Studies on the Transport of L-Tyrosine into an Adrenergic Clone of Mouse Neuroblastoma*

SUMMARY Studies on the transport of L-[U-14C]tyrosine into cultured mouse neuroblastoma clone which has high levels of tyrosine bydroxylase (EC 1.14.16. Z), are presented. This transport was by a saturable process, exhibited marked exchanging properties, and was inhibited by a reagent which attacks sulfhydryl groups but not by metabolic inhibitors. No major ion requirements were found for this transport, which was sensitive to changes in temperature but not to changes in pH in the range of 6 to 8. Molecules with close structural analogy were potent of


RESULTS
ISJect oj Culture Conditions on Tyrosine Transport-For the specific transport of tyrosine into NIE-115, we reported (1) that the transport constant, K [ (concentration of substrate at one-half maximum velocity of transport), was higher in cells cultured in IMedium I supplemented with 10% fetal calf serum as compared to Medium I without tyrosine and 0% fetal calf serum. The results of further studies on this observation (Table I) showed that the Kt was higher in cells cultured in Medium I with 10% fetal calf serum but that there was no significant difference in Kt values for cells cultured in Medium I, 0% fetal calf serum, versus Medium I without tyrosine, 0% fetal calf serum. The V,,, (maximum velocity of transport) per cell was also higher in cells grown in Medium I with 10% fetal calf serum. Since removal of serum from the medium effectively stops cell division in these cells (4, 16), these results probably reflect membrane changes associated with cell division as studied in detail by others (17, 18).
Our V,,, values per cell and per mg of protein as well as our K t values for the specific transport of tyrosine with the use of Medium I without tyrosine, 0% fetal calf serum, were higher than previously reported (1). We have no explanation for these differences.
We continued to use cells depleted of tyrosine in our experiments in order to be assured of studying initial rates.
Effect of Preloading Cells on Rate of Tyrosine Transport-Preincubation of NlE-115 cells in nonradioactive L-tyrosine (1. mM or 0.1 mM) reproducibly caused an increase in the initial rate of L-[U-'%]tyrosine transport (Fig. 1, A and B). An increase of lesser degree was seen with preincubation in L-phenylalanine or D-tyrosine.
The slower rat.e of tyrosine transport caused by pre- A and B, clone NlE-115 (pas-averaged 1.4 X lo6 and amount of protein per well averaged 210 pg. sage 18) was placed in Medium I without tyrosine and phenyl-For the control at zero time, the rate of tyrosine transport was alanine, 0% fetal calf serum, 24 hours prior to this experiment. 495 pmoles per min, and at 140 min it was 339 pmoles per min. The Separate groups of cells in duplicate were incubated in 1 ml ,of control was the same for Fig. 1 A and B. C, rat glioma C-6 (subphosphate-buffered saline for the control or 1 ml of phosphate-culture 56) was placed in Medium I without L-phenylalanine and buffered saline containing one of the following: 1 mM L-tyrosine L-tyrosine, 0% fetal calf serum, 24 hours prior to this experiment (O), 0.1 mM L-tyrosine (A), 1 mM L-phenylalanine (Cl), or 1 mM which was performed as described above for A and B except that n-tyrosine ( l ). Every 30 min during this experiment the 1 ml of the length of the tyrosine transport was 1 min. The average phosphate-buffered saline incubation solution for each condition number of cells per well was 1.8 X 105. 1 mM L-tyrosine (O), was replaced with fresh solution to avoid changes due to evapora-1 mM L-phenylalanine (A), and control (0). The curves are tion. At the indicated time points, the 1 ml of phosphate-buffered plotted through the average of duplicate values which are represaline solution was removed, and cells were tested for the transport sented by the horizontal bars bracketing the symbols. For those of tyrosine at 5 X lo+ M L-[UJ4C]tyrosine (final specific activity points which are not bracketed, duplicate values fall within the 24 mCi per mmole) during a 30-s transport assay at 37" aa described dimensions of the symbol.
incubation with Gphenylalanine at early time points has also been a reproducible phenomenon, With C-6 glioma (Fig. lC), preincubation only in L-tyrosine and not in L-phenylalanine had an effect on the initial rate of L-[U-Y?]tyrosine transport, and this effect was evident after 30 min of preincubation.
The maximum increase in the rate of tyrosine transport (picomoles per min) for NlE-115 preincubated in L-tyrosine (1 mM) was about 50% above the zero time value, and this was similar to the increase seen for C-6. p-Chloromercuribenzoic acid (0.1 mM), a reagent which attacks sulfhydryl groups, was a potent inhibitor of tyrosine transport (Table II).
L-Tyrosine (1 mM) added to the incubation medium with p-chloromercuribenzoic acid did not prevent the effect of this reagent (data not shown).
