially inhibited the Cl current (by 32.1 ± 20.4%, n = 7) (Fig. 5A). However, IgG
(10 µg/ml),
which is the immunoglobulin subclass for MRK16(40) , itself
caused partial inhibition of the current to a similar extent (by 34.3
± 13.1%, n = 3) (Fig. 5B). Even
in the cells preincubated with MRK16 (11 µg/ml) for 15 h, a
hypotonic challenge activated the Cl
current, and
MRK16 again partially inhibited the activated Cl
current (Fig. 5C). Extracellular application of
other anti-P-gp monoclonal antibodies, MRK17 (40) and
UIC2(41) , the epitopes of which are extracellular, also
produced partial inhibition effects at similar concentrations (data not
shown).
Figure 5:
Effects
of anti-P-glycoprotein monoclonal antibody, MRK16, on the
volume-sensitive Cl currents. Representative traces
of whole-cell Cl
currents recorded during application
of alternating 2-s pulses (from 0 to ±40 mV) before and after
hypotonic challenge in Intestine 407 cells without MRK16 pretreatment (A and B) or those with pretreatment with MRK16 (11
µg/ml) (C). MRK16 (27 µg/ml in A; 11
µg/ml in C) or the corresponding immunoglobulin
(IgG
, 10 µg/ml in B) was applied to the bath.
In C whole-cell recordings were started over 10 min after
washout of the antibody in the cells pretreated with MRK16 for 15 h. Arrowheads indicate the zero current level. Horizontal
lines above the current traces indicate the time of hypotonic
challenge or application of antibodies or
immunoglobulin.
Figure 6:
Effects of P-gp substrates on the
volume-sensitive Cl currents. A and B, representative traces of whole-cell Cl
currents recorded during application of alternating 2-s pulses
(from 0 to ±40 mV) and 5-s step pulse from -80 to
+20, +40, +60, and +80 mV (at asterisk)
before and after hypotonic challenge (at horizontal lines) in
Intestine 407 cells dialyzed for over 10 min with intrapipette
vincristine (250 µM, A) or daunomycin (250
µM, B). Arrowheads are the same as those
in Fig. 3. C, the mean current density of the peak
Cl
current at +80 mV in the presence of either
substrate was not statistically different from that in the absence of
substrate (control).
Figure 7:
Effects of P-gp inhibitors on the
volume-sensitive Cl currents. Verapamil (100
µM, A), nifedipine (100 µM, B), and DDFSK (100 µM, C) were applied
to the bath after (upper traces) or before hypotonic challenge (lower traces). Whole-cell Cl
currents were
recorded during application of alternative 2-s pulses from 0 to
±40 mV (or 0 to ±80 mV in 20-mV steps at asterisk). Arrowheads indicate the zero current
level. Horizontal lines above the current traces indicate the
time of the hypotonic challenge or the compound
application.
Figure 8:
Varying effects of a number of P-gp
inhibitors of volume-sensitive Cl currents. A, effects of bath application of verapamil (100
µM, n = 9), nifedipine (100
µM, n = 7), cyclosporin A (10
µM, n = 4), tamoxifen (1 µM, n = 4), or DDFSK (100 µM, n = 6) on the preactivated Cl
currents. The
percent of inhibition was evaluated from the peak Cl
current densities (at +40 mV) before (control, 130-150
pA/pF) and after application of the compound. B, effects of
pretreatment with extracellular verapamil (100 µM, n = 4), nifedipine (100 µM, n =
4), cyclosporin A (10 µM, n = 4),
tamoxifen (1 µM, n = 4), or DDFSK (100
µM, n = 4) on the swelling-induced
activation of Cl
currents. The percent of inhibition
was evaluated from the peak Cl
current densities (at
+40 mV) before and after (control, 80-100 pA/pF) washout of
the compound. Essentially similar results were obtained for the peak
Cl
current observed at +80 mV (n = 2-4).
Figure 9:
Effects of TPA or OAG on the
volume-sensitive Cl currents. Representative traces
of whole-cell Cl
currents recorded during application
of alternating 2-s pulses (0 to ±40 mV) in the absence or
presence of TPA (30 nM, A) or OAG (50
µM, B). The PKC activator was applied to the bath
after (a) or before (b) hypotonic challenge. Arrowheads and horizontal lines above the current
traces are the same as those in Fig. 7. The mean values of
preactivated volume-sensitive Cl
current density (at
+40 mV) before and after compound application were 157.4 ±
20.7 and 175.6 ± 29.9 pA/pF (n = 6) for TPA (A, a) as well as 186.7 ± 22.6 and 178.9
± 18.4 pA/pF (n = 5) for OAG (B, a), respectively. Those activated in the presence of TPA (A, b) and OAG (B, b) were 157.9 ± 13.4 (n = 5) and 183.7 ± 38.3 pA/pF (n = 3)
at +40 mV, respectively.
