 |
 Previous Article | Next Article 
The Journal of Neuroscience, December 15, 1998, 18(24):10257-10268
Cocaine Acts as an Apparent Competitive Inhibitor at the
Outward-Facing Conformation of the Human Norepinephrine Transporter:
Kinetic Analysis of Inward and Outward Transport
Nianhang
Chen1, 2 and
Joseph B.
Justice Jr2
1 Department of Pharmacology, Nanjing Medical
University, Nanjing 210029, People's Republic of China, and
2 Department of Chemistry, Emory University, Atlanta,
Georgia 30322
 |
ABSTRACT |
The inhibition by cocaine of inward and outward transport of
dopamine (DA) at the cloned human norepinephrine transporter (hNET) and
the relationship of the inhibitory patterns of cocaine to the
conformational requirements of the transporter were investigated. This
was done using rotating disk electrode voltammetry in transfected cells. The uphill uptake of external DA, the lack of inhibition by
internal substrates on DA uptake, and the accelerated exchange of
internal DA by external m-tyramine support a carrier
model in which the hNET alternates between outward-facing and
inward-facing conformations. Cocaine exhibited competitive inhibition
of DA uptake, which was insensitive to intracellular substrates. In contrast, the inhibition by cocaine of the
m-tyramine-induced DA efflux appeared noncompetitive
relative to intracellular DA, but competitive relative to extracellular
m-tyramine. Simultaneous measurement of
m-tyramine uptake and accompanying DA efflux at various
concentrations of intracellular DA showed that cocaine did not alter
the ratio of DA efflux to m-tyramine uptake. Moreover, cocaine displayed similar potency for inhibiting DA uptake and efflux.
Additionally, the inhibition profile of cocaine was unrelated to the
addition time of cocaine, simultaneously with or earlier than a
substrate. All of the findings are consonant with a competitive interaction between cocaine and substrates at the outward-facing conformation of the hNET. This action directly prevents the inward transport of external substrates, thereby inhibiting the outward transport of internal substrates by reducing the availability of the
inward-facing conformation. Consequently, the experimental inhibition
pattern of cocaine depends on the conformation of the hNET to which the
transported substrate is exposed.
Key words:
cocaine; dopamine; m-tyramine; norepinephrine
transporter; rotating disk voltammetry; uptake; efflux; kinetic
analysis
| |
INTRODUCTION |
The norepinephrine transporter (NET)
mediates uptake of synaptic norepinephrine (NE) into NE neurons. It
also transports structurally similar substrates including dopamine
(DA), tyramine, and amphetamine (Bönisch and Brüss, 1994 ;
Gu et al., 1994; Burnette et al., 1996; Justice et al., 1998). In the
conventional model for monoamine transporters, it is assumed that the
transporter alternates between its outward and inward facing
conformations (Rudnick, 1997). The outward-facing conformation exposes
the substrate site to the outside face of the membrane, and the
inward-facing conformation exposes the substrate site to the inside
face of the membrane. Like other members of monoamine transporter
family, the NET uses the energy of the transmembrane
Na+ gradient to drive the accumulation of a
substrate inside the cell (Bönisch and Brüss, 1994; Gu et
al., 1996). However, a reversal of transport direction can be induced
by inward transport of any substrate (Langeloh et al., 1987;
Levi and Raiteri, 1993; Burnette et al., 1996; Chen et al., 1998).
The psychostimulant cocaine interacts with NET as an effective
inhibitor both in vivo (Chen and Reith, 1994, 1995) and
in vitro (Graefe and Bönisch, 1988; Pacholczyk et al.,
1991; Gu et al., 1994; Masahiko et al., 1997). Although numerous
functional studies have been performed to address the molecular
mechanism of cocaine, there has been lack of agreement in the
literature concerning the interactions between cocaine and substrates
at catecholamine transporters. Earlier studies on isolated peripheral tissues reported that cocaine is a competitive inhibitor of the inward
transport of [3H]NE (Graefe and Bönisch,
1988) but is equally potent an inhibitor of
zero-[Na+]-induced [3H]NE
efflux when the tissue is preloaded with either low or high concentration of [3H]NE (Graefe and Fuchs, 1979),
resembling a noncompetitive pattern. Analogously, the discrepancy has
also been observed in studies on the inhibition by cocaine of another
catecholamine transporter, the dopamine transporter (DAT). In those
studies with brain tissue or synaptosomes, all patterns of inhibition
have been reported: competitive, noncompetitive, and uncompetitive
(Krueger, 1990; McElvain and Schenk, 1992; Wheeler et al., 1994;
Povlock and Schenk, 1997).
Catecholamine transporters are integral membrane proteins spanning the
membrane and exposed on both sides of membrane. They are not symmetric
in structure. Thus, cocaine, no matter how it approaches the
transporter, from one side of the membrane or from both sides, raises
the same question: on which transporter conformations does it act?
Depending on the specificity of cocaine for inward-facing and
outward-facing conformations, the inhibition pattern in different transport assays may vary from competitive to noncompetitive or mixed,
as elucidated for simple facilitated transport (Krupka and Devés,
1983; Stein, 1986; Devés and Krupka, 1989). Accordingly, the
inhibition pattern observed in a particular type of experiment may not
reflect the actual mechanism. Identification of the transporter conformational requirement of the action of cocaine is necessary to distinguish the mechanism of cocaine from its multiple inhibitory patterns.
In the present study, we performed kinetic analyses on inward and
outward DA transport via the human NET (hNET) to probe the mechanism of
cocaine. Two interrelated questions are addressed. First, what is the
real nature of the interaction between cocaine and substrate at the
hNET: competitive, noncompetitive, or uncompetitive? Second, which
conformation of the hNET does cocaine attack: outward-facing, inward-facing, or both? The results strongly suggest that cocaine interacts competitively with the outward-facing conformation of the hNET.
| |
MATERIALS AND METHODS |
Materials. Dopamine hydrochloride, cocaine hydrochloride (COC),
ouabain, [3H]H2O (1.0 mCi/gm), and
[14C]inulin (1.4 mCi/gm) were from Sigma (St,
Louis, MO). m-Tyramine (m-TYR) was from Research
Biochemicals (Natick, MA). The LLC-PK1 cell stably
expressing the human NE transporter (LLC-hNET) was a gift from Dr. Gary
Rudnick (Department of Pharmacology, Yale University). Because DA is
more stable at physiological pH than NE, we chose to use DA as a
substrate of the hNET throughout the transport assays.
Preparation of cell suspension. The culture of LLC-hNET
cells and the preparation of the cell suspensions were performed as described previously (Chen et al., 1998). The assay buffer contained (in mM) 120 NaCl, 4.7 KCl, 2.2 CaCl2,
1.2 MgSO4, 1.2 KH2PO4, 10 HEPES, and 10 glucose, pH
7.4.
Determination of DA content inside cells. Cells were
incubated with varying concentrations of DA under the same conditions as for rotating disk electrode (RDE) voltammetry experiments for 5-20
min. At each tested time, an aliquot (60 µl) of the cell suspension
was quickly transferred to a vial containing 1 ml ice-cold assay buffer
and centrifuged at 2000 × g for 1 min. The supernatant was removed and acidified by 1:10 volume of 0.1 M
perchloric acid, and the cells were lysed in 200 µl 0.1 M
perchloric acid. Samples of the supernatants and cell extracts were
analyzed for DA content by microbore HPLC with electrochemical
detection as described previously (Chen et al., 1998).
Assay of intracellular water space. Cells (~2 mg cell
protein per assay) were suspended in the assay buffer and incubated for
10 min at 37°C with [3H]H2O (1.5 µCi/ml) and [14C]inulin (0.3 µCi/ml) in a
final volume of 1 ml. The cell pellets were collected by centrifugation
at 1000 × g for 2 min and then lysed by 400 µl
Triton X-100 (0.1% in 5 mM Tris-HCl, pH 7.4). The
radioactivity of samples was measured by liquid scintillation counting.
