ORIGINAL ARTICLE
Annals of Nuclear Medicine Vol. 18, No. 3,227-234, 2004

Transceilular transport of radioiodinated 3-iodo-tz-methyl-L-tyrosine
across monolayers of kidney epithelial cell line LLC-PKINaoto SHIKANO,*Keiichi KAWAI,**Syuichi NAKAJIMA,*Akiko KUBODERA,***
Nobuo KUBOTA,*Nobuyoshi ISHIKAWA*and Hideo SAJI*****Department of Radiological Sciences, lbaraki Prefectural University of Health Sciences**School of Health Sciences, Faculty of Medicine, Kanazawa University***Faculty of Pharmaceutical Sciences, Science University of Tokyo****Graduate School of Pharmaceutical Sciences, Kyoto University

Objective: 3-[123I]iodo-o~-methyl-L-tyrosine ([123I]IMT) is an imaging agent for amino acidtransport. In order to obtain fundamental data related to tumor imaging with [123I]IMT and renal
physiological accumulation of [123I]IMT, we investigated the transport characteristics of [125I]IMTin porcine kidney epithelial cell line LLC-PKI using cell monolayers grown on microporous
membrane filters. Methods: LLC-PKI monolayers were created on a collagen-coated microporous(3/.tm) membrane (4.7 cm2). To examine transcellular transport (secretion and reabsorption) andaccumulation, the monolayers were incubated for up to 90 rain at 37~ with 18.5 kBq [125I]IMT in
Dulbecco's phosphate-buffered saline (pH 7.4) as an uptake solution. After incubation, transcellulartransport was assessed by quantifying the radioactivity of the solutions on each side of themonolayer. For the accumulation experiment, the cells were solubilized in NaOH solution, and theradioactivity was quantified. For the inhibition experiment, the inhibitor was added at a final
concentration of 1 mM. For the pH dependence experiment, the pH of the apical-side uptake
solution was varied from pH 5 to pH 8. Transport of [lgc]Tyr was examined for comparison.
Results: Bi-directional transcellular transport of [125I]IMT was observed, corresponding to secretion and reabsorption in proximal tubule. Accumulation of [125I]IMT from the basolateral side (1.62+_0.15%) and the apical side (2.62 + 0.35%) was observed at 90 rain. 2-Amino-bicyclo[2,2,1 ]heptane2-carboxylic acid (a specific inhibitor of system L), L-Tyr (mother compound of [ I25I]LMT) and 2aminoisobutyric acid (an inhibitor of system L and A) inhibited both directional transport (p < 0.01)
and accumulation (p < 0.01). 2-(Methylamino)isobutyric acid (a specific inhibitor of system A)
appeared to inhibit transport and accumulation, but the results were not significant. Decreasing
apical pH significantly enhanced accumulation of [125I]IMT from both sides (p < 0.001), whereas
accumulation of mother L-Tyr was significantly suppressed. Conclusions: The inhibition experiments suggest that the main contributor to [125I]IMT transport is system L, rather than Na +dependent transport, in both apical and basolateral membrane. [Iz5I]IMT was transported by thesystem that transported L-Tyr, but the observed pH dependence of transport suggests that different
mechanisms are involved in accumulation of [125I]IMT and [14C]Tyr.

Key words: amino acid ~ansport, systemL, epithelial cellline, LLC-PK1, 3-iodo-a-methyl-Ltyrosine
Received December 22, 2003, revision accepted February 5,
2004.
For reprint contact: Naoto Shikano, M.S., Department ofRadiological Sciences, Ibaraki PrefecturalUniversity of Health
Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 3000394, JAPAN.
E-mail: sikano@ipu.ac.jp

Vol. 18, No. 3, 2004

~TRODUCTION
3-[123I]Iodo-a-methyl-L-tyrosine ([123I]IMT, Fig. 1), a
non-metabolizable artificial amino acid, was developed
as an imaging agent for activity of L-Tyr transport. 1-3 The
majority of transport studies using [123I]IMT have examined transport in tumor cell lines4; e.g., rat C6 glioma

