Transcellular transport of organic cation across monolayers 1 of kidney epithelial cell line LLC-PK, HIDEYUKI SAITO, MICHIKO YAMAMOTO, KEN-ICHI INUI, AND RYOHEI HORI Department of Hospital Pharmacy, School of Medicine, Tokyo Medical and Dental University, Tokyo 113; and Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606, Japan Saito, Hideyuki, Michiko Yamamoto, Ken-Ichi Inui, and Ryohei Hori. Transcellular transport of organic cation across monolayers of kidney epithelial cell line LLC-PK,. Am. J. PhysioI. 262 (Cell PhysioL. 31): C59-C66, 1992.-Transcellular transport and the accumulation of [“Cl tetraethylammonium, a typical organic cation, by LLC-PK1 cell monolayers grown on microporous membrane filters were studied. Tetraethylammonium was accumulated progressively in the monolayers from the basolateral side and was transported unidirectionally to the apical side. The transcellular transport of tetraethylammonium was saturable, temperature dependent, and sensitive to the pH of the apical side of the monolayers. The apparent Michaelis constant and maximum velocity values for the transport were 67 PM and 222 pmol mg protein-’ min, respectively. Unlabeled tetraethylammonium, amiloride, procainamide, cimetidine, and choline inhibited the basolateral uptake and transcellular transport of [‘“Cl tetraethylammonium. The development of tetraethylammonium transport activity was observed in the differentiating cells. A sulfhydryl reagent inhibited the tetraethylammonium transport at both the basolateral and apical membranes of the LLC-PK1 cells. These findings suggest that these monolayers possess unidirectional transport systems for organic cations, corresponding to the secretion in the renal proximal tubules. l
tetraethylammonium transport; proton-organic port; tubular secretion; renal cell culture
cation
anti-
SECRETION OF DRUGS and xenobiotics is an important physiological function of the renal proximal tubules. In general, these compounds are negatively or positively charged organic species. The process of secreting organic cations through the proximal tubular cells is performed via unidirectional transcellular transport, i.e., the uptake of organic cation into the cells from blood across the basolateral membranes followed by the active extrusion across the brush-border membranes into the tubular fluid (21). Many investigations using plasma membrane vesicles isolated from the renal cortex have shown the mechanism for organic cation transport in both the brush-border (3, 7, 10, 24, 25, 27) and the basolateral membranes (18, 23-25, 27) from the kidney cortex. The transport mechanisms for organic cations have been particularly well characterized in the brush-border membranes and have demonstrated the presence of an H+organic cation antiport system, a process that exchanges cellular organic cation for tubular H+ (3, 7, 10, 24, 25, 27). Although studies in isolated basolateral membranes suggest a facilitated diffusion via an electrically conductive pathway for organic cations (18,23,25,27), an active transport process is not apparent. A better understanding of the mechanism of transepithelial transport of organic cations requires transport studies using intact tubular epithelia. 0363-6143/92
$2.00 Copyright
Cultured epithelial cells derived from the kidney have been useful in studying a variety of renal cellular functions, including transepithelial transport and the regulation of transport by hormones and drugs (6, 16). The pig kidney epithelial cell line LLC-PK1 (11) has been employed extensively as a model for the analysis of epithelial functions in the proximal tubules (1,9, 12-14). These cells form an oriented monolayer with microvilli and tight junctions and exhibit a unidirectional transport of electrolytes and some nutrients (1,9,20). We provided the first evidence to show that the apical membranes (corresponding to the brush-border membranes) of the LLC-PK, cells express the H+-organic cation antiport system (12). Cultured cell monolayers grown on microporous membrane filters have allowed the study of transepithelial transport under appropriate conditions (20). Recently, Roth-Ramel and co-workers (4, 5) have used LLC-PK, cell monolayers grown on permeable supports to examine the transepithelial transport of organic cations. However, the mechanisms for the transcellular transport of organic cations are not fully characterized. In this study, we attempted to clarify the cellular mechanism of the transcellular transport of tetraethylammonium by LLC-PK1 cell monolayers grown on collagen-coated microporous membrane filters. The results suggest that tetraethylammonium is transported across the monolayers from the basolateral-to-apical side, i.e., a unidirectional transcellular transport, and demonstrate the validity of LLC-PK, cell monolayers as a model for the renal proximal tubular secretion of organic cations. MATERIALS
AND
METHODS
CeLZ culture. LLC-PK1 cells obtained from the American Type Culture Collection (ATCC CRL-1392) were grown on plastic dishes (Falcon; Becton Dickinson, Lincoln Park, NJ) in Dulbecco”s modified Eagle’s medium (GIBCO, Life Technologies, Grand Island, NY), supplemented with 10% fetal calf serum (Microbiological Associates, Bethesda, MD) without antibiotics in an atmosphere of 5% C02-95% air at 37°C. Subculture was done every 7 days using 0.02% EDTA and 0.05% trypsin (9, 12-14). In general, loo-mm plastic dishes were inoculated with 1 X lo6 cells in 10 ml of complete culture medium. In this study, cells between the 210th and 220th passages were used. For the transport studies, LLC-PK1 cells were seeded on collagen-coated membrane filters (3-pm pores, 4.71 cm”-growth area) inside a Transwell cell culture chamber (Costar, Cambridge, MA) at a cell density of 5 x lo5 cells/cm”. Each Transwell chamber was placed in a 35-mm well of tissue culture plate with 2.6 ml of the outside medium (basolateral side) and 1.5 ml of the inside medium (apical side). The cell monolayers were fed fresh medium every 2 days. To evaluate the integrity of the monolayers, transepithelial electrical resistance was measured using Millicell-ERS (Milli-
0 1992 the American
Physiological
Society
c59
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C60
ORGANIC
CATION
TRANSPORT
pore, Bedford, MA) before the transport study. The transepithelial resistance ranged from 180 to 440 Q cm2 in the intact monolayers. Measurement of transepithelial transport and cellular accumulation. Transepithelial transport and accumulation of [ 14C]tetraethylammonium or cy-[ 14C]methyl-D-glucoside were measured using monolayer cultures grown in the Transwell chambers. The incubation medium was Dulbecco’s phosphatebuffered saline (pH 7.4) [PBS buffer made of (in mM) 137 NaCl, 3 KCl, 8 Na2HP04, 1.5 KH2P04, 1 CaC12, and 0.5 MgC1,], containing 5 mM D-ghCOSe, except for the experiment of a-methyl-D-glucoside transport. The pH of the medium was adjusted by adding a solution of HCl or NaOH. In Na+-free medium, NaCl and Na2HP04 of PBS buffer were replaced with choline chloride and K2HP04, respectively. In general experiments, after the removal of culture medium from both sides of the monolayers, the cell monolayers were preincubated with 2 ml of incubation medium in each side for 10 min at 37°C. Then, 2 ml of incubation medium containing [‘“Cl tetraethylammonium (50 ,uM, 7.4 kBq/ml) or a-[14C]methyl-D-glucoside (50 ,uM, 7.4 kBq/ml) and D-[“H]mannitol(4 PM, 29.6 kBq/ml) were added either to the basolateral or apical side, and 2 ml of radioactive free incubation medium were added to the opposite side. The monolayers were incubated for the specified period of time at 37°C. D-Mannitol, a compound that is not transported by the cells, was used to calculate paracellular fluxes and the extracellular trapping of tetraethylammonium and cymethyl-r>-glucoside. For transport measurements, an aliquot (50 ,ul) of the incubation medium in the other side was taken at the specified time, and the radioactivity was counted. For accumulation studies, the medium was removed by suction at the end of the incubation period, and the monolayers were rapidly washed two times with 2 ml of ice-cold incubation medium in each side. The filters with monolayers were detached from the chambers, the cells on the filters were solubilized in 0.5 ml of 1 N NaOH, and the radioactivity of each aliquot (100 ,ul) was counted. The radioactivity of the collected media and the solubilized cell monolayers was determined in 10 ml of ACS II (Amersham International, Buckinghamshire, UK) by liquid scintillation counting. Protein assay. The protein content of the solubilized cell monolayers was determined by the method of Bradford (2), using the Bio-Rad Protein Assay Kit with bovine y-globulin as a standard. The protein content of the intact monolayers was 0.8-0.9 mg/filter (4.71 cm”). Statistical analysis. Data were analyzed statistically using one-way analysis of variance followed by Fisher’s t test. Materials. [ 1-14C]tetraethylammonium bromide (0.15 GBq/ mmol) and D-[l-“H(N)] mannitol (1,110 GBq/mmol) were purchased from Du Pont-New England Nuclear Research Products (Boston, MA). Methyl(cw-o-[U-14C]gluco)pyranoside (5.33 GBq/mmol) was obtained from Amersham International. Tetraethylammonium bromide was obtained from Nacalai Tesque (Kyoto, Japan). Amiloride hydrochloride and choline chloride were obtained from Wako Pure Chemical Industries (Osaka, Japan). Cimetidine and cephalexin were kindly supplied by Fujisawa Pharmaceutical and Shionogi (both in Osaka, Japan), respectively. N1-methylnicotinamide, procainamide hydrochloride andp-chloromercuribenzene sulfonate (PCMBS) were purchased from Sigma Chemical (St. Louis, MO). All other chemicals used for the experiment were of the highest purity available. RESULTS
Transcellular transport and accumulation of tetraethylammonium. To determine whether the LLC-PK, cell monolayers grown on microporous membranes possessed
IN
RENAL
CELL
MONOLAYERS
a unidirectional transport activity for tetraethylammonium, transepithelial fluxes were measured by adding [“Cl tetraethylammonium and D- [3H] mannitol simultaneously either to the basolateral or apical side of the LLC-PK1 cell monolayers, and the appearance of radioactivity at the other side was examined. The transcellular transport of tetraethylammonium was calculated by subtracting the paracellular flux estimated by D- [3H] mannitol from the transepithelial flux of [ 14C]tetraethylammonium. The transepithelial fluxes of D- [“Hlmannitol from both sides to the other side of the monolayers were ~2% of the total radioactivity added per hour. Figure 1A shows the transcellular transport of [‘“Cltetraethylammonium from the basolateral-to-apical side and the apical-to-basolateral side. The basolateral-toapical transport of [‘“Cl tetraethylammonium was much greater than the apical-to-basolateral transport, and its rate was nearly constant for up to 60 min. The accumulation of [14C]tetraethylammonium by the LLC-PK1 cell monolayers was also measured at 60 min of incubation (Fig. IB ). The accumulation of [14C]tetraethylammonium from the basolateral side was U-fold higher than that from the apical side (basolateral, 3.43 t 0.27 nmolmg protein-‘. 60 min-‘; apical, 0.31 t 0.03 nmol*mg protein-l. 60 min-l). The amount of [“Cl tetraethylammonium trapped in the extracellular space was ~2% of the total [“Cl tetraethylammonium uptake. Table 1 shows the effect of temperature on the transcellular transport of [“Cl tetraethylammonium by the LLC-PK, cell monolayers. When the monolayers were incubated at 4OC, the basolateral-to-apical transport of [“Cl tetraethylammonium was reduced drastically compared with the transport at 37°C and did not differ from the apical-to-basolateral transport of the substrate; therefore, the basolateral-to-apical transport of tetraethylammonium by the monolayers was temperature dependent. These observations indicate that the LLC-PK, cell monolayers possessa unidirectional transport activity for tetraethylammonium; i.e., the LLC-PK, cell 0.6
5
15
30
60
MINUTES
Fig. 1. Transcellular transport (A) and accumulation (B) of tetraethylammonium by LLC-PK, cell monolayers. A: on 4th day after inoculation, LLC-PK, cell monolayers were incubated at 37°C with 50 PM [‘4C]tetraethylammonium (2 ml, pH 7.4) added either to basolateral (0) or apical (A) side of monolayers. Appearance of radioactivity in opposite side (2 ml, pH 7.4) was measured periodically. B: after 60-min incubation, monolayers were rapidly washed two times with 2 ml of ice-cold incubation medium in both sides, and radioactivity of solubilized cell monolayers was determined. Each point or column represents mean t SE of 3 monolayers.
