Membrane transport mechanisms of quinidine and procainamide in renal LLC-PK1 and intestinal LS180 cells.

Abstract

The aim of the present study was to compare the membrane transport mechanisms of procainamide with those of quinidine using renal epithelial LLC-PK(1) and intestinal epithelial LS180 cells. In LLC-PK(1) cells, the transcellular transport of 10 microM quinidine in the basolateral-to-apical direction was similar to that in the opposite direction, and 1 mM tetraethylammonium (TEA) did not affect the transcellular transport of the drug. On the other hand, the transcellular transport of 10 microM TEA and procainamide in LLC-PK(1) cells was directional from the basolateral side to the apical side. In addition, this directional transcellular transport of procainamide was diminished in the presence of 1 mM TEA. In LS180 cells, the temperature-dependent cellular uptake of 100 microM quinidine and procainamide was markedly increased by alkalization of the apical medium, and was inhibited significantly by 1 mM several hydrophobic cationic drugs, but not by TEA. The rank order of the inhibitory effects of hydrophobic cationic drugs on the uptake of procainamide in LS180 cells was imipramine>quinidine>diphenhydramine asymptotically equal topyrilamine>procainamide, which was consistent with that on the uptake of quinidine. These findings suggested that procainamide (but not quinidine) was transported by cation transport systems in renal epithelial cells, but that both procainamide and quinidine were taken up by another cation transport system in intestinal epithelial cells.

Full text

Page 1

We previously evaluated the renal excretion mechanism of
quinidine, which is a tertiary amine compound, using porcine
kidney epithelial LLC-PK1cells and P-glycoprotein (P-gp)-
expressed LLC-GA5-COL150 cells.1)The transepithelial
transport of quinidine in the basolateral-to-apical direction in
LLC-PK1cells was similar to that in the opposite direction.
In contrast, quinidine was transported actively in the baso-
lateral-to-apical direction in LLC-GA5-COL150 cells. The
results suggested that P-gp is mainly responsible for the
tubular secretion of quinidine in the kidney.1)We also evalu-
ated the intestinal absorption mechanism of quinidine using
human intestinal epithelial Caco-2 cells.2)The temperature-
dependent uptake of quinidine in Caco-2 cells grown on a
plastic dish was increased by alkalization of the apical
medium, and was inhibited by diphenhydramine and
imipramine. The results suggested that a cation transport sys-
tem was involved in the influx of quinidine at the apical
membrane in intestinal epithelial cells.2)
Procainamide, another tertiary amine compound with a
pKavalue of 9.23, is classified as a type IA antiarrhythmic
drug that works by decreasing conduction velocity, and pro-
longing tissue refractoriness.3)More than 80% of orally
administered procainamide is absorbed from the intestine in
humans.4)The Kp (octanol/buffer at pH 7.4) value of
procainamide is about 0.1, and approximately half of the
dose is excreted in the urine as unchanged drug.3—6)How-
ever, the mechanisms responsible for the membrane transport
of procainamide in intestinal and renal epithelial cells are
still unclear.
In the present study, the transport characteristics of pro-
cainamide in LLC-PK1cells were compared with those of
quinidine. In addition, we evaluated whether the transport
system for quinidine is present in another intestinal cell line,
LS180, as well as Caco-2. We also investigated whether the
transport system of procainamide in LS180 cells is the same
as that of quinidine.
