Advertisement
Research ArticleNeuropharmacology

Tetrabenazine Facilitates Exocytosis by Enhancing Calcium-Induced Calcium Release through Ryanodine Receptors

Ricardo de Pascual, Nuria Álvarez-Ortego, Cristóbal de los Ríos, Gema Jacob-Mazariego and Antonio G. García
Journal of Pharmacology and Experimental Therapeutics October 2019, 371 (1) 219-230; DOI: https://doi.org/10.1124/jpet.119.256560

Abstract

Vesicular monoamine transporter-2 is expressed in the presynaptic secretory vesicles membrane in the brain. Its blockade by tetrabenazine (TBZ) causes depletion of dopamine at striatal basal ganglia; this is the mechanism underlying its long-standing use in the treatment of Huntington’s disease. In the frame of a project aimed at investigating the kinetics of exocytosis from vesicles with partial emptying of their neurotransmitter, we unexpectedly found that TBZ facilitates exocytosis; thus, we decided to characterize such effect. We used bovine chromaffin cells (BCCs) challenged with repeated pulses of high K+. Upon repeated K+ pulsing, the exocytotic catecholamine release responses were gradually decaying. However, when cells were exposed to TBZ, responses were mildly augmented and decay rate delayed. Facilitation of exocytosis was not due to Ca2+ entry blockade through voltage-activated calcium channels (VACCs) because, in fact, TBZ mildly blocked the whole-cell Ca2+ current. However, TBZ mimicked the facilitatory effects of exocytosis elicited by BayK8644 (L-subtype VACC agonist), an effect blocked by nifedipine (VACC antagonist). On the basis that TBZ augmented the secretory responses to caffeine (but not those of histamine), we monitored its effects on cytosolic Ca2+ elevations ([Ca2+]c) triggered by caffeine or histamine. While the responses to caffeine were augmented twice by TBZ, those of histamine were unaffected; the same happened in rat cortical neurons. Hence, we hypothesize that TBZ facilitates exocytosis by increasing Ca2+ release through the endoplasmic reticulum ryanodine receptor channel (RyR). Confirming this hypothesis are docking results, showing an interaction of TBZ with RyRs. This is consonant with the existence of a healthy Ca2+-induced-Ca2+-release mechanism in BCCs.

SIGNIFICANCE STATEMENT A novel mechanism of action for tetrabenazine (TBZ), a drug used in the therapy of Huntington’s disease (HD), is described here. Such mechanism consists of facilitation by combining TBZ with the ryanodine receptor of the endoplasmic reticulum, thereby increasing Ca2+-induced Ca2+ release. This novel mechanism should be taken into account when considering the efficacy and/or safety of TBZ in the treatment of chorea associated with HD and other disorders. Additionally, it could be of interest in the development of novel medicines to treat these pathological conditions.

Introduction

The vesicular monoamine transporter (VMAT) uses a vesicle acidic pH gradient as a driving force to transport dopamine (DA), noradrenaline (NA), or serotonin [5-hydroxytryptamine (5-HT)] from the cytosol into the synaptic vesicle lumen (Henry et al., 1994). Two transporter subtypes have been characterized so far: VMAT1 localizes to the peripheral nervous system and central nervous system (CNS), while VMAT2 is expressed in vesicles at presynaptic nerve terminals of the CNS (Erickson et al., 1995; Benarroch, 2013). The classic blockers of VMAT are reserpine and tetrabenazine (TBZ). Reserpine unselectively and irreversibly blocks both VMAT1 and VMAT2, thereby depleting vesicle monoamine contents centrally and peripherally; as a consequence, reserpine, which was classically used as an antihypertensive medicine, causes depression, orthostatic hypotension, bradycardia, and diarrhea due to parasympathetic dominance. In contrast, by selectively blocking VMAT2 in the CNS, TBZ causes depletion of dopamine, NA, and 5-HT only centrally, thus precluding the peripheral side effects of reserpine. Since long, both drugs have been used in the treatment of chorea associated with Huntington’s disease (HD) and other hyperkinetic disorders; however, because of its higher safety, only TBZ is currently being used in the clinic (Jankovic, 2016).

For decades, reserpine has been widely used to deplete centrally the neurotransmitters NA and 5-HT to induce an animal model of depression and to develop antidepressive drugs (Hendley and Welch, 1975; Antkiewicz-Michaluk et al., 2014; Blasco-Serra et al., 2015; Ikram and Haleem, 2017). It has also extensively been used as a pharmacological tool to cause the depletion of NA from peripheral sympathetic nerve terminals (Mandela et al., 2010) and to elicit the depletion of NA and adrenaline from adrenal medullary chromaffin cells (CCs) (Dixon et al., 1975). Reserpine binds irreversibly to VMAT1 and VMAT2, thus blocking the sequestration of cytosolic monoamines, to gradually decrease their vesicle concentrations. Reserpine has also been used as an experimental tool to study how the vesicle content of catecholamines impinges in the final steps of exocytosis in adrenal CCs (Mundorf et al., 2000; Mark Wightman et al., 2018).

Following this research topic, we planned a study to investigate if TBZ, as reserpine does, could also depress the catecholamine release responses in bovine chromaffin cells (BCCs) repeatedly challenged with pulses of high potassium (K+). Although scarcely, VMAT2 is also expressed in CCs (Weihe et al., 1994), and thus, we were curious to know the effects of TBZ on those secretory responses. We were surprised to find that, rather than causing a gradual decrease of secretory responses as a consequence of VMAT2 blockade, TBZ caused a remarkable facilitation of catecholamine release. We report here the mechanism of action of such facilitation of exocytosis that seemed to be unrelated to VMAT2 blockade; rather, such effect is apparently linked to the ability of TBZ to release Ca2+ through the ryanodine receptor channel (RyR) of the endoplasmic reticulum (ER) of BCCs and rat embryo cortical neurons.

Materials and Methods

Preparation of Chromaffin Cell Cultures.

All experiments were carried out in accordance with the guidelines established by the National Council on Animal Care and were approved by the local Animal Care Committee of the Universidad Autónoma de Madrid. BCCs were isolated from calf adrenal medullary tissues obtained from a local slaughterhouse, isolated according to Moro et al. (1990) with some modifications (De Pascual et al., 2016), and plated at a density of 5 × 106 cells per 5 ml of Dulbecco’s modified Eagle’s medium on 6-cm-diameter Petri dishes (to study secretion) and on 12-mm-diameter glass coverslips at a density of 5 × 104 cells/coverslip to study calcium currents (ICa) through voltage-activated calcium channels (VACCs). Cells were kept for 1–4 days at 37°C in a water-saturated incubator in a 5% CO2/95% air atmosphere.

On-Line Monitoring of Catecholamine Release from Perifused Cell Populations.

Cells were scraped off carefully from the bottom of the Petri dish (5 × 106 cells per dish) with a rubber policeman and centrifuged at 100g for 10 minutes. The cell pellet was resuspended in 200 µl of Krebs-HEPES solution (composition: 144 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 11 mM glucose, 10 mM HEPES, 2 mM CaCl2 (pH 7.4) with NaOH). Cells were introduced in a 100-µl microchamber, which was previously filled with a filter for cell retention, and perifused at a rate of 2 ml/min. Both cells and perfusate were held at a temperature of 37°C. The liquid flowing from the superfusion chamber reached an electrochemical detector (model CH-9100; Metrohm AG, Herisau, Switzerland) placed just at the outlet of the microchamber, which monitors online the amount of catecholamines secreted under the amperometric mode (Green and Perlman, 1981; Borges et al., 1986; De Pascual et al., 2016). The catecholamines present in the perfusate were oxidized at +0.65 V, and the oxidation current was recorded with a frequency of 2 Hz to monitor the amount of total catecholamine secreted (Borges et al., 1986). Computer-controlled solenoid valves were used to rapidly exchange the solutions. Cells were intermittently stimulated at 1-minute intervals with a solution enriched in K+ (35 mM KCl with concomitant reduction of NaCl). Representative records of secretory responses shown in this report were accomplished by importing the data obtained in American Standard Code for Information Interchange format to the Origin 8.0 (Microcal) program.

Electrophysiological Recording of Ion Currents.