The inability to inhibit tyrosine transport by 2,4,6-trinitrobenzene sulfonic acid, which also attacks sulfhydryl groups (Table II)  Effect of Ions, pli, and Temperature-The specific transport of tyrosine was not dependent to any major extent on the presence in the incubation medium of sodium, potassium, calcium, or magnesium ions (Table III).
The small sodium dependency suggests that a small part of this tyrosine transport, occurs by a sodium-dependent system (for example, the alanine-preferring (A) system (7)). There was no change in the rate of tyrosine transport at pH 6, 7, or 8.
Temperature had a marked effect on this transport such that the rate of transport at 25" and I" was, respectively, 68% and 16% of that at 37". Since metabolic inhibitors did not affect this transport (Table II), the reduction in the rate of transport caused by a reduction in temperature may reflect physical changes in the membrane structure, an effect of temperature which changes the rate of sugar transport in Escherichia coli (19).
Inhibition of Specific Transport of Tyrosine by Amines, Amino Acids, and Analogs-Compounds were tested for their effects on the specific transport of tyrosine by using the Dixon method (20) to determine the inhibitor constant, Ki. A summary of the data is presented in Table IV. The most potent inhibitors of tyrosine transport (Ki values in the range of 2 to 7 X 1OP M) were compounds with close structural analogy to tyrosine such as L-3,4-dihydroxyphenylalanine, L-3-iodotyrosine, and L-phenylalanine (Fig. 2). Compounds wit,h Ki values in the range of 11 X 10-S M to 30 X 1O-5 M (Table IV) were not necessarily similar to tyrosine in structure although Q amino acids predominated. Tyramine, which is a decarboxylation product of tyrosine, had reduced ability to inhibit tyrosine transport. Some stereo-  specificity for the transport of tyrosine was indicated by the lesser potency of n-tyrosine to inhibit L-[ U-lJC]tyrosine transport. All compounds listed in Table IV were  confirmed competitive inhibition by L-alanine, L-leucine, cphenylalanine, n-tyrosine, L-tryptophan, and L-valine. The following compounds at 10 x 10v5 M did not inhibit transport of L-[ U-W]tyrosine at 2.5 X 10e5 M, L-arginine, L-glutamic acid, L-glutamine, L-lysine, and L-proline.
The structures of some of the least potent inhibitors (Ki values in the range of 35 to 50 X lo+ M) (Table IV and Fig. 2) indicate a correlation between the presence of an Q amino group and the ability of a compound to inhibit transport of tyrosine. Thus, L-2-amino-3-phenylpropionic acid (L-phenylalanine) was a very potent inhibitor of L-tyrosine transport, but DL-3-amino-3-phenylpropionic acid and 3-phenylpropionic acid were weak inhibitors, and L-2-hydroxy-3-phenylpropionic acid did not inhibit this transport.
Studies on Exit of Tyrosine-The loss of radioactivity from cells preloaded with L-[U-14C]tyrosine was much more rapid when nonradioactive tyrosine was present in the external medium at 37" (Fig. 3). This stimulation of exit occurred with NlE-115 cells which were not depleted of tyrosine and with C-6 glioma cells but did not occur when preloaded cells were incubated at 4' during the exit portion of the experiment. The stimulation of exit of L-[ U-14C]tyrosine from cells was dependent upon the concentration of nonradioactive tyrosine in the external medium (Fig. 4). Since the plateau in Fig. 4 occurred when 80% of the accumulated radioactivity had been excreted, the leveling off may reflect this limitation rather than the saturation of a process. However, the concentration of L-tyrosine in the external medium which gave half-maximum stimulation of exit was about 9 X 10m6 M, a value close to that found for the K t (Table I), although not equivalent to a KL as discussed below.
A number of compounds were tested for their effects on exit of radioactivity from cells which were preloaded for 4 min with of inhibitor, v = velocity of transport in absence of inhibitor, and Z = concentration of inhibitor. NlE-115 (subculture 15), 2.1 X lo6 cells, and 325 pg of protein per well were incubated for 4 min in 2.75 X lo+ M L-[UJ4C]tyrosine (final specific activity 41 mCi per mmole). After this preloading with radioactive tyrosine, the 0.2 ml of incubation medium was removed and was replaced with 1 ml of phosphate-buffered saline containing the indicated concentrations of nonradioactive tyrosine. After another 4 min, this 1 ml of medium was removed, and cells were harvested with 1 ml of 0.1 N NaOH and counted for radioactivity as described under "Methods and Materials." The results were calculated from the difference between the radioactivity recovered from cells incubated for 4 min with 1 ml of phosphate-buffered saline and the radioactivity recovered from cells after incubation with 1 ml of phosphatebuffered saline containing the nonradioactive tyrosine.  L-[ U-Wltyrosine (Table V). All compounds which markedly stimulated exit of radioactive tyrosine were inhibitors of tyrosine transport (Tables  IV and V). There was little correlation between the potency of a compound in stimulating exit and its potency in inhibiting transport. Thus, nL-3-amino-3-phenylpropionic acid which was not a potent inhibitor of tyrosine transport (Ki = 50 X 1OW M) had a marked effect on exit. L-Alanine, tyramine, and I-norepinephrine did not have an effect on exit (Table V) but did have an effect on tyrosine transport (Table IV). No compound was found that caused an inhibition of exit. Pate of Transported L-[ U-W] Tyrosine-About 2 % of the radioactive tyrosine which was transported into NlE-115 cells was incorporated into a trichloroacetic acid precipitate, and the great majority of the tyrosinc remained unmetabolized during a 4-min incubation at 37" (Table VI).
