Organic cation transport by rat hepatocyte basolateral membrane vesicles T. DWIGHT
McKINNEY
AND MELANIE
A. HOSFORD
Nephrology Section, Department of Medicine, Indiana University School of Medicine, and Medical Service, Veterans Affairs Medical Center, Indianapolis, Indiana 46202-5116 McKinney, T. Dwight, and Melanie A. Hosford Organic organic cation has been employed extensively to evalucation transport by rat hepatocyte basolateral membranevesi- ate the renal handling of organic cations. cles.Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G939G946, 1992.-Hepatocyte basolateral membrane possesses transport systemsfor mediated uptake of organic cations, the METHODS first step in the subsequentbiliary excretion and/or metabolism BLMV were preparedfrom 250- to 300-gmaleSprague-Dawof these compounds.The purposeof these studieswas to evalley rats (Harlan Sprague Dawley, Indianapolis, IN) by the uate potential mechanismsfor transport of this classof solutes acrossthis membraneby measuring3H-labeled tetraethylam- method of Blitzer and Donovan (5). Vesicles were stored in membranesuspensionbuffer consistingof 300 mM sucroseand monium ( [3H]TEA) transport into rat hepatocyte basolateral membranevesicles.[3H]TEA uptake wasstimulated by an out- 10 mM N-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid (HEPES), pH 7.5 at -70°C for subsequenttransport experiwardly directed proton gradient consistent with TEA-proton exchange.Proton gradient-stimulated [3H]TEA uptake was in- ments. For TEA transport studiesvesicleswere rapidly thawed in a hibited by quinidine and by the combination of valinomycin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) but 37°C water bath, washedand centrifuged three times at 47,000 not by CCCP alone or by N1-methylnicotinamide (NMN). An g for 5 min and resuspendedin the appropriate transport buffer outwardly directed TEA gradient also stimulated uptake of except in the taurocholate studiesin which vesicleswere left in [3H]TEA with values at early time points exceeding those at suspensionbuffer (5). Unlessspecifically noted, transport buffer equilibrium. This trans.stimulation or countertransport was consisted of either KC1 or NaCl (150 mM) and HEPES (10 saturable with an apparent Michaelis constant of 106 PM and mM), pH 7.4. The pH of KC1 buffers wasadjustedwith KOH, maximal velocity of 434 pmol mg-l .15 s-l. TEA countertrans- NaCl buffers with NaOH, mannitol buffers with tris(hydroxyport was &-inhibited by quinidine, cimetidine, and thiamine methyl)aminomethane (Tris) base,and N-methyl-D-glucamine and by low temperature, but not by NMN. Thiamine was also (NMDG) with HCl. In experiments examining the effect of an capable of trans.stimulating [3H]TEA uptake. An outwardly outwardly directed proton gradient 2-(N-morpholino)ethanedirected potassiumgradient enhancedand an inwardly directed sulfonic acid replaced HEPES as the buffer. Ethanol wasused potassiumgradient reducedTEA countertransport but had no to dissolvevalinomycin and carbonyl cyanide m-chlorophenyleffect on [3H]TEA uptake occurring in the absenceof other hydrazone (CCCP). In experiments utilizing these ionophores, electrochemicaldriving forces. Thesestudiesindicate that there an equalamount of ethanol (final concn 0.3%) wasaddedto the are at least two potential mechanismsin the hepatocyte baso- control vesicles.For countertransport or efflux experiments,the lateral membranefor transport of organic cations; organic cat- vesicleswere preincubated with 1 mM TEA or 55 PM [3H]TEA ion-organic cation exchange (countertransport) and organic (consisting of 5 PM C3H]TEA, 13.5 Ci/mmol, Amersham,Arcation-proton exchange.Furthermore, the resultsare consistent lington Heights, IL, and 50 PM nonradioactive TEA), respecwith the existence of more than one transporter with different tively, (or other compoundsas indicated in RESULTS) for 2 h at substrate affinities in each of these categories. Finally, these room temperature (23-25°C) before transport measurements results also suggestpotential mechanismsfor drug-to-drug in- were made.TEA transport was measuredat room temperature teractions occurring at the level of transport acrossthe hepato- by a rapid filtration method using 0.4 PM polycarbonate Nuclepore filters (Costar Nuclepore, Pleasanton, CA). [3H]taurocyte basolateralmembrane. cholate (1.5 Ci/mmol, New England Nuclear, Boston, MA) uptetraethylammonium; drugs; drug-to-drug interactions; coun- take wasmeasuredat 37°C. Vesicles(lo-16 mg/ml) werediluted tertransport; organic cation-proton exchange l:20 into reaction buffer, and the reaction stoppedat the desired time by dilution of an aliquot into 3 ml of ice-cold transport SIMILAR TO THE KIDNEY, the liver is known to possess buffer contained in a ten-place filtering manifold (Hoefer Scitransport systems for organic cations (26, 29). These entific, San Francisco, CA). This mixture was immediately filsystems are important in the disposition of both endogtered by vacuum at 440-550 Torr. The filter wassubsequently enous and exogenous organic cations. However, less is washedan additional three timeswith 3 ml of ice-coldtransport known about the hepatic handling of this class of combuffer and then placedin a scintillation vial to which was added 5 ml of Redy Solv HP (Beckman Instruments, Fullerton, CA). pounds compared with the kidney. Experiments with Radioactivity was determined by standard liquid scintillation membrane vesicles have been quite useful in defining many features of renal organic cation transport (15). spectroscopy. In each experiment, the radioactivity associated with blank filters or vesiclesexposedto radioactivity and However, only recently have they been utilized to study immediately filtered was subtracted from the experimental hepatic organic cation transport (21). Thus the present values. experiments were designed to confirm that hepatocyte Protein was determined by the Lowry method (13) using basolateral membrane vesicles (BLMV) could be em- bovine serumalbumin as a standard. Activities of the following ployed to elucidate potential mechanisms involved in enzymes were determined by establishedmethods on freshly mediated transport of organic cations by the rat liver. preparedmembranes:Na-K-adenosinetriphosphatase(ATPase; We chose to use radiolabeled tetraethylammonium 6), alkaline phosphatase(23), NADPH cytochrome c reductase (TEA) as the organic cation probe, in part, because this and succinate cytochrome c reductase (30), and acid phosl
G939 Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 13, 2019.