MATERIALS AND METHODS
Materials
(2.04GBq/mmol), [3H]quinidine (740GBq/mmol), [14C]pro-
cainamide hydrochloride (2.04GBq/mmol), and [3H]manni-
tol (740GBq/mmol) were purchased from American Radio-
labeled Chemicals Inc. (St. Louis, MO, U.S.A.). [14C]Mannitol
(1.96GBq/mmol) was purchased from Moravek Biochemi-
cals (Brea, CA, U.S.A.). TEA chloride and procainamide
hydrochloride were obtained from Nacalai Tesque Inc.
(Kyoto, Japan). Quinidine hydrochloride monohydrate was
purchased from Sigma Aldrich (St. Louis, MO, U.S.A.). All
other chemicals were of the highest purity available.
Cell Culture and Preparation of Monolayers
PK1cells at passage 197 and LS180 cells at passage 38 were
obtained from the American Type Culture Collection (Man-
assas, VA, U.S.A.). These cells were maintained by serial
passage in plastic dishes with Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum (Bio-
west Inc., Nuaille, France) in an atmosphere of 5% CO2–95%
air at 37°C.
LLC-PK1cells were seeded at a density of 5?105cells/
cm2on a 1.12cm2porous membrane (3mm pore size) in a
polyester membrane Transwell®-Clear insert (Costar, Cam-
bridge, MA, U.S.A.) to evaluate the transcellular transport of
cationic drugs. The seeded cells were maintained for 6d to
prepare differentiated cell monolayers. The maturity of the
monolayer was judged by transepithelial electrical resistance
(TEER). TEER was measured using a Millicell-ERS resist-
ance system (Millipore, Bedford, MA, U.S.A.). LLC-PK1cell
monolayers whose TEER was above 60W·cm2were used to
[14C]Tetraethylammonium (TEA) bromide
LLC-
August 2010 1407 Regular Article
Membrane Transport Mechanisms of Quinidine and Procainamide in
Renal LLC-PK1and Intestinal LS180 Cells
Miki MASAGO, Mari TAKAAI, Jumpei SAKATA, Asuka HORIE, Toshikazu ITO, Kazuya ISHIDA,
Masato TAGUCHI, and Yukiya HASHIMOTO*
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama; 2630 Sugitani, Toyama 930–0194,
Japan.
Received April 5, 2010; accepted May 21, 2010; published online May 26, 2010
The aim of the present study was to compare the membrane transport mechanisms of procainamide with
those of quinidine using renal epithelial LLC-PK1and intestinal epithelial LS180 cells. In LLC-PK1cells, the
transcellular transport of 10m mM quinidine in the basolateral-to-apical direction was similar to that in the oppo-
site direction, and 1mM tetraethylammonium (TEA) did not affect the transcellular transport of the drug. On
the other hand, the transcellular transport of 10m mM TEA and procainamide in LLC-PK1cells was directional
from the basolateral side to the apical side. In addition, this directional transcellular transport of procainamide
was diminished in the presence of 1mM TEA. In LS180 cells, the temperature-dependent cellular uptake of
100m mM quinidine and procainamide was markedly increased by alkalization of the apical medium, and was
inhibited significantly by 1mM several hydrophobic cationic drugs, but not by TEA. The rank order of the
inhibitory effects of hydrophobic cationic drugs on the uptake of procainamide in LS180 cells was
imipramine?quinidine?diphenhydramine?pyrilamine?procainamide, which was consistent with that on the
uptake of quinidine. These findings suggested that procainamide (but not quinidine) was transported by cation
transport systems in renal epithelial cells, but that both procainamide and quinidine were taken up by another
cation transport system in intestinal epithelial cells.
Key words
procainamide; quinidine; LLC-PK1cell; LS180 cell
Biol. Pharm. Bull. 33(8) 1407—1412 (2010)
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: yukiya@pha.u-toyama.ac.jp