Original Article 227

cells, 5 human GOS3 glioma cells 6 and human Ewing's
sarcoma cells. 7 In a recent study, we observed marked
accumulation of [125I]IMT in the renal cortex, 8 whereas amoderate accumulation of radiolabeled L-Tyr has been
observed. 9 These findings raised questions about themechanisms of [123I]IMT accumulation and membranetransport in a kidney epithelial cell line.
LLC-PKt is an epithelial cell line from a Hampshire pig
kidney. LLC-PKI cells are polarized epithelial cells withapical and basolateral membrane domains, l~ We presumed that transcellular transport studies with artificial
amino acids would be an interesting new application ofthis epithelial cell line. A confluent monolayer of LLCPK1 cells forms an oriented monolayer with tight junctions, and exhibits reabsorption of electrolytes and somenutrients via the microvilli of the apical membrane. 13
LLC-PKI has a hexose and phosphate transport systemwith characteristics similar to those observed in the proximal tubule.l~ Expression of amino acid transport systems L, A and ASC in LLC-PKI cells has been observed. 13
LLC-PKI cells are stable through more than 300 serial
passages, with no evidence of transformation. ~3LLC-PKI
retains several of the characteristics of differentiated renal
proximal tubular cells. It has been employed extensively
as a model for analysis of several functions in the proximal tubules. 14
In the present study, we attempted to clarify mechanisms of transcellular transport of [125I]IMT by LLC-PKI
cell monolayers grown on collagen-coated micro-porous
membrane filters (Fig. 2). 10

conventional chloramine-T method, as described elsewhere. 1.2 Synthesis of non-radioactive IMT was performed according to the method of Krummeich et al. 15[14C]Tyr and [14C]inulin were obtained from American
Radiolabeled Chemicals (USA).

Cell cultures and monolayer preparation
Cell line studies were performed using the methods described by Saitoh et al., with modifications, as follows.14
LLC-PKI cells were maintained by serial passages in 25-

Fig, 1 Chemicalstructures of 3-[123I]iodo-a-methyl-L-tyrosine([123I]IMT) and L-Tyr.

MATERIALS AND METHODS

Materials
Reagent-grade 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH), 2-(methylamino)isobutyric acid (MeAIB),
2-aminoisobutyric acid (AIB), a-methyl-L-tyrosine andChloramine-T were acquired from Sigma-Aldrich Japan
K.K. (Tokyo, Japan). Natural L-amino acids and otherchemicals of reagent grade were purchased from Kanto
Chemical Co., Tokyo, Japan. [125I]NaI (8.1 x 1019 Bq/mol) was obtained from Amersham Pharmacia Biotech
UK (Buckinghamshire, UK). A Nova-Pak C18 (3.9 • 300
mm; Waters, Milford, MA, USA) was used for separationand purification. Silica gel thin-layer chromatography kit(TLC, catalogue number Art. 5553) was obtained from
Merck (Darmstadt, Germany). LLC-PKI cells were obtained from Dainippon Pharmaceuticutical Co. (Osaka,
Japan). Plastic tissue culture flasks (surface area, 25 cm 2)
were purchased from Nalge Nunc International (Roskilde,
Denmark), and Transwell cell chambers (surface area,
4.71 cm 2) were purchased from Costar (Cambridge, MA,

USA).

Fig. 2 LLC-PKj cell monolayer grown on a permeable support
in Transwell cell chambers.

No-carrier-added [125I]IMT was prepared using the

Fig. 3 Transcellular transport of [125I]IMT(A) and accumulation at 90 min of incubation (B) at 6 days after inoculation.
Values represent the mean _+S.D. (n = 4-5 monolayers). *p <0.001.