Although this clone has very high levels of tyrosine hydroxylasc activity as measured by enzyme assay (3) the rate of tyrosine hydroxylation in intact cells is very slow," and, therefore, the formation of L-3,4-dihydroxyphcnylalanine from L-tyrosinn in living cells is not measurable with short incubation periods with the USC of a low specific activity precursor.

DI.SClJSSION
The data indicate that tyrosinc transport into mouse ncuroblastoma clone NIE-115 is by facilitated diffusion (6). Thus, this transport of tyrosine is by x saturable process (Table I) (1, 2), is not energy-dependent (Table II), is inhibited by a reagent which attacks sulfhydryl groups (Table II), is inhibited by molccules which are structurally analogous (Table IV, Fig. 2), and exhibits exchange with other molecules (Table V). In addition, the specific transport of tyrosine into these cultured cells is not dependent on sodium to a major degree and is not sensitive to pH (range 6 to 8). These latter characteristics, along with the very strong exchanging properties, are characteristics of the leu-tine-preferring (L) system described by 8). The inhibition of tyrosine transport into NlE-115 by such compounds as L-isoleucine, L-leucine, L-methionine, L-tryptophan, and ti-valine is consistent with the conclusion that tyrosine is transported into these cells by the L system and is somewhat similar to findings on the uptake of tyrosine by rat brain (22). N-Monomethylation of certain amino acids reduces or eliminates transport by the L system (23), and these results indicate the importance of the a! amino group for transport by this mechanism (24). Our results support this, since compounds which differed from L-phenylalanine in the a: position of the molecule showed marked diminution in their ability to inhibit tyrosine transport (Table IV and Fig. 2).
The stimulation of exit of a radioactively labeled compound from inside a cell by the same nonradioactively labeled compound outside a cell has been termed the tram effect (25) and is evidence in support of the mobile transport carrier hypothesis (6, 25). This hypothesis which does not fit all the data on transport (26, 27) states, in part, that the loaded and unloaded carriers cross the cell membrane and that the rate of transit of the loaded carrier can be different from that of the unloaded carrier.
In the mathematical formulation for a simple symmetrical mobile carrier, Stein has shown (6) that the trans effect will occur only when the ratio, r, of the rate of movement of loaded to unloaded carrier is greater than but not equal to unity.
Further, the concentration of substrate at one-half maximum velocity of transport (KI) would be equivalent to the concentration of substrate at one-half maximum stimulation of exit only when T = 1, a condition which would be incompatible with the observation of the trans effect for tyrosine.
Thus, the experimentally determined half-saturation concentration for stimulation of exit (Fig. 4) is not equivalent to a Kt.
The competitive inhibition of tyrosine transport by phenylalanine is evidence for a proposed mechanism for the reduced levels of catecholamines and their metabolites in phenylketonuria (28,29,30). Recently, it was shown that in rats in V&J dopamine and its metabolite 3-methoxy-4-hydroxyphenylacetic acid are derived from tyrosine (31, 32). The high concentrations of Lphenylalanine (millimolar range) obtained in the serum of phenylketonuric patients (33) is adequate to block a major portion of the transport of the catecholamine precursor, tyrosine, into the neuron and, therefore, reduce the synthesis of catecholamines.
That the transport of tyrosine into mouse neuroblastoma clone NlE-115 is similar to the leucine-preferring system described for other cell types corroborates our previous results (1,2) indicating a lack of specificity of tyrosine transport with cell type. It is also of interest to find that yet another cell type has this L system. However, an understanding of the characteristics of tyrosine transport will be useful to studies on the synthesis of catecholamines from the precursor tyrosine.
Thus, for esample, we have shown that in addition to being a potent inhibitor of tyrosine hydroxylation (34), L&iodotyrosine is a potent inhibitor of tyrosine transport.
With this knowledge of the characteristics of tyrosine transport, we have begun our studies on the hydroxylation of tyrosine after it has been transported into this clone.