Page 2

assess the transcellular transport of cationic drugs. All exper-
iments were carried out with the cells between passages 210
and 217. On the other hand, LS180 cells were seeded at a
density of 5?105cells/cm2on a 3.8cm2plastic dish using a
Falcon®multiwellTMplate (BD Bioscience, Franklin Lakes,
NJ, U.S.A.), and maintained for 7d. All experiments were
conducted with LS180 cells between passages 63 and 75.
Transcellular Transport of TEA, Quinidine, and Pro-
cainamide in LLC-PK1Cell Monolayers
lular transport of [14C]TEA, [3H]quinidine, and [14C]pro-
cainamide in LLC-PK1cell monolayers prepared on a porous
membrane was examined as described previously.1,7)In brief,
the monolayer was pre-incubated for 30min at 37°C with
culture medium (pH 6.3) containing 10mM unlabeled drug to
equilibrate the drug concentration. After the equilibration
period, the radio-labeled drug (0.33mCi/ml) was applied to
the apical chamber (0.75ml) to examine apical-to-basolateral
transcellular transport. [3H]Mannitol was used to estimate the
paracellular transport and extracellular trapping of [14C]TEA
and [14C]procainamide, and [14C]mannitol was used to esti-
mate that of [3H]quinidine.1,7,8)A volume (50ml) of medium
in the basolateral chamber (1.5ml) was then collected 30, 60,
and 90min after the radio-labeled drug was applied. Cells on
the porous membrane were collected following the last col-
lection of medium. Radioactivity in the medium and cells
was determined using a liquid scintillation counter, and
normalized against the initially applied doses. The time
course of the transport of cationic drugs in the opposite
(basolateral-to-apical) direction was examined in a similar
manner.
The transcellular transport of [14C]TEA, [3H]quinidine,
and [14C]procainamide were analyzed in a model-dependent
manner using NONMEM software running on a mainframe
UNIX machine at the Kyoto University Data Processing Cen-
ter, as described previously.1,7,8)When transcellular drug
transport is examined under the condition where the un-
labeled drug concentration in the monolayer is equilibrated
with that of the incubation medium in the apical and baso-
lateral chambers, transport data for a small amount of radio-
labeled drug can be analyzed using the linear pharmacoki-
netic model. That is, the following mass balance equations
were prepared for pharmacokinetic analysis:
The transcel-
(1)
(2)
(3)
where XA, XB, and XCare the amounts of radio-labeled drugs
in the apical chamber, basolateral chamber, and monolayer
determined at time t, respectively. VAand VBindicate the vol-
ume of the apical chamber (0.75ml) and the basolateral
chamber (1.5ml), respectively. VCindicates the cell volume
of cell monolayers obtained from sulfanilamide accumula-
tion experiments, as described previously (1.12ml/cm2?
1.12cm2?1.25ml).1)The influx and efflux clearance of
cationic drugs at the apical membrane of cells was desig-
