L-DOPA promotes striatal dopamine release through D1 receptors and reversal of dopamine transporter

Riccardo Viaro a,b, Francesco Longo a,1, Fabrizio Vincenzi c, Katia Varani c, Michele Morari a,*

Abstract

Previous studies have pointed out that L-DOPA can interact with D1 or D2 receptors independent of its conversion to endogenous dopamine. The present study was set to investigate whether L-DOPA modulates dopamine release from striatal nerve terminals, using a preparation of synaptosomes preloaded with [3H]DA. Levodopa (1 µM) doubled the K+-induced [3H]DA release whereas the D2/D3 receptor agonist pramipexole (100 nM) inhibited it. The L-DOPA-evoked facilitation was mimicked by the D1 receptor agonist SKF38393 (30–300 nM) and prevented by the D1/D5 antagonist SCH23390 (100 nM) but not the DA transporter inhibitor GBR12783 (300 nM) or the aromatic L-amino acid decarboxylase inhibitor benserazide (1 µM). Higher L-DOPA concentrations (10 and 100 µM) elevated spontaneous [3H]DA efflux. This effect was counteracted by GBR12783 but not SCH23390. Binding of [3H]SCH23390 in synaptosomes (in test tubes) revealed a dense population of D1 receptors (2105 fmol/mg protein). Both SCH23390 and SKF38393 fully inhibited [3H]SCH23390 binding (Ki 0.42 nM and 29 nM, respectively). L-DOPA displaced [3H]SCH23390 binding maximally by 44% at 1 mM. This effect was halved by addition of GBR12935 and benserazide. We conclude that L-DOPA facilitates exocytotic [3H]DA release through SCH23390-sensitive D1 receptors, independent of its conversion to DA. It also promotes non-exocytotic [3H]DA release, possibly via conversion to DA and reversal of DA transporter. These data confirm that L-DOPA can directly interact with dopamine D1 receptors and might extend our knowledge of the neurobiological mechanisms underlying L-DOPA clinical effects.

Keywords:
Dopamine release
D1 receptors
Dopamine transporter
L-DOPA SCH-23390
Synaptosomes

1. Introduction

Replacement therapy with levodopa (L-DOPA) remains the most effective treatment for Parkinson’s disease (PD). L-DOPA therapeutic action relies on its enzymatic conversion to dopamine (DA) by aromatic L-amino acid decarboxylase (AADC), which occurs in dopaminergic and non-dopaminergic striatal terminals or striatal neurons (Arai et al., 1994; Hefti et al., 1981; Ng et al., 1970). Nonetheless, different lines of evidence suggest that, in addition to serving as DA precursor, endogenous L-DOPA may act as a neurotransmitter or neuromodulator (Misu and Goshima, 1993). Indeed, neurons containing L-DOPA as end-product (i.e. positive for tyrosine hydroxylase while not expressing AADC) have been detected in the rat brain (Kitahama et al., 1988; Mons et al., 1989; Tison et al., 1989) and neurotransmitter-like release of L-DOPA has been found (Goshima et al., 1988; Misu et al., 1990). Consistently, L-DOPA has been found to stimulate D1, D2 or beta-adrenergic receptors without being converted to DA (Aceves et al., 1991; Goshima et al., 1986, 1991; Nakamura et al., 1994; Silva et al., 2006). Although these effects of L- DOPA have been registered both at pre- and postsynaptic levels, a significant number of studies have demonstrated that L-DOPA modulates DA and noradrenaline (Chang and Webster, 1995; Goshima et al., 1986; Misu et al., 1986), glutamate (Goshima et al., 1993) and GABA (Aceves et al., 1991) release in the brain, independent of its conversion to DA and purportedly through activation of presynaptic receptors. Nonetheless, one main limitation of these studies is that they were conducted in brain slices thus making quite difficult to dissect out presynaptic actions from network (polysynaptic) effects. For this reason, we set to investigate the neurochemical effects of L-DOPA in a preparation of superfused striatal synaptosomes preloaded with [3H]DA (Longo et al., 2017; Marti et al., 2003b; Mercatelli et al., 2019). The superfusion conditions emphasize L- DOPA effects on presynaptic receptors, minimizing indirect effects due to endogenous DA formed through L-DOPA uptake and decarboxylation into nerve terminals. In this preparation, we analyzed both spontaneous (Ca2+-independent) and K+-stimulated (Ca2+-dependent) [3H]DA release (Marti et al., 2003b), comparing the effect of L-DOPA with those of the D2/D3 receptor agonist pramipexole and D1 receptor agonist SKF38393. Moreover, since neurochemical data suggested that L-DOPA stimulated D1 receptors, a binding study in a synaptosomal preparation was conducted.