Recently, there has been an upsurge of interest in the
possible relation between the drug pump P-gp and the
Cl channel activity because of structural similarity
of P-gp to the cystic fibrosis transmembrane conductance regulator
(CFTR) Cl
channel, both of which belong to the ABC
(ATP-binding cassette) superfamily of transporters. In this connection,
two intriguing hypotheses have been proposed by the same group:
``pump/channel bifunctional hypothesis'' (18, 19) and ``PKC-mediated channel regulator
hypothesis''(28) . In the first hypothesis, P-gp is
assumed to be itself a volume-sensitive Cl
channel
molecule(18, 19) . However, virtually complete
inhibition of P-gp expression by antisense oligonucleotides against the
5`-end of the MDR1 gene failed to affect swelling-induced
activation of the Cl
channel in Intestine 407 cells ( Fig. 2and Fig. 3). This result is in good agreement with
an increasing number of recent papers, which report lack of correlation
between P-gp expression and the magnitude of the volume-sensitive
Cl
conductance(21, 22, 23, 24, 25) .
Also, in the present study four types of monoclonal antibodies directed
against P-gp (C219, MRK16, MRK17, and UIC2) were all found to be
ineffective in inhibiting the activity of the volume-sensitive
Cl
channel in Intestine 407 cells ( Fig. 4and Fig. 5). According to the bifunctional model, binding of the
substrate to the drug transporter P-gp should inhibit activation of
Cl
currents associated with osmotic swelling (19, 20) . However, as shown in Fig. 6,
Cl
currents of normal size were activated upon
osmotic swelling of Intestine 407 cells loaded with the substrate
(vincristine or daunomycin). Sensitivity of swelling-induced
Cl
currents to prior treatment with inhibitors for
P-gp-mediated drug transport (such as verapamil) has been thought to be
additional support for the bifunctional
hypothesis(18, 20) . In Intestine 407 cells, verapamil
never suppressed the volume-sensitive Cl
current (Fig. 7). This observation is definitely incompatible with the
bifunctional hypothesis. However, there remains the possibility that
P-gp and the volume-sensitive Cl
channel bear some
structural similarity or some functional correlation with each other,
since many other inhibitors for the drug pump function of P-gp (DDFSK,
tamoxifen, nifedipine, and cyclosporin A) suppressed, to a varying
extent, the volume-sensitive Cl
current in Intestine
407 cells ( Fig. 7and Fig. 8).
The second
``PKC-mediated regulator hypothesis'' is based on the
observation that swelling-induced Cl currents were
abolished by pretreatment with a PKC activator, TPA (30 nM, 2
min), in NIH-3T3 cells transfected with human MDR1 but not in
the parental cells lacking detectable P-gp expression(28) , and
also by the fact that the P-gp pump function is likely to be modulated
by phosphorylation due to PKC activation(48, 49) . In
the present study, however, both TPA and OAG failed to prevent
swelling-induced activation of the Cl
currents in
Intestine 407 cells (Fig. 9). This fact clearly rules out the
possibility that endogenous P-gp is involved in the modulation of the
volume-sensitive Cl
channel through PKC-mediated
phosphorylation of P-gp. Also, our previous studies showed that the
activity of the volume-sensitive Cl
channel in
Intestine 407 cells was not affected by treatment with PKC inhibitors,
polymyxin B (4) and H-7(33) . Therefore, it appears
that PKC-mediated phosphorylation of P-gp (or the Cl
channel) is not involved in the mechanism of activation or regulation
of volume-sensitive Cl
channels in the human
epithelial cells.
The present study clearly showed that P-gp, an ABC
transporter, is not the volume-sensitive Cl channel
molecule nor its PKC-mediated regulator. CFTR, which also belongs to
the ABC superfamily, is not responsible for the volume-sensitive
Cl
channel molecule, because the volume-sensitive
Cl
channel is not activated by cyclic
AMP(4) , has a halide selectivity distinct from that for CFTR (4) , and was found to be insensitive to glibenclamide (30
µM), (
)which is known to inhibit CFTR
Cl
channels(50, 51) . Another cloned
non-ligand-gated Cl
channel is CLC(52) .
Among the CLC members, CLC-2 was shown to exhibit volume
sensitivity(53) . However, the volume-sensitive Cl
channel in Intestine 407 cells is distinct from CLC-2 in the
voltage sensitivity, halide selectivity, and sensitivity to a stilbene
derivative Cl
channel
blocker(4, 33) . Thus, the molecular identification of
the volume-sensitive Cl
channel is still open to
investigation.