Inward currents through voltage-activated Na+ channels (INa) and VACCs (ICa) were recorded using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). Cells were perifused at 23 ± 2°C with a Tyrode solution containing 137 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES/NaOH (pH 7.4). Once the patch membrane was ruptured and the whole-cell configuration of the patch-clamp technique had been established, the cell was locally, rapidly, and constantly perifused with an extracellular solution of similar composition to the chamber solution, but containing 2 mM Ca2+ to monitor ICa. To measure ICa, cells were internally dialyzed with an intracellular solution containing 100 mM Cs-glutamate, 14 mM EGTA, 20 mM TEA-Cl, 10 mM NaCl, 5 mM Mg-ATP, 0.3 mM Na-GTP, and 20 mM HEPES/CsOH (pH 7.3). We used the protocol for ICa data acquisition; cells were voltage clamped at −80 mV, and step depolarizations with 50-ms depolarizing pulses were applied at 30-second intervals to minimize current rundown (Fenwick et al., 1982).

Data were acquired with a sample frequency of 20 kHz using the PULSE 8.74 software (Heka Elektronic, Lambrecht, Germany). Linear leak and capacitive components were subtracted by using a P/4 protocol, and series resistance was compensated by 80%. The data analysis was performed with Igor Pro (Wavemetrics, Lake Oswego, OR) and PULSE programs (Heka Elektronik).

Monitoring of Cytosolic Calcium Levels.

To monitor the changes of [Ca2+]c, cells were plated at a density of 2 × 105 cells per well into 96-well black plates, and the experiments were performed 48 hours later for BCCs or 7 days later for cortical neurons. Cells were loaded with a Krebs-HEPES solution containing 10 µM fluo-4-AM and 0.2% pluronic acid. Cells were incubated for 45 minutes at 37°C in the dark. After this incubation period, cells were washed twice with the Krebs-HEPES solution at room temperature in the dark. Changes in fluorescence (excitation 485 nm, emission 520 nm) were measured using a fluorescent plate reader (Fluostar; BMG Labtech, Offenburg, Germany). Basal levels of fluorescence were monitored before adding the stimulation solution (20 mM caffeine or 100 μM histamine) with an automatic dispenser. After stimulation of the cells, changes in fluorescence were measured for 60 seconds. To normalize fluo-4 signals, responses from each well were calibrated by measuring maximum and minimum fluorescence values. At the end of each experiment, 5% Triton X-100 (Fmax) was added, followed by 2 M MnCl2 (Fmin). Data were calculated as a percentage of Fmax-Fmin.

Molecular Docking.

Structure and conformational analysis of TBZ were acquired with the Monte Carlo method using Spartan’16 software (Wavefunction, Irvine, CA). The conformer with the lowest energy was subsequently refined with calculations based on the ab initio Hartree-Fock 6-31G** formula, and the three-dimensional structure obtained was selected for the docking studies. The structure of the binding domain of type 3 RyR was extracted from Protein Data Bank (http://www.rcsb.org; PDB ID: 4ERV) (Yuchi et al., 2012). Solvent molecules were removed from the protein. Docking calculations of TBZ on RyR were carried out using Molegro Virtual Docker in sufficiently large constrains onto the cavities found by the software, allowing the ligand to freely accommodate with the best pose. Different orientations of TBZ were reached in each cavity and ranked according to their energy scores by the MolDock Score algorithm. Among them, the pose shown in Fig. 8 represents the most highlighted one.

Chemicals.

The following chemicals were used: collagenase type I, cyclopiazonic acid (CPA), 2-aminoethyldiphenyl borinate (2-APB), ryanodine, and TBZ were from Sigma-Aldrich (Madrid, Spain); Dulbecco’s modified Eagle’s medium, bovine serum albumin fraction V, fetal calf serum, and antibiotics were from Gibco (Madrid, Spain). All other chemicals used were reagent grade from Merck and Panreac Química (Madrid, Spain). CPA, 2-APB, ryanodine, and TBZ (10−2 M) were prepared in dimethylsulfoxide and protected from light. Final drug concentrations were obtained by diluting the stock solutions directly into the extracellular solution. At these dilutions, dimethylsulfoxide had no effect on the parameters studied here.

Data Analysis.

Data are presented as single examples of original records to illustrate the experimental protocols and as averaged pooled data from several experiments, as means ± S.E.M. of the number of different experiments performed (number of cell batches, n) and different cultures used (N). Mathematical analysis was done using the GraphPad Prism software, version 5.01 (GraphPad Software, La Jolla, CA). The statistical significance between groups of experiments was estimated using a nonparametric Mann-Whitney rank sum test, or one-way analysis of variance followed by the Tukey or Kruskal-Wallis post hoc test, when appropriate.

Results

Facilitation of Catecholamine Release by TBZ.

Experiments were performed to test first how sequential pulsing with high K+ (35 mM, 5 seconds), applied at 1-minute intervals to BCCs trapped in a microchamber and perifused with Krebs-Hepes solution at 37°C, elicited the release of catecholamines, a response that was monitored online with an electrochemical detector. An example of those responses elicited by 22 intermittent K+ applications is displayed in Fig. 1A. In this experiment, the initial secretory response (P1) was 210 nA; in subsequent K+ pulses, the response gradually decayed to a value of 90 nA in P22.

Fig. 1.
Fig. 1.

Facilitation of exocytosis by TBZ. BCCs (5 × 106) trapped in a microchamber were constantly perifused with Krebs-HEPES at 37°C. After an initial equilibration period to get a stable baseline amperometric recording, cells were intermittently stimulated at 1-minute intervals with 5-second pulses of a solution enriched in K+ (35 mM, isosmotic reduction of Na+). Secretion of catecholamines was amperometrically monitored online and expressed in nanoamperes of current generated by oxidized amines [calibration bars in (A) and (B)]. (A) Example control experiment showing the secretory spike responses produced by 22 sequential K+ pulses. (B) Example experiment of secretory responses elicited by sequential K+ pulses before, during, and after cell perifusion with 3 μM TBZ (top horizontal line). (C) Averaged pooled data of experiments performed with the same protocols as those shown in (A) and (B); data in nanoamperes were normalized within each individual experiment as a percentage of P6 (ordinate). (D) Effects of various concentrations of TBZ (abscissa) on the K+ secretory responses; normalized data with resped K+ pulse P8 are plotted. Data in (C) and (D) are means ± S.E.M. of the number of cell batches (first number in parentheses) and cultures from different glands and days (second number in parentheses). **P < 0.01 with respect to control (one-way ANOVA with post hoc Kruskal-Wallis analysis).

In a similar experiment conducted with another batch of cells, the effect of TBZ at 3 μM on K+-elicited secretory responses was tested. The drug was applied 30 seconds before P7 and was perifused during the next nine K+ pulses until P16. During P7 to P10, the secretory response was augmented from 220 nA in P6 to around 260 nA in P8 to P10; after this enhanced plateau, secretion gradually decreased to a value of 220 nA in P16. Then, after TBZ withdrawal, the secretory response abruptly decayed to values of around 160 nA in P17 and P18 (Fig. 1B).

Pooled data of experiments performed with the previously described protocols are graphed in Fig. 1C. Within each individual experiment, data were normalized as a percentage of P6, the pulse preceding the exposure of cells to TBZ. The time course of control responses (pulses P6 to P22, means ± S.E.M. of 10 experiments from five different cultures) closely reproduces the time course of the example control experiment of Fig. 1A—namely, a gradual decay of the amplitude of secretory spike responses to 60% at P22. In the cells exposed to TBZ (3 μM), the responses are always above those of the control curve, including those that follow TBZ washout. Although the responses are only mildly enhanced by TBZ (26% above control, at P8), they are statistically significant (Fig. 1C).

Concerning the concentration-response relationship of the effects of TBZ on K+-elicited secretion, we found that at 1 and 3 μM, the drug exhibited a facilitatory action (109.9% ± 1.9% and 125.9% ± 5.4%, with respect to control response). However, at 10 μM, TBZ caused a mild inhibition (Fig. 1D).

Interactions of Tetrabenazine with BayK8644 and Nifedipine.

Catecholamine release from adrenal medullary CCs absolutely depends on the presence of Ca2+ ions in the extracellular milieu while the K+ depolarizing stimulus is applied (Douglas and Rubin, 1963). Exocytosis is triggered when the extracellular Ca2+ entering the cell through VACCs is abruptly increased at subplasmalemmal sites near the exocytotic machinery (Baker and Knight, 1978; Neher, 1998). Bovine CCs express L-subtype (α1D, Cav1.3), N-subtype (α2B, Cav2.2), and PQ-subtype (α2A, Cav2.1) of VACCs (García et al., 2006). All channels contribute to providing the necessary Ca2+ for exocytosis; however, L-subtype channels make a large contribution to K+-elicited secretion in BCCs that is facilitated by dihydropyridine (DHP) BayK8644 (BayK) and blocked by DHP nifedipine (García et al., 1984). Thus, we tested next the effects of these two DHPs on K+-elicited secretion as well as on the facilitation of such response by TBZ.