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ORGANIC
CATION
TRANSPORT
Table 1. Effect of temperature on transcellular transport of tetraethylammonium by LLC-PKI cell monolayers Transcellular Transport, pmol acmv2 - 60 min-’ Temperature,
“C
37 4
Apical to basolateral
Basolateral to apical
92.7t6.9 22.9-r-2.4
631.4t31.1 20.7k2.3
Values are means t SE of 3 monolayers. On 4th day after inoculation, LLC-PK1 cell monolayers were incubated at 37 or 4°C for 60 min with 50 PM [14C]tetraethylammonium (pH 7.4) added either to basolateral or apical side of monolayers. Appearance of radioactivity in opposite side (pH 7.4) was measured. 6 _
APICAL-BASAL
BASAL-APICAL
l
-I-Na
-10, G 5.2 -8 I2 a M -6
f -c
-4
g
-2
II r' 3
I
0
’
15
I
30 MINUTES
I
60
APICAL BASAL APICAL BASAL -Na -Na +Na +Na
”
Fig. 2. Transcellular transport (A) and accumulation (B) of a-methylD-glucoside by LLC-PK1 cell monolayers. On 6th day after inoculation, LLC-PK1 cell monolayers were preincubated at 37°C for 10 min (pH 7.4) with either phosphate-buffered saline or Na+-free medium as described in text and were then incubated at 37°C with 50 PM w[‘“C]methyl-D-glucoside (pH 7.4) added either to basolateral (A, A) or apical (0, 0) side of monolayers. Appearance of radioactivity at opposite side (pH 7.4) and accumulation were measured in presence (+Na) and absence (-Na) of Na’. Each point or column represents mean t SE of 3 monolayers.
monolayers accumulate tetraethylammonium progressively from the basolateral side of the monolayers and then extrude it in the apical side. Transcellular transport and accumulation of cu-methylo-glucoside. To ascertain that the unidirectional transport of tetraethylammonium was a specific phenomenon, the transcellular transport and the accumulation of amethyl-D-glucoside, a nonmetabolizable substrate for the Na+-dependent D-ghCOse transport system, were measured in the presence and absence of Na+. The transcellular transport of a-methyl-D-glucoside was measured by adding simultaneously cy-[‘*Cl methyl-D-glucoside and D[ 3H]mannitol either to the apical or basolateral side of the LLC-PK1 monolayers. The transcellular transport of a- [‘*C]methyl-D-glucoside by the LLC-PK1 cell monolayers appears in Fig. 2A. In the presence of Na’, the rate of apical-to-basolateral transport of a-methyl-Dglucoside increased with progressively longer periods of incubation, achieving 201.1 t 15.7 pmol/cm2 at 60 min. In contrast, the basolateral-to-apical transport showed minus values during the periods of incubation. Note that the net flux of a-methyl-D-glucoside represents the sum of the transcellular and paracellular fluxes across the
IN
RENAL
CELL
C61
MONOLAYERS
monolayers. If the flux across the cells is negligible and if there is a reuptake of the substrate that passesthrough the monolayers paracellularly, the transcellular transport corrected by the D-mannitol flux would have a minus value. The latter phenomenon can thus be explained by the reuptake of ar-methyl-D-glucoside at the apical surface of the LLC-PK1 cell monolayers. The transport in the absence of Na+ was negligible. Figure 2B shows the accumulation of a- [14C]methyl-D-glucoside by the LLCPK, cell monolayers in the presence and absence of Na+ at 60 min. The accumulation of cy-[ ‘*C]methyl-D-glucoside from the apical side was 9.35 t 0.42 nmol mg protein-‘. 60 min-‘, -25-fold greater than that from the basolateral side (0.37 t 0.01 nmol mg protein-’ 60 min-‘). In the absence of Na+, the accumulation from each side was extremely small, 0.014 and 0.051 nmolmg protein-‘.60 min-’ from the apical and basolateral sides, respectively. Concentration dependence of tetraethylammonium transport. Figure 3 shows the transepithelial transport of [‘*Cl tetraethylammonium from the basolateral-to-apical side of LLC-PK1 cell monolayers as a function of an increased concentration of substrate ranging from 0.025 to 1 mM at the basolateral side. The transport rates were estimated at 30 min, because findings were linear up to 60 min after incubation (Fig. 1A). The curve for the transcellular transport obtained by subtracting the paracellular flux from the total transepithelial flux was curvilinear, indicating a saturable process. The apparent Michaelis constant (Km) and maximum velocity ( Vmax) values of the transcellular transport of tetraethylammonium, estimated from the Michaelis-Menten equation using nonlinear least-squares analysis (28), were 67 PM and 222 pmol . mg protein-‘. min-’ (46.5 pmol .crnB2. min-l), respectively. Effect of cationic drugs on transcellular transport and accumulation of tetraethylammonium. The cis-inhibition of the transcellular transport and the accumulation of [‘*Cl tetraethylammonium by unlabeled tetraethylammonium was examined. As shown in Fig. 4A, the basolateral-to-apical transport of [‘*Cl tetraethylammonium l
5 7.E
0
TOTAL
A
PARACELLULAR
---
TRANSCELLULAR
T
z4
0
0.1
0.25
0.5 TEA
1 hM)
Fig. 3. Concentration dependence of tetraethylammonium (TEA) transport across LLC-PK1 cell monolayers. On 6th day after inoculation, LLC-PK1 cell monolayers were incubated at 37°C for 30 min with varying concentrations of [‘“CITEA (pH 7.4) added to basolateral side. Appearance of radioactivity in apical side (pH 7.4) was measured. Each point represents mean ~fr SE of 3 monolayers.
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ORGANIC
s z
05
15
30
CATION
TRANSPORT
P 1
60
MINUTES
Fig. 4. Effect of unlabeled TEA on transcellular transport (A) and accumulation (B) of [‘“CITEA by LLC-PK1 cell monolayers. On 6th day after inoculation, LLC-PK, cell monolayers were incubated at 37°C with 50 PM [‘“CITEA (pH 7.4) added to basolateral side in absence (0) and presence (0) of 2.5 mM TEA. Appearance of radioactivity in apical side (pH 7.4) and accumulation were measured. Each point or column represents mean -+ SE of 3 monolayers.