Intracellular water space was calculated as the difference between
total [3H]H2O space and extracellular
[14C]inulin space and expressed as microliter of
cell water per milligram of protein.
Measurement of rotating disk electrode voltammetry. For
one-electrode voltammetry, a Teflon-shielded glassy carbon working electrode (electrode 1, 3 mm diameter; Pine Instrument Company, Grove
City, PA) was mounted at the top of an electrochemical cell. An LC-4
Amperometric Detector (Bioanalytical Systems, Lafayette, IN) was used
as a potentiostat, and the output current was amplified (model 427;
Keithley Instruments, Cleveland, OH). The potential of the working
electrode was set at +400 mV relative to Ag/AgCl reference electrode.
For two-electrode voltammetry, a second Teflon-shielded glassy carbon
working electrode (electrode 2, 3 mm diameter) was fitted at the bottom
of the electrochemical cell. A dual channel microelectrode potentiostat
(model EI-400; Ensman Instrumentation, Bloomington, IN) was used
to control the potentials at the two separate working electrodes: +400
mV for electrode 1, and +650 mV for electrode 2. The resulting
oxidative current corresponded to DA signal at +400 mV and corresponded
to both DA and m-tyramine signals at +650 mV.
m-Tyramine was not oxidized at +400 mM. After the cell suspension was placed into the electrochemical cell, the
electrode 1 was introduced just below the surface of the solution and
rotated with an AFMSRX Analytical Rotator System (Pine
Instrument Company). All experiments were performed at an electrode
rotation rate of 4000 rpm at 37°C. Origin software (version 4.0;
MicroCal Software, Northampton, MA) was used for data acquisition as
described previously (Burnette et al., 1996; Chen et al., 1998)
Transport assays. In transport assays, cocaine was added
either simultaneously with, or 6 min earlier than a substrate, and the
initial transport rate over the first 10-15 sec was measured. The
simultaneous addition partially restricted cocaine outside the cell,
whereas the previous addition allowed cocaine to equilibrate across the
membrane during the assay. Comparison of results from both approaches
allowed evaluation of the relative contribution of external and
internal cocaine to the observed inhibition profile.
For uptake assays, cells were preincubated for 3-5 min and then 1 min
baseline was collected. Subsequently, DA was added to the cell
suspension, and the decrease in the DA signal was recorded. In some
experiments, cells were preincubated with or without 3 µM
m-tyramine in parallel at 37°C for 8 min. The incubation
was terminated by centrifugation at 1000 × g for 2 min. The resulting cell pellet was resuspended in assay buffer and then
used for uptake assays.
For efflux assays, cells were incubated with DA until the DA signal
decreased to a steady state. Subsequently, m-tyramine was
added and the increase in DA signal corresponding to this addition was
recorded until the signal ceased to increase. In the presence of
cocaine, m-tyramine-induced DA efflux was defined as the
difference between the total DA efflux in the presence of
m-tyramine plus cocaine and the nonspecific DA efflux in the presence of cocaine alone. The nonspecific DA efflux in the presence of
varying concentrations of cocaine at several fixed concentrations of
preloaded DA was separately determined in close generations of cells.
The Michaelis-Menten equation expressing the relationship between the
nonspecific DA efflux and [cocaine] at a fixed initial medium [DA]
([DA]o) was obtained by nonlinear fitting the data as a function of [cocaine] (3-300 µM). The linear
regression equation expressing the relationship between the nonspecific
DA efflux and initial [DA]o at a fixed [cocaine] was
obtained by fitting the data as a function of initial
[DA]o (0.5-4 µM). The nonspecific DA
efflux rate at a particular combination of [cocaine] and initial [DA]o was estimated according to the related equations.
Parallel measurements of the nonspecific DA efflux were included
periodically in the DA efflux assays, which gave values similar to
those estimated from the equations.
For simultaneous monitoring of m-tyramine uptake and DA
efflux, cells were incubated with DA until the DA signal decreased to a
steady state. Subsequently, m-tyramine (3 µM)
was added. The increase in the DA signal at electrode 1 and the signal
change at electrode 2 (a combination of increased DA signal and reduced m-tyramine signal) were recorded simultaneously until the DA
efflux signal reached its maximum. DA signal at electrode 1 was
subtracted from the mixed signal at electrode 2 to give the
m-tyramine signal. Because m-tyramine itself
caused a small signal decay by filming at the surface of the electrode,
the signal decay specifically for hNET-mediated m-tyramine
uptake was defined as the difference between the signal decay in the
absence and presence of 100 µM cocaine.
Data analysis and statistics. Initial transport rates were
obtained from linear regression analysis of the [DA]o or
medium [m-tyramine]
([m-tyramine]o) versus time over the
first 10-15 sec after an addition as described previously (Burnette et
al., 1996; Chen et al., 1998). Eadie-Hofstee transformation of the Michaelis-Menten equation was used to assess the apparent inhibition patterns of cocaine on the velocity of DA transport. The maximal rate
of transport (Vmax) and the
half-saturation concentration of a substrate
(Km) were estimated by nonlinear curve
fitting of the Michaelis-Menten equation to the rate data. If cocaine appeared as a competitive inhibitor of substrate transport, the half-saturation inhibition constant (Ki)
was estimated by fitting with the equation Km
obs = Km (1 + [cocaine]/Ki). Here Km
obs and Km are the concentrations of a
substrate to produce 50% of its maximal transport rate in the presence
and absence of cocaine, which were determined in parallel. In
experiments to address the effect of various concentrations of cocaine
on the transport of a fixed concentration of a substrate, the
competitive Ki of cocaine for inhibiting DA
uptake and m-tyramine-induced DA efflux were analyzed
according to the equation: v/v' = 1 + [cocaine]/{Ki(1+[S]/Ks)}, in which, v and v' are the initial transport rate
in the absence and presence of cocaine, respectively; [S]
is the concentration of the substrate (DA or m-tyramine);
Ks is the half-saturation concentration of the
substrate to be taken up or to induce DA efflux, and set at the average
values determined from separate saturation curves. IC50
values of cocaine for inhibiting m-tyramine uptake and
m-tyramine-induced DA efflux at each preloaded [DA] were
computed by nonlinear curve fitting the efflux rate data as a function
of [cocaine] with the logistic equation. This nonlinear regression
program was run with the rate value at [cocaine] = 0 fixed at the
tested value and the rate value at [cocaine] =  fixed at zero. In
experiments to simultaneously measure m-tyramine uptake and
DA efflux, the ratio of DA efflux to m-tyramine uptake was
fitted as a function of intracellular [DA]
([DA]i) using the equation: ratio = [DA]i/(K0.5 + [DA]i), derived from the six state transport model
(Justice and Reed, 1997). This equation can be rewritten as a special
example of the Michaelis-Menten expression: ratio = (Rmax[DA]i)/(K0.5 + [DA]i), in which Rmax is
the maximal ratio and equal to 1, and K0.5 is
the intracellular concentration of DA to produce half-maximal ratio. In
the presence of cocaine (3 µM), the predicted shift of
the curve of DA efflux/m-tyramine uptake ratio versus
[DA]i was obtained as follows: (1) estimate the DA efflux
solely caused by m-tyramine uptake by the equation efflux
rate+COC, estimated = uptake rate+COC,
experimental × efflux rate COC,
experimental/uptake rateCOC, experimental (2) fit
the efflux rate+COC, estimated as a function of
[DA]i to obtain its Michaelis-Menten expression; (3)
predict the efflux rate in the presence of putative interaction of
cocaine with internal DA (efflux rate+COC, predicted) from
the Michaelis-Menten expression of efflux rate+COC,
estimated modified for competitive, noncompetitive, and
uncompetitive inhibitions (Stein, 1986) (the Ki
of cocaine was set at 2 µM); and (4) estimate the
predicted ratio by replacing the experimental DA efflux rate with
predicted DA efflux rate from step 3, that is,
ratiopredicted = efflux rate+COC,
predicted/uptake rate+COC, experimental.