228 Naoto Shikano, Keiichi Kawai, Syuichi Nakajima, et al

Annals of Nuclear Medicine

Preparation of labeled compounds

A

B

Inhibitor

Inhibitor
I

ControlI

Na+ free -Na + free,~_~ll[,L-Tyr

4

Controli-I *Apical-to-basolateral

1

IMT

L-Tyr

Basolateral-to-apical IMT

BCH

BCH

AIB

AIB

MeAIB

MeAIB
-I

L-Arg
i

I

50

100

]

I

L-Arg

150

0

Transport of [125I]IMT(% of control)

t

14i

50

100

150

Accumulation of [12sI]IMT(%ofcontrol)

Fig. 4 Effects ofinhibitors (1 mM)ontranscellular transport (A)and accumulation (B) of [125I]IMT.
Each column represents mean + S.D. of 3 to 5 LLC-PKI cell monolayers grown on a permeable support.
*p < 0.01.
[125I]IMTA[14C]Tyr

B

Accumulation

Transport

~ 30

-7

30
"O

D

C
Accumulation

Transport

16

T

25-

E

-5

20-

- 14

"2

- 12
o

15-

e.

E

20-

@

-4
i.

10-

-10

I0-

L)

** /

5

8
*

0
[-.

I

i

i

i

5

6

7

8

pH of apical side
uptake solution

5

I

t

,

,

6

7

8

pH of apical side
uptake solution

d.

1

=

0

E-

i

i

5 6

7

..1.

pH of apical side

<
__

i

4

Apical-to-basolateral

I uptake solution

Basolateral-to-apical

f

i

94

6

6

I

7

8

=

9

pH of apical side
uptake solution.<

Fig. 5 Dependence of transcellular transport (A) and accumulation (B) of [i25I]IMT and dependenceof transcellular transport (C) and accumulation (D) of [lac]Tyr on apical pH. *p < 0.1301. Accumulationof [14C]Tyr(D) was significantly greater than accumulation of [t25I]IMT (B) (p < 0.001), via both apicalmembrane (t) and basolateral membrane (**). There was no significant difference between transcellulartransport of [125I]IMT(A) and that of (C) [L4C]Tyr.

cm 2 cell culture flasks. The cells were fed with Dulbecco's
modified Eagle's medium (Sigma-Aldrich Japan K.K.,
Tokyo, Japan) supplemented with L-glutamine (2 mM)
and 10% fetal bovine serum without antibiotics, in an
atmosphere of 7.5% CO2 and 95% air at 37~ (pH 7.4).
Subculturing was performed every 5 days using 0.02%
EDTA and 0.05% trypsin. To prepare monolayers on a
micro-pores support, cells were seeded at a density of 5 x
105 cells/cm 2 on polycarbonate membrane filter (pores, 3/zm) in Transwell cell chambers. The volume of medium
inside and outside the Transwell chamber was 1.5 and 2.6

Vol. 18, No. 3, 2004

ml, respectively. Fresh medium was replaced every 2
days. The cells were used between the 4th and 6th days.

Measurement of transepithelial transport and cellularaccumulation
Using monolayer cultures grown in Transwell chambers,
we measured transepithelial transport and accumulationof [125I]IMT. Dulbecco' s phosphate-buffered saline (PBS;
pH 7.4 adjusted by adding HCI or NaOH solution) conmining 137 mM NaCI, 3 mM KC1, 8 mM Na2HPO2, 1 mM
CaCI2, and 0.5 mM MgCI was used as the incubation