G940
HEPATIC
TETRAETHYLAMMONIUM
TRANSPORT 40
phatase (33). Chemicalswere obtained from Sigma Chemical (St. Louis, MO). In eachexperiment, all conditions were generally evaluated in quadruplicate. The mean of the results was calculated for that experiment. The resultsare presentedas meanst SE of experiments conducted under similar conditions. Statistical analysis wasdeterminedusing Student’s t test for paired observationsor analysis of variance followed by testing of meansby Fisher’s test. A P value < 0.05 was consideredto be statistically significant.
1
.c=*
RESULTS
To confirm that the methods used to prepare vesicles resulted in an enrichment in basolateral membranes, the activities of several enzyme markers were measured in five to seven different membrane preparations. These included Na-K-ATPase (basolateral membranes), alkaline phosphatase (canalicular membranes), acid phosphatase (lysosomes), NADPH cytochrome c reductase (endoplasmic reticulum), and succinate cytochrome c reductase (mitochondria). The following enrichment ratios (activity in final membrane preparation relative to that in the initial crude homogenate) were obtained: Na-K-ATPase, 32.8 t 6.2; alk a 1ine phosphatase, 1.6 & 0.2; acid phosphatase, 1.0 * 0.1; NADPH cytochrome c reductase, 0.8 t 0.1; succinate cytochrome c reductase, 0.08 t 0.01. Even though these results indicate the presence of other cellular constituents, they nevertheless indicate a marked enrichment in basolateral membranes in the final membrane preparation and are quite comparable to those reported by others (5, 11). Additionally, to determine if functional BLMV were present in our preparation, we measured [3H]taurocholate uptake in the presence and absence of an inwardly directed sodium gradient. Taurocholate uptake was markedly enhanced in the presence of a sodium gradient (data not shown) consistent with previous results (5, 11). Figure 1 depicts a representative experiment in which the membrane-associated radioactivity of 55 PM [3H]TEA was measured at early time points and at 2 h in the presence or absence of 1 mM nonradioactive TEA in the extravesicular medium (&-side). Although “coldn TEA reduced the membrane-associated radioactivity at early time points in this and other experiments, a substantial portion of activity remained suggesting that under these conditions (i.e., absence of imposed electrochemical gradients) only a portion of the [3H]TEA associated with the membranes may represent mediated transport1 In addition, membrane-associated radioactivity under these conditions was not reduced by 1 mM quinidine, cimetidine, or NMN (data not shown). Thus a series of experiments was undertaken to determine if uptake of TEA could be increased by imposition of electrochemical gradients. One mechanism by which organic cations could be taken up by hepatocytes is in exchange for protons, analogous to that demonstrated in brush-border membranes of the kidney of several species by numerous investigators (9,10,16,17,31,36). Indeed, it has been reported recently that the organic cation NMN can be transported across l In these particular experiments, uptake cannot be differentiated with certainty from binding of [3H]TEA to the membranes.
control
-
L I
0’
I
I
60
1 mMTEA
1 /r
120 set
2 hr
time
Fig. 1. Association of 3H-labeled tetraethylammonium ( [3H]TEA) with hepatic basolateral membrane vesicles (BLMV) in the absence of electrochemical gradients. Membrane-associated radioactivity was measured at indicated times with symmetric NaCl buffer with or without 1 mM TEA in extravesicular buffer. Representative experiment is depicted.
hepatic BLMV by this mechanism (21). In the latter report, an outwardly directed proton gradient markedly stimulated NMN transport with uptake at early time points exceeding those at equilibrium (“overshoot”). To test for this possibility, [3H]TEA uptake by BLMV was measured in the presence and absence of a transvesicular proton gradient. Figure 2 shows a representative experi-
-
0’
8.0-8.0
1
I
I
I
I
1
2
3
5
10 min
“-
2 hr
time
Fig. 2. Effect of proton gradient on incubated in KC1 buffers at pH 6.0 indicated times in KC1 buffers with is depicted. pHi, intravesicular pH;
[3H]TEA uptake. BLMV were preor 8.0, and uptake was measured at pH 8.0. Representative experiment pH,, extravesicular pH.
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HEPATIC
TETRAETHYLAMMONIUM
G941
TRANSPORT
40
10
Val
I 3
I
1 I-
5 min
2 hr
I
I
3
5
time
and NMN
on pH-stimulated
cantly inhibited proton gradient-stimulated uptake of 55 PM TEA at early time points but not at 2 h. In contrast to these results, under similar conditions NMN did not affect TEA uptake at early time points. Uptake at 2 h was slightly, but significantly, lower in the presence of NMN. Another mechanism by which organic cations could be transported across hepatic BLMV is by organic cationorganic cation exchange or countertransport. Moseley et al. (21) recently demonstrated that [3H JNMN uptake by hepatic BLMV could be stimulated by preloading vesicles with a high (tram) concentration of nonradioactive NMN. In the experiments depicted in Fig. 5, the effect of trans 1 mM TEA on the uptake of 55 PM [3H]TEA was examined. In addition, the effect of temperature was evaluated by conducting experiments at room temperature and at 4OC. Preloading BLMV with 1 mM TEA markedly stimulated uptake of [3H]TEA at early time points at 25OC with an overshoot in uptake at 30-120 s compared with that at 2 h. Trans.stimulation by TEA was abolished at 4’C with uptakes being similar to those observed under control conditions (absence of trans TEA). Figure 5 also demonstrates that uptake under control conditions was reduced at the lower temperature. In addition to TEA uptake TEA Uptake,
Time, min 3 5 10 120
34.34zk5.56 29.01*4.10 35.59k4.52 34.99dE2.79
I
180
minutes
ment and demonstrates that an outwardly directed proton gradient stimulates uptake at early time points compared with no proton gradient. Values at 2 h were similar in the presence or absence of the initial proton gradient. An outwardly directed proton gradient consistently stimulated TEA uptake compared with that observed in the absence of a proton gradient. Frequently, in the presence of a proton gradient there was an overshoot in uptake above equilibrium values as demonstrated in Fig. 2. Similar to a previous report of proton gradient-stimulated NMN uptake in these vesicles (21), proton gradient-stimulated TEA uptake was not enhanced by the proton ionophore CCCP consistent with the effect of the proton gradient not being due to a proton diffusion potential (Fig. 3). However, TEA uptake in the presence of an outwardly directed proton gradient was reduced by the combination of valinomycin and CCCP (Fig. 4). These results, which are similar to those reported for proton gradient-stimulated NMN uptake by these membranes (21), are consistent with the presence of proton-TEA exchange. To determine whether other organic cations could inhibit proton gradient-stimulated TEA uptake, we examined the effect of quinidine and NMN. As shown in Table 1, quinidine in a concentration of 1 mM signifi-
Control
/
Fig. 4. Effect of CCCP and valinomycin (val) on proton gradient-stimulated [3H]TEA uptake. BLMV were preincubated for 15 min in KC1 buffer at pH 6.0 with or without 2.7 PM val and 12.7 PM CCCP, and uptake of 52.5 PM 13H]TEA was measured at indicated times in KC1 buffer with pH 8.0. Representative experiment is depicted. Similar results were also obtained in a 2nd experiment.