nated as CLA→Cand CLC→A, respectively. The influx and ef-
flux clearance of cationic drugs at the basolateral membrane
of cells was designated as CLB→Cand CLC→B, respectively.
Paracellular transport clearance (CLA↔B) was estimated by
analyzing the transport profile of mannitol using the follow-
ing mass balance equations:
(4)
(5)
Cellular Uptake of Quinidine and Procainamide in
LS180 Cells
The cellular uptake of [3H]quinidine and
[14C]procainamide was examined in the presence of 100mM
unlabeled drug using LS180 cells grown on plastic dishes of
a multiwell plate. The composition of the incubation medium
was as follows: 125mM NaCl, 4.8mM KCl, 5.6mM D-glu-
cose, 1.2mM CaCl2, 1.2mM KH2PO4, 1.2mM MgSO4·7H2O
and 25mM 2-[4-(2-hydroxyethyl)-1-piperazyl]ethanesulfonic
acid (HEPES), and the pH of the medium was adjusted with
a solution of NaOH. In order to evaluate the effect of the ex-
tracellular pH on the cellular uptake of cationic drugs,
HEPES (neutral pH) was replaced with 2-(N-morpholino)
ethanesulfonic acid (pH 5.5). The cells were first pre-incu-
bated for 10min at 37 or 4°C with 2ml incubation medium
containing 100mM unlabeled drug to equilibrate the drug
concentration, followed by incubation for 5min with 1ml
fresh incubation medium. The incubation medium was re-
placed with 1ml fresh incubation medium containing radio-
labeled drug (0.2mCi/ml). After the cells were incubated
with radio-labeled drug for another 5—30min at 37 or 4°C,
they were immediately washed with ice-cold phosphate
buffer and collected. Radioactivity in the cells was deter-
mined as described above. Radio-labeled mannitol was used
to estimate the extracellular trapping of cationic drugs.8—11)
The cellular uptake of [3H]quinidine and [14C]procain-
amide was analyzed in a model-dependent manner using
NONMEM software, as described previously.9,10)The follow-
ing mass balance equations were prepared for the pharmaco-
kinetic analysis:
(6)
(7)
where XMand XCare the amount of radio-labeled drugs in
the incubation medium and the cells determined at time t,
respectively. VMindicates the volume of incubation medium
(1ml). VCindicates the cell volume (2.62ml/cm2?3.8cm2?
9.96ml), measured with sulfanilamide, as described previ-
ously.8)The influx and efflux clearances of cationic drugs
were designated as CLM→Cand CLC→M, respectively.
In order to evaluate the effect of extracellular Na?on the
cellular uptake of [3H]quinidine and [14C]procainamide,
dX
dt
CL
V
X
CL
V
X
CMC
M
M
CM
C
C
??
→→
⋅⋅
dX
dt
CL
V
X
CL
V
X
MMC
M
M
CM
C
C
???
→→
⋅⋅
dX
dt
CL
V
X
CL
V
X
BAB
A
A
AB
B
B
??
↔↔
⋅⋅
dX
dt
CL
V
X
CL
V
X
AAB
A
A
AB
B
B
???
↔↔
⋅⋅
dX
dt
CL
V
X
CL
V
X
CL
V
X
CL
V
X
CAC
A
A
BC
B
B
CA
C
C
CB
C
C
????
→→→→
⋅⋅⋅⋅
dX
dt
CL
V
X
CL
V
X
CL
V
X
CL
V
X
BBC
B
B
CB
C
C
AB
A
A
AB
B
B
?????
→→↔↔
⋅⋅⋅⋅
dX
dt
CL
V
X
CL
V
X
CL
V
X
CL
V
X
AAC
A
A
CA
C
C
AB
A
A
AB
B
B
?????
→→↔↔
⋅⋅⋅⋅
1408 Vol. 33, No. 8