Once more, we performed experiments of the type shown in Fig. 1. Normalized secretion (expressed as percentage of P1 in each separate batch of cells) is plotted in Fig. 2A, showing the decay of K+-elicited secretion in control cells, the drastic augmentation elicited by 1 μM BayK, and the milder increase elicited by 3 μM TBZ. The responses in the presence of BayK decayed rapidly toward control levels after its washout, in contrast to the responses after TBZ removal that decayed more slowly. Pooled data of peak responses (those indicated by the ellipse in Fig. 2A) are graphed in Fig. 2B. BayK nearly doubled the K+ secretory response (83.34% ± 6.1%) while TBZ augmented it by 38.05% ± 2.6%.

Fig. 2.
Fig. 2.

Interactions of TBZ with 1,4-dihydropyridine (DHP) derivatives BayK and nifedipine (Nife). Experiments displayed here were conducted with protocols similar to those represented in Fig. 1. (A) Drastic augmentation of K+-elicited secretion by 1 μM BayK that was applied from P7 to P16 (top horizontal bar). For the sake of comparison, the control and TBZ curves of Fig. 1C were replotted here. (B) Averaged pooled data of experiments performed with protocols shown in (A) (means ± S.E.M.). BayK nearly doubled the K+ secretory response (83.34% ± 6.1%), while TBZ augmented it by 38.05% ± 2.6%; the differences between both treatments is 45.29% ± 3.2% (one-way ANOVA, F = 73.35, Tukey post hoc comparison: ***P < 0.0001 control vs. BayK and TBZ; ***P < 0.0001 BayK vs. TBZ). (C) Effect of combined TBZ plus BayK, and of delayed addition of Nife on top of TBZ, on the K+ secretory responses; compounds were added as indicated by the bottom horizontal bar. (D) Data of the curve points identified with an ellipse in (C); TBZ augmented the K+ secretory responses by 45.3% ± 8.9%; this response was reduced by nifedipine to 45.3% ± 4% of the initial control; combined BayK and TBZ caused 87.7% ± 10.05% increase of secretion, and the differences between the two treatments (TBZ and TBZ+BayK) is 42.4% ± 3.1% (one-way ANOVA, F = 100.2, P < 0.0001, Tukey post hoc comparison: ***P < 0.0001 control vs. all pairs; ***P < 0.0001 TBZ vs. TBZ+BayK and TBZ+Nife; and ***P < 0.0001 TBZ+BayK vs. TBZ+Nife). Data are means ± S.E.M. of the number of cells and cultures shown in parentheses in (A) and (B).

Interactions between TBZ and DHPs were explored next. Figure 2C shows the drastic increase of secretion elicited by combined TBZ and BayK (3 and 1 μM, respectively); note that there was a combined tendency for the response to continue growing in the successive K+ pulses. If 3 μM nifedipine was added to cells previously being exposed to TBZ, the responses decreased drastically. Averaged pooled data (ellipse in Fig. 2C) on these different treatments are graphed in Fig. 2D. TBZ augmented the K+ secretory responses by 45.3% ± 8.9% (n = 9); this response was reduced by nifedipine to 45.3% ± 4% of the initial control (n = 6). On the other hand, combined BayK and TBZ caused a doubling of the response, a value similar to that obtained with BayK alone (87.7% ± 10.05%) (Fig. 2B). Once again we can see that the differences between the two treatments (42.4%), TBZ only and the combination of TBZ plus BayK, suggest that their effects do not follow the same mechanism of action.

Effects of TBZ on Sodium and Calcium Currents.

The experiments in Fig. 2 suggested that the enhancing effects of secretion elicited by TBZ could have an underlying BayK-like mechanism—that is, the prolongation of opening time of L-subtype channels with enhanced Ca2+ entry occurring during a depolarizing stimulus (Cena et al., 1989). In BCCs, BayK is known to augment Ca2+ entry to enhance secretion; also, the DHP augments the L-subtype component of the whole-cell Ca2+ current (ICa) of these cells (García et al., 2006). Hence, we tested next the effects of TBZ on ICa.

Cells were voltage clamped at −80 mV, and 50-ms depolarizing pulses were applied at 30-second intervals to generate maximal peak ICa currents (Fenwick et al., 1982). Figure 3A shows a control current composed of initial fast inactivating peak current that corresponds to Na+ current (INa; trace expanded) and an ensuing slow current that was stable upon the application of repeated pulses to 0 mV given at 30-second intervals, the Ca2+ current (ICa). Quantitative pooled data on peak INa (trace expanded) currents indicate that TBZ did not affect such current (Fig. 3B). When 3 μM TBZ was perifused, the blockade was nearly completely reversed upon TBZ washout (Fig. 3A). The degree of blockade was monitored at the initial peak current; in eight cells from three different cultures, peak ICa was blocked 12.7% ± 0.5% (paired t test, t = 7.528, P = 0.0001) by 3 μM TBZ (Fig. 3C).

Fig. 3.
Fig. 3.

Mild blockade of calcium currents, but not sodium currents, elicited by TBZ. Whole-cell inward Na+ (INa) and Ca2+ currents (ICa) were explored in voltage-clamped BCCs under the whole-cell configuration of the patch-clamp technique. Calcium at 2 mM was used as a charge carrier. The initial fast inactivating INa (extended area) and the ensuing slow inactivating ICa are identified by arrows in (A). Currents were elicited by 50-ms depolarizing pulses to 0 mV applied at 30-second intervals [protocol on top of (A)]. Upon breaking into the cell with the patch pipette, various pulses were given to stabilize the current. Subsequently, TBZ was applied, and 30 seconds thereafter four pulses were applied (total of 120 seconds with drug); finally, the drug was removed to allow current recovery. (A) Example INa (arrow INa) and ICa traces (arrow ICa) before (control), during cell exposure to TBZ, and after TBZ withdrawal in a cell stimulated with 50-ms pulses. (B) Pooled normalized data within each cell explored as percentage of the initial control INa amplitude. (C) Pooled normalized data within each cell explored as percentage of the initial control ICa amplitude; TBZ (3 μM) was blocked 12.7% ± 0.5% (paired test, t = 7.528, P = 0.0001). Data in (B) and (C) are means ± S.E.M. of the number of cells and cultures shown in parentheses.

Effects of TBZ on Secretion Elicited by Caffeine or Histamine Pulses.

At the ER Ca2+ store, two mechanisms have been implicated in the release of Ca2+ into the cytosol. This has been directly proven using Ca2+-sensitive aequorins targeted to the ER lumen. On the one hand, upon stimulation with caffeine, Ca2+ release occurred through ryanodine receptors; on the other hand, histamine also elicited Ca2+ release but through inositol 1,4,5-trisphosphate receptor (IP3R) channels (Alonso et al., 1999). Thus, we thought it interesting to discern whether the secretory responses elicited with repeated pulses of caffeine or histamine were affected by tetrabenazine, as those of K+ were.

In BCCs, caffeine pulses trigger catecholamine release responses linked to ER Ca2+ release through RyRs (Cheek et al., 1990); when repeatedly applied onto the same batch of cells, those responses undergo rapid inactivation because the ER Ca2+ store is gradually emptied (Lara et al., 1997). It seemed to us that this protocol could be used here to find out whether TBZ was affecting such responses.

A protocol similar to that used with K+ pulses (Fig. 1, A and B) was designed to test the secretory responses of BCCs challenged with 5-second pulses of 20 mM caffeine, applied at 1-minute intervals to the same batch of cells (22 pulses). An original amperometric record of the sequential secretion responses is displayed in Fig. 4A. The initial sharp response of 263 ± 34.3 nA was halved in P2; in subsequent pulses, the responses underwent a gradual decay to reach a plateau at a level of secretion that was only 25%–30% at caffeine pulses 15–22. In a different cell batch, cells were similarly challenged with 22 caffeine pulses; between pulses P7 and P16, 3 μM TBZ was introduced into the cell superfusion system. In the original example record of Fig. 4B, the responses decayed from 273 nA in P1 to 111.2 nA in P6. From P7 onward, cell exposure to TBZ modified the responses in two ways: 1) a prompt augmentation of secretory spiked to about 163.2 nA (P9), and 2) the decay of responses was prevented. Upon TBZ removal, the responses quickly faded to 79.5 nA in pulse P22.

Fig. 4.
Fig. 4.