Fig. 3. Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on proton gradient-stimulated [3H]TEA uptake. BLMV were preincubated for 15 min in KC1 buffer at pH 6.0 with or without 12.7 PM CCCP, and uptake of 55 PM [3H]TEA was measured at indicated times in KC1 buffer with pH 8.0. Data represent results of 3 separate experiments.
Table 1. Effect of quinidine
/
Quinidine
P
16.74k3.54 20.03k4.99 24.55k3.52 30.52t2.37
co.025 co.025 0.01 >0.45
pmol/mg Control 28.62k1.37 29.52k4.53 30.12t2.89 33.1ojc3.29
NMN 29.24zk2.57 27.37k3.63 27.06k2.74 27.70k3.62
P >0.85 HI.55 XI.55 co.05
Values are means zt SE; (n = 3 experiments for both quinidine and NMN). Basolateral membrane vesicles were preincubated in KC1 buffer at pH 6.0, and uptake of tetraethylammonium (TEA) was measured at the indicated times in KC1 buffer at pH 8.0 with or without 1 mM quinidine or NMN.
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HEPATIC
TETRAETHYLAMMONIUM
TRANSPORT
A 80 70
zi E
1
-
25” preload
-
4O preload I
-
0
m
60
25” control 4” control
I
I
60
120
/
set
/I
2 hr
time
Fig. 5. Countertransport experiments. [3H]TEA uptake was measured with symmetric KC1 buffer in control vesicles and ones preloaded with I mM TEA at the indicated times and temperature. Results from experiments with 3 separate membrane preparations are depicted. * P < 0.05 vs. other groups.
these results, when BLMV were preloaded with 55 PM [3H]TEA addition of 1 mM TEA to the extravesicular medium accelerated efflux of [3H]TEA from the vesicles (data not shown). After it was observed that [3H]TEA uptake was stimulated by a trans.concentration, other experiments were conducted to further characterize this process. The data shown in Fig. 6A indicate that TEA countertransport measured at 15 s is saturable.2 Analysis of these data by a Hanes-Woolf plot (Fig. 6B) yielded an apparent Michaelis constant (K,) of 106 PM and apparent maximal velocity ( Vmax)of 434 pmol/mg.3 Next, we determined if other organic cations in the uptake medium (c&side) inhibited TEA countertransport. As shown in Fig. 7, cimetidine and quinidine, commonly prescribed organic cation type drugs, in a cisconcentration of 1 mM markedly inhibited TEA countertransport at 60 and 120 s. Because uptake at 2 h was not affected, the inhibition observed at earlier time points did not result from loss of vesicle integrity. In contrast to these results, the endogenous organic cation NMN did not affect TEA countertransport. We also examined the effect of 1 mM cis- and transconcentrations of thiamine, a compound known to interact with organic cation transport pathways in hepatocytes (14,37), on TEA transport. As demonstrated in Fig. 8, G-thiamine markedly inhibited TEA countertrans2 Preliminary experiments demonstrated that [3H]TEA (55 and 850 PM) uptake under these conditions was linear for up to 20 s. 3 We did not determine the sidedness of the vesicles used in these studies. However, in the original description of the method employed to prepare these vesicles a minority were reported to be inside-out (5). It is possible that asymmetry in affinity for TEA exists between internal and external binding sites. If this is the case, the apparent kinetic values we report might be different from those that would be obtained in a totally right-side-out vesicle preparation.
:
tu ‘;3 z
10
8
.-0
I
Km = 106 @l Vmax = 434 pmolimg
1
0
I
200
’
I
I
I
600
400 TEA
I
1
800
1
1000
(PM)
Fig. 6. Kinetics of TEA countertransport. A: vesicles were preloaded with 1 mM TEA in KC1 buffer followed by measurement of 15-s uptake in the same buffer of [3H]TEA with concentrations indicated on abscissa. Results are from experiments with 4 different membrane preparations. B: Hanes-Woolf plot of data in A. Line represents linear regression of data using least-squares analysis.
port (TEA tram/thiamine cis). To determine whether the inhibition of TEA countertransport by thiamine was presumably due to competition with [3H]TEA in the extravesicular medium for transport, the ability of intravesicular thiamine to stimulate uptake of [3H]TEA was examined. As shown, preloading vesicles with 1 mM thiamine (thiamine trans) markedly stimulated uptake of 55 PM t3H]TEA at 60 and 120 s but not at 2 h compared with uptake of [3H]TEA measured without preloading with TEA or thiamine (control). As indicated by the lack of effect of NMN in the experiments shown in Fig. 7, not all organic cations we tested &-inhibited TEA countertransport. To further examine whether NMN might affect TEA transport in hepatic BLMV, we determined whether preloading vesicles with NMN might stimulate uptake of TEA. However, in two experiments depicted in Fig. 9, a tram 1 mM concentration of NMN did not enhance uptake of 55 PM [3H]TEA compared with a similar trans-concentration of TEA. Finally, to determine if TEA countertransport might
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HEPATIC
TETRAETHYLAMMONIUM
TEA trans
Y
Thiamine
1 l
I
60
-
l
I
120 set
I
I
G943
TRANSPORT
60
control
w
trans __t_)
Thiamine
cis
auinidine
I
2 hr
time
Fig. 7. Effect of other organic cations on TEA counter-transport. Vesicles were preloaded with 1 mM TEA in KC1 buffer, and uptake of 55 PM [3H]TEA was measured at indicated times in same buffer with or without (control) 1 mM concentrations of the other organic cations in the extravesicular media. NMN, R”-methylnicotinamide. Data represent results obtained in 6 different membrane preparations. * P < 0.01 vs. control.