Page 3

NaCl in incubation medium was replaced with N-methyl-
D-glucamine hydrochloride, and the pH of the medium was
adjusted to 7.4 by the addition of KOH. The effect of various
cationic drugs on the uptake of [3H]quinidine and [14C]pro-
cainamide in LS180 cells on plastic dishes of a multiwell
plate was evaluated at 37°C. That is, the cells were first pre-
incubated for 10min with 2ml incubation medium, followed
by 5min incubation with 1ml fresh incubation medium sup-
plemented with 1mM various cationic drugs. The incubation
medium was replaced with 1 ml incubation medium contain-
ing radio-labeled drug (0.2mCi/ml). After the cells were
incubated with radio-labeled drug for 5min, the amount of
radio-labeled drug in the cells was determined as described
above.
Statistical Analysis
Values are expressed as the mean?
S.E. In all figures, when error bars are not shown, they
are smaller than the symbol. Multiple comparisons were
performed using Scheffé’s test following one-way ANOVA
provided that the variances of groups were similar. If this
was not the case, Scheffé-type test was applied following
Kruskal–Wallis analysis. The statistical significance of
differences between two groups was tested using Student’s
t-test provided that the variances of the groups were similar.
If this was not the case, the Mann–Whitney U-test was ap-
plied. p?0.05 was considered to be statistically significant.
RESULTS
Transcellular Transport and Membrane Transport
Characteristics of TEA, Quinidine, and Procainamide in
Renal Epithelial LLC-PK1Cells
gated the transcellular transport of 100mM and 0.1mM quini-
dine in LLC-PK1cells, and reported that quinidine is not sig-
nificantly transported via the transport systems involved in
the directional transport of TEA.1)In the present study, we
first evaluated the transcellular transport of 10mM TEA and
quinidine across LLC-PK1cell monolayers (Figs. 1A—C),
and performed a pharmacokinetic analysis of the data on the
transcellular transport and cellular accumulation of radio-
labeled drug using a 3-compartment (apical and basolateral
chambers, and cell) model (Table 1). The transcellular trans-
port of 10mM TEA in the basolateral-to-apical direction was
much greater than that in the opposite direction (Fig. 1A).
The CLB→Cvalue was greater than the CLA→Cvalue, and the
CLC→Avalue was greater than the CLC→Bvalue (Table 1). On
the other hand, the transcellular transport of 10mM quinidine
in the basolateral-to-apical direction was similar to that in the
opposite direction (Fig. 1B). The CLB→Cvalue of quinidine
was similar to the CLA→Cvalue, and the CLC→Bvalue was
also similar to the CLC→Avalue. In addition, the transcellular
transport and membrane transport clearance of quinidine
were not affected by 1mM TEA (Fig. 1C). According to these
findings, it was reconfirmed that the transport systems for
TEA contribute little to the transcellular transport of quini-
dine in renal LLC-PK1cells.
We next evaluated the transcellular transport and mem-
brane transport clearance of 10mM procainamide in LLC-
PK1cell monolayers (Figs. 1D,E). Procainamide was trans-
ported directionally from the basolateral side to the apical
side (Fig. 1D). The estimated CLC→Avalue was greater than
any other clearance values (Table 1). Moreover, directional
transport of procainamide was diminished in the presence of
1mM TEA (Fig. 1E). The CLB→Cand CLC→Avalues were
decreased to 82.7% and 51.4% of the control values, respec-
Previously, we investi-
August 20101409
Fig. 1.
Drugs in LLC-PK1Cell Monolayers
(A) 10mM TEA, (B) 10mM quinidine, (C) 10mM quinidine with 1mM TEA, (D)
10mM procainamide, (E) 10mM procainamide with 1mM TEA. Open and closed circles
represent apical-to-basolateral (A→B) and basolateral-to-apical (B→A) transport, re-
spectively. Solid and dotted lines are simulation curves obtained by pharmacokinetic
analysis for drugs and mannitol transport, respectively. Open and closed columns repre-
sent cellular accumulation from the apical (A→C) and basolateral (B→C) sides, re-
spectively. Data are expressed as the mean?S.E. of 6—11 experiments.
Transcellular Transport and Cellular Accumulation of Cationic
Table 1.Influx and Efflux Clearance (ml/min/cm2) of Cationic Drugs in LLC-PK1Cell Monolayers
Drug Conc. (mM)
CLB→C
CLC→B
CLA↔B
CLC→A
CLA→C
TEA
Quinidine
?1mM TEA
Procainamide
?1mM TEA
10
10
0.515?0.015
1.633?0.056
1.673?0.009
0.284?0.005
0.235?0.004*
0.013?0.001
0.196?0.012
0.188?0.004
0.201?0.002
0.224?0.002*
0.024?0.002
0.084?0.004
0.083?0.002
0.055?0.007
0.078?0.002
0.401?0.009
0.228?0.010
0.206?0.003
0.348?0.004
0.179?0.005*
0.094?0.026
1.740?0.004
1.729?0.002
0.212?0.011
0.172?0.004*
10
Values are expressed as the mean?S.E. for 6—11 experiments. CLB→Cand CLC→Brepresent the influx and efflux clearance at the basolateral membrane, respectively. CLA→C
and CLC→Arepresent the influx and efflux clearance at the apical membrane, respectively. CLA↔Brepresents the paracellular transport clearance. ∗p<0.05; significantly different
from each control.

