A possible role for tyrosine kinases in the regulation of the neuronal dopamine transporter in mouse striatum

Neuroscience Letters 224 (1997) 201–205

A possible role for tyrosine kinases in the regulation of the neuronal dopamine transporter in mouse striatum J.R. Simon a , b , e ,*, D.J. Bare d, B. Ghetti a , d , e, J.A. Richter a,c,e a

Department of Psychiatry, Indiana University School of Medicine, Institute of Psychiatric Research, Indianapolis, IN 46202, USA b Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA c Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA d Department of Pathology and Laboratory Medicine (Division of Neuropathology), Indiana University School of Medicine, Indianapolis, IN 46202, USA e The Program in Medical Neurobiology, Indiana University School of Medicine, Institute of Psychiatric Research, Indianapolis, IN 46202, USA Received 5 November 1996; revised version received 30 January 1997; accepted 5 February 1997

Abstract The present investigation was undertaken to test the hypothesis that a reduction in the activity of protein tyrosine kinases would result in an alteration in dopamine transport. Genistein, a broad-spectrum inhibitor of protein tyrosine kinases, inhibited dopamine uptake into mouse striatal homogenates with an IC50 of 18 mM. The inhibition by genistein was rapid, reversible and somewhat selective, in that genistein did not inhibit the uptake of choline or GABA under similar conditions. Kinetic analyses indicated that genistein was a noncompetitive inhibitor. Another protein tyrosine kinase inhibitor, tyrphostin 23, also inhibited transport but was significantly less potent than genistein. Tyrphostin 25 and lavendustin A were without major effect on dopamine uptake. In addition, the inactive structural analog of genistein, genistin, had no significant effect on dopamine uptake. The inhibition of dopamine transport by 50 mM genistein was accompanied by a reduction in the level of a 110-kDa band of tyrosine phosphoprotein. It is suggested that protein tyrosine kinases play a role in the cascade of events which ultimately lead to regulation of neuronal dopamine transport.  1997 Elsevier Science Ireland Ltd. Keywords: Dopamine: Dopamine transporter; Tyrosine kinase; Genistein; Dopamine uptake

The primary mechanism for inactivation of dopamine (DA) following its release into the synapse is believed to be reuptake via the high affinity uptake system [12]. The high affinity uptake of dopamine into nerve terminals is mediated by the DA transporter (DAT) which is located in the nerve terminal plasma membrane. In recent years, cDNA for the DAT has been cloned from rat, bovine and human brain [9,14,22,26]. Based on the oligonucleotide sequence, the deduced protein structure appears to have 12 transmembrane spanning domains and several consensus phosphorylation sites for various serine/threonine kinases, e.g. protein kinase A (PKA), protein kinase C (PKC) and calcium/calmodulin kinase [10]. These putative phosphor-

* Corresponding author. Tel.: +1 317 2744730; fax: +1 317 2741365; e-mail: [email protected]

ylation sites may play a role in the regulation of transport activity. There is some evidence that protein kinase C activity may play a role in regulating the activity of the DAT in synaptoneurosomes from rat striatum [7]. Additionally, when the rat DAT was expressed in COS cells, activation of PKC by a phorbol ester resulted in a decrease in DA transport in a staurosporin-sensitive manner [15]. The possible involvement of PKA in regulating the DAT has also been investigated. In rat striatal preparations, activation of PKA had no effect on DA uptake [24] whereas in hypothalamic cell culture, PKA activation was found to enhance the uptake of DA [13]. In addition to the well studied serine/threonine phosphorylation of neuronal proteins (for review see [28]), tyrosine phosphorylation is also known to take place via the activity of protein tyrosine kinases (for review see [27]). Tyrosine