be conductive and/or affected by inorganic cation gradients, experiments were carried out with transvesicular potassium gradients and the potassium ionophore valinomycin. As shown in Fig. 10, with an outwardly directed potassium gradient and inwardly directed sodium gradient, TEA countertransport was enhanced compared with that occurring with symmetric potassium buffers. Furthermore, the reverse condition, i.e., inwardly directed potassium and outwardly directed sodium gradients was associated with the lowest countertransport. In addition, in these same experiments in the presence of an outwardly directed potassium gradient valinomycin enhanced and in the presence of an inwardly directed potassium gradient decreased TEA countertransport (Fig. 11). Results similar to those noted above were obtained when an NMDG gradient was used rather than sodium suggesting that the observations were due to potassium rather than to sodium (Fig. 12). In contrast to these countertransport experiments, sodium and potassium gradients had little effect on uptake of TEA in the absence of a trans.concentration of TEA (Fig. 13). These experiments were performed with gluconate salts of sodium and potassium. However, similar results were obtained with NaCl and KC1 (data not shown). DISCUSSION
The liver and kidneys are responsible for the disposition of a large number of endogenous and exogenous primary, secondary, and tertiary amines and quaternary ammonium compounds. Because the nitrogen of these substances carries a positive charge at physiological pH, they are collectively referred to as organic cations. Important organic cations include common drugs such as procainamide, cimetidine, dipyridamole, tricyclic antide-
1
60
I
120
I
I
I
2 hr
see
time
Fig. 8. Effect of thiamine on TEA transport. Vesicles were preloaded in KC1 buffer without (control and thiamine cis) or with 1 mM TEA or thiamine (TEA trams, TEA tram-thiamine cis, and thiamine tram) and uptake of 55 PM [3H]TEA in KC1 buffer was measured at the indicated times without (control, TEA tram, and thiamine tram) or with (thiamine cis and TEA tram-thiamine cis) 1 mM thiamine in the extravesicular medium. Results from a representative experiment are shown. 40
1:‘:1
,t 60
120 set
2 hr
time
Fig. 9. Effect of tram NMN on TEA uptake. Vesicles were preloaded with 1 mM TEA or NMN in KC1 buffer, and uptake of 55 PM [3H]TEA in KC1 buffer was measured at indicated times. Results represent experiments conducted with 2 different membrane preparations. Circles indicate experiments with one preparation and squares experiments with the other.
pressants, and neostigmine (and many others) and other compounds such as thiamine (18, 25). Once circulating compounds such as organic cations are taken up across the basolateral membrane of the hepatocyte they may undergo biotransformation and be returned to the blood
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GM4
HEPATIC
TETRAETHYLAMMONIUM
TRANSPORT 50
40
s E 2 E Q s m 4
30
20
a E 10
A
-
K/K .
Na/K
0
-
I
I
60 I 1
I 2 min time
1 I2 hr
+
50
y” % a 5 t-
NMDG/K i
set
+ val
11
2 hrs
Fig. 12. Effect of potassium and NMDG gradients and val on TEA countertransport. Vesicles were preloaded with 1 mM TEA in either KC1 (K/NMDG, K/NMDG + val) or NMDG (NMDG/K, NMDG/K + val) buffer, and uptake of 55 PM [3H]TEA at the indicated times was measured with KC1 t val 2.7 PM (NMDG/K, NMDG/K + val) or NMDG t va12.7 PM (K/NMDG, K/NMDG + val) as the extravesicular buffer. Data represent results of 1 experiment. in/out
50 m
WNa
q
WNa+val
§
Na/K
1
in/out K/Na
G E r g a
NMDG/K
__01__
time
Fig. 10. Effect of potassium and sodium gradients on TEA countertransport. Vesicles were preloaded with 1 mM TEA in either KC1 (K/Na, K/K) or NaCl (Na/K) buffer and uptake of 55 PM [3H]TEA at indicated times was measured with KC1 (Na/K, K/K) or NaCl (K/Na) as the extravesicular buffer. Data represent results obtained with 3 different membrane preparations. * P c 0.05compared with Na/K, tP < 0.05 compared with K/K.
60
120
__)I_
40
m
Na/K
+ val
3
WK
a
K/K +val
T
TT
40
30
20
10
0
-
I 1
I 2 min
J J-r
2 min 2 hr
time
2 hrs
time
Fig. 11. Effect of potassium and sodium gradients and val on TEA countertransport. Vesicles were preloaded with 1 mM TEA in either KC1 (K/Na) or NaCl (Na/K) buffer, and uptake of 55 FM [3H]TEA at the indicated times was measured with KC1 (Na/K) or NaCl (K/Na) with or without 2.7 PM val (- or + val) as the extravesicular buffer. Experiments performed in the absence of val are the same as those shown in Fig. 10. * P < 0.05 compared with results obtained with the same buffers without val.