Page 4

tively, by 1mM TEA (Table 1). These findings indicated that
the transport systems for TEA significantly contribute to the
transcellular transport of procainamide in renal LLC-PK1
cells.
Cellular Uptake of Quinidine and Procainamide in
Intestinal Epithelial LS180 Cells
reported that a cation transport system is involved in the
apical uptake of 100mM quinidine in human intestinal epithe-
lial Caco-2 cells.2,7)In the present study, we investigated
whether the cation transport system for quinidine is present
in another human intestinal epithelial cell line, LS180. Figure
2A shows the time course of the uptake of 100mM quinidine
at 37°C and 4°C in LS180 cells grown on plastic dishes.
The cellular uptake of quinidine was temperature-dependent,
and reached approximately 10% of the dose for 30min at
37°C (Fig. 2A). We performed a pharmacokinetic analysis of
the data on the cellular accumulation of quinidine using a
2-compartment (cell and medium) model (Table 2). The
CLM→Cand CLC→Mvalues of quinidine at 37°C were much
greater than those at 4°C (Table 2). We next investigated the
effect of extracellular Na?and pH on the initial (5-min) up-
take of quinidine in LS180 cells (Figs. 3A, 4A). No marked
difference was observed in the cellular uptake of quinidine in
the absence of extracellular Na?(Fig. 3A), whereas the cel-
lular uptake of quinidine was markedly greater at pH 7.5 than
at pH 5.5 (Fig. 4A). We also evaluated the effect of 1mM var-
ious cationic drugs on the cellular uptake of quinidine, which
was significantly inhibited by hydrophobic organic cations,
but not by typical hydrophilic cations, TEA and choline (Fig.
5A). The rank order of the inhibitory effects of hydrophobic
cationic drugs on the uptake of quinidine was imipramine?
quinidine?diphenhydramine?pyrilamine?procainamide
We have previously
(Fig. 5A). These findings suggested that quinidine was trans-
ported by the cation transport system in intestinal LS180
cells, as well as Caco-2 cells.
To compare the membrane transport characteristics of pro-
cainamide with those of quinidine in intestinal epithelial
cells, we also evaluated the uptake of 100mM procainamide
using LS180 cells. The cellular uptake of procainamide was
temperature-dependent, and reached approximately 4.4% of
the dose for 30min at 37°C (Fig. 2B). The CLM→Cand
CLC→Mvalues of procainamide at 37°C were much greater
than those at 4°C (Table 2). As shown in Figs. 3B and 4B,
1410 Vol. 33, No. 8
Fig. 2
LS180 Cells
(A) 100mM quinidine, (B) 100mM procainamide. Circles and triangles represent cel-
lular uptake of drugs at 37°C and 4°C, respectively. Solid lines represent simulation
curves obtained by pharmacokinetic analysis of the drug accumulation.
Time Course of the Uptake of Quinidine and Procainamide in
Fig. 3
cainamide in LS180 Cells
(A) 100mM quinidine, (B) 100mM procainamide. The cells were incubated with the
radio-labeled drug for 5min. Each column represents the mean?S.E. for 6 measure-
ments. ∗p<0.05; significantly different from Na?(?).
Effect of Extracellular Na+on the Uptake of Quinidine and Pro-
Fig. 4
cainamide in LS180 Cells
(A) 100mM quinidine, (B) 100mM procainamide. Open and hatched columns repre-
sent the uptake of drugs at pH 5.5 and 7.5, respectively. The cells were incubated with
the radio-labeled drug for 5min. Each column represents the mean?S.E. for 6 meas-
urements. ∗p<0.05; significantly different from pH 5.5.
Effect of Extracellular pH on the Uptake of Quinidine and Pro-
Fig. 5
cainamide in LS180 Cells
(A) 100mM quinidine, (B) 100mM procainamide. The cells were incubated with
radio-labeled drug for 5min in the presence of 1mM organic cations. Each column rep-
resents the mean?S.E. for 4—12 measurements. ∗p<0.05; significantly different from
the control.
Effect of Organic Cations on the Uptake of Quinidine and Pro-
Table 2.
cainamide in LS180 Cells
Influx and Efflux Clearance (ml/min/cm2) of Quinidine and Pro-
Drug Temperature (°C)
CLM→C
CLC→M
Quinidine37
4
37
4
7.542?0.140
1.897?0.025*
2.575?0.040
0.396?0.020*
0.647?0.029
0.335?0.003*
0.573?0.006
0.202?0.009*
Procainamide
Values are expressed as the mean?S.E. for 3—8 experiments. CLM→Cand CLC→M
represent the influx and efflux clearance in LS180 cells grown on plastic dishes, respec-
tively. ∗p<0.05; significantly different from 37°C.