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phosphorylation is believed to be involved in synaptogenesis and synaptic plasticity [4,8], as well as in the regulation of growth and guidance of axonal growth cones [3,11]. More recent evidence suggests the possible involvement of protein tyrosine kinases and tyrosine phosphorylation in long-term potentiation [20], in regulation of the NMDA receptor [29] and in the regulation of astroglial nitric oxide synthase [5]. The effects of protein tyrosine kinase inhibition have been investigated on various neurobiological systems. The specific inhibitor genistein [1] has been shown to block the induction of long-term potentiation in the hippocampus [20] and to facilitate neurite outgrowth in PC12 cells [19]. We have found that genistein increases the in vitro release of endogenous DA from mouse striatal slices [2]. In the present study, we investigated the potential involvement of protein tyrosine kinases in the regulation of DA transport in mouse striatal homogenates and have tested the hypothesis that a reduction in tyrosine kinase activity would result in an alteration in DA transport. Genistein, genistin and the tyrphostins were obtained from Sigma Chemical Co. (St. Louis, MO, USA); lavendustin and GBR12909 were obtained from Research Biochemicals Int. (Natick, MA, USA). The following tritiated substrates were purchased from New England Nuclear (Boston, MA, USA): 3,4-[ring2,5,6– 3H]dihydroxyphenylethylamine; g-[2,3– 3H(N)]aminobutyric acid; [methyl-3H]choline chloride. Mice were sacrificed by decapitation and the brains were removed and placed on a petri dish on ice. Striata were dissected bilaterally, weighed and homogenized in 160 volumes of 0.32 M sucrose. Uptake of dopamine was determined as previously described [23] with slight modifications. Briefly, 50 ml of the sucrose homogenate was added to 450 ml of Krebs–Ringer phosphate buffer (pH 7.4) containing approximately 10 nM [3H]dopamine (final specific activity, 37.5–56.5 Ci/mmol), and any drugs as indicated. Following a 2 min incubation at 30°C, uptake was terminated by centrifugation at 6000 × g for 15 min at 4°C. Supernatants were aspirated and the pellets were surfacewashed with 2 ml of ice-cold saline. Pellets were dissolved in 0.5 ml of 0.1 M NaOH and radioactivity was determined by liquid scintillation spectrometry following the addition of 5 ml of Cytoscint (ICN, Costa Mesa, CA, USA). Blank values for dopamine transport were determined by incubating samples in the presence of 0.5 mM GBR12909. Kinetic analyses of DA uptake under these conditions have yielded a Km value of 151 ± 7.6 nM (n = 11) and a Vmax value of 196 ± 10 pmol/2 min/mg protein (n = 11). For the uptake of GABA and choline, conditions identical to those for DA uptake were employed, except the final concentrations of the tritiated substrates were 5 nM (GABA final specific activity, 100 Ci/mmol; choline chloride final specific activity, 86.8 Ci/mmol). Blank values for choline and GABA uptake were determined by incubating samples at 4°C. The Krebs–Ringer phosphate buffer (pH 7.4) had the fol-