was depressed dramatically in the presence of 2.5 mM unlabeled compound. Furthermore, the presence of unlabeled tetraethylammonium inhibited the accumulation of [ 14C]tetraethylammonium from the basolateral side to 11% of the control value (Fig. 4B). These findings indicate that the basolateral uptake of [14C]tetraethylammonium was inhibited competitively by the unlabeled substrate, resulting in a marked depression of the subsequent transport of the substrate across the monolayers. These observations also suggest that a passive diffusion pathway may be a minor component of the uptake of tetraethylammonium across the basolateral membranes of the LLC-PK1 cell monolayers. To examine the substrate specificity of the basolateral organic cation transport system in the LLC-PK1 cells, we evaluated the effects of several cationic drugs added to the basolateral side on the transcellular transport and accumulation of [14C]tetraethylammonium from the basolateral side of the monolayers. As shown in Fig. 5, TRANSPORT
DRUGS AMILORIDE
(mfw 0 2.5 3
CIMETIDINE TETRAETHYLAMMONIUM
PROCAINAMIDE CHOLINE N’-METHYLNICOTINAMIDE N’-METHYLNICOTINAMIDE
CEPHALEXIN
. 25
(76 OF CONTROL)
IN
RENAL
MONOLAYERS
cationic drugs such as amiloride, cimetidine, tetraethylammonium, procainamide, and choline inhibited both the basolateral-to-apical transport and the accumulation of [14C]tetraethylammonium. On the other hand, an endogeneous cation N1-methylnicotinamide at 2.5 mM did not inhibit the transport and the accumulation, whereas at 10 mM it inhibited the transport without change of the accumulation. Cephalexin, a zwitterionic cephalosporin, had little inhibitory effect on the transport and the accumulation. Transcellular transport and accumulation of tetraethylammonium at various pH of apical side. We previously reported the H+ gradient-dependent transport of tetraethylammonium by apical membrane vesicles isolated from LLC-PK, cells (12). In this regard, we evaluated the effect of pH of the apical side on the transcellular transport and the accumulation of [14C]tetraethylammonium. As shown in Fig. 6A, when the pH of the apical incubation buffer was varied from 5.4 to 7.4 (pH of the basolateral side fixed at 7.4), the basolateral-to-apical transport of [ 14C]tetraethylammonium was significantly affected, being the greatest at pH 5.4 (1.56 t 0.16 nmol. crnw2.60 min-‘) and the lowest at pH 7.4 (0.50 t 0.08 nmol . crnw2.60 min-l). Conversely, the accumulation of [14C]tetraethylammonium was the lowest at pH 5.4 (0.69 t 0.02 nmol mg protein-l l 60 min-‘) and the greatest at pH 7.4 (3.81 t 0.15 nmolmg protein-l.60 min-‘) (Fig. 6B). These observations show that the transport of tetraethylammonium across the apical membranes can be stimulated by acidifying the medium on the apical side, i.e., inwardly directed H+ gradient acts as a driving force for the extrusion. Transcellular transport and accumulation of tetraethylammonium during growth in culture. We reported that several functions of the apical membranes of the LLCPKI cells, such as the activities of marker enzymes and Na+-dependent D-ghCOSe transport, developed significantly during growth in culture (9, 13). Thus the transcellular transport activity of [14C]tetraethylammonium was studied as a function of duration of culture. As shown l
ACCUMULATION 0
CELL
(% OF CONTROL) 50
100
B
Fig. 5. Effect of cationic drugs on transcellular transport (A) and accumulation (B) of TEA by LLC-PKI cell monolayers. On 6th day after inoculation, LLC-PK1 cell monolayers were incubated at 37°C for 60 min with 50 ,uM [‘“CITEA (pH 7.4) added to basolateral side in absence (control) and presence of other cationic drugs at same side. Appearance of radioactivity in apical side (pH 7.4) and accumulation were measured. Data were expressed as % of control value. Each column represents mean t SE of 3 monolayers.
10 . 25
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ORGANIC
CATION
TRANSPORT
RENAL
CELL
1.6
Days in Culture
-z 5 EO.8
Accumulation, protein-’ Apical
2 4 6
k
0
$0.4 Z
I
-5.4
I
1
5.9
6.4
APICAL
I
I
I
6.9
7.4
5.4
pH
1
1
5.9
6.4
APICAL
I
6.9
I
7.4 -
pH
Fig. 6. Transcellular transport (A) and accumulation (B) of TEA by LLC-PKI cell monolayers at various pH of apical side. On 4th day after inoculation, LLC-PK, cell monolayers were incubated at 37°C for 60 min with 50 PM [‘“CITEA (pH 7.4) added to basolateral side at various pH of apical side. Appearance of radioactivity in apical side and accumulation were measured. Each point represents mean k SE of 3 monolayers.
1.2
ro6 A4 02
oh 5
DAYS DAYS DAYS
15
30
C63
MONOLAYERS
Table 2. Growth dependence of transport and accumulation of tetraethylammonium by LLC-PKI cell monolayers
6
2.E z p
IN
60
MINUTES
Fig. 7. Transcellular transport of TEA from basolateral-to-apical side of LLC-PK1 cell monolayers during growth in culture. On and, 4th, and 6th days after inoculation, LLC-PK1 cell monolayers were incubated at 37°C with 50 PM [‘“CITEA (pH 7.4) added to basolateral side of monolayers. Appearance of radioactivity in apical side (pH 7.4) was measured periodically. Each point represents mean t SE of 3 monolayers.
in Fig. 7, the transcellular transport of [14C]tetraethylammonium by the monolayers from the basolateral-toapical side increased markedly according to the duration of culture. Both the accumulation and the total amount of the uptake and appearance of [14C]tetraethylammonium in the apical side are summarized in Table 2. The accumulation of [“Cl tetraethylammonium from the basolateral side of the monolayers cultured for 2 days did not differ significantly from that of the monolayers cultured for 4 days, whereas a significant decrease in accumulation was observed in the monolayers on the 6th day of inoculation. The total amount of [14C]tetraethylammonium that crossed the basolateral membranes increased with duration of culture. In contrast, accumulation from the apical side was unaffected during culture. These findings suggest that the transcellular transport of tetraethylammonium by LLC-PK, cells is a characteristic of differentiating cultures similar to the development of Na+-dependent hexose transport (1, 13).
0.24t0.03 0.31~0.03 0.20*0.02
nmol . mg - 60 min-’ Basolateral
3.11t0.19 3.43t0.27 2.21t0.20*
Total Amount, nmol - mg protein-l 60 min-’
-
5.14t0.25 7.32+0.38-t 8.15kO.4O"f
Values are means k SE of 3 monolayers. LLC-PK1 cell monolayers were incubated at 37°C for 60 min with 50 FM [14C]tetraethylammonium (pH 7.4) added either to basolateral or apical side. Appearance of radioactivity in opposite side (pH 7.4) and accumulation were measured. Total amount of [“Cl tetraethylammonium transferred across basolateral membranes was calculated by adding transcellular transport (basolateral-to-apical side) to accumulation of [14C]tetraethylammonium. * P < 0.05 and f- P < 0.01, significant differences from value of 2 days using analysis of variance followed by Fisher’s t test.
Effect of PCMBS on transcellular transport and accumulation of tetraethylammonium. We previously reported that sulfhydryl groups are essential for the H’-organic cation antiport system of the renal brush-border membranes (8) and in LLC-PK, apical membranes (12). In this study, we examined the effect of PCMBS, a sulfhydry1 reagent, on the transcellular transport and accumulation of tetraethylammonium. As summarized in Table 3, when the apical surface of the monolayers was pretreated with 0.1 mM PCMBS, the transcellular transport of [14C]tetraethylammonium was decreased to 15% of the control value while the accumulation was increased to 145% of the control value. Pretreatment of the basolateral surface of the monolayers with PCMBS (0.1 mM) also inhibited the transport and the accumulation of [14C]tetraethylammonium to 73 and 53% of the control values, respectively. The most profound inhibition of the [“Cl tetraethylammonium transport was observed when both sides of the monolayers were pretreated with PCMBS while the accumulation did not differ significantly from the control. The simultaneous inhibition of the basolateral uptake and apical efflux of [14C]tetraethylammonium by PCMBS pretreatment may result in an accumulation of the substrate as much as that of control. These observations demonstrate that sulfhydryl groups may play an important role in both the apical and basolateral transport systems for tetraethylammonium in the LLC-PK1 cells. DISCUSSION
The results demonstrate that LLC-PK, cell monolayers possessfunctionally unidirectional transport systems for tetraethylammonium in both the basolateral and apical membranes. In contrast to tetraethylammonium, a-methyl-D-glucoside accumulated within the LLC-PK, cells from the apical side in the presence of Na+ and appeared at. the basolateral side of the monolayers, indicating an opposite direction for tetraethylammonium transport. These findings support the conclusion that the LLC-PK1 cell monolayers possess the bidirectional transport ability responsible for the reabsorptive and secretory functions of the renal proximal tubular epithelium.
