Anal. Chem. 1996, 68, 2932-2938
Human Norepinephrine Transporter Kinetics Using Rotating Disk Electrode Voltammetry W. B. Burnette,† M. Danna Bailey,† Shola Kukoyi,† R. D. Blakely,‡ C. G. Trowbridge,† and J. B. Justice, Jr.*,†
Department of Chemistry, Emory University, Atlanta, Georgia 30322, and Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37235
Rotating disk electrode (RDE) voltammetry is applied to the measurement of the transport of the catecholamine neurotransmitters norepinephrine (4-(2-amino-1-hydroxyethyl)-1,2-benzenediol, NE) and dopamine (3,4-dihydroxyphenethylamine, DA) in suspensions of LLC-NET cells, a line of porcine kidney cells expressing the human norepinephrine transporter (hNET). Initial rate of transport was assessed by following the initial decrease in neurotransmitter after its addition to the cell suspension, as measured by the decrease in oxidation current at +0.45 V vs Ag/AgCl. The initial rate of norepinephrine uptake was saturable, with Vmax and KM of 197 ( 17 amol min-1 cell-1 and 1.64 ( 0.46 µM, respectively. The RDE method also allows observation of outward transport (efflux) of the DA or NE previously taken up by the cells. Outward transport was induced by the addition of either d-amphetamine (d-AMPH) or p-tyramine (4-hydroxyphenethylamine, p-TYR), which are also substrates for the NE transporter. The technique was also used to monitor accelerated NE uptake by cells preloaded with p-TYR, a phenomenon distinguishing carriers from channels. Together, these findings document the utility of RDE for the nonisotopic measurement of neurotransmitter influx and efflux from transfected mammalian cells. The plasma membrane transporter for the catecholamine neurotransmitter norepinephrine (NE) is an integral membrane protein whose primary function appears to be the removal of NE from the extracellular fluid, terminating its action. The role of NE in depression and other physiological processes has made its transport a subject of intense study.1-3 In vitro assays of catecholamine transport are usually performed by measuring the total accumulation of radiolabeled substrate into a cell or tissue preparation over a period of incubation; a single measurement is used to estimate the average rate of transport over the period of incubation. The interval may be sufficient for the substrate to accumulate intracellularly, giving rise to significant outward transport. Under such conditions, initial rate measurements will reflect net bidirectional, rather than unidirectional, substrate flux. †
Emory University. Vanderbilt University. (1) Bunney, W. E., Jr.; Davis, J. M. Arch. Gen. Psychiatry 1965, 13, 509-522. (2) Rudorfer, M. V.; Sherer, M. A.; Lane, E. A.; Golden, R. N.; Linnoila, M.; Potter, W. Z. J. Clin. Psychopharmacol. 1991, 11, 22-27. (3) Veith, R. C.; Lewis, N.; Linares, O. A.; Barnes, R. F.; Raskind, M. A.; Villacres, E. C.; Murburg, M. M.; Ashleigh, E. A.; Castillo, S.; Peskind, E. R.; Pascualy, M.; Halter, J. B. Arch. Gen. Psychiatry 1994, 51, 411-422. ‡
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Recently, a novel application of a well-established electrochemical method, rotating disk electrode voltammetry (RDE), was introduced, which allows measurement of fast initial rates and also provides a record of the complete time course of uptake.4 The method has been applied to the study of the dopamine transporter in tissue homogenates and synaptosomal preparations.5-8 In those studies, measurements of the initial time course of DA loss from the medium were made, rather than measurement of accumulation into tissue at the end of an incubation period. The decrease in DA concentration was shown to be linear with respect to time, a characteristic consistent with unidirectional transport. Such experiments can more closely approximate zero trans conditions9 (initial absence of substrate opposite (trans) the side of the measurement) than most assays of radiolabeled catecholamine transport. The advantages of zero trans conditions are that it is well-defined mathematically and that the kinetic data complement the data obtained under equilibrium exchange conditions.9 The difference in the kinetic information obtained under these different experimental conditions can be understood by considering the simplified four-state transporter model illustrated in Figure 1. The transporter has two empty conformations, Tout and Tin, “facing” either outside the cell or intracellularly, respectively. Similarly, the transporter bound with substrate S also may “face” inward or outward. For uptake to occur, the substrate in the medium outside the cell binds to the outward conformation, followed by a change in conformation to the inward face with subsequent dissociation from the transporter. For additional uptake to occur, the transporter must return to the outward-facing