Whereas the secretory responses to caffeine pulsing were enhanced by TBZ, those of histamine were unaffected. Experimental protocols are similar to those of Fig. 1, but here cells were challenged with 5-second pulses of 20 mM caffeine or 100 μM histamine, applied at 1-minute intervals. (A) Original example record of the secretory responses elicited by caffeine (pulses P1 to P22 in the abscissa; secretory responses in nanoamperes per pulse, calibration bars). (B) As in (A) but 3 μM TBZ was cell perifused during caffeine pulses P7 to P16. (C) Cells challenged with 5-second pulses of 100 μM histamine applied at 1-minute intervals. (D) A similar experiment with histamine pulses and cell exposure to TBZ at pulses P7 to P16. (E) Pooled data of control experiments performed with caffeine or histamine; data are normalized as percentage of P1 within each individual experiment. (F) Pooled data from experiments on cell stimulation with caffeine or histamine, and introduction of TBZ as indicated. In the case of caffeine, maximum enhanced secretion by TBZ was achieved at P8 (28.04% ± 2.03%), and the lowest increase was at pulse P16 (21.22 ± 1.89). Mann-Whitney no parametric test: ***P = 0.0006; **P = 0.0043. Data on pulse secretion are means ± S.E.M. of the individual number of experiments and cell cultures shown in parentheses.

Through the stimulation of H1 receptors, histamine also elicits the secretion of catecholamines by stimulating the release of ER Ca2+ via IP3Rs into the cytosol (Cheek et al., 1994; de Pascual and García, 2007; Milla et al., 2011). Thus, experiments similar to those performed with caffeine were carried out by challenging the cells with 5-second pulses of 100 μM histamine. The example record in Fig. 4C shows the fast gradual decay of the histamine-elicited secretory responses, likely due to H1 receptor desensitization. The example trace in Fig. 4D was taken from a similar experiment in which TBZ was applied at pulses P7 to P16; the drug did not affect the decay of histamine secretory responses at all.

Pooled data from five to eight experiments conducted in three to four cell cultures in cells challenged with caffeine or histamine are graphed in Fig. 4, E and F. Normalized overlapping curves on pulse number versus secretion responses indicated a very close time course of secretion decay in both cells challenged with caffeine or histamine (Fig. 4E). On the other hand, pooled data indicate that while TBZ augmented and maintained stabilization of the caffeine secretory responses, the histamine responses were unaffected by the drug. The maximum increase occurred with caffeine at P9 (28.04% ± 2.03%; n = 6, P < 0.001). In five experiments, TBZ did not affect the histamine-evoked secretion (Fig. 4F).

Pharmacological Manipulation of the ER Calcium Store and Its Effect on Caffeine and Histamine Secretory Responses.

To test whether the effects of TBZ on the secretory responses of cells challenged with caffeine or histamine were linked to the ER Ca2+ store, we pharmacologically intervened on such store at three ionic receptor sites: inhibition of sarco-endoplasmic reticulum calcio ATPase (SERCA) with CPA (Goeger et al., 1988; Seidler et al., 1989), inhibition of the RyR with ryanodine (Fleischer et al., 1985; Pessah et al., 1986; Lattanzio et al., 1987), or blockade of the IP3R channel with 2-APB (Maruyama et al., 1997); these interventions are known to deplete the ER Ca2+ store, but through different mechanisms. Thus, experiments with protocols similar to those described earlier with caffeine and histamine were conducted.

Figure 5A shows that CPA caused a sharp decrease of the caffeine responses (with or without TBZ) with a very fast time course; upon CPA washout, the responses recovered to the expected control level. Ryanodine (10 μM) also elicited a prompt secretion decrease of the caffeine responses applied in the presence or absence of TBZ. Data are quantitatively expressed in Fig. 5B. The data on TBZ-elicited enhanced secretion are normalized as a percentage of control (P9) and were calculated from the experiments of Fig. 4. In the presence of CPA, TBZ+CPA, ryanodine, or TBZ+ryanodine, the caffeine secretory responses were drastically decreased by 69.3% ± 3.2%, 65.3% ± 5.1%, 84.3% ± 3.1%, and 90.2% ± 1.3%, respectively (Fig. 5B).

Fig. 5.
Fig. 5.

Calcium depletion of the ER by SERCA inhibition with 5 μM CPA depresses the secretory responses to caffeine or histamine. (A) Time course of secretory responses to sequential caffeine pulses (20 mM caffeine, for 5 seconds, applied at 1-minute intervals); drugs [CPA and TBZ at 3 μM, ryanodine (RYA) at 10 μM] were applied from pulses 7 to 16 (top horizontal bar). (B) In the presence of CPA, TBZ+CPA, RYA, or TBZ+RYA, the caffeine secretory responses were drastically decreased by 69.3% ± 3.2%, 65.3% ± 5.1%, 84.3% ± 3.1%, and 90.2% ± 1.3%, respectively (one-way ANOVA, F = 198.7, P < 0.0001; Tukey post hoc comparison: ***P < 0.0001 control vs. all pairs; ***P < 0.0001 TBZ vs. RYA and TBZ+RYA; and ***P < 0.0001 CPA vs. TBZ+CPA, RYA, and TBZ+RYA). (C) Time course of secretory responses elicited by sequential pulses of histamine (100 μM for 5 seconds applied at 1-minute intervals) and the effects of various compounds (CPA and 2-APB at 10 μM) on those responses, added from pulses P7 to P16 (top horizontal bar). (D) The histamine secretory responses were decreased in the presence of CPA (44.2% ± 3.2%), TBZ+CPA (43.2% ± 3.2%), 2-APB (84.7% ± 4.5%), and TBZ+2-APB (88.5% ± 4.2%) (one-way ANOVA, F = 321.6, P < 0.0001; Tukey post hoc comparison: ***P < 0.0001 control vs. all pairs; ***P < 0.0001 CPA vs. 2-APB and TBZ+2-APB; and ***P < 0.0001 TBZ+CPA vs. 2-APB and TBZ+2-APB). (B and D) Averaged pooled data normalized as percentage of P9 (control response); data correspond to pulse P9. Data in (B) and (D) are means ± S.E.M. of the number of cell batches (n) and cultures (N), shown in parentheses on top of each column.

Experiments with histamine pulses also showed a pronounced inhibition of secretion responses in the presence of CPA (44.2% ± 3.2%), TBZ+CPA (43.2% ± 3.2%), 2-APB (84.7% ± 4.5%), and TBZ+2-APB (88.5% ± 4.2%) (Fig. 5, C and D). Thus, these experiments provide proof that the secretory responses of caffeine and histamine, when applied either alone or in the presence of TBZ, are mediated by Ca2+ release from the ER Ca2+ store. They also indicate that TBZ may be selectively acting on the RyR calcium channel of the ER.

Effects of TBZ on the Cytosolic Elevations of Calcium Elicited by Caffeine or Histamine.

That caffeine triggers ER Ca2+ release into the cytosol of BCCs through RyRs has been indirectly studied with cytosolic Ca2+ fluorescence probes (Cheek et al., 1990, 1993) and directly studied with ER-targeted aequorins (Alonso et al., 1999). Similar approaches have been used to study the release of ER Ca2+ through IP3Rs of BCCs (Stauderman and Pruss, 1990; Artalejo et al., 1993; Alonso et al., 1999). Here, we studied the effects of TBZ on ER Ca2+ release through RyRs (caffeine) or IP3Rs (histamine) in populations of BCCs loaded with fluo-4.

Cells were incubated at 37°C in the dark with TBZ at 1–10 μM, with ryanodine (10 μM), or with combined Rya+TBZ. Figure 6A shows example traces of the time course of [Ca2+]c elevations triggered by 20 mM caffeine. In control cells, there was a rapid [Ca2+]c elevation that gradually faded off surely because the ER Ca2+ was being depleted and the cytosolic Ca2+ elevations are cleared by mitochondria and by plasmalemmal Ca2+ efflux pumps (Alonso et al., 1999; Villalobos et al., 2002). In the presence of TBZ at 1, 3, and 10 μM, the caffeine-elicited Ca2+ elevations were notably augmented, and although the [Ca2+]c transient also faded off gradually, the [Ca2+]c was higher at the end of the recording period (60 seconds) with respect to control. On quantitative terms, the peak [Ca2+]c elevations were augmented more than twice in the presence of 1, 3, and 10 μM TBZ (228.4% ± 26.4%, 197.3% ± 17.4%, and 236.8% ± 27.2%, respectively) (Fig. 6B). Obviously, in the presence of ryanodine, which causes full depletion of ER Ca2+ (Alonso et al., 1999), either alone or combined with TBZ, the caffeine response was abolished (7.68% ± 3.08% and 5.72% ± 2.6%, respectively) (Fig. 6, A and B).