lowing composition: 126 mM NaCl, 4.75 mM KCl, 1.3 mM CaCl2, 15.8 mM Na2HPO4, 1.4 mM MgCl2 and 10 mM dextrose. When drugs were present during the uptake assay, they were added from 50-fold concentrated stock solutions prepared with 50% DMSO. The final concentration of DMSO during the uptake was thus 1%, a concentration which, in preliminary studies, was determined to have no effect on DA uptake. Protein was determined on an aliquot of the homogenate by the method of Lowry et al. [17] using BSA for standards. Western blot analyses were performed on samples which were generated under the identical conditions and at the same time as those samples used in the measurement of [3H]DA transport. Briefly, samples were incubated as described above (in the absence of [3H]DA), and following centrifugation at 6000 × g for 15 min, the pellets were extracted with 50 ml of a modified RIPA lysis buffer consisting of 50 mM Tris–HCl (pH 7.4); 150 mM sodium chloride; 1.0% (v/v) Nonidet-P40; 0.5% (w/v) sodium deoxycholate; 0.1% (w/v) sodium dodecyl sulfate (SDS); 0.11 TIU/ml aprotinin; 0.01% (w/v) leupeptin. The samples were kept on ice for 30 min and centrifuged at 10 000 × g for 10 min at 4°C. The supernatant was retained and stored at −70°C. Aliquots of the samples were assayed for protein concentration. Extracted proteins (25 mg) were separated by electrophoresis on 7.5% SDS polyacrylamide mini-gels and electrotransferred to a nitrocellulose membrane (MiniProtean II system of Bio-Rad, Richmond, CA, USA). In all cases, equivalent protein loading was confirmed by staining of the blots with Ponceau S. The membrane was blocked for 3 h with 2% BSA in rinsing buffer containing 10 mM Tris– HCl (pH 7.4), 150 mM NaCl, 0.05% (v/v) Tween 20, and 0.05% (v/v) Nonidet-P40, and then incubated for 12 h in the blocking buffer with the mouse monoclonal anti-phosphotyrosine antibody 4G10 at 1.0 mg/ml (Upstate Biotechnology Inc., Lake Placid, NY, USA). The membrane was washed thoroughly in rinsing buffer, incubated with a horseradish peroxidase-conjugated secondary antibody, washed again and processed for protein detection using enhanced chemiluminescence (ECL-Amersham, Arlington Heights, IL, USA). Phosphoproteins were visualized on Kodak XOMAT AR film with exposures adjusted such that the response was within the linear range of the film. Total band density was measured using an AIC microimage analysis system (Analytical Imaging Concepts, Inc., Atlanta, GA, USA). High range (mol. wt. 36 000–205 000) unstained protein molecular weight markers were used (Sigma Chemical Co., St. Louis, MO, USA). The specificity of the antiphosphotyrosine antibody used in this study was demonstrated previously in our laboratory [2]. All animal use procedures were conducted in strict accordance with the NIH ‘Guide to the Care and Use of Laboratory Animals’ and were approved by the Indiana University Institutional Animal Care and Use Committee. The uptake of DA was measured at various times, from

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Fig. 1. Dixon plot for genistein inhibition of DA uptake. Striatal homogenates were incubated in the presence of varying concentrations of [3H]DA (18 nM, B; 48 nM, X; 108 nM, O)and in the absence and presence of varying concentrations of genistein. Ordinate values are in (pmol/2 min/ mg protein)−1. The experiment was repeated three times, and the data shown are from a single experiment.

0–2 min, both in the absence and presence of 20 mM genistein. At all times investigated, the inhibition of DA uptake caused by genistein was equivalent, and amounted to approximately 41–45%. When genistein was removed (by centrifugation and resuspension) following a 10 min preexposure, inhibition of DA transport was no longer demonstrable, indicating a rapidly reversible effect of the drug (data not shown). The rapidity of the genistein effect is consistent with the relatively high lipid solubility of this agent. Genistein produced a concentration-dependent reduction in DA transport over a concentration range of 5–50 mM, with inhibition of uptake ranging from approximately 20% at 5 mM genistein to about 80% at 50 mM genistein. In these experiments, the estimated IC50 was determined to be 18 mM. Kinetic analyses of the genistein inhibition of DA uptake revealed a Ki of 16.3 ± 1.2 mM (n = 3). The observation of a common intercept on the abscissa in this particular plot, suggests that the inhibition is non-competitive with DA (Fig. 1). In addition to genistein, three other protein tyrosine kinase inhibitors were investigated for their ability to alter DA uptake. Tyrphostin 23, although considerably less potent than genistein, was capable of inhibiting DA transport at concentrations ranging from 50–200 mM, whereas lavendustin A and tyrphostin 25 were without effect at 50 and 100 mM, respectively (Table 1). Genistin, a non-active structural analogue of genistein, was also without effect on DA transport at concentrations up to 200 mM (data not shown). When choline or GABA uptake was examined in the absence and presence of 20 mM genistein, no changes were observed in the transport of either substrate, suggesting that the genistein inhibition of DA uptake was not merely a