conformation. With no substrate initially present on the inside, return of the empty transporter (Tin f Tout) is the only return path possible. It has been shown for several transporters that the return of the empty form is the slow step in limiting the rate of transport.9 Under zero trans conditions, uptake is as described above. However, as the intracellular concentration of substrate increases, and at equilibrium, the transporter can return loaded with substrate (TSin f TSout). The faster return, coupled with a fixed number of carrier sites, results in an increased availability of outwardly facing transporter and, in turn, a faster unidirectional rate of inward transport than in the abscense of Sin, at any given (4) Schenk, J. O.; Patterson, T. A.; McElvain, J. S. Trends Anal. Chem. 1990, 9, 325-330. (5) McElvain, J. S.; Schenk, J. O. Biochem. Pharmacol. 1992, 43, 2189-2199. (6) Meiergerd, S. M.; Hooks, S. M.; Schenk, J. O. J. Neurochem. 1994, 63, 1277-1281. (7) Meiergerd, S. M.; McElvain, J. S.; Schenk, J. O. Biochem. Pharmacol. 1994, 47, 1627-1634. (8) Meiergerd, S. M.; Schenk, J. O. J. Neurochem. 1994, 63, 1683-1692. (9) Stein, W. D. Transport and Diffusion Across Cell Membranes; Academic Press: Orlando, FL, 1986; Chapter 3. S0003-2700(96)00022-4 CCC: $12.00
© 1996 American Chemical Society
Figure 1. Four-state model of a simple transporter. Vertical arrows represent binding and dissociation of substrate S with transporter T. Horizontal arrows represent changes in conformation between transporter states facing ouside the cell and states facing inside the cell. Transporter is not implied to cross the cell membrane but rather to present appropriate binding sites to each compartment at different states of the transport cycle.
Sout. The effect is known as trans acceleration. A tracer amount of labeled substrate is typically used to observe transport since exchange of unlabeled material results in no observable net transport at equilibrium. The effect can also be observed by preloading the cells with a second substrate, as described in the present work. In vitro studies of catecholamine transport using tissue prepartions or synaptosomes are complicated by the fact that more than one catecholamine transporter may be present. It is known that the NE transporter has a higher apparent affinity for DA than for NE itself.10,11 The existence of two or more transporters having different kinetic properties but capable of interacting with the same neurotransmitter can lead to a complex time dependence. Complications of this kind can be avoided by the use of cell lines expressing a single transporter.12 Such cell lines also make it easier to achieve intracellular zero trans conditions. An additional problem with tissue slice preparations is that the neurotransmitter must diffuse through the tissue to reach transporter sites not at the surface of the tissue. It has been shown that this can produce artifacts in the data.13 Near et al.14 found two different apparent affinities for the uptake of DA in striatal chopped tissue, while a single site (KM ) 0.13 µM) was found in striatal synaptosomes, a preparation in which the uptake sites are more accessible to the bathing medium. Finally, vesicular amine transport pathways are also present in neuronal preparations, adding an additional compartment to considerations of substrate flux. The human NE and DA transporters have now been cloned11,15 and their amino acid sequences determined. These transporters show a high degree of sequence identity or similarity and are believed to belong to a large family of neurotransmitter transporters whose endogenous substrates include biogenic amines and amino acids.16 Recombinant DNA technology has been used to express transporters in cultured mammalian cells, where they do not occur naturally.11,15,17,18 The modified cell line used in the present work19,20 contains functional NE transporters in the cell (10) Giros, B.; Wang, Y.-M.; Suter, S.; McLeskey, S. B.; Pifl, C.; Caron, M. G. J. Biol. Chem. 1994, 269, 15985-15988. (11) Pacholczyk, T.; Blakely, R. D.; Amara, S. G. Nature 1991, 350, 350-353. (12) Bonisch, H.;Bruss, M. Ann. N. Y. Acad. Sci. 1994, 733, 193-202. (13) Green, A. L. J. Pharm. Pharmacol. 1976, 28, 265-274. (14) Near, J. A.; Bigelow, J. C.; Wightman, R. M. J. Pharmacol. Exp. Ther. 1988, 245, 921-927. (15) Giros, B.; El Mestikawy, S.; Godinot, N.; Zheng, K.; Han, H.; Yang-Feng, T.; Caron, M. G. Mol. Pharmacol. 1992, 42, 383-390. (16) Nelson, N.; Lill, H. J. Exp. Biol. 1994, 196, 213-228. (17) Blakely, R. D.; Moore, K. M.; Qian, Y. Molecular Biology and Function of Carrier Proteins; Rockefeller University Press: New York, 1993; pp 283300. (18) Buck, K. J.; Amara, S. G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1258412588. (19) Gu, H.; Wall, S. C.; Rudnick, G. J. Biol. Chem. 1994, 269, 7124-7130.