Fig. 6.
Fig. 6.

TBZ augments the cytosolic Ca2+ elevations ([Ca2+]c) elicited by caffeine but not those of histamine. Cells plated in 96-well black plates were loaded with fluo-4 acetoxymethyl esther. After 10-minute incubation, cells were challenged with 20 mM caffeine [arrow in (A)] or 100 μM histamine [arrow in (C)]. The [Ca2+]c curves are expressed as increases of arbitrary fluorescence units normalized as a percentage of maximum [Ca2+]c increase (ordinate). (A) Example records of caffeine-elicited [Ca2+]c in the absence (control) or the presence of the drug treatments indicated in each trace. (B) Pooled data on the normalized [Ca2+]c elevations were augmented more than twice in the absence (control) or presence of 1, 3, and 10 μM TBZ (228.4% ± 26.4%, 197.3% ± 17.4%, and 236.8% ± 27.2%); obviously, in the presence of ryanodine, which causes full depletion of ER Ca2+ either alone or combined with TBZ, the caffeine response was abolished (7.68% ± 3.08% and 5.72% ± 2.6%, respectively; one-way ANOVA, F = 34.41, P < 0.0001; Tukey post hoc comparison: **P < 0.001; ***P < 0.0001, control vs. all pairs). (C) Example records of histamine-elicited [Ca2+]c in the absence (control) or presence of the drug treatments indicated in each trace. (D) Pooled data on the normalized [Ca2+]c elevations where TBZ does not alter the histamine-evoked [Ca2+]c transients. In the presence of 2-APB and combined TBZ+2-APB, the histamine response was abolished (3.63% ± 3.1% and 0.04% ± 4.7%, respectively; one-way ANOVA, F = 58.56, P < 0.0001; Tukey post hoc comparison: ***P < 0.0001, control vs. all pairs). Data are normalized as percentage of control; they are the means ± S.E.M. of the number of wells and cultures shown in parentheses (first and second number, respectively).

Experiments with protocols similar to those of caffeine were done using histamine. Figure 6C shows a family of [Ca2+]c elevation curves triggered by histamine (100 μM) applied in the absence (control) and presence of the various treatments (protocols similar to those used in the caffeine experiments). These histamine-triggered Ca2+ signals differed from those of caffeine in two aspects: 1) the peak [Ca2+]c elevation was smaller, and 2) the decay of [Ca2+]c was very slow. This could find an explanation in the fact that the mitochondrial Ca2+ uniporter has a low affinity for Ca2+; if the histamine Ca2+ elevation is lower, mitochondria may not see such local [Ca2+]c, and hence, the Ca2+ clearance will take place only at the expense of the low-rate plasmalemmal Ca2+ pumps (Villalobos et al., 2002). Another drastic difference of histamine responses in comparison with those of caffeine is the fact that TBZ at 1–10 μM did not affect the [Ca2+]c elevations triggered by histamine (Fig. 6, C and D). As expected, the blocker of IP3R 2-APB (at 10 μM) alone or combined with TBZ abolished the histamine responses (3.63% ± 3.1% and 0.04% ± 4.7%, respectively) (Fig. 6, C and D).

Effects of TBZ on the [Ca2+]c Elevations Triggered by Caffeine or Histamine in Cortical Neurons.

The proof of concept in which TBZ is used to treat the HD-associated chorea symptoms is based on its ability to inhibit VMAT2 at central dopaminergic neurons. Therefore, we considered it of interest to explore whether TBZ was also targeting RyRs (and not IP3Rs) in primary cultures of rat embryo cortical neurons. Thus, experiments similar to those performed with BCCs (Fig. 6) were conducted in neurons loaded with fluo-4.

Figure 7A shows a family of traces of the [Ca2+]c elevations triggered by caffeine in control neurons and in neurons previously exposed to TBZ (1–10 μM), ryanodine (10 μM), or combined ryanodine+TBZ. The control Ca2+ signal reached a peak and then a gradual decay followed. In the presence of TBZ, the peak was more than doubled and also decayed to reach the control [Ca2+]c level by the end of the 60-second recording period. Pooled results using this protocol are graphed in Fig. 7B. Note the augmentation by TBZ (1, 3, and 10 μM) of the caffeine-elicited Ca2+ signal (219.2% ± 22.3%, 214.6% ± 24.7%, and 218.7% ± 33%, respectively) and the abolition of the responses by ryanodine, alone or combined with TBZ (2.53% ± 10.9% and 4.58% ± 7.6%, respectively).

Fig. 7.
Fig. 7.

Augmentation by TBZ of the [Ca2+]c elevations triggered by caffeine, but not those of histamine, in rat embryo cortical neurons. Neurons were loaded with fluo-4, and experiments similar to those carried out with BCCs (Fig. 6) were performed. Neuron challenging with 20 mM caffeine [arrow in (A)] or 100 μM histamine [arrow in (C)] in the absence (control) or presence of TBZ (at 1–10 μM), ryanodine (Rya; 10 μM), 2-APB (10 μM), combined Rya+TBZ, or 2-APB+TBZ. (A) Family of caffeine-elicited [Ca2+]c traces monitored as normalized arbitrary fluorescence units (AFUs; ordinate); neuron treatments are labeled at the right part of each trace. (B) Pooled data of peak [Ca2+]c elevations; note the augmentation by TBZ (1, 3, and 10 μM) of the caffeine-elicited Ca2+ signal (219.2% ± 22.3%, 214.6% ± 24.7%, and 218.7% ± 33%, respectively) and the abolition of the responses by ryanodine, alone or combined with TBZ (2.53% ± 10.9% and 4.58% ± 7.6%, respectively; one-way ANOVA, F = 37.09, P < 0.0001; Tukey post hoc comparison: ***P < 0.0001, control vs. all pairs). (C) Family of histamine-elicited [Ca2+]c traces monitored as normalized AFUs (ordinate). (D) Pooled data on the normalized [Ca2+]c elevations by histamine showing that TBZ did not modify the histamine-elicited [Ca2+]c transients; in the presence of 2-APB and combined TBZ+2-APB, the histamine response was abolished (8.72% ± 11% and 2.01% ± 1.95%, respectively; one-way ANOVA, F = 93.20, P < 0.0001; Tukey post hoc comparison: ***P < 0.0001 control vs. all pairs). Data in (B) and (D) are means ± S.E.M. of the number of wells and cultures shown in parentheses on top of each column.

The family of [Ca2+]c traces displayed in Fig. 7C were generated by histamine in the absence (control) or presence of the different treatments. Of note is the very mild decay of the response in contrast to caffeine. This graph and pooled data of Fig. 7D show that TBZ did not modify the histamine-elicited [Ca2+]c elevations. They also show that 2-APB alone or combined with TBZ abolished the histamine-evoked responses (8.72% ± 11% and 2.01% ± 1.95%, respectively).

Molecular Docking Study of TBZ Coupling with the Ryanodine Receptor.

An explanation of the pharmacological mechanism observed with TBZ could be that it directly interacts with the RyR. A prediction of such interaction may be appreciated by molecular docking analysis using the X-ray structure of the type 3 RyR deposited in the Protein Data Bank (http://www.rcsb.org; PDB ID: 4ERV) (Yuchi et al., 2012). The complex TBZ-RyR with the lowest energy occurred where RyR posed in a binding pocket located at the RyR Ca2+-binding domain (Fig. 8A), connected to several amino acids by hydrogen bonds (2.20 Å with Arg 2599 and 2.94 Å with the skeleton NH of Leu 2721) and by van der Waals interactions with Ala 2749 and Lys 2703 (Fig. 8B).

Fig. 8.
Fig. 8.

Docking of tetrabenazine-ryanodine receptor complex. (A) Pose of TBZ (sticks, CPK-colored) in the type 3 RyR Ca2+-binding domain (main interacting residues in gray). (B) H-bonds (Arg 2599 and Leu 2721) and van der Waals contacts (Ala 2719 and Lys 2703) formed between amino acids (CPK-colored thin sticks) and TBZ (CPK-colored sticks).