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ORGANIC
CATION
Table 3. Effect of PCMBS on transcellular by LLC-PK, cell monolayers
Pretreatment
PBS (control) 0.01 mM PCMBS 0.1 mM PCMBS 0.01 mM PCMBS 0.1 mM PCMBS
0.01 mM 0.1 mM
PCMBS PCMBS
Added
to
Both Apical Apical Basolateral Basolateral Both Both
TRANSPORT
transport
IN
RENAL
CELL
and accumulation
Transport, nmol - cm-’ 60 mine1 1.46t0.18 1.54t0.14 0.22+0.02-f1.47to.11 1.07t0.01"
1.63-sO.09 0.16+O.OOt
The kinetic parameters of the basolateral-to-apical transport of tetraethylammonium across the LLC-PK1 cell monolayers were calculated; the apparent K, and Vmax values were 67 PM and 222 pmolmg protein-‘. min-l, respectively. It should be noted that the transcellular transport of tetraethylammonium is a consequence of two processes, basolateral uptake and apical efflux. Thus these apparent K, and Vmax values reflect the transport characteristics of both the basolateral and apical membranes. The LLC-PK1 cell monolayers accumulated tetraethylammonium progressively from the basolateral but not the apical side of the monolayers. Furthermore, the accumulation from the basolateral side was inhibited by unlabeled substrate and by cationic drugs such as amiloride, cimetidine, and procainamide, which are known to interact with organic cation transport systems in the renal basolateral membranes (18, 23, 24, 27). Thus it may be suggested that the uptake of tetraethylammonium from the basolateral side represents a function of the transport system localized in the basolateral membranes of the LLC-PK1 cells. In the present study, we found that N1-methylnicotinamide at a concentration of 2.5 mM did not influence the transport and accumulation of [14C]tetraethylammonium (50 PM) by the LLC-PK, cell monolayers, although at PO mM it inhibited the transport without change of the accumulation. Therefore, it could be suggested that the interaction of N1methylnicotinamide with the basolateral transport system for organic cations was relatively weak compared with other cationic drugs and that Nl-methylnicotinamide inhibited the efflux of tetraethylammonium via the apical transport system after entering the cells. Wright and Wunz (27) reported that Nl-methylnicotinamide inhibited tetraethylammonium uptake into the renal brush-border membranes but not into the basolatera1 membranes, suggesting there may be a qualitative difference in the structural requirements associated with substrate recognition by the transport systems of the two membranes. Assuming the intracellular water content of 7 pl/mg protein (4), the intracellular tetraethylammonium concentration at the apical pH of 7.4 was -lo-fold higher than the concentration (50 PM) in the basolateral me-
% Control Value 100 105 15 101 73 112 11
MONOLAYERS
of tetraethylammonium
Accumulation, nmol - mg protein-’ - 60 min-’ 2.11t0.02 2.37t0.04 3.05+0.05t 1.95t0.21 1.12tO.O6f2.19kO.04 1.8lt0.08*
%Control Value 100 112 145 92 53 104 86
dium (intracellular concentration at 60 min on the 4th day after inoculation, 490-540 PM; Figs. 1 and 6, Table 2). This may be near an equilibrium value that could be expected by a cell interior negative potential. We demonstrated previously that tetraethylammonium is transported across the renal basolateral membranes via a carrier-mediated system, which is stimulated by a negative membrane potential (25). Therefore, it could be suggested that the basolateral uptake of tetraethylammonium is mediated via a cell potential-dependent transport system. However, present data do not exclude the possibility that tetraethylammonium might be bound to intracellular anionic sites. It has been well documented that tetraethylammonium is transported across the renal brush-border membranes by a H+-organic cation antiport system (3, 10, 25, 27). We demonstrated that the LLC-PK, cells possess this antiport system in their apical membranes (12). However, there is no evidence to show the H+-organic cation antiport system functions in the intact LLC-PK, cells. In this study, the transcellular transport of tetraethylammonium by the LLC-PK, cell monolayers was dependent on the pH of the medium on the apical side with the greatest transport occurring at 5.4, the most acidic pH. This finding suggests that, in intact LLC-PK1 cells, the H+ gradient directed inwardly from the exterior of the cells could serve as a driving force for tetraethylammonium transport at the apical membranes, i.e., a H+tetraethylammonium antiport system. In the isolated perfused tubule, the stimulatory effect of acidic luminal pH on the secretion of tetraethylammonium was demonstrated (3). The inwardly directed H+ gradient in the proximal tubule can be generated predominantly by the Na+-H+ exchanger in the brush-border membranes, which is dependent on the electrochemical gradient of Na+ produced by the Na+-K+-ATPase in the basolateral membranes (15). Such an exchanger has been demonstrated in the apical membranes of LLC-PK1 cells (19). Therefore, the H+ gradient produced by a Na’-H+ exchange system in the apical membranes of the LLC-PK1 cells might have been responsible for tetraethylammonium transport at pH 7.4 in the present study. Recently, McKinney et al. (17) demonstrated that intact LLC-PK, cells possess a mechanism in their apical membranes for
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ORGANIC
CATION
TRANSPORT
the mediated transport of tetraethylammonium and that H+-organic cation exchange does not appear to be predominant. The discrepancy might be explained by a difference in experimental conditions in that McKinney et al. (17) used LLC-PK1 cell monolayers grown on plastic wells. The basolateral-to-apical transport of tetraethylammonium by the LLC-PK, cell monolayers was found to increase significantly during growth in culture. There was no significant difference in the accumulation of tetraethylammonium from the basolateral side between days 2 and 4, while there was an appreciable decrease in the accumulation on day 6. The same magnitude of the basolateral uptake and apical efflux of the substrate indicates a parallel development of the transport activity of tetraethylammonium at both the basolateral and apical membranes of the LLC-PK1 cells during 4 days in culture. In the monolayers cultured for 6 days, the transport activity of tetraethylammonium in the apical membranes was further increased, but the transport activity in the basolateral membranes was not or less increased, resulting in a lower accumulation in the cells. The total amount of tetraethylammonium that crossed the basolateral membranes increased with the duration of the culture. The protein content of the monolayers did not increase appreciably in cells cultured for 2-6 days (0.8 0.9 mg protein/filter). Thus the development of tetraethylammonium transport activity was not due to an increase in the density of cells on the filter. The development of apical membrane enzyme activity and Na’dependent hexose transport in the LLC-PK, cells has been described in differentiating cultures (1, 13). Mechanisms for the development of the organic cation transport system in the LLC-PK, cells should be further investigated. The transport of tetraethylammonium across the LLC-PK, cell monolayers was inhibited by the sulfhydryl reagent PCMBS, placed on both the basolateral and apical sides of the monolayers. Because of its hydrophilicity, PCMBS cannot permeate across the cell membranes (26); therefore, it does not react with sulfhydryl groups at the internal surface of the LLC-PK1 cells. Accordingly, these findings suggest that sulfhydryl groups are essential to the organic cation transport system at both the basolateral and apical membranes of the LLC-PK, cells and that these functional sulfhydryl groups of the transporters are localized at the external side of the cells. Essential sulfhydryl groups for the organic cation transport system have been demonstrated (8, 22). We have reported that PCMBS specifically inhibits the H+ gradient-dependent transport of tetraethylammonium in the renal brush-border membranes and that the functional sulfhydryl groups may be localized on the external side of the brush-border membrane vesicles (8). Considering the membrane vesicles to be oriented right-side-out, it is suggested that the functional sulfhydryl groups of the organic cation transporter are localized at the luminal surface of the tubular cells, consistent with the present findings. In conclusion, our results suggest that tetraethylammonium is transported across the LLC-PK1 cell monolayers from the basolateral to the apical side, i.e., a
IN
RENAL
CELL
C65
MONOLAYERS
unidirectional transcellular transport that is saturable, temperature dependent, and sensitive to pH of the apical side. This transport activity is dependent on cell differentiation. Moreover, it is evident that functional sulfhydry1 groups are essential to the transport of tetraethylammonium at both the basolateral and apical membranes. These groups are localized at least in part on the external surface of the cells. Further studies with LLCPK1 cell monolayers will provide the information required to understand the precise mechanisms involved in the transcellular transport of organic cations. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. Address for reprint requests: K. Inui, Dept. of Hospital Pharmacy, School of Medicine, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. Received
26 February
1991; accepted
in final
form
17 July
1991.
REFERENCES 1. Amsler, K., and J. S. Cook. Development of Na+-dependent hexose transport in a cultured line of porcine kidney cells. Am. J. Physiol. 242 (Cell Physiol. 11): C94-ClOl, 1982. 2. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. 3. Dantzler, W. H., 0. H. Brokl, and S. H. Wright. Brush-border TEA transport in intact proximal tubules and isolated membrane vesicles. Am. J. Physiol. 256 (Renal FZuid EZectroZyte Physiol. 25): F290-F297,1989. 4. Fauth, C., B. Rossier, and F. Roth-Ramel. Transport of tetraethylammonium by a kidney epithelial cell line (LLC-PK1). Am. J. Physiol. 254 (Renal Fluid Electrolyte Physiol. 23): F351-F357, 1988. 5. Fouda, A.-K., C. Fauth, and F. Roth-Ramel. Transport of organic cations by kidney epithelial cell line LLC-PK1. J. Pharmacol. Exp. Ther. 252: 286-292, 1990. 6. Handler, J. S. Studies of kidney cells in culture. Kidney Int. 30: 208-215,1986. 7. Holohan, P. D., and C. R. Ross. Mechanisms of organic cation transport in kidney plasma membrane vesicles. 2. ApH studies. J. Pharmacol. Exp. Ther. 216: 294-298, 1981. R., H. Maegawa, T. Okano, M. Takano, and K. Inui. 8. Hori, Effect of sulfhydryl reagents on tetraethylammonium transport in rat renal brush border membranes. J. PharmacoZ. Exp. Ther. 241: lOlO-1016,1987. 9. Hori, R., K. Yamamoto, H. Saito, M. Kohno, and K. Inui. Effect of aminoglycoside antibiotics on cellular functions of kidney epithelial cell line (LLC-PK1): a model system for aminoglycoside nephrotoxicity. J. PharmacoZ. Exp. Ther. 230: 742-748, 1984. 10. Hsyu, P.-H., and K. M. Giacomini. The pH gradient-dependent transport of organic cations in the renal brush border membrane. Studies with acridine orange. J. BioZ. Chem. 262: 3964-3968, 1987. 11. Hull, R. N., W. R. Cherry, and G. W. Weaver. The origin and characteristics of a pig kidney cell strain, LLC-PK1. In Vitro RockviZZe 12: 670-677, 1976. 12. Inui, K., H. Saito, and R. Hori. H+-gradient-dependent active transport of tetraethylammonium cation in apical membrane vesicles isolated from kidney epithelial cell line LLC-PK,. Biochem. J. 227: 199-203,1985. 13. Inui, K., H. Saito, T. Iwata, and R. Hori. Aminoglycosideinduced alterations in apical membranes of kidney epithelial cell line (LLC-PK1). Am. J. Physiol. 254 (Cell Physiol. 23): C251-C257, 1988. 14. Inui, K., H. Saito, M. Takano, T. Okano, S. Kitazawa, and R. Hori. Enzyme activities and sodium-dependent active D-ghCOSe transport in apical membrane vesicles isolated from kidney epithelial cell line (LLC-PK1). Biochim. Biophys. Acta 769: 514-518, 1984. 15. Kinsella, J. L., and P. S. Aronson. Properties of the Na+-H+
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16. 17.
18.
19. 20.
‘21. 22.