membrane but lacks the mechanisms found in the NE nerve terminal for the synthesis, storage, exocytotic release, and metabolism of NE. The absence of these complicating processes, in combination with the rapid voltammetric measurement of neurotransmitter concentration over the complete time course of an experiment, makes it possible to investigate catecholamine transporter kinetics more thoroughly than previously possible. The present report demonstrates the feasibility of using the RDE methodology to examine catecholamine transporters expressed in stable cell lines and illustrates the utility of the method for making observations not readily performed by other methods. EXPERIMENTAL SECTION Reagents. Dopamine hydrochloride, L-norepinephrine hydrochloride, and p-tyramine hydrochloride were obtained from Sigma. All buffer and analyte solutions were prepared in distilled water, and Krebs/Ringer/HEPES (KRH) buffer (120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM 4-(2-hydroxyethyl)-1-piperizineethanesulfonic acid (HEPES), 10 mM D-glucose, pH 7.4) was sterilized by filtering through 0.2 µM membrane filter units (Nalgene, Rochester, NY). All media components were used as received without further purification. Rotating Disk Electrode Voltammetry. RDE voltammetric measurements were made with an AFMSRX analytical rotator system (Pine Instrument Co., Grove City, PA), equipped with a 3 mm glassy carbon electrode, a Pt auxiliary electrode, and a Ag/ AgCl reference electrode. 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). All experiments were performed at an electrode rotation rate of 4000 rpm and an applied potential of +0.45 V vs Ag/AgCl. The temperature was maintained at 37 °C. Voltammetric measurements were acquired at a frequency of 128 Hz, averaged, and recorded at a frequency of 8 Hz on a 486 PC through a DT 2801A interface board (Data Translation, Marlboro MA), controlled by Origin data acquisition software (MicroCal Software, Northampton, MA). Cell Culture. LLC-NET cells, a line of porcine kidney cells expressing the human NE transporter, were obtained from Dr. Gary Rudnick19 (Department of Pharmacology, Yale University). The cells were maintained in a growth medium made from 1 L of Eagle’s minimum essential medium (Fisher Scientific, Pittsburgh, PA), supplemented with 100 mL of 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 10 mL of a 10 000 units/ mL penicillin/streptomycin solution (both from Life Technologies, Grand Island, NY), and made 2 mM in L-glutamine. Cells were grown to confluence in Falcon cell culture dishes (150 mm × 25 mm) in a 5% CO2 atmosphere at 37 °C. Transport Assays. Cells were washed with 20 mL of KRH buffer and harvested by scraping in two 2.5 mL volumes of room temperature KRH, which were combined. The harvested cells were transferred to a single glass test tube and centrifuged at 2500 rpm (1000g) for 2 min. The pellet was divided for use in four replicate transport assays. One of three different experiments was then performed: (1) substrate dependence of transport, (2) induced efflux of preloaded NE and DA in response to either p-TYR or d-AMPH, or (3) acceleration of NE transport by preloaded p-TYR. At the end of each experiment, the cell (20) Melikian, H. E.; McDonald, J. K.; Gu, H.; Rudnick, G.; Moore, K. R.; Blakely, R. D. J. Biol. Chem. 1994, 269, 12290-12297.
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suspension was used for cell counting with a hemocytometer.21 Kinetics of NE and DA Transport. The pellet was resuspended in 1.5 mL of room temperature KRH buffer and transferred to a 1.5 mL centrifuge tube. The centrifuge tube was placed in a water bath at 37 °C, and an O2 stream was directed over the surface of the suspension for cell viability. After 5 min, a 300 µL aliquot was placed in the electrochemical cell, while the rest of the suspension remained in the water bath. With the rotating glassy carbon electrode just below the surface of the solution, the potential was applied and the current observed for stability for 90 s prior to baseline acquisition. Following a 60 s period of baseline signal acquisition, 6 µL of NE or DA in 120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, and 1.2 mM MgSO4 was added to the suspension. After the addition, data were recorded for an additional 4 min. Four replicates were performed at each concentration (100 nM to 15 mM). Data were not corrected for nonspecific binding or transport. Stimulated Efflux of NE and DA. The pellet was resuspended in 2.5 mL of KRH buffer saturated with a 95% O2/5% CO2 mixture and placed in a 12 mm × 75 mm test tube. The suspension was incubated in a 37 °C water bath under O2 for 5 min, after which a 400 µL aliquot was placed in the electrochemical cell under an O2 stream. The RDE was introduced, rotation begun, and the potential applied. The current was monitored for 90 s, followed by a 60 s period of baseline signal acquisition. Eight microliters of NE or DA was added to bring the concentration to 1 µM, and the decrease in NE or DA was recorded for 6 min. The final 60 s was used to establish a baseline for measurement of subsequent