Original Article229

medium. In Na+-free medium, we replaced the NaCi andNa2HPO4 normally present in PBS with, respectively,
choline chloride and K2HPO4. In general experiments, thecell monolayers were preincubated with 2 ml of incubation medium on each side for 10 min at 37~ after theremoval of culture medium from both sides of the monolayers. Then, we added 2 ml of incubation medium containing [125I]IMT (18.5 kBq) either to the basolateral orapical side, and added 2 ml of incubation medium to the
opposite side. We incubated the monolayers for a specified
period of time at 37~ or 4~ WE used [14C]inulin (18.5
kBq), a compound that is not transported by the cells, toevaluate paracellular fluxes and extracellular trapping ofradioactivity. We added, L-Tyr, IMT, BCH, AIB, MeAIB
or L-Arg at a final concentration of 1 mM for inhibition
studies. Cells were incubated for 90 min at 37~ with 18.5
kBq [125I]IMT. The pH (hypothetic urine pH) of apicalside incubation medium was adjusted to 5.0, 6.0, 7.0 or 8.0
by adding HCI or NaOH solution for the pH dependence
experiment. We fixed the pH of the basolateral side at 7.4(hypothetic blood pH). We incubated the cells for 90 min
at 37~ with 18.5 kBq [~25I]IMT or [14C]Tyr added to theapical or basolateral side.
For transport measurements, at the specified time, analiquot (50/.tl) of the incubation medium on the opposite
side was obtained, and the radioactivity was measured.
For accumulation studies, we removed the medium bysuction at the end of the incubation period, and thenrapidly washed the monolayers twice with 2 ml of icecold incubation medium on each side. We solubilized
the cells on the filters in 0.5 ml of 1 N NaOH, and thenmeasured the radioactivity of the aliquots (I00/zl) of the
incubation medium on each side. We used an ARC1000M well-type scintillation counter (Aloka), and an
LS6500 liquid scintillation counter (Beckman Instruments, Fullerton, CA, USA) in Clear-sol II (Nacalai
Tesque Inc., Kyoto, Japan) to determine the 1251and 14C
radioactivity of the collected media and the solubilized
cell monolayers.
Statistical analysis
The values obtained in each experiment were expressed as
the mean + S.D. Statistical comparisons between groups
were performed using Student's t test.
RESULTS

Apical membrane

Basolateral membrane
Fig. 6 Principal amino acid transport systems expressed in intact
kidney epithelial cells.

of the administered dose of [14C]inulin leaked to the
opposite side of the LLC-PKI monolayer.
As shown in Figure 3, apical-to-basolateral transportof [125I]IMT (29.01 + 5.17%) was much greater thanbasolateral-to-apical transport (6.95 + 0.17%) for up to 90
min (Fig. 3A). Accumulation of [z25I]IMT from thebasolateral side (1.62 + 0.16%) and apical side (2.62 +
0.35%) was observed at 90 min of incubation (Fig. 3B).
In the examination of substrate specificity of the basolateral and apical amino acid transport system in LLCPKI cells, at 1 mM, the amino acids BCH, L-Tyr, AIB
and IMT inhibited [125I]IMT transport and accumulation from both sides (p < 0.01). MeAIB exhibited an
inhibitory effect, but this was not significant. Na+-depen dent transport was not the main contributor to the transport from the basolaterai to apical side, or the transport
from the apical to basolateral side. L-Arg, a basic aminoacid, did not inhibit [125I]IMT transport or accumulation(Fig. 4).
We evaluated effects of apical-side pH on transcellulartransport and accumulation of [125I]IMT. When pH ofapical incubation buffer was increased from 5.0 to 8.0 (pHof basolateral side fixed at 7.4), accumulation of [1251]IMTfrom both basolateral and apical side significantly decreased (p < 0.001), whereas basolateral-to-apical andapical-to-basolateral transport were not significantly affected (Fig. 5 A, B). When pH of apical incubation buffer
was increased from 5.0 to 8.0, accumulation of [14C]Tyrfrom both basolateral and apical sides significantly increased (p < 0.001), whereas basolateral-to-apical andapicai-to-basolateral transport of [14C]Tyr was not significantly affected (Fig. 5 C, D).
DISCUSSION[125I]IMT labeling efficiency and radiochemical purities
were greater than 80% and 95%, respectively. Specificradioactivity was greater than 8.1 x 1019 Bq/mol.
For monolayer preparation, apical-to-basolateral andbasolateral-to-apical leak of [lac]inulin was <0.26% and< 0.07% of the total [14C]inulin, respectively, up to 120
min. The amount of [14C]inulin trapped in the extracellular space from both sides was <0.003% of the total[14C]inulin. Because of formation of tight junctions, little