Fig. 13. Effect of potassium and sodium gradients and val on TEA uptake. Vesicles were preloaded with either potassium gluconate (K/Na, K /Na + val, K/K, K/K + val) or sodium gluconate (Na/K, Na/K + val) buffer and uptake of 55 PM [3H]TEA at indicated times was measured with potassium gluconate & va12.7 PM (Na/K, Na/K + val, K/K, K/K + val) or sodium gluconate t val 2.7 PM (K/Na, K/Na + val) as the extravesicular buffer. Data represent results of experiments with 4 different membrane preparations. *P
McKINNEY
AND MELANIE
A. HOSFORD
Nephrology Section, Department of Medicine, Indiana University School of Medicine, and Medical Service, Veterans Affairs Medical Center, Indianapolis, Indiana 46202-5116 McKinney, T. Dwight, and Melanie A. Hosford Organic organic cation has been employed extensively to evalucation transport by rat hepatocyte basolateral membranevesi- ate the renal handling of organic cations. cles.Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G939G946, 1992.-Hepatocyte basolateral membrane possesses transport systemsfor mediated uptake of organic cations, the METHODS first step in the subsequentbiliary excretion and/or metabolism BLMV were preparedfrom 250- to 300-gmaleSprague-Dawof these compounds.The purposeof these studieswas to evalley rats (Harlan Sprague Dawley, Indianapolis, IN) by the uate potential mechanismsfor transport of this classof solutes acrossthis membraneby measuring3H-labeled tetraethylam- method of Blitzer and Donovan (5). Vesicles were stored in membranesuspensionbuffer consistingof 300 mM sucroseand monium ( [3H]TEA) transport into rat hepatocyte basolateral membranevesicles.[3H]TEA uptake wasstimulated by an out- 10 mM N-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid (HEPES), pH 7.5 at -70°C for subsequenttransport experiwardly directed proton gradient consistent with TEA-proton exchange.Proton gradient-stimulated [3H]TEA uptake was in- ments. For TEA transport studiesvesicleswere rapidly thawed in a hibited by quinidine and by the combination of valinomycin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) but 37°C water bath, washedand centrifuged three times at 47,000 not by CCCP alone or by N1-methylnicotinamide (NMN). An g for 5 min and resuspendedin the appropriate transport buffer outwardly directed TEA gradient also stimulated uptake of except in the taurocholate studiesin which vesicleswere left in [3H]TEA with values at early time points exceeding those at suspensionbuffer (5). Unlessspecifically noted, transport buffer equilibrium. This trans.stimulation or countertransport was consisted of either KC1 or NaCl (150 mM) and HEPES (10 saturable with an apparent Michaelis constant of 106 PM and mM), pH 7.4. The pH of KC1 buffers wasadjustedwith KOH, maximal velocity of 434 pmol mg-l .15 s-l. TEA countertrans- NaCl buffers with NaOH, mannitol buffers with tris(hydroxyport was &-inhibited by quinidine, cimetidine, and thiamine methyl)aminomethane (Tris) base,and N-methyl-D-glucamine and by low temperature, but not by NMN. Thiamine was also (NMDG) with HCl. In experiments examining the effect of an capable of trans.stimulating [3H]TEA uptake. An outwardly outwardly directed proton gradient 2-(N-morpholino)ethanedirected potassiumgradient enhancedand an inwardly directed sulfonic acid replaced HEPES as the buffer. Ethanol wasused potassiumgradient reducedTEA countertransport but had no to dissolvevalinomycin and carbonyl cyanide m-chlorophenyleffect on [3H]TEA uptake occurring in the absenceof other hydrazone (CCCP). In experiments utilizing these ionophores, electrochemicaldriving forces. Thesestudiesindicate that there an equalamount of ethanol (final concn 0.3%) wasaddedto the are at least two potential mechanismsin the hepatocyte baso- control vesicles.For countertransport or efflux experiments,the lateral membranefor transport of organic cations; organic cat- vesicleswere preincubated with 1 mM TEA or 55 PM [3H]TEA ion-organic cation exchange (countertransport) and organic (consisting of 5 PM C3H]TEA, 13.5 Ci/mmol, Amersham,Arcation-proton exchange.Furthermore, the resultsare consistent lington Heights, IL, and 50 PM nonradioactive TEA), respecwith the existence of more than one transporter with different tively, (or other compoundsas indicated in RESULTS) for 2 h at substrate affinities in each of these categories. Finally, these room temperature (23-25°C) before transport measurements results also suggestpotential mechanismsfor drug-to-drug in- were made.TEA transport was measuredat room temperature teractions occurring at the level of transport acrossthe hepato- by a rapid filtration method using 0.4 PM polycarbonate Nuclepore filters (Costar Nuclepore, Pleasanton, CA). [3H]taurocyte basolateralmembrane. cholate (1.5 Ci/mmol, New England Nuclear, Boston, MA) uptetraethylammonium; drugs; drug-to-drug interactions; coun- take wasmeasuredat 37°C. Vesicles(lo-16 mg/ml) werediluted tertransport; organic cation-proton exchange l:20 into reaction buffer, and the reaction stoppedat the desired time by dilution of an aliquot into 3 ml of ice-cold transport SIMILAR TO THE KIDNEY, the liver is known to possess buffer contained in a ten-place filtering manifold (Hoefer Scitransport systems for organic cations (26, 29). These entific, San Francisco, CA). This mixture was immediately filsystems are important in the disposition of both endogtered by vacuum at 440-550 Torr. The filter wassubsequently enous and exogenous organic cations. However, less is washedan additional three timeswith 3 ml of ice-coldtransport known about the hepatic handling of this class of combuffer and then placedin a scintillation vial to which was added 5 ml of Redy Solv HP (Beckman Instruments, Fullerton, CA). pounds compared with the kidney. Experiments with Radioactivity was determined by standard liquid scintillation membrane vesicles have been quite useful in defining many features of renal organic cation transport (15). spectroscopy. In each experiment, the radioactivity associated with blank filters or vesiclesexposedto radioactivity and However, only recently have they been utilized to study immediately filtered was subtracted from the experimental hepatic organic cation transport (21). Thus the present values. experiments were designed to confirm that hepatocyte Protein was determined by the Lowry method (13) using basolateral membrane vesicles (BLMV) could be em- bovine serumalbumin as a standard. Activities of the following ployed to elucidate potential mechanisms involved in enzymes were determined by establishedmethods on freshly mediated transport of organic cations by the rat liver. preparedmembranes:Na-K-adenosinetriphosphatase(ATPase; We chose to use radiolabeled tetraethylammonium 6), alkaline phosphatase(23), NADPH cytochrome c reductase (TEA) as the organic cation probe, in part, because this and succinate cytochrome c reductase (30), and acid phosl
G939 Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 13, 2019.