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TRANSPORT
exchanger in renal microvillus membrane vesicles. Am. J. Physiol. 238 (Renal Fluid EZectroZyte Physiol. 7): F46LF469, 1980. Kreisberg, J. I., and P. D. Wilson. Renal cell culture. J. Electron Microsc. Tech. 9: 235-263, 1988. McKinney, T. D., C. DeLeon, and K. V. Speeg, Jr. Organic cation uptake by a cultured renal epithelium. J. Cell. Physiol. 137: 513-520,1988. Montrose-Rafizadeh, C., F. Mingard, H. Murer, and F. Roth-Ramel. Carrier-mediated transport of tetraethylammonium across rabbit renal basolateral membrane. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F243-F251, 1989. Moran, A. Sodium-hydrogen exchange system in LLC-PK, epithelium. Am. J. Physiol. 252 (Cell Physiol. 21): C63-C67, 1987. Mullin, J. M., L. Fluk, and A. Kleinzeller. Basal-lateral transport and transcellular flux of methyl a-D-glucoside across LLCPK, renal epithelial cells. Biochim. Biophys. Acta 885: 233-239, 1986. Rennick, B. R. Renal tubule transport of organic cations. Am. J. Physiol. 240 (Renal Fluid Electrolyte Physiol. 9): F83-F89, 1981. Sokol, P. P., P. D. Holohan, and C. R. Ross. Essential disulfide and sulfhydryl groups for organic cation transport in renal brushborder membranes. J. Biol. Chem. 261: 3282-3287,1986.
IN
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23. Sokol, P. P., and T. D. McKinney. Mechanism of organic cation transport in rabbit renal basolateral membrane vesicles. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1599-F1607, 1990. 24. Takano, M., K. Inui, T. Okano, and R. Hori. Cimetidine transport in rat renal brush border and basolateral membrane vesicles. Life Sci. 37: 1579-1585, 1985. 25. Takano, M., K. Inui, T. Okano, H. Saito, and R. Hori. Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles. Biochim. Biophys. Acta 773: 113-124,1984. 26. Vansteveninck, J., R. I. Weed, and A. Rothstein. Localization of erythrocyte membrane sulfhydryl groups essential for glucose transport. J. Gen. Physiol. 48: 617-632, 1965. 27. Wright, S. H., and T. M. Wunz. Transport of tetraethylammonium by rabbit renal brush-border and basolateral membrane vesicles. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F1040-F1050,1987. 28. Yamaoka, K., Y. Tanigawara, T. Nakagawa, and T. Uno. A pharmacokinetic analysis program (MULTI) for microcomputer. J. Pharmacobio-Dyn. 4: 879-885, 1981.
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tetraethylammonium transport; proton-organic port; tubular secretion; renal cell culture
cation
anti-
SECRETION OF DRUGS and xenobiotics is an important physiological function of the renal proximal tubules. In general, these compounds are negatively or positively charged organic species. The process of secreting organic cations through the proximal tubular cells is performed via unidirectional transcellular transport, i.e., the uptake of organic cation into the cells from blood across the basolateral membranes followed by the active extrusion across the brush-border membranes into the tubular fluid (21). Many investigations using plasma membrane vesicles isolated from the renal cortex have shown the mechanism for organic cation transport in both the brush-border (3, 7, 10, 24, 25, 27) and the basolateral membranes (18, 23-25, 27) from the kidney cortex. The transport mechanisms for organic cations have been particularly well characterized in the brush-border membranes and have demonstrated the presence of an H+organic cation antiport system, a process that exchanges cellular organic cation for tubular H+ (3, 7, 10, 24, 25, 27). Although studies in isolated basolateral membranes suggest a facilitated diffusion via an electrically conductive pathway for organic cations (18,23,25,27), an active transport process is not apparent. A better understanding of the mechanism of transepithelial transport of organic cations requires transport studies using intact tubular epithelia. 0363-6143/92
$2.00 Copyright
Cultured epithelial cells derived from the kidney have been useful in studying a variety of renal cellular functions, including transepithelial transport and the regulation of transport by hormones and drugs (6, 16). The pig kidney epithelial cell line LLC-PK1 (11) has been employed extensively as a model for the analysis of epithelial functions in the proximal tubules (1,9, 12-14). These cells form an oriented monolayer with microvilli and tight junctions and exhibit a unidirectional transport of electrolytes and some nutrients (1,9,20). We provided the first evidence to show that the apical membranes (corresponding to the brush-border membranes) of the LLC-PK, cells express the H+-organic cation antiport system (12). Cultured cell monolayers grown on microporous membrane filters have allowed the study of transepithelial transport under appropriate conditions (20). Recently, Roth-Ramel and co-workers (4, 5) have used LLC-PK, cell monolayers grown on permeable supports to examine the transepithelial transport of organic cations. However, the mechanisms for the transcellular transport of organic cations are not fully characterized. In this study, we attempted to clarify the cellular mechanism of the transcellular transport of tetraethylammonium by LLC-PK1 cell monolayers grown on collagen-coated microporous membrane filters. The results suggest that tetraethylammonium is transported across the monolayers from the basolateral-to-apical side, i.e., a unidirectional transcellular transport, and demonstrate the validity of LLC-PK, cell monolayers as a model for the renal proximal tubular secretion of organic cations. MATERIALS
AND
METHODS
CeLZ culture. LLC-PK1 cells obtained from the American Type Culture Collection (ATCC CRL-1392) were grown on plastic dishes (Falcon; Becton Dickinson, Lincoln Park, NJ) in Dulbecco”s modified Eagle’s medium (GIBCO, Life Technologies, Grand Island, NY), supplemented with 10% fetal calf serum (Microbiological Associates, Bethesda, MD) without antibiotics in an atmosphere of 5% C02-95% air at 37°C. Subculture was done every 7 days using 0.02% EDTA and 0.05% trypsin (9, 12-14). In general, loo-mm plastic dishes were inoculated with 1 X lo6 cells in 10 ml of complete culture medium. In this study, cells between the 210th and 220th passages were used. For the transport studies, LLC-PK1 cells were seeded on collagen-coated membrane filters (3-pm pores, 4.71 cm”-growth area) inside a Transwell cell culture chamber (Costar, Cambridge, MA) at a cell density of 5 x lo5 cells/cm”. Each Transwell chamber was placed in a 35-mm well of tissue culture plate with 2.6 ml of the outside medium (basolateral side) and 1.5 ml of the inside medium (apical side). The cell monolayers were fed fresh medium every 2 days. To evaluate the integrity of the monolayers, transepithelial electrical resistance was measured using Millicell-ERS (Milli-
0 1992 the American
Physiological
Society
c59
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C60
ORGANIC
CATION
TRANSPORT
pore, Bedford, MA) before the transport study. The transepithelial resistance ranged from 180 to 440 Q cm2 in the intact monolayers. Measurement of transepithelial transport and cellular accumulation. Transepithelial transport and accumulation of [ 14C]tetraethylammonium or cy-[ 14C]methyl-D-glucoside were measured using monolayer cultures grown in the Transwell chambers. The incubation medium was Dulbecco’s phosphatebuffered saline (pH 7.4) [PBS buffer made of (in mM) 137 NaCl, 3 KCl, 8 Na2HP04, 1.5 KH2P04, 1 CaC12, and 0.5 MgC1,], containing 5 mM D-ghCOSe, except for the experiment of a-methyl-D-glucoside transport. The pH of the medium was adjusted by adding a solution of HCl or NaOH. In Na+-free medium, NaCl and Na2HP04 of PBS buffer were replaced with choline chloride and K2HP04, respectively. In general experiments, after the removal of culture medium from both sides of the monolayers, the cell monolayers were preincubated with 2 ml of incubation medium in each side for 10 min at 37°C. Then, 2 ml of incubation medium containing [‘“Cl tetraethylammonium (50 ,uM, 7.4 kBq/ml) or a-[14C]methyl-D-glucoside (50 ,uM, 7.4 kBq/ml) and D-[“H]mannitol(4 PM, 29.6 kBq/ml) were added either to the basolateral or apical side, and 2 ml of radioactive free incubation medium were added to the opposite side. The monolayers were incubated for the specified period of time at 37°C. D-Mannitol, a compound that is not transported by the cells, was used to calculate paracellular fluxes and the extracellular trapping of tetraethylammonium and cymethyl-r>-glucoside. For transport measurements, an aliquot (50 ,ul) of the incubation medium in the other side was taken at the specified time, and the radioactivity was counted. For accumulation studies, the medium was removed by suction at the end of the incubation period, and the monolayers were rapidly washed two times with 2 ml of ice-cold incubation medium in each side. The filters with monolayers were detached from the chambers, the cells on the filters were solubilized in 0.5 ml of 1 N NaOH, and the radioactivity of each aliquot (100 ,ul) was counted. The radioactivity of the collected media and the solubilized cell monolayers was determined in 10 ml of ACS II (Amersham International, Buckinghamshire, UK) by liquid scintillation counting. Protein assay. The protein content of the solubilized cell monolayers was determined by the method of Bradford (2), using the Bio-Rad Protein Assay Kit with bovine y-globulin as a standard. The protein content of the intact monolayers was 0.8-0.9 mg/filter (4.71 cm”). Statistical analysis. Data were analyzed statistically using one-way analysis of variance followed by Fisher’s t test. Materials. [ 1-14C]tetraethylammonium bromide (0.15 GBq/ mmol) and D-[l-“H(N)] mannitol (1,110 GBq/mmol) were purchased from Du Pont-New England Nuclear Research Products (Boston, MA). Methyl(cw-o-[U-14C]gluco)pyranoside (5.33 GBq/mmol) was obtained from Amersham International. Tetraethylammonium bromide was obtained from Nacalai Tesque (Kyoto, Japan). Amiloride hydrochloride and choline chloride were obtained from Wako Pure Chemical Industries (Osaka, Japan). Cimetidine and cephalexin were kindly supplied by Fujisawa Pharmaceutical and Shionogi (both in Osaka, Japan), respectively. N1-methylnicotinamide, procainamide hydrochloride andp-chloromercuribenzene sulfonate (PCMBS) were purchased from Sigma Chemical (St. Louis, MO). All other chemicals used for the experiment were of the highest purity available. RESULTS