Secretion and reabsorption are not, respectively, reverse
reabsorption and reverse secretion, because the nature ofefflux and influx via a membrane is not the same in allcases. In intact renal proximal tubule cells, basolateral
membrane transport involves the L, A and ASC systems,
whereas, in apical membrane, the b ~247B ~ B ~ A and
ASC systems contribute to neutral amino acid transport

230 Naoto Shikano, Keiichi Kawai, Syuichi Nakajima, et al

Annals of Nuclear Medicine

B

Amino acid

A

(a)

(b)

Sa = Esec - lreab~

Ra = Ireab - Esee

Apical membrane
Rb = Ereab - IsecSb = Isec - Ereab
9

LLC-PK1 Cell
Accumulative secretion

Accumulative reabsorption

A. . . . Sb-Sa>OAreab = Ra - Rb > 0

I t

Basolateralmembrane

9

Amino acidC

9 .o__.

o.o.,o .

"I.

.I

o.

9

9 ..

9 io"

9

9

.'e-.....

- .,-,....

Accumulative secretion

Non-accumulative secretion[total > Etotal, Ireab < Esec, Isec > Ereabttotal < Etotal, Ireab < Esec, Isec > Ereabo llP_

ele 9

. 1__

9 ~149149

eele 9elee

9 e o~ 9** ~oo9 v99149

Accumulative reabsorption

Non-accumulative reabsorption]total > Etotal, Ireab > Esec, [sec < Ereab

Itotal < Etotal, Ireab > Esec, Isec < EreabFig. 7 Scheme showingamino acid pathway in LLC-PKI (A). Simplifiedmodel of amino acid transport
via apical and basolateral membrane (B) and (C). Q: amino acid. Length of arrow corresponds tovelocity of transport of amino acids.

(Fig. 6). 16 System b ~ is a Na+-independent transportsystem involved in transport of neutral amino acids (Leu,
Ile, Val, Phe, Tyr, Trp, His, Met, Gin, Asn Thr, Cys, Ser
and Ala) and basic amino acids (Arg, Lys and Orn) in
apical membrane. 17 System B ~ is the main neutral aminoacid transport system predominantly involved in reabsorption of aromatic amino acids on the apical side ofproximal tubule cells, but it has not been fully characterized at the molecular level. 18,19 Palacin et al. havesuggested the presence of an Na+-dependent transportsystem (system B ~247on the apical side of proximal tubule cells. 2~ Recently, cDNA of a system T transporter
designated TATI (T-type amino acid transporter 1) wascloned. 21 Expression of TAT1 has been detected inbasolateral membrane of proximal tubule cells.
Tumor cell line studies have revealed that systems L, A,
T and B~ (and/or B~ play roles in [123I]IMT transport)
Studies of [123I]IMT uptake in tumor cells have shownthat the majority of transport takes place via the Na +independent system L (>70%), and that relatively minor
uptake takes place via the Na§
system B ~247(<20%). Lahoutte et al. reported that system T also mediates [123I]IMT transport into U266 human myeloma
cells.22 [123I]IMT transport via system T accounted for up