All curve fitting was done with the Origin nonlinear fitting and
plotting software. All results were expressed as mean ± SE. Statistics consisted of one-way ANOVA followed by the Newman-Keuls test and various Student's t test, as appropriate. The
accepted level of significance was 0.05.
| |
RESULTS |
Intracellular DA concentrations
The intracellular water content of hNET-LLC cells was 3.17 ± 0.16 µl/mg protein (n = 17). Intracellular DA
concentrations were estimated according to the cellular DA content and
the cell water space. The cellular DA levels were similar at 5 and 10 min after the addition of DA, and then they tended to decrease (Fig. 1A). Significant
reduction in cellular DA levels was seen at 20 min after the addition
of DA (Fig. 1A). This loss of internal DA was more
appreciable at low initial [DA]o than higher initial [DA]o (Fig. 1A). The COMT
inhibitor tropolone (1 mM) significantly increased the
cellular DA level when initial [DA]o was 1 µM, indicating metabolism of intracellular DA by
catechol-O-methyltransferase (COMT) (Eshleman et al., 1997;
Chen et al., 1998). However, it had no effect on the cellular DA level
when initial [DA]o was 6 µM (data not
shown), possibly because of the saturation of the COMT at higher
internal DA concentrations. The DA level in cells incubated without DA
was undetectable. The DA level in the wash supernatants was only
detectable in assays with high initial [DA]o (4-6
µM), and was <30 nM.
View larger version (38K):
[in this window]
[in a new window]
|
Figure 1.
Relationship of intracellular DA concentration
with time and initial medium DA concentrations. A,
Change in [DA]i with the incubation time at each initial
[DA]o. Data are presented as percentage of the
[DA]i at 5 min. Intracellular DA degradation was
significant at 20 min, especially when the initial [DA]o
was lower. B, Linear relationship between
[DA]i and initial [DA]o at 10 min. The
straight line represents the result of the least squares
linear regression. Similar linear relationships were observed at 5, 15, and 20 min with slopes being 65 ± 2, 76 ± 4, and 62 ± 4, respectively (r = 0.99; p < 0.0001 at each time point). Values are mean ± SE of four
experiments, each of which was performed with six concentrations of DA
and four time points on the same generation of cells.
*p < 0.05 versus the 100% at 5 min (one sample
t test).
|
|
Over the range of initial [DA]o tested (0.5 µM-6 µM), the [DA]o,
measured by RDE voltammetry, decreased to its steady state within 3-10
min after its addition. At the steady state, [DA]o varied
from 24 to 700 nM, whereas [DA]i varied from
44 to 517 µM. Comparisons of [DA]o and
[DA]i at the steady state indicate that the hNET builds
up a 700-1800-fold DA gradient across the membrane. Within a time
range comparable to that for the RDE voltammetry experiments (maximal
20 min), the [DA]i was linear with the initial [DA]o (Fig. 1B). To ensure maximal
uptake of extracellular DA and minimal degradation of intracellular DA
in the efflux experiments, we initiated DA efflux by adding
m-tyramine between 5 and 10 min after the addition of DA,
during which the steady state of [DA]o was established,
and the [DA]i was relatively stable. The linear regression equation, obtained from plotting [DA]i at 10 min after the addition of DA as a function of initial
[DA]o (Fig. 1B), was used to estimate
the intracellular DA concentrations in all efflux experiments.
Saturation kinetic characteristics of inward and outward transport
of dopamine
For DA dependence of the transport, the initial rates of DA uptake
and subsequent m-tyramine (10 µM)-induced DA
efflux were measured in the same cell suspensions. DA transport was
saturable and followed the hyperbolic relationship described by
Michaelis-Menten kinetics (Fig.
2A, B). The
Hill slopes were 0.88 ± 0.1 for DA uptake, and 1.08 ± 0.07 for DA efflux. Although the Vmax values were
similar between inward and outward transport of DA, the
Km for m-tyramine-induced DA efflux
was much higher than that for DA uptake (0.88 and 396 µM,
respectively, Fig. 2A, B).
View larger version (22K):
[in this window]
[in a new window]
|
Figure 2.
Hyperbolic relationships between the initial rate
of the DA transport and the substrate concentration. For DA dependence
of the transport, the initial rate of DA uptake and subsequent
m-tyramine-induced DA efflux were measured in the same
cell suspensions. The DA efflux was initiated by
m-tyramine (m-TYR) after
[DA]o decreased to the steady state. Additions of DA or
m-tyramine were made at one concentration per cell
suspension. The solid curve was fit by the Michaelis-Menten equation.
A, Dependence of the DA uptake on the initial
[DA]o. B, Dependence of the
m-tyramine-induced DA efflux on [DA]i at a
fixed initial [m-tyramine]o (10 µM). Note that the Km relative
to [DA]i is very high, although the
Vmax is close to that for DA uptake.
C, Dependence of the m-tyramine-induced
DA efflux on the initial [m-tyramine]o at
a fixed [DA]i (84 µM). Values are mean ± SE of three to six experiments, each of which was performed with
eight concentrations of DA or five concentrations of
m-tyramine on the same generation of cells.
|
|
For m-tyramine dependence of the outward DA transport, the
cells was preloaded with 84 µM DA (resulting from an
incubation of cells with 1 µM [DA]o)
and DA efflux was induced by various initial medium concentrations of
m-tyramine. The efflux rate was also saturable with initial
[m-tyramine]o and well fitted by the Michaelis-Menten expression (Fig. 2C). The Hill slope was
1.11 ± 0.06. The Km value for
m-tyramine to induce DA efflux was similar to its
Ki to inhibit DA uptake (1.95 ± 0.16 vs
1.63 ± 0.22 µM, n = 4).
Effect of cocaine on inward transport of dopamine
Cocaine added simultaneously with DA
To investigate the inhibition pattern of the external cocaine,
cocaine was added simultaneously with DA to minimize the entry of
external cocaine into cells during the early phase of DA uptake. Initial rates of DA transport into LLC-hNET cells devoid of endogenous substrates were determined at various concentrations of DA and cocaine
in the medium. The Eadie-Hofstee plot of the data showed that the
inhibition pattern of cocaine was competitive (Fig.
3A). It raised
Km with no change in Vmax
(Table 1). The observed
Km (Km obs) showed
a high degree of linearity with [cocaine] (Fig. 3A,
inset). The graphically estimated competitive
Ki of cocaine was 0.79 ± 0.04 µM, similar to the value of 0.82 ± 0.02 µM (n = 4) determined from the separate
inhibition curves of cocaine for the uptake of 1 µM
DA.
View larger version (31K):
[in this window]
[in a new window]
|
Figure 3.
Competitive inhibition by cocaine
(COC) of DA uptake. Eadie-Hofstee plots were used to
analyze the inhibition patterns. The straight solid line
represents the result of the least squares linear regression. Each
panel shows a representative experiment with five levels of initial
[DA]o and indicated levels of [cocaine] on the same
generation of cells. Each experiment was performed three times with
similar results. A, Effect of cocaine when added
simultaneously with DA. Cocaine increased the Km
obs (slope of the curve) without changing the
Vmax (y intercept of
the curve). Inset shows replot of Km
obs obtained from the Eadie-Hofstee plot as a function of the
concentration of cocaine. The straight dotted line
represents the predicted shift in Km obs by
the competitive model. B, Effect of cocaine when added 6 min earlier than DA. Earlier addition of cocaine did not change the
inhibition pattern of cocaine. C, Effect of cocaine when
added together with 200 µM ouabain 6 min earlier than DA.
Inactivation of Na+,K+-ATPase
with ouabain inhibited the DA uptake in a mixed competitive and
noncompetitive manner, with Km increasing
and Vmax decreasing, but it did not change
the inhibition pattern of cocaine. This experiment was run in parallel
with the experiment in B.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Kinetic parameters determined from saturation analyses of
the initial rate of dopamine uptake in the absence and presence of
cocaine
|
|
Cocaine added 6 min earlier than DA
To explore the inhibition pattern of equilibrated cocaine, a 6 min
preincubation of cells with cocaine was performed. Within this
preincubation period, cocaine was expected to reach its binding plateau
(Calligaro and Eldefrawi, 1987) and to distribute across the membrane
with intracellular cocaine concentration higher than extracellular
cocaine concentration (Sershen et al., 1982; Sulzer and Rayport,
1990).