Transcellular transport and accumulation of tetraethylammonium. To determine whether the LLC-PK, cell monolayers grown on microporous membranes possessed
IN
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MONOLAYERS
a unidirectional transport activity for tetraethylammonium, transepithelial fluxes were measured by adding [“Cl tetraethylammonium and D- [3H] mannitol simultaneously either to the basolateral or apical side of the LLC-PK1 cell monolayers, and the appearance of radioactivity at the other side was examined. The transcellular transport of tetraethylammonium was calculated by subtracting the paracellular flux estimated by D- [3H] mannitol from the transepithelial flux of [ 14C]tetraethylammonium. The transepithelial fluxes of D- [“Hlmannitol from both sides to the other side of the monolayers were ~2% of the total radioactivity added per hour. Figure 1A shows the transcellular transport of [‘“Cltetraethylammonium from the basolateral-to-apical side and the apical-to-basolateral side. The basolateral-toapical transport of [‘“Cl tetraethylammonium was much greater than the apical-to-basolateral transport, and its rate was nearly constant for up to 60 min. The accumulation of [14C]tetraethylammonium by the LLC-PK1 cell monolayers was also measured at 60 min of incubation (Fig. IB ). The accumulation of [14C]tetraethylammonium from the basolateral side was U-fold higher than that from the apical side (basolateral, 3.43 t 0.27 nmolmg protein-‘. 60 min-‘; apical, 0.31 t 0.03 nmol*mg protein-l. 60 min-l). The amount of [“Cl tetraethylammonium trapped in the extracellular space was ~2% of the total [“Cl tetraethylammonium uptake. Table 1 shows the effect of temperature on the transcellular transport of [“Cl tetraethylammonium by the LLC-PK, cell monolayers. When the monolayers were incubated at 4OC, the basolateral-to-apical transport of [“Cl tetraethylammonium was reduced drastically compared with the transport at 37°C and did not differ from the apical-to-basolateral transport of the substrate; therefore, the basolateral-to-apical transport of tetraethylammonium by the monolayers was temperature dependent. These observations indicate that the LLC-PK, cell monolayers possessa unidirectional transport activity for tetraethylammonium; i.e., the LLC-PK, cell 0.6
5
15
30
60
MINUTES
Fig. 1. Transcellular transport (A) and accumulation (B) of tetraethylammonium by LLC-PK, cell monolayers. A: on 4th day after inoculation, LLC-PK, cell monolayers were incubated at 37°C with 50 PM [‘4C]tetraethylammonium (2 ml, pH 7.4) added either to basolateral (0) or apical (A) side of monolayers. Appearance of radioactivity in opposite side (2 ml, pH 7.4) was measured periodically. B: after 60-min incubation, monolayers were rapidly washed two times with 2 ml of ice-cold incubation medium in both sides, and radioactivity of solubilized cell monolayers was determined. Each point or column represents mean t SE of 3 monolayers.
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ORGANIC
CATION
TRANSPORT
Table 1. Effect of temperature on transcellular transport of tetraethylammonium by LLC-PKI cell monolayers Transcellular Transport, pmol acmv2 - 60 min-’ Temperature,
“C
37 4
Apical to basolateral
Basolateral to apical
92.7t6.9 22.9-r-2.4
631.4t31.1 20.7k2.3
Values are means t SE of 3 monolayers. On 4th day after inoculation, LLC-PK1 cell monolayers were incubated at 37 or 4°C for 60 min with 50 PM [14C]tetraethylammonium (pH 7.4) added either to basolateral or apical side of monolayers. Appearance of radioactivity in opposite side (pH 7.4) was measured. 6 _
APICAL-BASAL
BASAL-APICAL
l
-I-Na
-10, G 5.2 -8 I2 a M -6
f -c
-4
g
-2
II r' 3
I
0
’
15
I
30 MINUTES
I
60
APICAL BASAL APICAL BASAL -Na -Na +Na +Na
”
Fig. 2. Transcellular transport (A) and accumulation (B) of a-methylD-glucoside by LLC-PK1 cell monolayers. On 6th day after inoculation, LLC-PK1 cell monolayers were preincubated at 37°C for 10 min (pH 7.4) with either phosphate-buffered saline or Na+-free medium as described in text and were then incubated at 37°C with 50 PM w[‘“C]methyl-D-glucoside (pH 7.4) added either to basolateral (A, A) or apical (0, 0) side of monolayers. Appearance of radioactivity at opposite side (pH 7.4) and accumulation were measured in presence (+Na) and absence (-Na) of Na’. Each point or column represents mean t SE of 3 monolayers.
monolayers accumulate tetraethylammonium progressively from the basolateral side of the monolayers and then extrude it in the apical side. Transcellular transport and accumulation of cu-methylo-glucoside. To ascertain that the unidirectional transport of tetraethylammonium was a specific phenomenon, the transcellular transport and the accumulation of amethyl-D-glucoside, a nonmetabolizable substrate for the Na+-dependent D-ghCOse transport system, were measured in the presence and absence of Na+. The transcellular transport of a-methyl-D-glucoside was measured by adding simultaneously cy-[‘*Cl methyl-D-glucoside and D[ 3H]mannitol either to the apical or basolateral side of the LLC-PK1 monolayers. The transcellular transport of a- [‘*C]methyl-D-glucoside by the LLC-PK1 cell monolayers appears in Fig. 2A. In the presence of Na’, the rate of apical-to-basolateral transport of a-methyl-Dglucoside increased with progressively longer periods of incubation, achieving 201.1 t 15.7 pmol/cm2 at 60 min. In contrast, the basolateral-to-apical transport showed minus values during the periods of incubation. Note that the net flux of a-methyl-D-glucoside represents the sum of the transcellular and paracellular fluxes across the
IN
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C61
MONOLAYERS
monolayers. If the flux across the cells is negligible and if there is a reuptake of the substrate that passesthrough the monolayers paracellularly, the transcellular transport corrected by the D-mannitol flux would have a minus value. The latter phenomenon can thus be explained by the reuptake of ar-methyl-D-glucoside at the apical surface of the LLC-PK1 cell monolayers. The transport in the absence of Na+ was negligible. Figure 2B shows the accumulation of a- [14C]methyl-D-glucoside by the LLCPK, cell monolayers in the presence and absence of Na+ at 60 min. The accumulation of cy-[ ‘*C]methyl-D-glucoside from the apical side was 9.35 t 0.42 nmol mg protein-‘. 60 min-‘, -25-fold greater than that from the basolateral side (0.37 t 0.01 nmol mg protein-’ 60 min-‘). In the absence of Na+, the accumulation from each side was extremely small, 0.014 and 0.051 nmolmg protein-‘.60 min-’ from the apical and basolateral sides, respectively. Concentration dependence of tetraethylammonium transport. Figure 3 shows the transepithelial transport of [‘*Cl tetraethylammonium from the basolateral-to-apical side of LLC-PK1 cell monolayers as a function of an increased concentration of substrate ranging from 0.025 to 1 mM at the basolateral side. The transport rates were estimated at 30 min, because findings were linear up to 60 min after incubation (Fig. 1A). The curve for the transcellular transport obtained by subtracting the paracellular flux from the total transepithelial flux was curvilinear, indicating a saturable process. The apparent Michaelis constant (Km) and maximum velocity ( Vmax) values of the transcellular transport of tetraethylammonium, estimated from the Michaelis-Menten equation using nonlinear least-squares analysis (28), were 67 PM and 222 pmol . mg protein-‘. min-’ (46.5 pmol .crnB2. min-l), respectively. Effect of cationic drugs on transcellular transport and accumulation of tetraethylammonium. The cis-inhibition of the transcellular transport and the accumulation of [‘*Cl tetraethylammonium by unlabeled tetraethylammonium was examined. As shown in Fig. 4A, the basolateral-to-apical transport of [‘*Cl tetraethylammonium l
5 7.E
0
TOTAL
A
PARACELLULAR
---
TRANSCELLULAR
T
z4
0
0.1
0.25
0.5 TEA
1 hM)
Fig. 3. Concentration dependence of tetraethylammonium (TEA) transport across LLC-PK1 cell monolayers. On 6th day after inoculation, LLC-PK1 cell monolayers were incubated at 37°C for 30 min with varying concentrations of [‘“CITEA (pH 7.4) added to basolateral side. Appearance of radioactivity in apical side (pH 7.4) was measured. Each point represents mean ~fr SE of 3 monolayers.