Vol. 18, No. 3, 2004

to 43.8% of all uptake into U266 cells. 22 Studies of human
TAT1 show that it transports three aromatic amino acids(Tyr, Phe and Trp) in an Na+-independent manner. It isnoteworthy that a-methyl-L-tyrosinewas not a substrate. 21
Tyr is a substrate of system b~ transport. 17 However, it
has also been shown that the Na+-independent carriersystems b~ and y+ do not play roles in [123I]IMT uptake. 3
It is unclear whether system y+L is involved in [123I]IMT
uptake (Fig. 6), 3
In the present study, [125I]IMT concentration of intracellular fluid via apical membrane at 90 min in pH 7.4 was
9.5 times that of its uptake solution, and that of [125I]IMT
via basal membrane was 5.9 times its uptake solution. For[lgC]Tyr, the values were 56.3 times (via basolaterai
membrane) and 9.1 times (via basal membrane) that of its
uptake solution, respectively. This indicates that uptakeof [12sI]IMT and [14C]Tyr is due to carrier-mediatedtransport against an outwardly directed amino acid concentration gradient, rather than facilitated diffusion. But
the higher accumulation of [L4C]Tyr,relative to [125I]IMT,
were contrary to prediction, because renal physiologicalaccumulation of radiolabeled [123I]IMT is reportedly
higher than that of radiolabeled Tyr. Recently, we also
found probenecid-sensitive rapid urinary secretion andOriginal Article 231

marked accumulation of [~25I]IMT in an S2-1ike segmentof mouse kidney.8 Figure 3A does not indicate [125I]IMT
secretion but [I~5I]IMT reabsorption, in net function oftransceilular transport by LLC-PKI monolayer. Rabito et
al. reported that LLC-PKI shows close similarity to thepars recta of the renal proximal tubule, which contains $2
and $3 cells. 1oThe lack of accumulative p-aminohippurate
secretion 1~and pathological observation of transmission
electron micrograph of confluent LLC-PKI monolayerl~suggest that LLC-PKt cell shows similarity to $3 cells.
Natural amino acids are reabsorbed via S 1, $2 and $3
cells, and secreted via $2 cells.
Figure 7A is a scheme showing a hypothetical aminoacid pathway in transcellular transport with LLC-PKj.
Here, Ireab and E s e c were velocity of reabsorptive influxand velocity of secretive efflux in apical membrane. Isecand E r e a b were velocity of secretive influx and velocityof reabsorptive efflux in basolateral membrane. Itotal andE t o t a l are given by two equations: Itotal = I r e a b + Isec;and E t o t a l = E s e c + E r e a b . Figure 7B and Figure 7C show
some conditions of transcellular transport of substrates
based on this idea. If Itotal > Etotal, transceilular transportof substrate is accumulative (Fig. 7C (a), (b), (c), (f), (g)and (h)). If I t o t a l < E t o t a l , transcellular transport ofsubstrate is not accumulative (Fig. 7C (d), (e), (i) and (j)).
On the other hand, if I r e a b < E s e c and I s e c > E r e a b ,transcellular transport of substrate is secretive (Fig. 7C(a), (b), (c), (d) and (e)). If Ireab > E s e c and Isec < E r e a b ,transcellular transport of substrate is reabsorptive (Fig.
7C (f), (g), (h), (i) and (j)). In the present study with LLCPK1 monolayer in normal uptake condition, transport of[125I]IMT and [lac]Tyr occurred as accumulative reabsorption (apical-to-basolateral transcellular transport).
The mechanisms of the apicai-to-basolaterai transcellular transport (reabsorption) of [125I]IMT (Fig. 3) are
explained in Figure 7B (b) and Figure 7C (f). The reabsorptive transport velocity in basolateral membrane (Rb =E r e a b - Isec) is a rate-determining step velocity of reabsorption. According to the data shown in Figure 3A, Rb =0.322% dose/min/4.7-cm 2. The reabsorptive transport
velocity in apical membrane Ra = I r e a b < E s e c (>0)
should be larger than the velocity Rb, because of accumulative transport (Fig. 7B (b)). For accumulation velocity inreabsorption, the following equation defines the relationship between each membrane transport velocity and A r e a b :transport velocity in basolateral membrane Sb = I s e c E r e a b (>0) should be larger than the velocity Sa, becauseof accumulative transport (Fig. 7B (a)). The accumulation
velocity in secretion A s e c is given by the following
equation: A s e c = S b - S a = (lsec - E r e a b ) - ( E s e c - Ireab )= ( I r e a b + I s e c ) - ( E s e c + E r e a b ) = I t o t a l - E t o t a l > O.