Page 5

the initial (5-min) cellular uptake of procainamide was essen-
tially Na?-independent, but was significantly increased at pH
7.5 as compared with pH 5.5. In addition, the cellular uptake
of procainamide was also inhibited by hydrophobic organic
cations, but not by TEA and choline (Fig. 5B). The rank
order of the inhibitory effects of hydrophobic cationic drugs
on the uptake of procainamide was imipramine?quinidine?
diphenhydramine?pyrilamine?procainamide, which was con
sistent with that on the uptake of quinidine (Figs. 5A,B).
These findings suggested that the cation transport system for
procainamide in intestinal LS180 cells is similar/identical to
that for quinidine.
DISCUSSION
The present study had two major findings. First, pro-
cainamide (but not quinidine) was transported by the cation
transport systems in renal epithelial LLC-PK1cells. Second,
both procainamide and quinidine were transported by another
cation transport system in intestinal epithelial LS180 cells.
During the secretion of organic cations in the renal proxi-
mal tubules, cations are translocated across the basolateral
membrane, and then are released across the luminal mem-
brane.12)The porcine kidney epithelial cell line LLC-PK1
possesses the morphological structure and function similar
to those of renal proximal tubular cells, and has been widely
used to elucidate the transport properties of cationic
drugs.1,13—16)The membrane transport characteristics of
TEA, a prototypical substrate of organic cation transport sys-
tems in the kidney, have been well evaluated using LLC-PK1
cells.1,14,17)TEA was transported directionally across LLC-
PK1cell monolayers from the basolateral side to the apical
side.1,14)The unidirectional transport of TEA was tempera-
ture-dependent and saturable, and was inhibited by organic
cations, such as cimetidine and choline.14)The basolateral
uptake of TEA in LLC-PK1cells was dependent on the
inside negative potential, and was decreased by lowering
basolateral medium pH.15,18)On the other hand, the apical
efflux of TEA in LLC-PK1cells was markedly stimulated by
acidification of the apical medium.14,17)Recent studies have
demonstrated that organic cation transporter 2 (OCT2) is
expressed on the basolateral membrane of epithelial cells in
renal proximal tubules, and that it translocates TEA in an
electrogenic manner.12,19)In addition, multidrug and toxin
extrusion 1 (MATE1) is localized to the brush-border mem-
brane of renal proximal tubules, and is a TEA/proton
antiporter.12,19)
Takano et al. investigated the transport of procainamide in
LLC-PK1cell monolayers.16)They reported that the uptake of
procainamide in LLC-PK1cells grown on plastic dishes was
temperature-dependent and saturable, and was inhibited by
cimetidine. The apical uptake of procainamide was greater at
alkaline pH than acidic pH, and increased when intracellular
pH was decreased, indicating the involvement of a H?/pro-
cainamide antiport system in the apical membrane of LLC-
PK1cells. In addition, the basolateral-to-apical transport of
procainamide across LLC-PK1cell monolayers was greater
than transport in the opposite direction, and was inhibited by
cimetidine.16)To our knowledge, the present study is the first
report which provides apical and basolateral membrane
transport clearance values of procainamide in renal epithelial
LLC-PK1cells. The directional transport of procainamide
across LLC-PK1cell monolayers was less than that of TEA
(Figs. 1A,D), and the CLB→Cand CLC→Avalues of pro-
cainamide were smaller than those of TEA (Table 1). The
findings suggested that procainamide is transported by
cation-specific transport systems in renal LLC-PK1cells, but
that the intrinsic activity and/or affinity are low as compared
with TEA.
We previously evaluated the cellular uptake of 100mM
quinidine in intestinal epithelial Caco-2 cells, which are
widely used as an experimental model to study the intestinal
absorption of various drugs.2,7)The cellular uptake of quini-
dine in Caco-2 cells grown on plastic dishes was tempera-
ture-dependent, and markedly increased by alkalization of
the apical medium at 37°C.2)In addition, the uptake of quini-
dine was inhibited by hydrophobic organic cations, and the
rank order of the inhibitory effects of hydrophobic cationic
drugs was imipramine?quinidine?diphenhydramine.2,7)In
the present study, to confirm the involvement of the cation
transport system in intestinal absorption of quinidine, we in-
vestigated the membrane transport mechanism of quinidine
using another intestinal epithelial cell line, LS180. This cell
line possesses characteristics of the small intestine (e.g., ex-
pression of microvillus), and has also been used as an in vitro
model of the intestine.8—11,20)The cellular uptake of quini-
dine in LS180 cells was temperature- and pH-dependent, and
was inhibited by hydrophobic cationic drugs with the follow-
ing rank order of inhibitory effect: imipramine?quinidine?
diphenhydramine?pyrilamine?procainamide (Figs. 2—5).
These findings indicated that the cation transport system is
present not only in Caco-2 cells, but also in LS180 cells.
In the present study, we demonstrated that procainamide is
transported by the TEA-insensitive cation transport system in
human intestinal epithelial LS180 cells. Procainamide may
be actively taken up by the cation transport system not only
in the human intestine, but also in the rabbit intestine. Ka-
tsura et al. investigated the transport mechanism of pro-
cainamide using rabbit intestinal brush-border membrane
vesicles.21)The uptake of procainamide in the vesicles was
stimulated by an outward H?gradient, and the stimulation
was reduced by the addition of carbonyl cyanide p-(trifluo-
romethoxy)phenylhydrazone (FCCP), a protonophore. In ad-
dition, the initial uptake of procainamide was inhibited by
tertiary amines, such as diphenhydramine and triethylamine,
but not by TEA. They thought that the transport of pro-
cainamide at the intestinal brush-border membrane is medi-
ated by the H?/tertiary amine antiport system.21)Further
studies will be needed to identify the specific transport sys-
tem for cationic drugs in the rabbit and/or human intestine.
In conclusion, cation transport systems in renal epithelial
LLC-PK1cells transport procainamide, but not quinidine. In
contrast, another cation transport system in intestinal epithe-
lial LS180 cells takes up both quinidine and procainamide.
Acknowledgements
Grants-in-Aid for Scientific Research from Japan Society for
the Promotion of Sciences (JSPS) and from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT)
of Japan.
This work was supported in part by
August 20101411