Preincubation of the cells with 2 µM cocaine also
inhibited DA uptake in a competitive manner (Fig. 3B). Its
Ki was slightly lower than that obtained with
concurrent addition of cocaine and DA (Table 1) but did not reach
statistical significance. To inspect the impact of the intracellular
[Na+] on the inhibition pattern of cocaine, we
examined the effect of cocaine on DA uptake at higher intracellular
[Na+] by partially inhibiting the
Na+,K+-ATPase with ouabain. The
presence of ouabain (200 µM) in the medium strongly
reduced DA uptake in a mixed competitive and noncompetitive manner,
with Km increasing and
Vmax decreasing (Table 1). However, ouabain
failed to change the inhibition pattern of cocaine (Fig. 3C). Under this condition, cocaine still affected DA
uptake by elevating the Km without effect on
Vmax (Table 1). The Ki of cocaine in the presence of ouabain was similar to its
Ki in the absence of ouabain (Table 1).
To investigate the contribution of internal substrates to the
inhibitory effect of equilibrated cocaine on DA uptake, we tested the
initial rates of DA uptake in cells containing no m-tyramine or in cells preloaded with m-tyramine (3 µM)
in the absence and presence of 1 µM cocaine. Preloading
of cells with m-tyramine did not significantly reduce the
initial rate of DA uptake (Table 2).
Moreover, equilibrated cocaine showed identical effects on the DA
uptake into cells preloaded without and with m-tyramine (Table 2).
View this table:
[in this window]
[in a new window]
|
Table 2.
Effect of equilibrated cocaine on the initial rate of DA
uptake in the LLC-hNET cells preloaded with or without
m-tyramine
|
|
Effect of cocaine on outward transport of dopamine
DA efflux at various [DA]i and a fixed initial
[m-tyramine]o
With the initial [m-tyramine]o fixed at
10 µM, initial rates of internal DA transport out of the
cells were determined at various concentrations of intracellular DA and
initial medium cocaine. The typical time course is shown in Figure
4. Cocaine reduced the initial efflux
rate at all tested preloading concentrations of DA (Fig. 4, compare
A, B). The DA efflux in the presence of cocaine
alone (Fig. 4C) results from diffusion of internal DA that
is normally offset by uptake (Chen et al., 1998). Inhibition of hNET by
cocaine made this efflux visible. At higher preloading [DA], this
nonspecific efflux rate constituted an appreciable part of DA efflux
rate observed in the presence of both m-tyramine and cocaine
(Fig. 4, compare B, C). The regression equations
expressing the relationship between cocaine-induced apparent DA efflux
and initial [DA]o (0.5-4 µM) were: initial
rate (picomoles per second per milligram) = 0.001 + 0.178 [DA]o at 3 µM [cocaine]
(r = 0.999; n = 3), 0.022 + 0.24 [DA]o at 10 µM [cocaine]
(r = 0.997; n = 3), and  0.02 + 0.349 [DA]o at 30 µM [cocaine]
(r = 0.999; n = 3), respectively. The
initial rate of cocaine-induced apparent DA efflux at each initial
[DA]o was calculated from the equation and subtracted
from the initial rate of the DA efflux in the presence of both
m-tyramine and cocaine.
View larger version (20K):
[in this window]
[in a new window]
|
Figure 4.
Time course of m-tyramine
(m-TYR)-induced DA efflux at various intracellular
concentrations of DA. DA was added to an initial medium concentration
of 0.5-4 µM and allowed to be taken up until
[DA]o decreased to near steady state, at which time, the
intracellular DA concentrations ranged from 44 to 321 µM.
The DA efflux was subsequently initiated by 10 µM
m-tyramine in the absence and presence of 30 µM cocaine (COC). Each panel shows a
representative experiment with indicated levels of initial
[DA]o on the same generation of cells. Each experiment
was performed three times with similar results. The
arrow denotes the addition time of DA,
m-tyramine, cocaine, or m-tyramine plus
cocaine. The values at the end of each curve denote the initial medium
DA concentration. A, DA efflux in the presence of
m-tyramine alone. B, DA efflux in the
presence of both m-tyramine and cocaine. Cocaine reduced
the initial rate of the DA efflux, although cocaine itself caused an
apparent DA efflux (C). This experiment was run
in parallel with the experiment in A. C,
DA efflux in the presence of cocaine alone. The initial rate of the DA
efflux in the presence of cocaine was slower but proportional to the
initial [DA]o in a linear manner. The initial rate of DA
efflux in the presence of 30 µM cocaine alone at 3 µM initial [DA]o was not measured in this
particular experiment, but it can be estimated from the linear
regression equation obtained from this experiment (see Results). In
other experiments, measurement of the apparent DA efflux induced by
cocaine at 3 µM initial [DA]o gave values
similar to those estimated from the regression equation. For estimation
of the initial rate of m-tyramine-induced DA efflux in
the presence of cocaine, the subtraction was made at each initial
[DA]o as follows: RateTYR,COC = RateTYR+COC RateCOC.
|
|
The design of the experiment allowed assessment of the inhibition
pattern of cocaine in two ways. First, the IC50 values of cocaine to inhibit m-tyramine-induced DA efflux were
estimated at several fixed [DA]i (Table
3). An increment in [DA]i
did not significantly change the IC50 value of cocaine
(Table 3). It can be seen that the steady state [DA]o
before initiation of the DA efflux was <400 nM (Table 3),
which was too low to significantly affect the effect of
m-tyramine (10 µM) or cocaine (3-30
µM). Second, the kinetic parameters for the DA efflux
were estimated at several fixed [cocaine]. An increase in [cocaine]
reduced Vmax without significantly changing
Km (Table 4). Both
results indicated noncompetitive inhibition of DA efflux by cocaine. In
addition, a similar conclusion can be obtained from the Eadie-Hofstee
transformation of the efflux data (Fig.
5A).
View this table:
[in this window]
[in a new window]
|
Table 4.
Effect of cocaine on kinetic parameters determined from
saturation analysis of the initial rate of
m-tyramine-induced dopamine efflux
|
|
View larger version (34K):
[in this window]
[in a new window]
|
Figure 5.
Inhibitory patterns of cocaine
(COC) on m-tyramine-induced DA efflux.
Eadie-Hofstee plots were used to analyze the inhibition patterns. The
straight solid line represents the result of the least
squares linear regression. Each panel shows a representative experiment
with five levels of [substrate] on the same generation of cells. Each
experiment was performed three times with similar results.
A, Noncompetitive inhibition pattern of cocaine relative
to [DA]i (44-321 µM). The protocol applied
is depicted in Figure 4. Inset shows replot of
Km obs obtained from the Eadie-Hofstee plot
as a function of cocaine concentration. The straight dotted
line represents the average value of Km
obs obtained from this experiment. B, Competitive
inhibition pattern of cocaine relative to initial
[m-tyramine]o (1-100 µM).
The protocol applied is depicted in Figure 6. Inset
shows replot of Km obs obtained from the
Eadie-Hofstee plot as a function of the concentration of cocaine. The
straight dotted line represents the predicted shift in
Km obs by the competitive model.
|
|
DA efflux at various initial
[m-tyramine]o and a
fixed [DA]i
With [DA]i fixed at 84 µM, the initial
efflux rates were determined at various initial medium concentrations
of m-tyramine and cocaine. The typical time course was shown
in Figure 6. High concentrations (30 and
100 µM) of m-tyramine released ~70% of DA
taken up by the cells (Fig. 6A), which was compatible
with the DA recovery (~64% of added DA) from the cells determined by HPLC at 5-10 min after the addition of DA. This suggests that almost
all intracellular DA could be released by high concentrations of
m-tyramine. Cocaine reduced the initial efflux rate more
appreciably at lower initial [m-tyramine]o
than at higher initial [m-tyramine]o (Fig.