According to the data shown in Figure 3B, A r e a b =0.029% dose/min/4.7-cm 2. Thus, Ra = A r e a b + R b =0.029 + 0.322 = 0.351% dose/min/4.7-cm2.
An alternative explanation of the mechanisms of thebasolateral-to-apical transcellular transport (secretion) of[J25I]IMT (Fig. 3) is shown in Figure 7B (a) and Figure 7C(a). The secretive transport velocity in apical membrane
S a = E s e c - I r e a b (= 0.077% dose/min/4.7-cm2) is a ratedetermining step velocity of secretion. The secretive

According to the data shown in Figure 3B, A s e c = 0.018%
dose/min/4.7-cm2. Thus, Sb = A s e c + Sa = 0.018 + 0.077= 0.095% dose/min/4.7-cm2.
In the present inhibition study, bi-directional transcellular transport (secretion and reabsorption) and accumulation of [125I]IMT were observed in the LLC-PKj cellmonolayers (Figs. 3, 4 and 5). Transport of [125I]IMT was
inhibited by BCH, L-Tyr and AIB, which are substrates ofsystem L. The amino acid BCH markedly inhibited both
directional accumulation and transport (p < 0.01) of[1251]IMT. System L uptake has been defined as uptake ofan amino acid in Na§
buffer which is subject toinhibition by an excess of BCH. 23,24The size of the difference between uptake of [125I]IMT in buffer with Na + andwithout Na § suggests that Na+-dependent transport is not
the main pathway. These results suggest that transportof neutral amino acids by LLC-PKI ceils is dominated by
Na+-independent transport (i.e., system L), as indicatedby the inhibitory effect of BCH under Na+-free conditions. This is consistent with a previous report of system
L-mediated transport of L-dopa, a neutral amino acid, in
LLC-PKI cells. 25 The decreases in accumulation andtransport of [125I]IMT caused by the inhibitors shown in
Figure 4 are explained by the models in Figure 7B (e) and(j). Inhibited influx and efflux via apical and basolateral
membrane reduce transport and accumulation of [~25I]IMT.
Shotweli et al. studied pH dependence of activity of theneutral amino acid transport systems L, A and ASC in
Chinese hamster ovary cells. 26 Lowered pH enhanced
uptake of substrates via system L, and substrate uptake via
system A was abolished when the pH was lowered. 26 In
the present study, transcellular transport (not significant)and accumulation of [125I]IMT from both directions wereenhanced by lowered pH (p < 0.001) (Fig. 5A). These
results indicate that system L transport and accumulationof [125I]IMT across the apical membrane can be stimulated by acidifying the medium on the apical side.
The pH-dependent accumulation of [125I]IMT and[14C]Tyr shown in Figure 5B and Figure 5D suggests that
pH of extra-cellular fluid on the apical side could affecttransport system activity in the membrane in contact withthe extra-cellular fluid (apical-to-basolateral accumulation), and opposite side of the extra-cellular fluid (basolateral-to-apical accumulation). Despite formation oftight junctions, the slight leakage of the administereddose of [14C]inulin to the opposite side of the LLC-PKj
monolayer suggests that hydrogen ions in apical-side
uptake solution also leak to the opposite side. It is generally considered most likely that lower pH stimulates
influx via system L transporter in both membranes. Stimu-

232 NaotoShikano, Keiichi Kawai, Syuichi Nakajima, et al

Annals o f Nuclear Medicine

Areab = Ra - Rb = (Ireab - Esec) - (Ereab - Isec) =( I r e a b + Isec) - ( E s e c + E r e a b ) = I t o t a l - E t o t a l > O.