G940
HEPATIC
TETRAETHYLAMMONIUM
TRANSPORT 40
phatase (33). Chemicalswere obtained from Sigma Chemical (St. Louis, MO). In eachexperiment, all conditions were generally evaluated in quadruplicate. The mean of the results was calculated for that experiment. The resultsare presentedas meanst SE of experiments conducted under similar conditions. Statistical analysis wasdeterminedusing Student’s t test for paired observationsor analysis of variance followed by testing of meansby Fisher’s test. A P value < 0.05 was consideredto be statistically significant.
1
.c=*
RESULTS
To confirm that the methods used to prepare vesicles resulted in an enrichment in basolateral membranes, the activities of several enzyme markers were measured in five to seven different membrane preparations. These included Na-K-ATPase (basolateral membranes), alkaline phosphatase (canalicular membranes), acid phosphatase (lysosomes), NADPH cytochrome c reductase (endoplasmic reticulum), and succinate cytochrome c reductase (mitochondria). The following enrichment ratios (activity in final membrane preparation relative to that in the initial crude homogenate) were obtained: Na-K-ATPase, 32.8 t 6.2; alk a 1ine phosphatase, 1.6 & 0.2; acid phosphatase, 1.0 * 0.1; NADPH cytochrome c reductase, 0.8 t 0.1; succinate cytochrome c reductase, 0.08 t 0.01. Even though these results indicate the presence of other cellular constituents, they nevertheless indicate a marked enrichment in basolateral membranes in the final membrane preparation and are quite comparable to those reported by others (5, 11). Additionally, to determine if functional BLMV were present in our preparation, we measured [3H]taurocholate uptake in the presence and absence of an inwardly directed sodium gradient. Taurocholate uptake was markedly enhanced in the presence of a sodium gradient (data not shown) consistent with previous results (5, 11). Figure 1 depicts a representative experiment in which the membrane-associated radioactivity of 55 PM [3H]TEA was measured at early time points and at 2 h in the presence or absence of 1 mM nonradioactive TEA in the extravesicular medium (&-side). Although “coldn TEA reduced the membrane-associated radioactivity at early time points in this and other experiments, a substantial portion of activity remained suggesting that under these conditions (i.e., absence of imposed electrochemical gradients) only a portion of the [3H]TEA associated with the membranes may represent mediated transport1 In addition, membrane-associated radioactivity under these conditions was not reduced by 1 mM quinidine, cimetidine, or NMN (data not shown). Thus a series of experiments was undertaken to determine if uptake of TEA could be increased by imposition of electrochemical gradients. One mechanism by which organic cations could be taken up by hepatocytes is in exchange for protons, analogous to that demonstrated in brush-border membranes of the kidney of several species by numerous investigators (9,10,16,17,31,36). Indeed, it has been reported recently that the organic cation NMN can be transported across l In these particular experiments, uptake cannot be differentiated with certainty from binding of [3H]TEA to the membranes.
control
-
L I
0’
I
I
60
1 mMTEA
1 /r
120 set
2 hr
time
Fig. 1. Association of 3H-labeled tetraethylammonium ( [3H]TEA) with hepatic basolateral membrane vesicles (BLMV) in the absence of electrochemical gradients. Membrane-associated radioactivity was measured at indicated times with symmetric NaCl buffer with or without 1 mM TEA in extravesicular buffer. Representative experiment is depicted.
hepatic BLMV by this mechanism (21). In the latter report, an outwardly directed proton gradient markedly stimulated NMN transport with uptake at early time points exceeding those at equilibrium (“overshoot”). To test for this possibility, [3H]TEA uptake by BLMV was measured in the presence and absence of a transvesicular proton gradient. Figure 2 shows a representative experi-
-
0’
8.0-8.0
1
I
I
I
I
1
2
3
5
10 min
“-
2 hr
time
Fig. 2. Effect of proton gradient on incubated in KC1 buffers at pH 6.0 indicated times in KC1 buffers with is depicted. pHi, intravesicular pH;
[3H]TEA uptake. BLMV were preor 8.0, and uptake was measured at pH 8.0. Representative experiment pH,, extravesicular pH.
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HEPATIC
TETRAETHYLAMMONIUM
G941
TRANSPORT
40
10
Val
I 3
I
1 I-
5 min
2 hr
I
I
3
5
time
and NMN
on pH-stimulated
cantly inhibited proton gradient-stimulated uptake of 55 PM TEA at early time points but not at 2 h. In contrast to these results, under similar conditions NMN did not affect TEA uptake at early time points. Uptake at 2 h was slightly, but significantly, lower in the presence of NMN. Another mechanism by which organic cations could be transported across hepatic BLMV is by organic cationorganic cation exchange or countertransport. Moseley et al. (21) recently demonstrated that [3H JNMN uptake by hepatic BLMV could be stimulated by preloading vesicles with a high (tram) concentration of nonradioactive NMN. In the experiments depicted in Fig. 5, the effect of trans 1 mM TEA on the uptake of 55 PM [3H]TEA was examined. In addition, the effect of temperature was evaluated by conducting experiments at room temperature and at 4OC. Preloading BLMV with 1 mM TEA markedly stimulated uptake of [3H]TEA at early time points at 25OC with an overshoot in uptake at 30-120 s compared with that at 2 h. Trans.stimulation by TEA was abolished at 4’C with uptakes being similar to those observed under control conditions (absence of trans TEA). Figure 5 also demonstrates that uptake under control conditions was reduced at the lower temperature. In addition to TEA uptake TEA Uptake,
Time, min 3 5 10 120
34.34zk5.56 29.01*4.10 35.59k4.52 34.99dE2.79
I
180
minutes
ment and demonstrates that an outwardly directed proton gradient stimulates uptake at early time points compared with no proton gradient. Values at 2 h were similar in the presence or absence of the initial proton gradient. An outwardly directed proton gradient consistently stimulated TEA uptake compared with that observed in the absence of a proton gradient. Frequently, in the presence of a proton gradient there was an overshoot in uptake above equilibrium values as demonstrated in Fig. 2. Similar to a previous report of proton gradient-stimulated NMN uptake in these vesicles (21), proton gradient-stimulated TEA uptake was not enhanced by the proton ionophore CCCP consistent with the effect of the proton gradient not being due to a proton diffusion potential (Fig. 3). However, TEA uptake in the presence of an outwardly directed proton gradient was reduced by the combination of valinomycin and CCCP (Fig. 4). These results, which are similar to those reported for proton gradient-stimulated NMN uptake by these membranes (21), are consistent with the presence of proton-TEA exchange. To determine whether other organic cations could inhibit proton gradient-stimulated TEA uptake, we examined the effect of quinidine and NMN. As shown in Table 1, quinidine in a concentration of 1 mM signifi-
Control
/
Fig. 4. Effect of CCCP and valinomycin (val) on proton gradient-stimulated [3H]TEA uptake. BLMV were preincubated for 15 min in KC1 buffer at pH 6.0 with or without 2.7 PM val and 12.7 PM CCCP, and uptake of 52.5 PM 13H]TEA was measured at indicated times in KC1 buffer with pH 8.0. Representative experiment is depicted. Similar results were also obtained in a 2nd experiment.