NE or DA efflux. To induce efflux, 8 µL of unbuffered electrolyte as a control, 2 µM d-AMPH, or 1 µM p-TYR was added to the suspension. Neither compound is oxidized at the potential used (+0.45 V vs Ag/AgCl). Efflux was monitored for 5 min. Acceleration of NE Transport by p-TYR. As in the above procedures, the current was monitored for 90 s, followed by 60 s of recording to establish a baseline current. To preload the cells, 8 µL of p-TYR was added (or 8 µL of electrolyte solution as a control), bringing the concentration to 400 nM. Two minutes later, 8 µL of NE was added to bring the NE concentration to 400 nM. The decrease in NE oxidation current was then recorded for 3 min. Uptake of Structural Analogs. Initial uptake rates of 1 µM solutions of analogs of DA and NE were obtained as described for kinetics of DA and NE. Data were normalized to the rate of DA uptake (n ) 2-4). The following structures were tested (listed in order of Figure 11): norepinephrine, dopamine, 6-hydroxydopamine, octopamine, 4-methoxy-3-hydroxyphenethylamine, 3,4-dihydroxynorephedrine, N-methyldopamine, 5-hydroxyphenethylamine, 3-methoxy-4-hydroxyphenethylamine, normetanephrine, 3,4-dihydroxybenzylamine, isoproterenol, 3,4-dihydroxyphenylglycol, metanephrine, and dipropyldopamine. Data Analysis. The baseline current preceding addition of catecholamine was extrapolated and subtracted from the raw current vs time record. A linear regression was performed on 10 s of the baseline-corrected clearance profile, using the interval from 1 to 11 s after the point of the maximum rate of increase in the oxidation current (Figure 2). A calibration factor for conver(21) Hockfield, S.; Carlson, S.; Evans, C.; Levitt, P.; Pintar, J.; Silberstein, L. Selected Methods for Antibody and Nucleic Acid Probes; Cold Spring Harbor Laboratory Press: Plainview, NY, 1993; Chapter 1.
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Figure 2. Baseline-subtracted data (b) showing the initial rise in the DA oxidation current following an addition of DA to bring concentration to 300 nM in a suspension of LLC-NET cells (arrow). The current reaches a maximum approximately 500 ms after the addition. The solid line is the linear regression used to extrapolate back to the theoretical value of the current at the point of the maximum rate of change in the signal (∼300 ms after the DA addition), shown by the asterisk.
sion of current to concentration was obtained by extrapolation of the regression line back to time of maximum rate of current increase. The initial rate of substrate clearance from the extracellular medium, v0, was calculated using the slope of the regression and the calibration factor. Initial rates were normalized to cell count by dividing the rate by the mean of the four cell counts, expressed in units of 10-18 mol-1 min-1 cell. Control experiments in nontransfected LLC-PK1 cells showed negligible change in oxidation current during the first 10 s after the addition of DA, and therefore control rates were not subtracted from rates measured in transfected cells. To obtain kinetic parameter estimates for DA and NE transport, initial rate data were first fit to the Eadie-Hofstee equation, a linear transformation of the Michaelis-Menten rate expression:
v0 ) Vmax - KM(v0/[NE])
(1)
where the y-intercept is Vmax, the maximal rate of transport, the slope is KM, the apparent affinity for transport, and [NE] is the initial NE concentration. The intercept and slope obtained from the linear fit of the data to this equation were then used as initial estimates for fitting to the Michaelis-Menten function:
v0 ) (Vmax[NE])/(KM + [NE])
(2)
Initial rates of efflux of preloaded NE and DA, caused by the addition of 1 µM p-TYR or 2 µM d-AMPH, were determined as follows: data from each efflux experiment were corrected for baseline and converted to concentration using the calibration derived from the addition of NE or DA. The concentration profile following addition of p-TYR or d-AMPH was corrected by subtraction of the average control profile following addition of buffer alone. Initial rates of NE and DA efflux were estimated by linear regression over the 20 s following addition of p-TYR or d-AMPH. In the trans acceleration experiment, the effect of prior exposure of the cells to 400 nM p-TYR on the initial rate of NE clearance was determined by comparing the mean initial rates of the control and p-TYR groups using a two-population t-test.
Figure 3. Representative time course of the transport of NE into LLC-NET cells. Concentration vs time profile, after baseline subtraction, for the addition and clearance of 300 nM NE. Inset: expanded view of the initial decay phase following the addition of NE, showing the range of the data used to estimate the initial rate (indicated by arrows). Solid circles represent concentration vs time data; solid line was obtained by linear regression.
RESULTS AND DISCUSSION Kinetics of NE and DA Transport. Figure 2 shows the rapid rise in current that occurs following addition of DA to a 300 µL suspension of LLC-NET cells. The time from addition of substrate (arrow) to the maximum in the signal is