6B). The slow rise in the early phase of the apparent
DA efflux caused by cocaine alone stood in sharp contrast to the steep
rising in the early phase of the DA efflux induced by the same
concentrations of m-tyramine (Fig. 6, compare A,
C). The Michaelis-Menten equation expressing the
relationship between initial rate of cocaine-induced apparent DA efflux
and initial medium [cocaine] was: initial rate (picomoles per second
per milligram) = 0.26[cocaine]/(3.0 + [cocaine]) with a Hill number
1.02 ± 0.025 (n = 6). Thus, the maximal initial
rate of cocaine-induced DA efflux was only 3% of that induced by
m-tyramine (0.26 vs 7.7 pmol · sec1 · mg1) at
84 µM [DA]i, suggesting that the
contribution of inhibiting reuptake of external DA by
m-tyramine to the observed DA efflux is negligible.
View larger version (18K):
[in this window]
[in a new window]
|
Figure 6.
Time course of DA efflux induced by various
concentrations of m-tyramine (m-TYR) at a
fixed intracellular concentration of DA. DA was added to an initial
medium concentration of 1 µM and allowed to be taken up
until [DA]o decreased to near steady state, at which
time, [DA]i was 84 µM. The DA efflux was
subsequently initiated by various concentrations of
m-tyramine (1-100 µM) in the absence and
presence of 10 µM cocaine. Each panel shows a
representative experiment with five levels of initial
[m-tyramine]o or [cocaine] on the same
generation of cells. Each experiment was performed three times with
similar results. The arrow denotes the addition time of
DA, m-tyramine, cocaine, or m-tyramine
plus cocaine. The values at the end of each curve denote the initial
medium m-tyramine concentration. A, DA
efflux in the presence of m-tyramine alone.
B, DA efflux in the presence of both
m-tyramine and cocaine. Cocaine reduced the initial rate
of the DA efflux more appreciably at lower
[m-tyramine]o than at higher
[m-tyramine]o. C, DA efflux
in the presence of cocaine alone. For estimation of the initial rate of
m-tyramine-induced DA efflux in the presence of cocaine,
the subtraction was made at each [cocaine] as follows:
RateTYR,COC = RateTYR+COC RateCOC.
|
|
The Eadie-Hofstee plot of the data showed that the inhibition pattern
produced by cocaine was competitive (Fig. 5B);
Vmax is unchanged and Km
increased with medium [cocaine] (Table 4). The graphically estimated
competitive Ki for cocaine was 2.36 ± 0.43 µM. Based on the competitive model,
Ki values for cocaine to inhibit
m-tyramine (10 µM)-induced DA efflux at
various [DA]i were estimated. The
Ki values at all levels of [DA]i
were similar and close to the graphically estimated
Ki (Table 4). However, these
Ki values for cocaine to inhibit
m-tyramine-induced DA efflux were significantly higher than
its Ki to inhibit DA uptake (Table 1;
p < 0.01; unpaired t test).
Concurrent effect of cocaine on inward transport of
m-tyramine and outward transport of dopamine
To clarify which conformation of the hNET cocaine attacks, we used
a two-electrode approach to examine the simultaneous uptake of
m-tyramine and the efflux of DA induced by
m-tyramine in a single run. The typical time course for the
original signals and the normalized concentration profiles in the
absence and presence of cocaine are shown in Figure
7.
View larger version (29K):
[in this window]
[in a new window]
|
Figure 7.
Simultaneous measurement of
m-tyramine (m-TYR) uptake and
accompanying DA efflux by two-electrode RDE voltammetry. DA was added
to an initial medium concentration of 1 µM and allowed to
be taken up until [DA]o decreased to near steady state,
at which time, [DA]i was 84 µM. DA efflux
was initiated by 3 µM m-tyramine in the
absence and presence of 3 µM cocaine
(COC). Data shown are from a representative experiment
on the same generation of cells. Each experiment was performed six
times with similar results. The arrow denotes the
addition time of DA, m-tyramine, or
m-tyramine plus cocaine. A, Oxidation
signals measured in the absence of cocaine. Electrode 1 recorded the DA
signal while electrode 2 recorded both DA and m-tyramine
signals. B, Oxidation signals measured in the presence
of 3 µM cocaine. C, Concurrent changes in
medium DA and m-tyramine signals in the absence of
cocaine, determined by subtraction of the DA signal at electrode 1 from
the mixed signal at electrode 2 plotted in A after
correction for relative response. D, Concurrent changes
in medium DA and m-tyramine signals in the presence of 3 µM cocaine, determined as in C. For
estimation of the initial rate of m-tyramine uptake in
the absence and presence of 3 µM cocaine, the subtraction
was made as follows: m-tyramine uptake rate = total decay rate decay rate with 100 µM cocaine
(data not shown). Comparison of C with D
reveals that both m-tyramine uptake and accompanying DA
efflux are reduced by cocaine.
|
|
m-Tyramine uptake and accompanying DA efflux in the
absence of cocaine
Change in the [DA]i (Fig.
8A) did not modify the
initial rate of m-tyramine uptake, reminiscent of the
insensitivity of DA uptake to the intracellular m-tyramine
(Table 2). The slightly lower rate value at highest [DA]i
did not reach significance when compared with that at lowest
[DA]i (12.8 ± 0.8 pmol · sec1 · mg1 at
14.9 µM [DA]i vs 11.9 ± 0.9 pmol · sec1 · mg1 at
242 µM [DA]i; p > 0.05; n = 6; paired t test). In contrast, m-tyramine-induced DA efflux significantly increased with
[DA]i (Fig. 8B). The ratios of DA
efflux to m-tyramine uptake were <1 at all tested
[DA]i. However, the ratio increased with
[DA]i and followed the relationship defined by
Michaelis-Menten kinetic models (Fig.
9), with Rmax = 1.01 ± 0.10 and K0.5 = 340 ± 48 µM. Thus, theoretically, with the intracellular DA
approaching infinitely high concentration, the efflux rate of internal
DA would equal the uptake rate of external m-tyramine.
View larger version (26K):
[in this window]
[in a new window]
|
Figure 8.
Inhibition by cocaine of m-tyramine
uptake and m-tyramine-induced DA efflux at various
intracellular concentrations of DA. The initial medium concentration of
m-tyramine was 3 µM. The protocol applied
is depicted in Figure 7. A, Inhibition of
m-tyramine uptake. Intracellular DA has no significant
effect on m-tyramine uptake or on the inhibitory effect
of cocaine. B, Inhibition of
m-tyramine-induced DA efflux. The DA efflux increases
with [DA]i but is almost equally inhibited by cocaine at
most [DA]i (59, 38, 56, 51, 54, 55, 56, and 73% of
control at 14.9, 24.8, 44.5, 64.2, 84, 123.5, 163, and 242 µM [DA]i, respectively). Values are
mean ± SE of six experiments, each of which was performed with
eight concentrations of DA on the same generation of cells.
**p < 0.01 versus control.
|
|
View larger version (25K):
[in this window]
[in a new window]
|
Figure 9.
The ratio of DA efflux to
m-tyramine uptake at various intracellular concentrations of
DA. Cocaine did not shift the ratio curve. The dotted
lines represent predicted shifts of the ratio curve in the
presence of 3 µM cocaine, based on no interaction,
competitive interaction, noncompetitive interaction, or uncompetitive
interaction of cocaine with intracellular DA at the inward-facing
conformation of the hNET (hNETin). The predicted
ratio of DA efflux to m-tyramine uptake for each model
was estimated as described in Materials and Methods.
|
|
Cocaine added simultaneously with m-tyramine
This was examined because the noncompetitive pattern of cocaine on
the DA efflux was found with respect to [DA]i when
cocaine and m-tyramine were added at the same time. Cocaine
(3 µM), added simultaneously with m-tyramine,
significantly reduced both m-tyramine uptake and
accompanying DA efflux. In agreement with the results obtained from
one-electrode measurement of DA uptake and efflux, DA preloading did
not significantly modify the inhibitory effect of cocaine either on the
m-tyramine uptake or on the accompanying DA efflux (Fig.