Discussion

The central finding in this investigation is the facilitation of the catecholamine release responses elicited by TBZ in BCCs challenged with sequential brief K+ pulses. As these responses depend on Ca2+ entry through VACCs (Douglas and Rubin, 1963; García et al., 2006; Mahapatra et al., 2012), their facilitation by TBZ could be due to an augmented Ca2+ entry through VACCs. As BCCs express the L-subtype (20%), N-subtype (30%), and PQ-subtype (50%) of VACCs, enhanced Ca2+ entry could be associated with a selective effect of TBZ on one of those channel subtypes. Under conditions of BCC depolarization with high K+ concentrations for several seconds, the N- and PQ-subtype channels undergo fast inactivation in comparison with slower L-subtype channel inactivation (Villarroya et al., 1999; Hernández-Guijo et al., 2001); this explains why K+-elicited secretion from BCCs is mostly triggered by Ca2+ entry through L-subtype channels and that this response is augmented by BayK, which delays channel inactivation and is blocked by nifedipine and other dihydropyridine derivatives (García et al., 1984). As expected, BayK enhanced and nifedipine diminished the K+-elicited secretory responses independently of the presence or absence of TBZ (Fig. 2), indicating that facilitation of secretion by TBZ could be due to either enhanced Ca2+ entry through VACCs or Ca2+ mobilization from intracellular Ca2+ stores. We can discard the first option on the basis that TBZ did not augment ICa; on the contrary, the drug caused a mild current inhibition (Fig. 3).

That the ER Ca2+ store contributes to the shaping of the [Ca2+]c transients and the secretory responses in BCCs has been proven with different experimental approaches. The most conspicuous has been the depletion of the ER Ca2+ store with the SERCA blocker thapsigargin, which depresses the depolarization-evoked secretory responses in BCCs (Nowicky and Duchen, 1998; Pan and Fox, 2000; Cuchillo-Ibáñez et al., 2002). This is consonant with other experimental approaches showing that challenging of BCCs with repeated caffeine pulses causes secretory responses by ER Ca2+ release through RyRs, which inactivate as the ER Ca2+ store is emptied; additionally, the K+ secretory responses are also decreased upon caffeine challenging (Lara et al., 1997). This is in agreement with the observation that the K+ challenging of BCCs transfected with ER-targeted aequorin causes an ER Ca2+ decrease, which is direct proof of the presence of Ca2+-induced Ca2+ release (CICR) (Alonso et al., 1999); this is proof that this CICR mechanism contributes to the augmentation of K+ secretory responses, likely due to more sustained [Ca2+]c elevations in depolarized BCCs. Here comes our hypothesis in the present study in the sense that TBZ could facilitate the K+ secretory responses through the higher or more prolonged activation of the CICR mechanism.

In support of such a hypothesis comes the clear-cut experiment showing that TBZ caused a notable and sustained facilitation of the caffeine secretory responses (Fig. 4). Of interest was the fact that this drug did not affect the histamine secretory responses that are known to be mediated by IP3R stimulation and ER Ca2+ release (Stauderman and Pruss, 1990; Artalejo et al., 1993; Alonso et al., 1999) (Fig. 4). These two experiments strongly suggest that TBZ selectively enhances secretion through the CICR mechanism and helps to discard other targets such as cytoskeletal proteins, Ca2+-dependent intracellular vesicle transport, proteins of exocytosis, or other signaling pathways that facilitate vesicle priming and exocytosis, such as PKC activation by phorbol esters (Gillis et al., 1996; Yang et al., 2002) or cAMP-dependent PKA (Carabelli et al., 2003). In support of this explanation comes the observation that ER Ca2+ depletion with SERCA blocker CPA drastically reduced all secretory responses to caffeine and histamine in both the absence and presence of TBZ (Fig. 5).

This hypothesis is strengthened by the more direct experimental approach to [Ca2+]c change monitoring in BCCs challenged with caffeine or histamine. In the case of caffeine, the [Ca2+]c elevations are entirely due to ER Ca2+ release through RyRs of BCCs (Cheek et al., 1990, 1993; Alonso et al., 1999). Otherwise, histamine also releases ER Ca2+ into the cytosol of BCCs but through IP3Rs (Stauderman and Pruss, 1990; Artalejo et al., 1993; Alonso et al., 1999). We found that while TBZ caused a drastic augmentation of the caffeine-elicited [Ca2+]c elevations (Fig. 6, A and B), those generated by histamine were not modified by TBZ. This strongly supports the view that TBZ acts on the RyR to facilitate the CICR mechanism and to augment the caffeine-elicited (and the K+-elicited) exocytotic release of catecholamines (Figs. 1 and 2). The docking experiment of Fig. 8 strengthens this view.

By facilitating the release of ER Ca2+ through the depolarization-triggered CICR mechanism of BCCs (Alonso et al., 1999), TBZ may just maintain a mild [Ca2+]c elevation in between K+ pulses, so that the Ca2+-dependent transport of new secretory vesicles from a reserve vesicle pool could be enhanced to refill the ready-release vesicle pool located at subplasmalemmal exocytotic active sites (Rettig and Neher, 2002). This mechanism was elegantly demonstrated first at the laboratory of Erwin Neher in single voltage-clamped BCCs using histamine to elicit a mild [Ca2+]c elevation (von Rüden and Neher, 1993), using electrical pulses to elicit exocytosis. Later on, experiments on intact BCC populations challenged with physiologic acetylcholine or nicotine also showed a Ca2+-dependent vesicle transport facilitated by mild elevations of residual [Ca2+]c or by ER Ca2+ release (Arnaiz-Cot et al., 2008; de Diego et al., 2008). In this context, it seems that TBZ indirectly secures and maintains healthy secretory responses upon repeated depolarization of K+ stimuli by facilitating ER Ca2+ release mediated by RyR activation, thus maintaining mild sustained [Ca2+]c elevations to boost vesicle transport and the refilling, with new vesicles, of the secretory machinery at subplasmalemmal sites. This view is additionally reinforced by docking studies, which predict a direct interaction of TBZ with the phosphorylation domain of the RyR (Fig. 8). Thus, the RyR would be affecting the phosphorylation/dephosphorylation-dependent regulation of RyR exerted by both kinases and phosphatases.

In neurons, the ER Ca2+ store may act as a Ca2+ sink (when partially empty) or as a Ca2+ source (when filled) (Tsien and Tsien, 1990); this is also true for BCCs (Lara et al., 1997; Milla et al., 2011). In this context, it seems that TBZ may regulate the ER Ca2+ release during cell depolarization through the RyR, as shown by the experiments on [Ca2+]c monitoring in Fig. 7. This facilitation of exocytosis is a novel mechanism of action of TBZ, independent of its well established mechanism of VMAT2 blockade and the depletion of central catecholamines, mainly of dopamine at the basal ganglia. Because the expression of VMAT2 is extremely scarce in BCCs (Weihe et al., 1994), it seems that the facilitation by TBZ of depolarization-elicited (and caffeine-elicited) catecholamine release in the cells is unrelated to VMAT blockade. A last comment on the potential clinical projection of basic findings reported here deserves attention.

In 2008, the US Food and Drug Administration approved TBZ for HD-associated chorea. Approval was based on clinical trials showing the drug was effective; however, side effects included increased risk of depression, suicidality, akathisia, anxiety, and Parkinsonism, among other tolerability issues (Huntington Study Group, 2006; Yero and Rey, 2008; Jankovic and Roos, 2014). In April 2017, the Food and Drug Administration approved a deuterated form of TBZ, deutetrabenazine, which also inhibits VMAT2. An indirect comparison on the tolerability of TBZ versus deutetrabenazine concluded that the latter had a more favorable profile of adverse events (Claassen et al., 2017). This may be due to a better pharmacokinetic profile and lesser dose of deutetrabenazine required to treat HD chorea (Dean and Sung, 2018). However, the greater toxicity of TBZ could also be associated with its new target described here—namely, the ER ryanodine receptor and enhanced ER Ca2+ release into the cytosol. It is generally accepted that cell Ca2+ overload has neurotoxic effects that could lead to synaptic deficits and the adverse events of TBZ. Whether deutetrabenazine shares the ER Ca2+-mobilizing effect with its parent compound could be interesting to study.

Limitations of our study mainly deal with more direct approaches to better define the potential binding of TBZ to the ryanodine receptor. Also, experiments with ER-targeted aequorins, such as those we performed in a previous report (Alonso et al., 1999), will allow the direct monitoring of the changes undergone by the ER Ca2+ store upon cell stimulation with high K+ plus TBZ. Although these experiments may add value to our present work, we think that the present pharmacological study provides a quite solid collection of data to support the view that, beyond its well described inhibitory effect of VMAT2, TBZ also elicits the liberation of ER Ca2+ into the cytosol through an entirely different action—namely, potentiation of the CICR mechanism of BCCs.