Fig. 3. Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on proton gradient-stimulated [3H]TEA uptake. BLMV were preincubated for 15 min in KC1 buffer at pH 6.0 with or without 12.7 PM CCCP, and uptake of 55 PM [3H]TEA was measured at indicated times in KC1 buffer with pH 8.0. Data represent results of 3 separate experiments.
Table 1. Effect of quinidine
/
Quinidine
P
16.74k3.54 20.03k4.99 24.55k3.52 30.52t2.37
co.025 co.025 0.01 >0.45
pmol/mg Control 28.62k1.37 29.52k4.53 30.12t2.89 33.1ojc3.29
NMN 29.24zk2.57 27.37k3.63 27.06k2.74 27.70k3.62
P >0.85 HI.55 XI.55 co.05
Values are means zt SE; (n = 3 experiments for both quinidine and NMN). Basolateral membrane vesicles were preincubated in KC1 buffer at pH 6.0, and uptake of tetraethylammonium (TEA) was measured at the indicated times in KC1 buffer at pH 8.0 with or without 1 mM quinidine or NMN.
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HEPATIC
TETRAETHYLAMMONIUM
TRANSPORT
A 80 70
zi E
1
-
25” preload
-
4O preload I
-
0
m
60
25” control 4” control
I
I
60
120
/
set
/I
2 hr
time
Fig. 5. Countertransport experiments. [3H]TEA uptake was measured with symmetric KC1 buffer in control vesicles and ones preloaded with I mM TEA at the indicated times and temperature. Results from experiments with 3 separate membrane preparations are depicted. * P < 0.05 vs. other groups.
these results, when BLMV were preloaded with 55 PM [3H]TEA addition of 1 mM TEA to the extravesicular medium accelerated efflux of [3H]TEA from the vesicles (data not shown). After it was observed that [3H]TEA uptake was stimulated by a trans.concentration, other experiments were conducted to further characterize this process. The data shown in Fig. 6A indicate that TEA countertransport measured at 15 s is saturable.2 Analysis of these data by a Hanes-Woolf plot (Fig. 6B) yielded an apparent Michaelis constant (K,) of 106 PM and apparent maximal velocity ( Vmax)of 434 pmol/mg.3 Next, we determined if other organic cations in the uptake medium (c&side) inhibited TEA countertransport. As shown in Fig. 7, cimetidine and quinidine, commonly prescribed organic cation type drugs, in a cisconcentration of 1 mM markedly inhibited TEA countertransport at 60 and 120 s. Because uptake at 2 h was not affected, the inhibition observed at earlier time points did not result from loss of vesicle integrity. In contrast to these results, the endogenous organic cation NMN did not affect TEA countertransport. We also examined the effect of 1 mM cis- and transconcentrations of thiamine, a compound known to interact with organic cation transport pathways in hepatocytes (14,37), on TEA transport. As demonstrated in Fig. 8, G-thiamine markedly inhibited TEA countertrans2 Preliminary experiments demonstrated that [3H]TEA (55 and 850 PM) uptake under these conditions was linear for up to 20 s. 3 We did not determine the sidedness of the vesicles used in these studies. However, in the original description of the method employed to prepare these vesicles a minority were reported to be inside-out (5). It is possible that asymmetry in affinity for TEA exists between internal and external binding sites. If this is the case, the apparent kinetic values we report might be different from those that would be obtained in a totally right-side-out vesicle preparation.
:
tu ‘;3 z
10
8
.-0
I
Km = 106 @l Vmax = 434 pmolimg
1
0
I
200
’
I
I
I
600
400 TEA
I
1
800
1
1000
(PM)
Fig. 6. Kinetics of TEA countertransport. A: vesicles were preloaded with 1 mM TEA in KC1 buffer followed by measurement of 15-s uptake in the same buffer of [3H]TEA with concentrations indicated on abscissa. Results are from experiments with 4 different membrane preparations. B: Hanes-Woolf plot of data in A. Line represents linear regression of data using least-squares analysis.
port (TEA tram/thiamine cis). To determine whether the inhibition of TEA countertransport by thiamine was presumably due to competition with [3H]TEA in the extravesicular medium for transport, the ability of intravesicular thiamine to stimulate uptake of [3H]TEA was examined. As shown, preloading vesicles with 1 mM thiamine (thiamine trans) markedly stimulated uptake of 55 PM t3H]TEA at 60 and 120 s but not at 2 h compared with uptake of [3H]TEA measured without preloading with TEA or thiamine (control). As indicated by the lack of effect of NMN in the experiments shown in Fig. 7, not all organic cations we tested &-inhibited TEA countertransport. To further examine whether NMN might affect TEA transport in hepatic BLMV, we determined whether preloading vesicles with NMN might stimulate uptake of TEA. However, in two experiments depicted in Fig. 9, a tram 1 mM concentration of NMN did not enhance uptake of 55 PM [3H]TEA compared with a similar trans-concentration of TEA. Finally, to determine if TEA countertransport might
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HEPATIC
TETRAETHYLAMMONIUM
TEA trans
Y
Thiamine
1 l
I
60
-
l
I
120 set
I
I
G943
TRANSPORT
60
control
w
trans __t_)
Thiamine
cis
auinidine
I
2 hr
time
Fig. 7. Effect of other organic cations on TEA counter-transport. Vesicles were preloaded with 1 mM TEA in KC1 buffer, and uptake of 55 PM [3H]TEA was measured at indicated times in same buffer with or without (control) 1 mM concentrations of the other organic cations in the extravesicular media. NMN, R”-methylnicotinamide. Data represent results obtained in 6 different membrane preparations. * P < 0.01 vs. control.