8).
By using the Michaelis-Menten expression of the DA efflux modified for
the inhibition mechanisms under consideration, we compared the
experimental ratio curve with those predicted from the models. The
reasoning is as follows. Cocaine could inhibit
m-tyramine-induced DA efflux by two ways: (1) competing with
m-tyramine for the outward-facing hNET, thereby reducing the
amount of the inward-facing hNET available for internal DA; and (2)
directly interacting with internal DA at the inward-facing hNET. If
cocaine does not interact with the inward-facing hNET, the reduction in
DA efflux in the presence of cocaine would be solely caused by the
reduction in m-tyramine uptake. In this case, the ratio of
DA efflux to m-tyramine uptake at any [DA]i
will be the same as that in the absence of cocaine. If cocaine has an
additional effect on the inward-facing hNET, it would cause further
reduction in DA efflux, that is, the DA efflux would decrease more than
predicted from the reduction in m-tyramine uptake, resulting
in a lower ratio. In this case, the ratio curve in the presence of
cocaine would shift to the right relative to that in the absence of
cocaine, with its shape varying with the putative inhibition patterns
of cocaine at the inward-facing hNET (Fig. 9). Thus, the shift would be
more significant at lower [DA]i for a competitive
inhibition, more significant at higher [DA]i for an
uncompetitive inhibition, and equal (in terms of percent inhibition) at
all [DA]i for a noncompetitive inhibition.
When added simultaneously with m-tyramine, cocaine did not
change the m-tyramine uptake to DA efflux ratio at all
tested [DA]i (Fig. 9). For the ratio curve in the
presence of cocaine, the Rmax was 0.95 ± 0.10, and the K0.5 was 319 ± 48 µM, which were close to the values in the absence of
cocaine. All models for an inhibition at the inward-facing conformation
needed a Ki value of 100 µM to
obtain a ratio curve close to that without cocaine.
Cocaine added 6 min earlier than m-tyramine
This experiment was designed to test whether cocaine could affect
m-tyramine-induced DA efflux by interacting with internal DA
if it was allowed to equilibrate with the transporters or to fully
enter the cell. Cocaine was added to various medium concentrations at a
time point when the DA (1 µM) uptake reached equilibrium (4 min after DA addition), and incubated with the DA-preloaded cells
for 6 min before the addition of m-tyramine (3 µM). Under this condition, after initiation of the DA
efflux by m-tyramine, the intracellular [cocaine] could be
between 1 and 25 µM (assuming 2.5-fold concentrated
in the cell; Sulzer and Rayport, 1990), or between 4 and 40 µM (assuming fourfold concentrated in the cell; Sershen
et al., 1982). Despite this possible intracellular accumulation of
cocaine, the DA efflux/m-tyramine uptake ratio remained
unchanged (Table 5). In addition, the
IC50 of cocaine for inhibiting m-tyramine uptake
was similar to its IC50 for inhibiting accompanying DA
efflux (Table 5).
The competitive Ki of cocaine for inhibiting
m-tyramine (3 µM)-induced DA efflux was
2.57 ± 0.36 µM. This value was close to its
Ki for inhibiting m-tyramine (10 µM)-induced DA efflux when cocaine was added
simultaneously with m-tyramine (2-2.5 µM, Table 3). The Ki of cocaine for inhibiting
m-tyramine was not available because of the lack of
information about the Km of
m-tyramine uptake. The filming at the electrode surface by
mtyramine varies in degree with the concentration of
m-tyramine, which hampered our attempts to measure the
Km of m-tyramine uptake into
DA-preloaded cells under the present electrochemical conditions.
| |
DISCUSSION |
In the present study, the uphill inward transport of external DA,
the acceleration of outward transport of internal DA by external
substrates, and the lack of inhibition of inward transport by high
concentrations of intracellular substrates, are compatible with an
alternating access transporter model (Stein, 1986; Rudnick et al.,
1997). Thus, kinetic analysis derived from this model was used to
address the inhibition mechanism of cocaine at the hNET. The results
strongly support a competitive action of cocaine at the outward-facing
conformation of the hNET.
In uptake assays, if cocaine is a competitive hNET inhibitor at
inward-facing conformation only (Fig.
10B, either
left or right) or at both inward-facing and
outward-facing conformations, it should show a noncompetitive or mixed
inhibition pattern on the zero trans uptake of the external DA, because
the external DA could not displace an inhibitor bound to the
inward-facing hNET. Furthermore, the inhibition should be weakened by
internal substrates displacing the inhibitor from the inward-facing
conformation. Finally, the inhibition kinetics would vary with the
amount of the inhibitor binding the inward-facing conformation from
inside. In the last case, the binding amount of intracellular cocaine could be increased by allowing more cocaine to enter the cell through
longer incubation or by enhancing the binding affinity of intracellular
cocaine through elevating the intracellular [Na+]
with ouabain. None of the above-mentioned events happened in our study.
The inhibition by cocaine of DA uptake appeared entirely competitive,
which was not modified by a change in the intracellular concentration
of substrate, cocaine, or Na+.
View larger version (34K):
[in this window]
[in a new window]
|
Figure 10.
Illustration of the models for interactions of
cocaine with different conformations of the hNET. A,
Competitive interaction at the outward-facing conformation. Cocaine
could bind the outward-facing conformation from either outside
(left) or inside (right). The binding of
cocaine prevents the binding of external DA. B,
Competitive interaction at the inward-facing conformation. Cocaine
could bind the inward-facing conformation from either inside
(left) or outside (right). The binding of
cocaine prevents the binding of internal DA. C,
Noncompetitive interaction at the inward-facing conformation. Cocaine
could bind the inward-facing conformation from either inside
(left) or outside (right). The binding of
cocaine does not prevent the binding of internal DA.
|
|
The competitive inhibition by cocaine of DA uptake shown in the present
study agrees with previous NET transport studies using [3H]NE in isolated peripheral tissues (Graefe and
Bönisch, 1988) but differs from DAT transport studies using the
same RDE voltammetry approach in rat brain tissue suspensions (McElvain
and Schenk, 1992; Povlock and Schenk, 1997). It should be pointed out
that the competitive pattern of cocaine observed in zero trans entry experiments, such as the present DA uptake assay, does not necessarily represent a competitive mechanism at the transporter. This pattern may
also be observed for a noncompetitive inhibitor primarily confined to
the outward-facing conformation of that transporter (Deves and Krupka,
1989). It has been presumed for catecholamine transporters that the
transporter-substrate complex returns faster than the unloaded
transporter (Schömig et al., 1988; Zimányi et al., 1989).
If the reorientation of the unloaded hNET is the rate-limiting step in
the process of DA uptake, the addition of external DA may cause the
hNET to be trapped in the inward-facing conformation during initial
uptake, out of reach of an inhibitor primarily binding to the
outward-facing conformation of the hNET. As a result, increasing the
external concentration of DA could overcome the effect of even a
noncompetitive inhibitor, making the pattern competitive. We explored
this possibility by testing the effect of internal substrates on
cocaine inhibition. Under the assumption that the internal
substrate-loaded transporter returns faster than the unloaded one
(Stein, 1986), the internal substrate should enhance the cocaine
inhibition (if it is caused by a noncompetitive mechanism at the
outward-facing conformation) by increasing the availability of the
outward-facing conformation of the hNET to cocaine. However, cocaine
inhibition was not significantly modified by the internal substrates.
Therefore, a noncompetitive mechanism of cocaine at the outward-facing
conformation of the hNET seems unlikely.
Because formation of the inward-facing hNET is a necessary prerequisite
for the outward transport of internal DA, more direct information about
the role of inward-facing hNET in the action of cocaine can be obtained
from the efflux assays. The process of m-tyramine-induced
outward transport consists of two steps (for simplicity, the steps for
cotransported ions were not included), according to the simple
transporter model. External m-tyramine binds to the
outward-facing state of the hNET, followed by translocation to the
inward-facing state and release of m-tyramine inside. Then, internal DA binds, followed by reorientation of the complex and release
of DA outside. In the hNET-expressing non-neuronal cells devoid of
exocytotic release and vesicular storage of internal amines, the
m-tyramine-induced DA efflux is most likely related to the
above process. This idea is reinforced by the similarity between the
Km of m-tyramine for inducing DA
efflux and its Ki for inhibiting DA uptake, as
well as by the one-phase Hill slopes for the concentration-response
curves of m-tyramine-induced DA efflux.