2. Results

Basal synaptosomal [3H]DA efflux was 0.021 ± 0.001 pmol/ mg tissue /min (n = 68) and corresponded to a fractional release (FR; tritium efflux expressed as percentage of the tritium content in the filter at the onset of the corresponding collection period) of 6.79 ± 0.15%. A 2 min pulse of 10 mM K+ evoked a tritium overflow of 0.006 ± 0.001 pmol/mg tissue/min (n = 24), which was attenuated by ~70% in the absence of Ca2+ (Fig. 1A). Firstly, the effect of striatal D2 receptor activation was evaluated using the D2/D3 receptor agonist pramipexole (W4,12.10 = 67.02, p < 0.0001; Fig. 1A). Pramipexole (100 nM) halved the K+-induced tritium overflow and this effect was prevented by pre- treatment with the D2/D3 receptor antagonist amisulpride (100 nM), which was per se ineffective. In parallel, the effect of D1 receptor activation was also assessed (W5,13.68 = 8.57, p < 0.0001; Fig. 1B). The D1 receptor agonist SKF38393 (30–300 nM) increased [3H]DA overflow, and pre-treatment with SCH23390 (100 nM), ineffective per se, suppressed this modulation. Superfusion of synaptosomes with L-DOPA qualitatively replicated SKF38393 profile (W4,11.53 = 11.30, p = 0.0006; Fig. 1C). In fact, L-DOPA (1 μM) doubled tritium overflow, an effect prevented by SCH23390 (100 nM). The DA transporter (DAT) inhibitor GBR12783 was per se ineffective (W5,13.44 = 20.46, p < 0.0001; Fig. 1D) and did not attenuate the increase of [3H]DA overflow evoked by L- DOPA (1 μM). This effect was unaltered also in the presence of the AADC inhibitor, benserazide, indicating it was not due to the conversion of L- DOPA into DA by AADC.
In order to more thoroughly evaluate the contribution of L-DOPA to synaptosomal DA release, L-DOPA effect on spontaneous [3H]DA efflux was also monitored. Striatal [3H]DA efflux in our preparation was previously shown to be essentially unaffected by Ca2+ removal and tetrodotoxin application (Marti et al., 2003b). L-DOPA increased tritium efflux in a concentration-dependent manner (W3,9.41 = 24.97, p < 0.0001; Fig. 2A) being effective at concentrations higher than 1 µM. GBR12783 (300 nM) prevented the response to 10 µM L-DOPA (W5,12.73 = 29.38, p < 0.0001; Fig. 2B) and attenuated that to 100 µM L-DOPA (W5,13.06 = 21.11, p < 0.0001; Fig. 2C) while SCH23390 (1 µM) was ineffective. GBR12783 and SCH23390 alone did not affect spontaneous tritium efflux at the concentrations tested.
The SCH23390-sensitive effect of L-DOPA (1 µM) on stimulated tritium overflow suggested an interaction of L-DOPA with D1/D5 receptors. To confirm this interaction, binding experiments were performed. Saturation binding experiments of [3H]SCH23390 were performed in mouse striatal synaptosomes to investigate the affinity (KD) and density (Bmax) of D1/D5 dopamine receptors. The saturation curve of [3H]SCH23390 and the relative Scatchard plot revealed a KD value of 0.72 ± 0.05 nM and a receptor density of 2105 ± 69 fmol/mg protein (Fig. 3A-B). Competition binding experiments were performed in mouse striatal synaptosomes to evaluate the possible interaction of L- DOPA with D1 receptors in comparison with the D1/D5 antagonist SCH23390 and the D1/D5 agonist SKF38393 as reference compounds. As expected, SCH23390 showed a good affinity towards D1/D5 receptors with a Ki value of 0.42 ± 0.02 nM (Fig. 3C). The D1 agonist SKF38393 inhibited [3H]SCH23390 binding with a Ki of 29.1 ± 2.0 nM. Interestingly, L-DOPA was able to partially displace [3H]SCH23390 with a 44% maximal inhibition of specific binding at 1 mM (Fig. 3C). Since binding experiments were performed in test tubes, the possibility was investigated that endogenous DA could contribute to SCH23390 displacement from D1 receptors. Therefore, displacement curve was retested in the presence of GBR12783 (to block DAT) and benserazide (to block AADC). Under these conditions, L-DOPA still displaced [3H] SCH23390 binding, although less effectively and at higher concentrations than in the absence of DAT and AADC inhibitors.