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ORGANIC
s z
05
15
30
CATION
TRANSPORT
P 1
60
MINUTES
Fig. 4. Effect of unlabeled TEA on transcellular transport (A) and accumulation (B) of [‘“CITEA by LLC-PK1 cell monolayers. On 6th day after inoculation, LLC-PK, cell monolayers were incubated at 37°C with 50 PM [‘“CITEA (pH 7.4) added to basolateral side in absence (0) and presence (0) of 2.5 mM TEA. Appearance of radioactivity in apical side (pH 7.4) and accumulation were measured. Each point or column represents mean -+ SE of 3 monolayers.
was depressed dramatically in the presence of 2.5 mM unlabeled compound. Furthermore, the presence of unlabeled tetraethylammonium inhibited the accumulation of [ 14C]tetraethylammonium from the basolateral side to 11% of the control value (Fig. 4B). These findings indicate that the basolateral uptake of [14C]tetraethylammonium was inhibited competitively by the unlabeled substrate, resulting in a marked depression of the subsequent transport of the substrate across the monolayers. These observations also suggest that a passive diffusion pathway may be a minor component of the uptake of tetraethylammonium across the basolateral membranes of the LLC-PK1 cell monolayers. To examine the substrate specificity of the basolateral organic cation transport system in the LLC-PK1 cells, we evaluated the effects of several cationic drugs added to the basolateral side on the transcellular transport and accumulation of [14C]tetraethylammonium from the basolateral side of the monolayers. As shown in Fig. 5, TRANSPORT
DRUGS AMILORIDE
(mfw 0 2.5 3
CIMETIDINE TETRAETHYLAMMONIUM
PROCAINAMIDE CHOLINE N’-METHYLNICOTINAMIDE N’-METHYLNICOTINAMIDE
CEPHALEXIN
. 25
(76 OF CONTROL)
IN
RENAL
MONOLAYERS
cationic drugs such as amiloride, cimetidine, tetraethylammonium, procainamide, and choline inhibited both the basolateral-to-apical transport and the accumulation of [14C]tetraethylammonium. On the other hand, an endogeneous cation N1-methylnicotinamide at 2.5 mM did not inhibit the transport and the accumulation, whereas at 10 mM it inhibited the transport without change of the accumulation. Cephalexin, a zwitterionic cephalosporin, had little inhibitory effect on the transport and the accumulation. Transcellular transport and accumulation of tetraethylammonium at various pH of apical side. We previously reported the H+ gradient-dependent transport of tetraethylammonium by apical membrane vesicles isolated from LLC-PK, cells (12). In this regard, we evaluated the effect of pH of the apical side on the transcellular transport and the accumulation of [14C]tetraethylammonium. As shown in Fig. 6A, when the pH of the apical incubation buffer was varied from 5.4 to 7.4 (pH of the basolateral side fixed at 7.4), the basolateral-to-apical transport of [ 14C]tetraethylammonium was significantly affected, being the greatest at pH 5.4 (1.56 t 0.16 nmol. crnw2.60 min-‘) and the lowest at pH 7.4 (0.50 t 0.08 nmol . crnw2.60 min-l). Conversely, the accumulation of [14C]tetraethylammonium was the lowest at pH 5.4 (0.69 t 0.02 nmol mg protein-l l 60 min-‘) and the greatest at pH 7.4 (3.81 t 0.15 nmolmg protein-l.60 min-‘) (Fig. 6B). These observations show that the transport of tetraethylammonium across the apical membranes can be stimulated by acidifying the medium on the apical side, i.e., inwardly directed H+ gradient acts as a driving force for the extrusion. Transcellular transport and accumulation of tetraethylammonium during growth in culture. We reported that several functions of the apical membranes of the LLCPKI cells, such as the activities of marker enzymes and Na+-dependent D-ghCOSe transport, developed significantly during growth in culture (9, 13). Thus the transcellular transport activity of [14C]tetraethylammonium was studied as a function of duration of culture. As shown l
ACCUMULATION 0
CELL
(% OF CONTROL) 50
100
B
Fig. 5. Effect of cationic drugs on transcellular transport (A) and accumulation (B) of TEA by LLC-PKI cell monolayers. On 6th day after inoculation, LLC-PK1 cell monolayers were incubated at 37°C for 60 min with 50 ,uM [‘“CITEA (pH 7.4) added to basolateral side in absence (control) and presence of other cationic drugs at same side. Appearance of radioactivity in apical side (pH 7.4) and accumulation were measured. Data were expressed as % of control value. Each column represents mean t SE of 3 monolayers.
10 . 25
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ORGANIC
CATION
TRANSPORT
RENAL
CELL
1.6
Days in Culture
-z 5 EO.8
Accumulation, protein-’ Apical
2 4 6
k
0
$0.4 Z
I
-5.4
I
1
5.9
6.4
APICAL
I
I
I
6.9
7.4
5.4
pH
1
1
5.9
6.4
APICAL
I
6.9
I
7.4 -
pH
Fig. 6. Transcellular transport (A) and accumulation (B) of TEA by LLC-PKI cell monolayers at various pH of apical side. On 4th day after inoculation, LLC-PK, cell monolayers were incubated at 37°C for 60 min with 50 PM [‘“CITEA (pH 7.4) added to basolateral side at various pH of apical side. Appearance of radioactivity in apical side and accumulation were measured. Each point represents mean k SE of 3 monolayers.
1.2
ro6 A4 02
oh 5
DAYS DAYS DAYS
15
30
C63
MONOLAYERS
Table 2. Growth dependence of transport and accumulation of tetraethylammonium by LLC-PKI cell monolayers
6
2.E z p
IN
60
MINUTES
Fig. 7. Transcellular transport of TEA from basolateral-to-apical side of LLC-PK1 cell monolayers during growth in culture. On and, 4th, and 6th days after inoculation, LLC-PK1 cell monolayers were incubated at 37°C with 50 PM [‘“CITEA (pH 7.4) added to basolateral side of monolayers. Appearance of radioactivity in apical side (pH 7.4) was measured periodically. Each point represents mean t SE of 3 monolayers.
in Fig. 7, the transcellular transport of [14C]tetraethylammonium by the monolayers from the basolateral-toapical side increased markedly according to the duration of culture. Both the accumulation and the total amount of the uptake and appearance of [14C]tetraethylammonium in the apical side are summarized in Table 2. The accumulation of [“Cl tetraethylammonium from the basolateral side of the monolayers cultured for 2 days did not differ significantly from that of the monolayers cultured for 4 days, whereas a significant decrease in accumulation was observed in the monolayers on the 6th day of inoculation. The total amount of [14C]tetraethylammonium that crossed the basolateral membranes increased with duration of culture. In contrast, accumulation from the apical side was unaffected during culture. These findings suggest that the transcellular transport of tetraethylammonium by LLC-PK, cells is a characteristic of differentiating cultures similar to the development of Na+-dependent hexose transport (1, 13).
0.24t0.03 0.31~0.03 0.20*0.02
nmol . mg - 60 min-’ Basolateral
3.11t0.19 3.43t0.27 2.21t0.20*
Total Amount, nmol - mg protein-l 60 min-’
-
5.14t0.25 7.32+0.38-t 8.15kO.4O"f
Values are means k SE of 3 monolayers. LLC-PK1 cell monolayers were incubated at 37°C for 60 min with 50 FM [14C]tetraethylammonium (pH 7.4) added either to basolateral or apical side. Appearance of radioactivity in opposite side (pH 7.4) and accumulation were measured. Total amount of [“Cl tetraethylammonium transferred across basolateral membranes was calculated by adding transcellular transport (basolateral-to-apical side) to accumulation of [14C]tetraethylammonium. * P < 0.05 and f- P < 0.01, significant differences from value of 2 days using analysis of variance followed by Fisher’s t test.
Effect of PCMBS on transcellular transport and accumulation of tetraethylammonium. We previously reported that sulfhydryl groups are essential for the H’-organic cation antiport system of the renal brush-border membranes (8) and in LLC-PK, apical membranes (12). In this study, we examined the effect of PCMBS, a sulfhydry1 reagent, on the transcellular transport and accumulation of tetraethylammonium. As summarized in Table 3, when the apical surface of the monolayers was pretreated with 0.1 mM PCMBS, the transcellular transport of [14C]tetraethylammonium was decreased to 15% of the control value while the accumulation was increased to 145% of the control value. Pretreatment of the basolateral surface of the monolayers with PCMBS (0.1 mM) also inhibited the transport and the accumulation of [14C]tetraethylammonium to 73 and 53% of the control values, respectively. The most profound inhibition of the [“Cl tetraethylammonium transport was observed when both sides of the monolayers were pretreated with PCMBS while the accumulation did not differ significantly from the control. The simultaneous inhibition of the basolateral uptake and apical efflux of [14C]tetraethylammonium by PCMBS pretreatment may result in an accumulation of the substrate as much as that of control. These observations demonstrate that sulfhydryl groups may play an important role in both the apical and basolateral transport systems for tetraethylammonium in the LLC-PK1 cells. DISCUSSION
The results demonstrate that LLC-PK, cell monolayers possessfunctionally unidirectional transport systems for tetraethylammonium in both the basolateral and apical membranes. In contrast to tetraethylammonium, a-methyl-D-glucoside accumulated within the LLC-PK, cells from the apical side in the presence of Na+ and appeared at. the basolateral side of the monolayers, indicating an opposite direction for tetraethylammonium transport. These findings support the conclusion that the LLC-PK1 cell monolayers possess the bidirectional transport ability responsible for the reabsorptive and secretory functions of the renal proximal tubular epithelium.