In the present study, cocaine inhibited m-tyramine-induced
DA efflux noncompetitively relative to intracellular DA, but
competitively relative to extracellular m-tyramine. The
results are consistent with a predominant binding of cocaine at the
outward-facing form of the hNET that is inaccessible to internal DA but
accessible to the external m-tyramine. Consequently, cocaine
could reduce the inward transport of m-tyramine and,
thereby, the amount of inward-facing hNET available for the internal
DA, resulting in a reduction in Vmax relative to
[DA]i; but it could not displace DA from the
inward-facing hNET, allowing an unchanged Km
relative to [DA]i. In addition, the higher
Ki of cocaine for inhibiting the efflux may be
partly caused by its inability to directly prevent the binding of
intracellular DA to the inward-facing conformation.
At least two other interpretations could be raised for the apparently
noncompetitive pattern of cocaine inhibition on
m-tyramine-induced DA efflux with respect to intracellular
DA. First, cocaine could quickly diffuse into the cell and bind to a
DA-bound inward-facing form of the hNET (Fig. 10C,
left). Second, external cocaine and internal DA could
simultaneously bind to the inward-facing form, one from each
compartment (Fig. 10C, right). In both cases, the inhibition by cocaine of m-tyramine-induced DA efflux should
have shown mixed competitive and noncompetitive patterns relative to extracellular m-tyramine, because the external
m-tyramine displaced only cocaine bound to the
outward-facing hNET, not that bound to the inward-facing hNET. However,
increasing the external concentration of m-tyramine
competitively attenuated the inhibition by cocaine of the DA efflux,
questioning noncompetitive action of cocaine at the inward-facing conformation.
The higher Km for outward transport of internal
DA may be related to a lower binding affinity of internal DA for the
hNET caused by a different conformation or caused by the unfavorable ionic composition of the intracellular side or a slower return rate
constant for the loaded hNET. It is unclear whether DA could bind to
negatively charged proteins or get trapped in acidic compartments within the cell. If this occurs, it would lower the free concentration of intracellular DA, leading to an overestimation of the
Km.
In a different approach, we measured m-tyramine uptake and
accompanying DA efflux simultaneously. This approach allowed us to
investigate the conformational requirement of the transporter of
cocaine even without knowledge of its inhibitory nature. Two observations from this experiment rule out the possibility that cocaine
may act at the inward-facing conformation of the hNET. First, in
contrast to a shift in the DA efflux/m-tyramine uptake ratio
curve predicted from the putative interactions between cocaine and
internal DA at inward-facing conformation, cocaine did not change the
shape and position of the ratio curve across a wide range of
[DA]i. Second, the IC50 value for cocaine to
inhibit m-tyramine-induced DA efflux is similar to the
IC50 value for it to inhibit m-tyramine uptake.
Both observations also suggest that cocaine inhibits
m-tyramine-induced DA efflux solely by inhibiting the inward
transport of m-tyramine.
Although the kinetics of inhibition can reveal which conformation
cocaine attacks, it does not indicate the exact location of the
cocaine-binding site, i.e., outside or inside face of the membrane. Our
data seem in favor of the binding of cocaine at the outside face of the
membrane. This is based on an assumption that less cocaine may enter
the cell during the first 15 sec than 6 min and the fact that a 6 min
preincubation of cells with cocaine failed to significantly enhance the
inhibitory effect of cocaine on inward or outward transport. However,
because cocaine is actually a lipophilic weak base entering the cell
quickly, the observed failure may also be simply caused by the
possibility that cocaine enters cells fast enough to reach a
significant intracellular concentration in less than a few seconds.
One mechanism underlying the lack of action of cocaine at the
inward-facing conformation may be that the low
[Na+] and high [K+] inside
the cell greatly impair the binding affinity of internal cocaine to the
hNET, as has been indicated for the binding of cocaine analogs to the
DAT (Chen et al., 1997). Thus, the intracellular concentration of
cocaine, although perhaps higher than outside, may be far below its
Ki for inhibiting inward-facing hNET and could
not compete with the extremely high concentration of internal DA for
the inward-facing hNET. If this possibility stands, cocaine might show
an apparently noncompetitive component in its inhibition on amine
uptake by the NE terminals, because the axoplasm concentration of
substrates in NE terminals may be much lower than observed in the
LLC-hNET cells. Nevertheless, other mechanisms may also play a role,
because elevation of intracellular [Na+] with
ouabain failed to change the inhibition profile of cocaine on DA
uptake. In this context, discrete regions of the hNET may be required
for the generation of the outward-facing and inward-facing conformations so that the inward-facing hNET does not form a binding pocket to incorporate cocaine.
The asymmetrical inhibition feature of cocaine is of importance in
several respects. First, it may be useful for dissecting conformational
changes during the transport cycle, in particular, when the transporter
is exposed to alterations in the membrane potential, the ionic
gradient, and pH, as well as site-directed mutagenesis. Second, kinetic
analysis of interactions of cocaine with other inhibitors at the hNET
may be helpful for exploring the conformation specificity of the latter
compounds. Third, identification of the incorporation sites of cocaine
analogs within the primary sequence may lend insights into domains
exposed in outward-facing conformation of the hNET. A combination of
structural and kinetic data will be necessary to establish a
comprehensive understanding of the transporter mechanism and its inhibition.
| |
FOOTNOTES |
Received May 18, 1998; revised Sept. 23, 1998; accepted Sept. 25, 1998.
This study was supported by National Institute on Drug Abuse (NIDA)
Grant R03 DA10896. J.B.J. is a recipient of an NIDA Research Scientist
Award (K02 DA00179). We thank Brian Reed for help with the
two-electrode voltammetry experiments.
Correspondence should be addressed to Dr. Nianhang Chen, Department of
Chemistry, Emory University, Atlanta, GA 30322-2210.
| |
REFERENCES |
-
Bönisch H,
Brüss M
(1994)
The noradrenaline transporter of the neuronal plasma membrane.
Ann NY Acad Sci
733:193-202[Web of Science][Medline].
-
Burnette WB,
Bailey MD,
Kukoyi S,
Blakely RD,
Trowbridge CG,
Justice Jr JB
(1996)
Human norepinephrine transporter kinetics using rotating disk electrode voltammetry.
Anal Chem
68:2932-2938[Medline].
-
Calligaro DO,
Eldefrawi ME
(1987)
Central and peripheral cocaine receptors.
J Pharmacol Exp Ther
243:61-68[Abstract/Free Full Text].
-
Chen NH,
Reith MEA
(1994)
Effect of locally applied cocaine, lidocaine, and various uptake blockers on monoamine transmission in the ventral tegmental area of freely moving rats: a microdialysis study on monoamine interactions.
J Neurochem
63:1701-1703[Web of Science][Medline].
-
Chen NH,
Reith MEA
(1995)
Autoregulation and monoamine interactions in the ventral tegmental area in the absence and presence of cocaine: a microdialysis study in freely moving rats.
J Pharmacol Exp Ther
271:1597-1610[Abstract/Free Full Text].
-
Chen NH,
Ding JH,
Wang YL,
Reith MEA
(1997)
Modeling of the interaction of Na+ and K+ with the binding of the cocaine analog [125I]3
 -(iodophenyl)tropan-2-carboxylic acid isopropyl ester to the dopamine transporter.
J Neurochem
68:1968-1981[Web of Science][Medline]. -
Chen NH,
Trowbridge CG,
Justice Jr JB
(1998)
Voltammetric studies on mechanisms of dopamine efflux in the presence of substrates and cocaine from cells expressing human norepinephrine transporter.
J Neurochem
71:653-665[Web of Science][Medline].