lation of the apical-side membrane by contact with theextra-cellular fluid is greater than that of the opposite side
membrane. This mechanism explains the increase in[125I]IMT accumulation in low pH conditions (Fig. 5B).
Figure 7C (b) shows the results for secretion in low pH,
and Figure 7C (g) shows the results for reabsorption inlower pH. The increasing accumulation with lower pH
may be due to the difference in the magnitude of stimulation between the two membranes.
Also in the present study, accumulation of [14C]Tyrfrom both directions was diminished by lowered pH (p <0.001) (Fig. 5B). This suggests that system A contributes to accumulation of [lac]Tyr. These findings suggest
differences in mechanisms of accumulation between[125I]IMT and [14C]Tyr. It appears most likely that lower
pH diminishes influx via system A transporter in both
membranes. Figure 7C (c) shows results for secretion in
low pH, and Figure 7C (h) shows results for reabsorption
in low pH. The decrease of [lac]Tyr accumulation shownin Figure 5D in lower pH conditions can be explainedusing this model. The decreasing accumulation with lower
pH may be due to the difference in magnitude of abolishment between the two membranes.
The high functional expression of system L transport inboth sides of the membranes may be a representative
characteristic by which LLC-PKI can be distinguishedfrom intact normal renal proximal cells. Incidentally,
several characteristics of LLC-PKI cells are comparable
to those of tumor cell lines. It has been reported that LLCPK1 cells grow rapidly in monolayer cultures. Furthermore, they are stable through more than 300 serial
passages, with no evidence that the cells undergo transformation. 13 The finding that system L is conspicuously
up-regulated in many kinds of tumors and transformed
cell lines is consistent with the observed increase in
uptake of amino acids for rapid cell growth and proliferation. 23 On the other hand, studies of transport systems in
the apical membrane of LLC-PKI cells have shown thepresence of system A and ASC, 1~ but have not identifiedNa§
neutral amino acid transport systems (B~247
and/or B~ of the type responsible for Leu reabsorption by
the proximal tubule in vivo. l~ L27 These properties aredifferent from those of intact epithelial cells in proximal
tubule. As a tool for studying artificial amino acid transport in epithelia, transcellular transport studies constitutean interesting new application of LLC-PKI cells. However, investigation of the mechanism of high [125I]IMT
accumulation with the present experimental system would
be facilitated by using intact kidney proximal tubule cells($2) from the renal cortex.
CONCLUSIONS
We investigated transcellular transport of [125I]IMT in
LLC-PKI (porcine kidney epithelial cell line) monolayersgrown on collagen-coated microporous membrane filters.

Vol. 18, No. 3, 2004

We observed bi-directional transcellular transport of[125I]IMT, by LLC-PKI cell monolayers, corresponding
to secretion and reabsorption in proximal tubule. Thistransport was shared with transport of L-Tyr, a mothercompound of drug design. The main transport pathway of[125I]IMT in LLC-PKI cells was system L. Na§
dent systems contributed a smaller amount to the total
uptake. Differences in mechanisms of accumulation between [125I]IMT and [Inc]Tyr are suggested by their respective pH dependence.
ACKNOWLEDGMENTS
We wish to thank Toshio Miyamoto, Yuri Aisawa, Takuya Doi
and Daisuke Izumi (lbaraki Prefectural University) for their
excellent technical assistance. This work was supported by
Grants-in-Aid for Scientific Research (#10770451, #14770498,#13557075 and #15659283) from the Ministry of Education,
Science, Sports and Culture of Japan and the Japan Society for
the Promotion of Science. Financial support was also provided
by Ibaraki Prefectural University Project Research (9808-3,
0118-1 and 0220-1 ) and Ibaraki Prefectural University Grantsin-Aid for the Encouragement for Young Scientists 2001,2002
and 2004.
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