be conductive and/or affected by inorganic cation gradients, experiments were carried out with transvesicular potassium gradients and the potassium ionophore valinomycin. As shown in Fig. 10, with an outwardly directed potassium gradient and inwardly directed sodium gradient, TEA countertransport was enhanced compared with that occurring with symmetric potassium buffers. Furthermore, the reverse condition, i.e., inwardly directed potassium and outwardly directed sodium gradients was associated with the lowest countertransport. In addition, in these same experiments in the presence of an outwardly directed potassium gradient valinomycin enhanced and in the presence of an inwardly directed potassium gradient decreased TEA countertransport (Fig. 11). Results similar to those noted above were obtained when an NMDG gradient was used rather than sodium suggesting that the observations were due to potassium rather than to sodium (Fig. 12). In contrast to these countertransport experiments, sodium and potassium gradients had little effect on uptake of TEA in the absence of a trans.concentration of TEA (Fig. 13). These experiments were performed with gluconate salts of sodium and potassium. However, similar results were obtained with NaCl and KC1 (data not shown). DISCUSSION
The liver and kidneys are responsible for the disposition of a large number of endogenous and exogenous primary, secondary, and tertiary amines and quaternary ammonium compounds. Because the nitrogen of these substances carries a positive charge at physiological pH, they are collectively referred to as organic cations. Important organic cations include common drugs such as procainamide, cimetidine, dipyridamole, tricyclic antide-
1
60
I
120
I
I
I
2 hr
see
time
Fig. 8. Effect of thiamine on TEA transport. Vesicles were preloaded in KC1 buffer without (control and thiamine cis) or with 1 mM TEA or thiamine (TEA trams, TEA tram-thiamine cis, and thiamine tram) and uptake of 55 PM [3H]TEA in KC1 buffer was measured at the indicated times without (control, TEA tram, and thiamine tram) or with (thiamine cis and TEA tram-thiamine cis) 1 mM thiamine in the extravesicular medium. Results from a representative experiment are shown. 40
1:‘:1
,t 60
120 set
2 hr
time
Fig. 9. Effect of tram NMN on TEA uptake. Vesicles were preloaded with 1 mM TEA or NMN in KC1 buffer, and uptake of 55 PM [3H]TEA in KC1 buffer was measured at indicated times. Results represent experiments conducted with 2 different membrane preparations. Circles indicate experiments with one preparation and squares experiments with the other.
pressants, and neostigmine (and many others) and other compounds such as thiamine (18, 25). Once circulating compounds such as organic cations are taken up across the basolateral membrane of the hepatocyte they may undergo biotransformation and be returned to the blood
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GM4
HEPATIC
TETRAETHYLAMMONIUM
TRANSPORT 50
40
s E 2 E Q s m 4
30
20
a E 10
A
-
K/K .
Na/K
0
-
I
I
60 I 1
I 2 min time
1 I2 hr
+
50
y” % a 5 t-
NMDG/K i
set
+ val
11
2 hrs
Fig. 12. Effect of potassium and NMDG gradients and val on TEA countertransport. Vesicles were preloaded with 1 mM TEA in either KC1 (K/NMDG, K/NMDG + val) or NMDG (NMDG/K, NMDG/K + val) buffer, and uptake of 55 PM [3H]TEA at the indicated times was measured with KC1 t val 2.7 PM (NMDG/K, NMDG/K + val) or NMDG t va12.7 PM (K/NMDG, K/NMDG + val) as the extravesicular buffer. Data represent results of 1 experiment. in/out
50 m
WNa
q
WNa+val
§
Na/K
1
in/out K/Na
G E r g a
NMDG/K
__01__
time
Fig. 10. Effect of potassium and sodium gradients on TEA countertransport. Vesicles were preloaded with 1 mM TEA in either KC1 (K/Na, K/K) or NaCl (Na/K) buffer and uptake of 55 PM [3H]TEA at indicated times was measured with KC1 (Na/K, K/K) or NaCl (K/Na) as the extravesicular buffer. Data represent results obtained with 3 different membrane preparations. * P c 0.05compared with Na/K, tP < 0.05 compared with K/K.
60
120
__)I_
40
m
Na/K
+ val
3
WK
a
K/K +val
T
TT
40
30
20
10
0
-
I 1
I 2 min
J J-r
2 min 2 hr
time
2 hrs
time
Fig. 11. Effect of potassium and sodium gradients and val on TEA countertransport. Vesicles were preloaded with 1 mM TEA in either KC1 (K/Na) or NaCl (Na/K) buffer, and uptake of 55 FM [3H]TEA at the indicated times was measured with KC1 (Na/K) or NaCl (K/Na) with or without 2.7 PM val (- or + val) as the extravesicular buffer. Experiments performed in the absence of val are the same as those shown in Fig. 10. * P < 0.05 compared with results obtained with the same buffers without val.
Fig. 13. Effect of potassium and sodium gradients and val on TEA uptake. Vesicles were preloaded with either potassium gluconate (K/Na, K /Na + val, K/K, K/K + val) or sodium gluconate (Na/K, Na/K + val) buffer and uptake of 55 PM [3H]TEA at indicated times was measured with potassium gluconate & va12.7 PM (Na/K, Na/K + val, K/K, K/K + val) or sodium gluconate t val 2.7 PM (K/Na, K/Na + val) as the extravesicular buffer. Data represent results of experiments with 4 different membrane preparations. *P