In conclusion, on the basis of the results here presented, the following conclusions can be established (Fig. 9): 1) TBZ potentiates exocytosis triggered by cell depolarization and caffeine, but not histamine; 2) such potentiating is not due to augmented Ca2+ entry; 3) rather, it depends on a functional, full ER Ca2+ store, because it is due to the facilitation of ER Ca2+ release through the RyR; 4) this facilitation is not linked to VMAT2 blockade; and 5) enhanced ER Ca2+ release also occurs in neurons. This could have clinical relevance in the context of TBZ use to treat HD chorea or other distonic disorders, from the point of view of either efficacy and/or side effects.

Fig. 9.
Fig. 9.

Scheme showing the mechanism of action of TBZ involved in the augmentation of the exocytotic release of catecholamines triggered by K+-induced depolarization of the BCCs. (1) K+ depolarization opens the L-subtype of VACC; (2) this leads to a sudden transient increase of the cytosolic Ca2+ concentration ([Ca2+]c) that (3) triggers the rapid release of catecholamines from large dense-cored vesicles; (4) augmented [Ca2+]c also triggers the CICR through the RyR to further augment the exocytotic catecholamine response (3).

Acknowledgments

We thank Blanca Oriz Reyzabal for drawing the summary outline. We also thank the continued support of Fundación Teófilo Hernando (Madrid, Spain).

Authorship Contributions

Participated in research design: de Pascual, García.

Conducted experiments: de Pascual, Álvarez-Ortego, Jacob-Mazariego.

Performed data analysis: de Pascual, Álvarez-Ortego, de los Ríos, Jacob-Mazariego.

Wrote or contributed to the writing of the manuscript: de Pascual, García.

Footnotes

    • Received March 11, 2019.
    • Accepted June 4, 2019.

  • This work was supported by the Ministerio de Ciencia, Innovación y Universidades [Grant SAF 2016-78892-R], Spain; European Union Horizon 2020 Research and Innovation Programme under the Marie Skoldowska-Curie Grant Agreement [Grant 766124]; and Cátedra Nutrinfant Universidad Autónoma de Madrid/Alter.

  • https://doi.org/10.1124/jpet.119.256560.

Abbreviations

2-APB
2-aminoethyldiphenyl borinate
BayK
BayK8644
BCC
bovine chromaffin cell
CC
chromaffin cell
CICR
Ca2+-induced Ca2+ release
CNS
central nervous system
CPA
cyclopiazonic acid
DHP
dihydropyridine
ER
endoplasmic reticulum
HD
Huntington’s disease
5-HT
5-hydroxytryptamine
IP3R
inositol 1,4,5-trisphosphate receptor
NA
noradrenaline
RyR
ryanodine receptor
SERCA
sarco-endoplasmic reticulum calcio ATPase
TBZ
tetrabenazine
VACC
voltage-activated calcium channel
VMAT
vesicular monoamine transporter