-
Devés R,
Krupka RM
(1989)
Inhibition kinetics of carrier systems.
Methods Enzymol
171:113-133[Web of Science][Medline].
-
Eshleman AJ,
Stewart E,
Evenson AK,
Mason JN,
Blakely RD,
Janowsky A,
Neve KA
(1997)
Metabolism of catecholamines by catechol-O-methyltransferase in cells expressing recombinant catecholamine transporters.
J Neurochem
69:1459-1466[Web of Science][Medline].
-
Graefe K-H,
Fuchs G
(1979)
On the mechanism of neuronal efflux of axoplasmic 3H-noradrenaline.
In: Catecholamines: basic and clinical frontiers (Usdin E,
Kopin IJ,
Barchas J,
eds), pp 268-270. New York: Pergamon.
-
Graefe K-H,
Bönisch H
(1988)
The transport of amines across the axonal membranes of noradrenergic and dopaminergic neurons.
In: Catecholamines I (Trendelenbury U,
Weiner N,
eds), pp 193-245. New York: Sringer-Verlag.
-
Gu H,
Wall S,
Rudnick G
(1994)
Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence.
J Biol Chem
269:7124-7130[Abstract/Free Full Text].
-
Gu H,
Wall SC,
Rudnick G
(1996)
Ion coupling stoichiometry for the norepinephrine transporter in membrane vesicles from stably transfected cells.
J Biol Chem
271:6911-6916[Abstract/Free Full Text].
-
Justice Jr JB,
Reed B
(1997)
Dual electrode characterization of induced efflux of dopamine by m-tyramine via the norepinephrine transporter.
Soc Neurosci Abstr
23:695.
-
Justice Jr JB,
Danek KS,
Kable JW,
Barker EL,
Blakely RD
(1998)
Voltammetric approaches to kinetics and mechanism of the norepinephrine transporter.
Adv Pharmacol
42:191-194.
-
Krueger BK
(1990)
Kinetics and block of dopamine uptake in synaptosomes from rat caudate nucleus.
J Neurochem
55:260-267[Web of Science][Medline].
-
Krupka RM,
Devés R
(1983)
Kinetics of inhibition of transport systems.
Int Rev Cytol
84:303-352[Web of Science][Medline].
-
Langeloh A,
Bönisch H,
Trendelenburg U
(1987)
The mechanism of the 3H-noradrenaline releasing effect of various substrates of uptake: multifactorial induction of outward transport.
Naunyn Schmiedebergs Arch Pharmacol
336:602-610[Web of Science][Medline].
-
Levi G,
Raiteri M
(1993)
Carrier-mediated release of neurotransmitters.
Trends Neurosci
16:415-419[Web of Science][Medline].
-
Masahiko T,
Groshan K,
Blakely RD,
Richelson E
(1997)
Pharmacological profile of antidepressants and related compounds at human monoamine transporters.
Eur J Pharmacol
340:249-258[Web of Science][Medline].
-
McElvain JS,
Schenk JO
(1992)
A multisubstrate mechanism of striatal dopamine uptake and its inhibition by cocaine.
J Neurochem
43:2189-2199.
-
Pacholczyk T,
Blakely RD,
Amara SG
(1991)
Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter.
Nature
350:350-354[Medline].
-
Povlock SL,
Schenk JO
(1997)
A multisubstrate kinetic mechanism of dopamine transport in the nucleus accumbens and its inhibition by cocaine.
J Neurochem
69:1093-1105[Web of Science][Medline].
-
Rudnick G
(1997)
Mechanisms of biogenic amine neurotransmitter transporters.
In: Neurotransmitter transporters: structure, function, and regulation (Reith MEA,
ed), pp 73-100. Totowa, NJ: Humana.
-
Schömig K,
Körber M,
Bönisch H
(1988)
Kinetic evidence for a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.
Naunyn Schmiedebergs Arch Pharmacol
337:626-632[Web of Science][Medline].
-
Sershen H,
Reith MEA,
Lajtha A
(1982)
Comparison of the properties of central and peripheral binding sites for cocaine.
Neuropharmacology
21:469-474[Web of Science][Medline].
-
Sulzer D,
Rayport S
(1990)
Amphetamine and other psychostimulants reduce pH gradients in midbrain dopaminergic neurons and chromaffin granules: a mechanism of action.
Neuron
5:797-808[Web of Science][Medline].
-
Stein WD
(1986)
In: Transport and diffusion across cell membranes. San Diego: Academic.
-
Wheeler DD,
Edwards AM,
Chapman BM,
Ondo JG
(1994)
Effects of cocaine on sodium dependent dopamine uptake in rat striatal synaptosomes.
Neurochem Res
19:49-56[Web of Science][Medline].
-
Zimányi I,
Lajtha A,
Reith MEA
(1989)
Comparison of characteristics of dopamine uptake and mazindol binding in mouse striatum.
Naunyn Schmiedebergs Arch Pharmacol
340:626-632[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/182410257-12$05.00/0
This article has been cited by other articles:
|
|
|
|
 
S. Apparsundaram, D. J. Stockdale, R. A. Henningsen, M. E. Milla, and R. S. Martin
Antidepressants Targeting the Serotonin Reuptake Transporter Act via a Competitive Mechanism
J. Pharmacol. Exp. Ther.,
December 1, 2008;
327(3):
982 - 990.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Parnas, J. D. Gaffaney, M. F. Zou, J. R. Lever, A. H. Newman, and R. A. Vaughan
Labeling of Dopamine Transporter Transmembrane Domain 1 with the Tropane Ligand N-[4-(4-Azido-3-[125I]iodophenyl)butyl]-2{beta}-carbomethoxy-3{beta}-(4-chlorophenyl)tropane Implicates Proximity of Cocaine and Substrate Active Sites
Mol. Pharmacol.,
April 1, 2008;
73(4):
1141 - 1150.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. A. Vaughan, D. S. Sakrikar, M. L. Parnas, S. Adkins, J. D. Foster, R. A. Duval, J. R. Lever, S. S. Kulkarni, and A. Hauck-Newman
Localization of Cocaine Analog [125I]RTI 82 Irreversible Binding to Transmembrane Domain 6 of the Dopamine Transporter
J. Biol. Chem.,
March 23, 2007;
282(12):
8915 - 8925.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. K. Henry, E. M. Adkins, Q. Han, and R. D. Blakely
Serotonin and Cocaine-sensitive Inactivation of Human Serotonin Transporters by Methanethiosulfonates Targeted to Transmembrane Domain I
J. Biol. Chem.,
September 26, 2003;
278(39):
37052 - 37063.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. H. Sitte, B. Hiptmair, J. Zwach, C. Pifl, E. A. Singer, and P. Scholze
Quantitative Analysis of Inward and Outward Transport Rates in Cells Stably Expressing the Cloned Human Serotonin Transporter: Inconsistencies with the Hypothesis of Facilitated Exchange Diffusion
Mol. Pharmacol.,
April 16, 2001;
59(5):
1129 - 1137.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
E. S. Vizi
Role of High-Affinity Receptors and Membrane Transporters in Nonsynaptic Communication and Drug Action in the Central Nervous System
Pharmacol. Rev.,
March 1, 2000;
52(1):
63 - 90.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-G. Chen and G. Rudnick
Permeation and gating residues in serotonin transporter
PNAS,
February 1, 2000;
97(3):
1044 - 1049.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Chen, J. V. Ferrer, J. A. Javitch, and J. B. Justice Jr.
Transport-dependent Accessibility of a Cytoplasmic Loop Cysteine in the Human Dopamine Transporter
J. Biol. Chem.,
January 21, 2000;
275(3):
1608 - 1614.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Chen, C. G. Trowbridge, and J. B. Justice Jr.
Cationic Modulation of Human Dopamine Transporter: Dopamine Uptake and Inhibition of Uptake
J. Pharmacol. Exp. Ther.,
September 1, 1999;
290(3):
940 - 949.
[Abstract]
[Full Text]
|
|
|
|
|
Top
Abstract
Introduction