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C64
ORGANIC
CATION
Table 3. Effect of PCMBS on transcellular by LLC-PK, cell monolayers
Pretreatment
PBS (control) 0.01 mM PCMBS 0.1 mM PCMBS 0.01 mM PCMBS 0.1 mM PCMBS
0.01 mM 0.1 mM
PCMBS PCMBS
Added
to
Both Apical Apical Basolateral Basolateral Both Both
TRANSPORT
transport
IN
RENAL
CELL
and accumulation
Transport, nmol - cm-’ 60 mine1 1.46t0.18 1.54t0.14 0.22+0.02-f1.47to.11 1.07t0.01"
1.63-sO.09 0.16+O.OOt
The kinetic parameters of the basolateral-to-apical transport of tetraethylammonium across the LLC-PK1 cell monolayers were calculated; the apparent K, and Vmax values were 67 PM and 222 pmolmg protein-‘. min-l, respectively. It should be noted that the transcellular transport of tetraethylammonium is a consequence of two processes, basolateral uptake and apical efflux. Thus these apparent K, and Vmax values reflect the transport characteristics of both the basolateral and apical membranes. The LLC-PK1 cell monolayers accumulated tetraethylammonium progressively from the basolateral but not the apical side of the monolayers. Furthermore, the accumulation from the basolateral side was inhibited by unlabeled substrate and by cationic drugs such as amiloride, cimetidine, and procainamide, which are known to interact with organic cation transport systems in the renal basolateral membranes (18, 23, 24, 27). Thus it may be suggested that the uptake of tetraethylammonium from the basolateral side represents a function of the transport system localized in the basolateral membranes of the LLC-PK1 cells. In the present study, we found that N1-methylnicotinamide at a concentration of 2.5 mM did not influence the transport and accumulation of [14C]tetraethylammonium (50 PM) by the LLC-PK, cell monolayers, although at PO mM it inhibited the transport without change of the accumulation. Therefore, it could be suggested that the interaction of N1methylnicotinamide with the basolateral transport system for organic cations was relatively weak compared with other cationic drugs and that Nl-methylnicotinamide inhibited the efflux of tetraethylammonium via the apical transport system after entering the cells. Wright and Wunz (27) reported that Nl-methylnicotinamide inhibited tetraethylammonium uptake into the renal brush-border membranes but not into the basolatera1 membranes, suggesting there may be a qualitative difference in the structural requirements associated with substrate recognition by the transport systems of the two membranes. Assuming the intracellular water content of 7 pl/mg protein (4), the intracellular tetraethylammonium concentration at the apical pH of 7.4 was -lo-fold higher than the concentration (50 PM) in the basolateral me-
% Control Value 100 105 15 101 73 112 11
MONOLAYERS
of tetraethylammonium
Accumulation, nmol - mg protein-’ - 60 min-’ 2.11t0.02 2.37t0.04 3.05+0.05t 1.95t0.21 1.12tO.O6f2.19kO.04 1.8lt0.08*
%Control Value 100 112 145 92 53 104 86
dium (intracellular concentration at 60 min on the 4th day after inoculation, 490-540 PM; Figs. 1 and 6, Table 2). This may be near an equilibrium value that could be expected by a cell interior negative potential. We demonstrated previously that tetraethylammonium is transported across the renal basolateral membranes via a carrier-mediated system, which is stimulated by a negative membrane potential (25). Therefore, it could be suggested that the basolateral uptake of tetraethylammonium is mediated via a cell potential-dependent transport system. However, present data do not exclude the possibility that tetraethylammonium might be bound to intracellular anionic sites. It has been well documented that tetraethylammonium is transported across the renal brush-border membranes by a H+-organic cation antiport system (3, 10, 25, 27). We demonstrated that the LLC-PK, cells possess this antiport system in their apical membranes (12). However, there is no evidence to show the H+-organic cation antiport system functions in the intact LLC-PK, cells. In this study, the transcellular transport of tetraethylammonium by the LLC-PK, cell monolayers was dependent on the pH of the medium on the apical side with the greatest transport occurring at 5.4, the most acidic pH. This finding suggests that, in intact LLC-PK1 cells, the H+ gradient directed inwardly from the exterior of the cells could serve as a driving force for tetraethylammonium transport at the apical membranes, i.e., a H+tetraethylammonium antiport system. In the isolated perfused tubule, the stimulatory effect of acidic luminal pH on the secretion of tetraethylammonium was demonstrated (3). The inwardly directed H+ gradient in the proximal tubule can be generated predominantly by the Na+-H+ exchanger in the brush-border membranes, which is dependent on the electrochemical gradient of Na+ produced by the Na+-K+-ATPase in the basolateral membranes (15). Such an exchanger has been demonstrated in the apical membranes of LLC-PK1 cells (19). Therefore, the H+ gradient produced by a Na’-H+ exchange system in the apical membranes of the LLC-PK1 cells might have been responsible for tetraethylammonium transport at pH 7.4 in the present study. Recently, McKinney et al. (17) demonstrated that intact LLC-PK, cells possess a mechanism in their apical membranes for
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ORGANIC
CATION
TRANSPORT
the mediated transport of tetraethylammonium and that H+-organic cation exchange does not appear to be predominant. The discrepancy might be explained by a difference in experimental conditions in that McKinney et al. (17) used LLC-PK1 cell monolayers grown on plastic wells. The basolateral-to-apical transport of tetraethylammonium by the LLC-PK, cell monolayers was found to increase significantly during growth in culture. There was no significant difference in the accumulation of tetraethylammonium from the basolateral side between days 2 and 4, while there was an appreciable decrease in the accumulation on day 6. The same magnitude of the basolateral uptake and apical efflux of the substrate indicates a parallel development of the transport activity of tetraethylammonium at both the basolateral and apical membranes of the LLC-PK1 cells during 4 days in culture. In the monolayers cultured for 6 days, the transport activity of tetraethylammonium in the apical membranes was further increased, but the transport activity in the basolateral membranes was not or less increased, resulting in a lower accumulation in the cells. The total amount of tetraethylammonium that crossed the basolateral membranes increased with the duration of the culture. The protein content of the monolayers did not increase appreciably in cells cultured for 2-6 days (0.8 0.9 mg protein/filter). Thus the development of tetraethylammonium transport activity was not due to an increase in the density of cells on the filter. The development of apical membrane enzyme activity and Na’dependent hexose transport in the LLC-PK, cells has been described in differentiating cultures (1, 13). Mechanisms for the development of the organic cation transport system in the LLC-PK, cells should be further investigated. The transport of tetraethylammonium across the LLC-PK, cell monolayers was inhibited by the sulfhydryl reagent PCMBS, placed on both the basolateral and apical sides of the monolayers. Because of its hydrophilicity, PCMBS cannot permeate across the cell membranes (26); therefore, it does not react with sulfhydryl groups at the internal surface of the LLC-PK1 cells. Accordingly, these findings suggest that sulfhydryl groups are essential to the organic cation transport system at both the basolateral and apical membranes of the LLC-PK, cells and that these functional sulfhydryl groups of the transporters are localized at the external side of the cells. Essential sulfhydryl groups for the organic cation transport system have been demonstrated (8, 22). We have reported that PCMBS specifically inhibits the H+ gradient-dependent transport of tetraethylammonium in the renal brush-border membranes and that the functional sulfhydryl groups may be localized on the external side of the brush-border membrane vesicles (8). Considering the membrane vesicles to be oriented right-side-out, it is suggested that the functional sulfhydryl groups of the organic cation transporter are localized at the luminal surface of the tubular cells, consistent with the present findings. In conclusion, our results suggest that tetraethylammonium is transported across the LLC-PK1 cell monolayers from the basolateral to the apical side, i.e., a
IN
RENAL
CELL
C65
MONOLAYERS
unidirectional transcellular transport that is saturable, temperature dependent, and sensitive to pH of the apical side. This transport activity is dependent on cell differentiation. Moreover, it is evident that functional sulfhydry1 groups are essential to the transport of tetraethylammonium at both the basolateral and apical membranes. These groups are localized at least in part on the external surface of the cells. Further studies with LLCPK1 cell monolayers will provide the information required to understand the precise mechanisms involved in the transcellular transport of organic cations. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. Address for reprint requests: K. Inui, Dept. of Hospital Pharmacy, School of Medicine, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. Received
26 February
1991; accepted
in final
form
17 July
1991.
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16. 17.
18.
19. 20.
‘21. 22.
ORGANIC
CATION
TRANSPORT
exchanger in renal microvillus membrane vesicles. Am. J. Physiol. 238 (Renal Fluid EZectroZyte Physiol. 7): F46LF469, 1980. Kreisberg, J. I., and P. D. Wilson. Renal cell culture. J. Electron Microsc. Tech. 9: 235-263, 1988. McKinney, T. D., C. DeLeon, and K. V. Speeg, Jr. Organic cation uptake by a cultured renal epithelium. J. Cell. Physiol. 137: 513-520,1988. Montrose-Rafizadeh, C., F. Mingard, H. Murer, and F. Roth-Ramel. Carrier-mediated transport of tetraethylammonium across rabbit renal basolateral membrane. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F243-F251, 1989. Moran, A. Sodium-hydrogen exchange system in LLC-PK, epithelium. Am. J. Physiol. 252 (Cell Physiol. 21): C63-C67, 1987. Mullin, J. M., L. Fluk, and A. Kleinzeller. Basal-lateral transport and transcellular flux of methyl a-D-glucoside across LLCPK, renal epithelial cells. Biochim. Biophys. Acta 885: 233-239, 1986. Rennick, B. R. Renal tubule transport of organic cations. Am. J. Physiol. 240 (Renal Fluid Electrolyte Physiol. 9): F83-F89, 1981. Sokol, P. P., P. D. Holohan, and C. R. Ross. Essential disulfide and sulfhydryl groups for organic cation transport in renal brushborder membranes. J. Biol. Chem. 261: 3282-3287,1986.
IN
RENAL
CELL
MONOLAYERS
23. Sokol, P. P., and T. D. McKinney. Mechanism of organic cation transport in rabbit renal basolateral membrane vesicles. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1599-F1607, 1990. 24. Takano, M., K. Inui, T. Okano, and R. Hori. Cimetidine transport in rat renal brush border and basolateral membrane vesicles. Life Sci. 37: 1579-1585, 1985. 25. Takano, M., K. Inui, T. Okano, H. Saito, and R. Hori. Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles. Biochim. Biophys. Acta 773: 113-124,1984. 26. Vansteveninck, J., R. I. Weed, and A. Rothstein. Localization of erythrocyte membrane sulfhydryl groups essential for glucose transport. J. Gen. Physiol. 48: 617-632, 1965. 27. Wright, S. H., and T. M. Wunz. Transport of tetraethylammonium by rabbit renal brush-border and basolateral membrane vesicles. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F1040-F1050,1987. 28. Yamaoka, K., Y. Tanigawara, T. Nakagawa, and T. Uno. A pharmacokinetic analysis program (MULTI) for microcomputer. J. Pharmacobio-Dyn. 4: 879-885, 1981.
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