result of a non-specific membrane effect, and was at least somewhat selective for DA transport (Table 2). The effect of 50 mM genistein on the tyrosine phosphorylation of prominent striatal phosphoproteins was quantitatively evaluated by immunoblotting with anti-phosphotyrosine antibodies, followed by densitometry. Tissue proteins were extracted after the usual procedure for the assay of DA uptake either in the presence or absence of genistein. Several phosphotyrosine-modified proteins were detected in striatal tissue extracts ranging from approximately 180–30 kDa (Fig. 2). The two most prominent phosphoproteins detected were proteins of an approximate Mr of 180 000 and 110 000 as previously described for the striatum and other brain areas [2,8]. The 110-kDa immunoreactivity migrated as a broad band and appears to represent more then a single protein [2]. A densitometric comparison of autoradiographic bands in three separate experiments revealed that treatment with genistein on average reduced the level of protein phosphorylation of the 110-kDa band by 20% (Fig. 2). No significant changes were evident in the other lower molecular weight bands, suggesting some selectivity of the kinase inhibitor. No phosphoprotein band was detected in the region where the DAT would be expected to migrate (60–80 kDa). The results of the present study clearly indicate that genistein, an inhibitor of protein tyrosine kinases, inhibits DA transport in mouse striatal preparations. This inhibition by genistein is rapid, reversible and somewhat selective for the DA transporter, as it is ineffective in inhibiting the GABA and choline transporters under conditions similar to those used for DA transport. Furthermore, the inhibition of DA transport is accompanied by a reduction in the level of tyrosine phosphorylation of a 110-kDa protein band. Table 1 Effect of protein tyrosine kinase inhibitors on DA uptake in mouse striatal homogenates Inhibitor Tyrphostin 23 25 mM 50 mM 100 mM 200 mM Tyrphostin 25 25 mM 50 mM 100 mM Lavendustin A 0.25 mM 0.5 mM 1.0 mM 25 mM 50 mM

DA uptake (percent control) 93 82* 69* 42* 106 107 108 112 121 116 114 96

Striatal homogenates were incubated at 30°C for 2 min in the presence of 10 nM [3H]DA in the absence and presence of the indicated concentrations of inhibitor. Values represent mean % of control uptake for 3–4 determinations. The mean control value was 8.54 ± 0.49 pmol/2 min/mg protein (n = 9). *P , 0.05 compared to control.

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Table 2 Effect of genistein on choline, GABA and DA uptake in mouse striatal homogenates Condition

Choline uptake

GABA uptake

DA uptake

Control Genistein (20 mM)

0.48 ± 0.05 0.45 ± 0.01

1.3 ± 0.15 1.1 ± 0.14

11.9 ± 0.41 6.6 ± 0.16*

Striatal homogenates were incubated at 30°C for 2 min in the presence of either 5 nM [3H]choline chloride, 5 nM [3H]GABA or 10 nM [3H]DA in the absence and presence of 20 mM genistein. Uptake values are reported as pmol/2 min/mg protein and represent means ± SEM from 3–4 experiments. *P , 0.05.