3. Discussion

Previous studies in striatal slices showed that L-DOPA concentration- dependently facilitated spontaneous and stimulus-evoked DA release (Chang and Webster, 1995; Misu et al., 1986). Interestingly, when slices were treated with an AADC inhibitor the L-DOPA profile became biphasic, low concentrations (30 nM) facilitating and higher ones (1 µM) inhibiting impulse-evoked DA release (Misu et al., 1986). In the present study, pramipexole inhibited impulse-evoked synaptosomal DA release via amisulpride-sensitive D2 receptors whereas L-DOPA elevated it via SCH23390-sensitive D1 receptors. Therefore, it could be speculated that beside the well-characterized D2 autoreceptors, dopaminergic terminals are endowed also with facilitatory D1 autoreceptors. Indeed, rare presynaptic D1 receptors have been detected on dopaminergic striatal nerve terminals (Hersch et al., 1995), and D1 receptors have been found to co- localize not only with choline acetyltransferase-positive (i.e. cholinergic) and glutamic acid decarboxylase-positive (i.e. GABAergic) but, less intensely, also with striatal tyrosine hydroxylase-positive (i.e. dopaminergic and noradrenergic) striatal nerve terminals (Wu et al., 2006). Moreover, activation of presynaptic D1 receptors stimulated Ca2+ levels in striatal synaptosomes in a SCH23390-sensitive manner (Wu et al., 2006). However, it is well known that striatal D1 receptors are mainly postsynaptic or located presynaptically on non-dopaminergic nerve terminals (heteroreceptors) (Fremeau et al., 1991; Hersch et al., 1995; Wu et al., 2006), thus a significant contribution of postsynaptic D1 receptors would account for the dense D1 receptor binding observed in our synaptosomal preparation. Indeed, in this synaptosomal preparation postsynaptic membranes often remain attached to the “active site” of presynaptic terminals (Gulyassy et al., 2020; Miklosi et al., 2018´ ), suggesting that postsynaptic D1 receptors might be involved in the L-DOPA- induced [3H]DA release. Interestingly, stimulation of postsynaptic D1 receptors on medium-sized spiny neurons elevated striatal glutamate release via inhibition of retrograde endocannabinoid release (Andre et al., 2010). Whether this is also the case for DA release in our preparation remains to be determined. Therefore, although we cannot dissect out whether pre- or postsynaptic SCH23390-sensitive D1 receptors mediated the effect of L-DOPA, it clearly appears that L-DOPA can directly interact with D1 receptors without its conversion to dopamine. Whether such an effect contributes to the therapeutic and side-effects of L-DOPA is controversial. The effective concentrations of L-DOPA stimulating exocytotic DA release in the present study are similar to those (1 µM) measured in the CSF of PD patients after oral (Olanow et al., 1991) or i.v. (Woodward et al., 1993) L-DOPA administration, and very close to those measured in the striatum of marmosets (442 nM) orally administered with a therapeutic dose of L-DOPA (Zhang et al., 2003). However, previous studies showed that L-DOPA-mediated behaviors are inconsistently affected by AADC blockers. In some studies, L-DOPA-mediated contralateral circling (Melamed et al., 1984; Treseder et al., 2000) or dyskinesia (Buck and Ferger, 2008) were markedly reduced (albeit not suppressed) by blockade of central AADC whereas in others L-DOPA- induced turning was unaffected (Alachkar et al., 2010; Nakamura et al., 1994; Nakazato and Akiyama, 1989; Treseder et al., 2001). Moreover, L- DOPA was reported to induce dyskinesia independently of modulation of striatal DA levels (Navailles et al., 2011; Nevalainen et al., 2011; Porras et al., 2014), possibly via stimulation of somato-dendritic and presynaptic D1 receptors at striato-nigral medium-sized spiny neurons, leading to enhanced firing and/or nigral GABA release (Mela et al., 2012; Robertson and Robertson, 1989; Yamamoto et al., 2006). In fact, L-DOPA stimulation of [3H]GABA release (EC50 1 µM) from nigral slices of DA-denervated rats was observed in the presence of an AADC inhibitor, i.e. independent of its conversion to DA (Aceves et al., 1991).
Binding experiments in our synaptosomal preparation, however, revealed, a negligible binding of L-DOPA to D1 receptors, in agreement with previous study in D1-transfected cells (Toll et al., 1998). In our preparation, Ki values of SCH233390 (0.42 nM) and SKF38393 (29.1 nM) were in line with those previously reported in the rodent brain (SCH23390, 0.12–0.80 nM; SKF 38393, 18–41 nM) (Andersen, 1988; Neumeyer et al., 2003; Qandil et al., 2003; Watts et al., 1993). L-DOPA displaced [3H]SCH23390 binding but, different from classical D1 receptor ligands, only partially (40%). This profile is reminiscent of allosteric negative modulation (May et al., 2007) although the possibility that L-DOPA behaves as a negative allosteric modulator does not easily reconcile with its facilitatory effect on [3H]DA release. It is also possible that the SCH23390-sensitive D1 receptor identified in our preparation does not belong to the classical D1A receptor subtype, since more than one D1 receptor subtype has been cloned (Tiberi et al., 1991) and D1 receptors can heteromerize with DA and non-DA receptors (Casado- ´ Anguera et al., 2019; Fuxe et al., 2015) or couple with different G-proteins (Wang et al., 1995) generating receptor entities with unique pharmacological profiles (Undie et al., 1994). Nonetheless, we must consider that release and binding experiments were conducted under substantially different experimental conditions. Specifically, L-DOPA binding to D1 receptors was assessed in test tubes, where binding might be modulated by other molecules released and accumulated in the extracellular milieu. In fact, the [3H]SCH23390 binding displacement induced by L-DOPA was halved in the presence of DAT and AADC inhibitors, suggesting that occupation of D1 receptors is partly mediated by endogenous DA formed from L-DOPA and released from DA terminals through the reversal of DAT. Indeed, DAT reversal of spontaneous [3H] DA efflux by L-DOPA was observed in the present synaptosomal preparation. Different from stimulus-evoked tritium overflow, spontaneous [3H]DA efflux is Ca2+-insensitive and only slightly (~15%) tetrodotoxin-sensitive (Marti et al., 2003b), suggesting it mainly reflects non vesicular release and leakage from synaptosomes. Misu and coworkers (Misu et al., 1986) showed that, different from the stimulus- evoked DA release, the increase in spontaneous efflux induced by L- DOPA was prevented by an AADC inhibitor, i.e. relied on L-DOPA conversion to DA. Consistently, we found that GBR12783 was ineffective on stimulus-evoked [3H]DA overflow but prevented the elevation of [3H] DA efflux induced by high L-DOPA concentrations. These data support the view that L-DOPA is taken up into nerve terminals, decarboxylated to DA by AADC, and released via DAT reversal, confirming the in vivo evidence that L-DOPA can promote DA release through non vesicular mechanisms (De Deurwaerd`ere et al., 2017). Specifically, in vivo microdialysis showed that reverse dialysis of 5 µM L-DOPA into SN increased local DA levels via non vesicular mechanisms (Thorr´e et al., 1998), and systemic administration of L-DOPA (3–12 mg/Kg) elevated in vivo hippocampal and prefronto-cortical DA also after removal of Ca2+ from the perfusion medium, i.e. under conditions of impaired vesicular release (Miguelez et al., 2016). Altogether, these findings indicate that DAT reversal might occur in vivo at therapeutic L-DOPA concentrations, causing DA to stimulate D1 receptors.
In conclusion, this study presents the first evidence that low L-DOPA concentrations facilitate synaptosomal exocytotic DA release through direct stimulation of D1 receptors. Higher concentrations L-DOPA stimulate non vesicular DA release via L-DOPA conversion to DA and DAT reversal. These data add to previous evidence that L-DOPA can act as a neurotransmitter, and may offer new insights into the neurobiological mechanisms underlying L-DOPA therapeutic and side-effects, in particular L-DOPA-induced dyskinesia (Bastide et al., 2015; De Deurwaerd`ere et al., 2017).