In the conditions used for in vitro uptake assays, any apparent inhibition of transport could be a result of stimulated release of the substrate. Thus, it is important to document that the putative inhibition of uptake is not, in fact, a stimulation of efflux. In the case of genistein, we have previously shown that this agent is, in fact, capable of stimulating the release of endogenous DA from striatal slices [2], but at much higher concentrations than those utilized in the present study. In our previous study on DA release, significant effects of genistein were not observed at concentrations less than 100 mM, whereas in the present study, the IC50 for genistein inhibition of DA uptake was approximately 18 mM. Thus, it is unlikely that the inhibition of radiolabeled DA uptake is a result of a genistein-induced increase in DA efflux, but rather appears to be the result of an effect of genistein on the activity of the DAT. Conversely, the DAreleasing effect of higher genistein concentrations was not a result of DAT inhibition [2]. In addition, genistin, an inactive structural analog of genistein, was ineffective in altering DA uptake, further suggesting a specific role for protein tyrosine kinases, and ruling out an effect related to the general structure of isoflavone compounds. The apparent non-competitive nature of the genistein inhibition of DA uptake also argues against the notion that genistein is merely increasing the efflux of DA. If this were the case, one would expect isotopic dilution of the [3H]DA by endogenous stores, and an ‘apparent’ increase in the Km of transport. The lack of change in Km coupled with an alteration in Vmax of DA uptake is consistent with the notion that the effects of genistein are a result of either a reduced number of transporters operating at a normal rate, or a normal compliment of transporters operating at a reduced rate. Experiments designed to determine the effects of genistein on ligand binding to the DAT (in the absence of transport) may help to resolve which of these possibilities is correct. The lack of significant inhibition of DA uptake by some of the other protein tyrosine kinase inhibitors was unexpected. Tyrphostin 25 was ineffective in inhibiting DA transport at concentrations up to 100 mM, despite its known ability to inhibit protein tyrosine kinase activity [6,18]. One possible explanation for its lack of inhibitory effect on DA uptake may be related to the potential chemical instability of the compound. It has been observed that

the inhibition of protein tyrosine kinase activity by tyrphostin 25 corresponds to the chemical formation, over many hours, of products derived from the parent tyrphostin [21]. In the present experiments, the tyrphostin solutions were made fresh daily and incubation with inhibitor during the uptake assay was for only a brief period (2 min at 30°C). Under these conditions, there would not be sufficient time for the active moiety to be generated. Additionally, lack, or relative lack of inhibition of DA uptake by tyrphostin 25, and especially by lavendustin A, may be explained by the relative selectivity of the various protein tyrosine kinase inhibitors. Thus, genistein is considered to be a broad-spectrum, relatively non-selective protein tyrosine kinase inhibitor [16], and as such, may be better suited to inhibit an appropriate tyrosine kinase(s) in a multi-component pathway leading to the inhibition of the DAT. Whereas genistein has been shown to be capable of inhibiting some serine/ threonine kinases [1,20,25], it is most likely that the inhibitory effects observed in the present study would be limited to the tyrosine kinases. Thus, Akiyama et al. [1] reported that the concentration of genistein required to inhibit PKA or PKC by 50% was greater than 100 mg/ml (.370 mM), whereas that required to inhibit protein tyrosine kinases by 50% was in the range of 6–8 mg/ml (22–29 mM). The possibility that protein tyrosine kinases are not involved in the genistein-induced inhibition of DA uptake should be considered. Thus, it is possible that the alterations seen in the tyrosine phosphorylation on western blots in the present study are unrelated to the inhibition of DA uptake. If this is indeed the case, then inhibition of DA uptake by genistein would have to be attributable to some other effect

Fig. 2. Effect of genistein on the level of phosphorylation on tyrosine residues in striatal extract proteins evaluated by immunoblotting with anti-phosphotyrosine antibodies. Tissue proteins were extracted after the usual procedure for the assay of dopamine uptake. Proteins (25 mg) were separated on 7.5% SDS-polyacrylamide mini-gels and electrotransferred to nitrocellulose membrane. Equivalent protein loading was confirmed by staining with Ponceau S (not shown). The membrane was probed with the primary antibody 4G10 and immunoreactive bands were visualized on X-ray film using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Mr, migration of unstained protein molecular weight standards ( × 10−3). C, control; G, genistein treated. pp180 and pp110 were the two prominent striatal phosphoproteins which upon analysis demonstrated a decrease in phosphotyrosine immunoreactivity. This decrease of phosphotyrosine caused by genistein was reproduced in three independent experiments.

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