4. Experimental procedure

4.1. Animal subjects

Young adult (8–10-week-old) male C57BL/6J mice (20–25 g) obtained from a colony set at the LARP facility of the University of Ferrara were used. Mice were housed with free access to food and water and kept under environmentally controlled conditions (12-h light/dark cycle with light on between 07:00 and 19:00). The experiments complied with the ARRIVE guidelines, were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC) and were approved by the Italian Ministry of Health and Ethical Committee of the University of Ferrara (license #ECE79.2.EXT.3). All efforts were made to minimize the number of animals used and their suffering.

4.2. Synaptosome preparation and [3H]DA analysis

Mice were anesthetized and sacrificed via cervical dislocation, and striatum was quickly excised to prepare synaptosomes, as previously described (Marti et al., 2001, 2003b; Morari et al., 1998). Striatum was homogenized in ice-cold 0.32 M sucrose (pH 7.4) with a Teflon-glass homogenizer and centrifuged at 800 × g for 10 min at 4 ◦C. The supernatant was then centrifuged at 11,000 × g for 20 min at 4 ◦C, the pellet resuspended in 1.5 mL oxygenated (95% O2, 5% CO2) Krebs solution (mM: NaCl 118.5, KCl 4.7, CaCl2 1.2, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 10) containing ascorbic acid (0.05 mM) and disodium EDTA (0.03 mM). Synaptosomes were incubated at 37 ◦C with 50 nM [3H]-DA (specific activity 40 Ci/mmol; Perkin-Elmer, Boston, MA, USA) for 25 min, after which 12 mL of pre-oxygenated Krebs were added (Longo et al., 2017; Marti et al., 2003a). One millilitre aliquots of the suspension (~0.35 mg protein) were slowly injected into nylon syringe filters (outer diameter 13 mm, 0.45 µM pore size, internal volume ~100 µL; Teknokroma, Barcelona, Spain), maintained at 36.5 ◦C in a thermostatic bath and superfused (0.4 mL/min) with a pre-carbogenated Krebs solution. Under these experimental conditions, spontaneous [3H]DA efflux was essentially unaffected by reuptake. Filters were washed for 20 min, after which sample collection was started (every 3 min). The effect of drugs was evaluated on both spontaneous and K+- stimulated neurotransmitter outflow. In this case, drugs were added to the perfusion medium 6 (agonist) or 9 (antagonist) min before a 10 mM K+ pulse (120 sec) and maintained until the end of the experiment. [3H] DA levels in the samples were measured by liquid scintillation spectrophotometry. Sample superfusate (1.2 mL/sample) and filter retained (dissolved with 1 mL of 1 M NaOH followed by 1 M HCl) were opportunely mixed with Ultima Gold XR scintillation fluid (Packard Instruments B.V., Groningen, The Netherlands) and radioactivity in the samples and in the filters was measured using a Perkin Elmer Tri Carb 2810 TR scintillation counter.

4.3. Saturation and competition binding experiments

Saturation binding experiments to D1 dopamine receptors were carried out by using [3H]SCH23390 as radioligand (specific activity 84.3 Ci/mmol) (Trampus et al., 1991). Mouse striatal synaptosomes were incubated for 60 min at 30 ◦C with different concentrations (0.1 nM-10 nM) of [3H]-SCH23390 in 50 mM Tris-HCl pH 7.4, 5 mM MgCl2. Non-specific binding was determined in the presence of 1 µM SCH23390 (Tocris, Bristol, UK) and was always < 10% of the total binding. Competition binding experiments were performed incubating mouse striatal synaptosomes with 1 nM of [3H]SCH23390 in 50 mM Tris-HCl pH 7.4, 5 mM MgCl2 for 60 min at 30 ◦C in the presence of increasing concentrations of SCH23390 (0.01 nM-1 µM), SKF38393 (0.1 nM-10 µM) or L-DOPA (1 nM-1 mM). Non-specific binding was determined in the presence of 1 µM SCH 23,390 and was always < 10% of the total binding. At the end of the incubation time, bound and free radioactivity was separated by filtering the assay mixture through Whatman GF/B glass fiber filters using a Brandel cell harvester (Brandel Instruments, Unterfohring, Germany). The filter bound radioactivity was counted ¨ using a Perkin Elmer Tri Carb 2810 TR scintillation counter. The protein concentration was determined according to a Bio-Rad method with bovine albumin as standard reference. Inhibitory binding constant values, Ki were calculated from the IC50 values according to the Cheng & Prusoff equation Ki = IC50/(1 + [C*]/KD*), where [C*] is the concentration of the radioligand and KD* its dissociation constant (Cheng and Prusoff, 1973).

4.4. Data and statistical analysis

Data, means ± SEM of 6 determinations per group, were expressed as absolute content (pmol/mg tissue/min), percent of basal tritium efflux (Fig. 2) or K+-evoked tritium overflow (Fig. 1). Tritium efflux was calculated as fractional release (FR, i.e. tritium efflux expressed as percentage of the tritium content in the filter at the onset of the corresponding collection period) whereas K+-evoked tritium overflow was calculated as net FR, i.e. tritium overflow as percent of the tritium content in the filter at the onset of the corresponding collection period. All values displayed a normal distribution (Kolmogorov-Smirnov test) but violate the assumption of homogeneity of variance (Bartlett’s test). Therefore, statistical analysis was performed (Prism software; San Diego, CA, USA) by Welch’s ANOVA on percent (Fig. 1) or area-under- the curve (AUC; Fig. 2) values followed by the Dunnett’s T3 test for multiple comparisons (Dunnett, 1980). Binding curves are representative of 4 independent experiments performed in duplicate. P values < 0.05 were considered statistically significant.

4.5. Materials

Amisulpride, benserazide, GBR12783 dihydrochloride, L-DOPA, SCH23390 hydrochloride and SKF38393 were purchased from Tocris Bioscience (Bristol, UK). Pramipexole hydrochloride was purchased from McTony Bio&Chem (Vancouver, Canada), [3H]DA and [3H] SCH23390 from Perkin Elmer (Boston, MA, USA). All drugs were freshly dissolved in Krebs solution just prior to use.

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