References

  1. ↵
    1. Alonso MT,
    2. Barrero MJ,
    3. Michelena P,
    4. Carnicero E,
    5. Cuchillo I,
    6. García AG,
    7. García-Sancho J,
    8. Montero M, and
    9. Alvarez J
    (1999) Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin. J Cell Biol 144:241–254.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Antkiewicz-Michaluk L,
    2. Wąsik A,
    3. Możdżeń E,
    4. Romańska I, and
    5. Michaluk J
    (2014) Antidepressant-like effect of tetrahydroisoquinoline amines in the animal model of depressive disorder induced by repeated administration of a low dose of reserpine: behavioral and neurochemical studies in the rat. Neurotox Res 26:85–98.
    OpenUrl
  3. ↵
    1. Arnáiz-Cot JJ,
    2. de Diego AM,
    3. Hernández-Guijo JM,
    4. Gandía L, and
    5. García AG
    (2008) A two-step model for acetylcholine control of exocytosis via nicotinic receptors. Biochem Biophys Res Commun 365:413–419.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Artalejo AR,
    2. García AG, and
    3. Neher E
    (1993) Small-conductance Ca(2+)-activated K+ channels in bovine chromaffin cells. Pflugers Arch 423:97–103.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Baker PF and
    2. Knight DE
    (1978) Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nature 276:620–622.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Benarroch EE
    (2013) Monoamine transporters: structure, regulation, and clinical implications. Neurology 81:761–768.
    OpenUrl
  7. ↵
    1. Blasco-Serra A,
    2. Escrihuela-Vidal F,
    3. González-Soler EM,
    4. Martínez-Expósito F,
    5. Blasco-Ausina MC,
    6. Martínez-Bellver S,
    7. Cervera-Ferri A,
    8. Teruel-Martí V, and
    9. Valverde-Navarro AA
    (2015) Depressive-like symptoms in a reserpine-induced model of fibromyalgia in rats. Physiol Behav 151:456–462.
    OpenUrl
  8. ↵
    1. Borges R,
    2. Sala F, and
    3. García AG
    (1986) Continuous monitoring of catecholamine release from perfused cat adrenals. J Neurosci Methods 16:289–300.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Carabelli V,
    2. Giancippoli A,
    3. Baldelli P,
    4. Carbone E, and
    5. Artalejo AR
    (2003) Distinct potentiation of L-type currents and secretion by cAMP in rat chromaffin cells. Biophys J 85:1326–1337.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Ceña V,
    2. Stutzin A, and
    3. Rojas E
    (1989) Effects of calcium and Bay K-8644 on calcium currents in adrenal medullary chromaffin cells. J Membr Biol 112:255–265.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Cheek TR,
    2. Moreton RB,
    3. Berridge MJ,
    4. Stauderman KA,
    5. Murawsky MM, and
    6. Bootman MD
    (1993) Quantal Ca2+ release from caffeine-sensitive stores in adrenal chromaffin cells. J Biol Chem 268:27076–27083.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Cheek TR,
    2. Murawsky MM, and
    3. Stauderman KA
    (1994) Histamine-induced Ca2+ entry precedes Ca2+ mobilization in bovine adrenal chromaffin cells. Biochem J 304:469–476.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Cheek TR,
    2. O’Sullivan AJ,
    3. Moreton RB,
    4. Berridge MJ, and
    5. Burgoyne RD
    (1990) The caffeine-sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis. FEBS Lett 266:91–95.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Claassen DO,
    2. Carroll B,
    3. De Boer LM,
    4. Wu E,
    5. Ayyagari R,
    6. Gandhi S, and
    7. Stamler D
    (2017) Indirect tolerability comparison of deutetrabenazine and tetrabenazine for Huntington disease. J Clin Mov Disord 4:3.
    OpenUrl
  15. ↵
    1. Cuchillo-Ibáñez I,
    2. Olivares R,
    3. Aldea M,
    4. Villarroya M,
    5. Arroyo G,
    6. Fuentealba J,
    7. García AG, and
    8. Albillos A
    (2002) Acetylcholine and potassium elicit different patterns of exocytosis in chromaffin cells when the intracellular calcium handling is disturbed. Pflugers Arch 444:133–142.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Dean M and
    2. Sung VW
    (2018) Review of deutetrabenazine: a novel treatment for chorea associated with Huntington’s disease. Drug Des Devel Ther 12:313–319.
    OpenUrl
  17. ↵
    1. de Diego AM,
    2. Tapia L,
    3. Alvarez RM,
    4. Mosquera M,
    5. Cortés L,
    6. López I,
    7. Gutiérrez LM,
    8. Gandía L, and
    9. García AG
    (2008) A low nicotine concentration augments vesicle motion and exocytosis triggered by K(+) depolarisation of chromaffin cells. Eur J Pharmacol 598:81–86.
    OpenUrlCrossRefPubMed
  18. ↵
    1. De Pascual R,
    2. Colmena I,
    3. Ruiz-Pascual L,
    4. Baraibar AM,
    5. Egea J,
    6. Gandía L, and
    7. García AG
    (2016) Regulation by L channels of Ca(2+)-evoked secretory responses in ouabain-treated chromaffin cells. Pflugers Arch 468:1779–1792.
    OpenUrl
  19. ↵
    1. de Pascual R and
    2. García AG
    (2007) Ouabain augments and maintains the catecholamine release responses evoked by repetitive pulses of potassium, caffeine or histamine in perifused bovine chromaffin cells. Eur J Pharmacol 568:99–105.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Dixon WR,
    2. Garcia AG, and
    3. Kirpekar SM
    (1975) Release of catecholamines and dopamine beta-hydroxylase from the perfused adrenal gland of the cat. J Physiol 244:805–824.
    OpenUrlPubMed
  21. ↵
    1. Douglas WW and
    2. Rubin RP
    (1963) The mechanism of catecholamine release from the adrenal medulla and the role of calcium in stimulus-secretion coupling. J Physiol 167:288–310.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Erickson JD,
    2. Eiden LE,
    3. Schafer MK, and
    4. Weihe E
    (1995) Reserpine- and tetrabenazine-sensitive transport of (3)H-histamine by the neuronal isoform of the vesicular monoamine transporter. J Mol Neurosci 6:277–287.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Fenwick EM,
    2. Marty A, and
    3. Neher E
    (1982) Sodium and calcium channels in bovine chromaffin cells. J Physiol 331:599–635.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Fleischer S,
    2. Ogunbunmi EM,
    3. Dixon MC, and
    4. Fleer EA
    (1985) Localization of Ca2+ release channels with ryanodine in junctional terminal cisternae of sarcoplasmic reticulum of fast skeletal muscle. Proc Natl Acad Sci USA 82:7256–7259.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. García AG,
    2. García-De-Diego AM,
    3. Gandía L,
    4. Borges R, and
    5. García-Sancho J
    (2006) Calcium signaling and exocytosis in adrenal chromaffin cells. Physiol Rev 86:1093–1131.
    OpenUrlCrossRefPubMed
  26. ↵
    1. García AG,
    2. Sala F,
    3. Reig JA,
    4. Viniegra S,
    5. Frías J,
    6. Fontériz R, and
    7. Gandía L
    (1984) Dihydropyridine BAY-K-8644 activates chromaffin cell calcium channels. Nature 309:69–71.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Gillis KD,
    2. Mossner R, and
    3. Neher E
    (1996) Protein kinase C enhances exocytosis from chromaffin cells by increasing the size of the readily releasable pool of secretory granules. Neuron 16:1209–1220.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Goeger DE,
    2. Riley RT,
    3. Dorner JW, and
    4. Cole RJ
    (1988) Cyclopiazonic acid inhibition of the Ca2+-transport ATPase in rat skeletal muscle sarcoplasmic reticulum vesicles. Biochem Pharmacol 37:978–981.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Green DJ and
    2. Perlman RL
    (1981) On-line measurement of catecholamine secretion. Anal Biochem 110:270–276.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Hamill OP,
    2. Marty A,
    3. Neher E,
    4. Sakmann B, and
    5. Sigworth FJ
    (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Hendley ED and
    2. Welch BL
    (1975) Electroconvulsive shock: sustained decrease in norepinephrine uptake affinity in a reserpine model of depression. Life Sci 16:45–54.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Henry JP,
    2. Botton D,
    3. Sagne C,
    4. Isambert MF,
    5. Desnos C,
    6. Blanchard V,
    7. Raisman-Vozari R,
    8. Krejci E,
    9. Massoulie J, and
    10. Gasnier B
    (1994) Biochemistry and molecular biology of the vesicular monoamine transporter from chromaffin granules. J Exp Biol 196:251–262.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Hernández-Guijo JM,
    2. Maneu-Flores VE,
    3. Ruiz-Nuno A,
    4. Villarroya M,
    5. García AG, and
    6. Gandía L
    (2001) Calcium-dependent inhibition of L, N, and P/Q Ca2+ channels in chromaffin cells: role of mitochondria. J Neurosci 21:2553–2560.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Huntington Study Group
    (2006) Tetrabenazine as antichorea therapy in Huntington disease: a randomized controlled trial. Neurology 66:366–372.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Ikram H and
    2. Haleem DJ
    (2017) Repeated treatment with reserpine as a progressive animal model of depression. Pak J Pharm Sci 30:897–902.
    OpenUrl
  36. ↵
    1. Jankovic J
    (2016) Dopamine depleters in the treatment of hyperkinetic movement disorders. Expert Opin Pharmacother 17:2461–2470.
    OpenUrl
  37. ↵
    1. Jankovic J and
    2. Roos RA
    (2014) Chorea associated with Huntington’s disease: to treat or not to treat? Mov Disord 29:1414–1418.
    OpenUrl
  38. ↵
    1. Lara B,
    2. López MG,
    3. Villarroya M,
    4. Gandía L,
    5. Cleeman L,
    6. Morad M, and
    7. García AG
    (1997) A caffeine-sensitive Ca2+ store modulates K+-evoked secretion in chromaffin cells. Am J Physiol 272:C1211–C1221.
    OpenUrl
  39. ↵
    1. Lattanzio FA Jr.,
    2. Schlatterer RG,
    3. Nicar M,
    4. Campbell KP, and
    5. Sutko JL
    (1987) The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes. J Biol Chem 262:2711–2718.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Mahapatra S,
    2. Calorio C,
    3. Vandael DH,
    4. Marcantoni A,
    5. Carabelli V, and
    6. Carbone E
    (2012) Calcium channel types contributing to chromaffin cell excitability, exocytosis and endocytosis. Cell Calcium 51:321–330.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Mandela P,
    2. Chandley M,
    3. Xu YY,
    4. Zhu MY, and
    5. Ordway GA
    (2010) Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles. Neurochem Int 56:760–767.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Mark Wightman R,
    2. Domínguez N, and
    3. Borges R
    (2018) How intravesicular composition affects exocytosis. Pflugers Arch 470:135–141.
    OpenUrl
  43. ↵
    1. Maruyama T,
    2. Kanaji T,
    3. Nakade S,
    4. Kanno T, and
    5. Mikoshiba K
    (1997) 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem 122:498–505.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Milla J,
    2. Montesinos MS,
    3. Machado JD,
    4. Borges R,
    5. Alonso E,
    6. Moreno-Ortega AJ,
    7. Cano-Abad MF,
    8. García AG, and
    9. Ruiz-Nuño A
    (2011) Ouabain enhances exocytosis through the regulation of calcium handling by the endoplasmic reticulum of chromaffin cells. Cell Calcium 50:332–342.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Moro MA,
    2. López MG,
    3. Gandía L,
    4. Michelena P, and
    5. García AG
    (1990) Separation and culture of living adrenaline- and noradrenaline-containing cells from bovine adrenal medullae. Anal Biochem 185:243–248.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Mundorf ML,
    2. Troyer KP,
    3. Hochstetler SE,
    4. Near JA, and
    5. Wightman RM
    (2000) Vesicular Ca(2+) participates in the catalysis of exocytosis. J Biol Chem 275:9136–9142.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Neher E
    (1998) Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20:389–399.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Nowicky AV and
    2. Duchen MR
    (1998) Changes in [Ca2+]i and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons. J Physiol 507:131–145.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Pan CY and
    2. Fox AP
    (2000) Rundown of secretion after depletion of intracellular calcium stores in bovine adrenal chromaffin cells. J Neurochem 75:1132–1139.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Pessah IN,
    2. Francini AO,
    3. Scales DJ,
    4. Waterhouse AL, and
    5. Casida JE
    (1986) Calcium-ryanodine receptor complex. Solubilization and partial characterization from skeletal muscle junctional sarcoplasmic reticulum vesicles. J Biol Chem 261:8643–8648.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Rettig J and
    2. Neher E
    (2002) Emerging roles of presynaptic proteins in Ca++-triggered exocytosis. Science 298:781–785.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Seidler NW,
    2. Jona I,
    3. Vegh M, and
    4. Martonosi A
    (1989) Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum. J Biol Chem 264:17816–17823.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Stauderman KA and
    2. Pruss RM
    (1990) Different patterns of agonist-stimulated increases of 3H-inositol phosphate isomers and cytosolic Ca2+ in bovine adrenal chromaffin cells: comparison of the effects of histamine and angiotensin II. J Neurochem 54:946–953.
    OpenUrlPubMed
  54. ↵
    1. Tsien RW and
    2. Tsien RY
    (1990) Calcium channels, stores, and oscillations. Annu Rev Cell Biol 6:715–760.
    OpenUrlCrossRefPubMed
  55. ↵
    1. Villalobos C,
    2. Nuñez L,
    3. Montero M,
    4. García AG,
    5. Alonso MT,
    6. Chamero P,
    7. Álvarez J, and
    8. García-Sancho J
    (2002) Redistribution of Ca2+ among cytosol and organella during stimulation of bovine chromaffin cells. FASEB J 16:343–353.
    OpenUrlCrossRefPubMed
  56. ↵
    1. Villarroya M,
    2. Olivares R,
    3. Ruíz A,
    4. Cano-Abad MF,
    5. de Pascual R,
    6. Lomax RB,
    7. López MG,
    8. Mayorgas I,
    9. Gandía L, and
    10. García AG
    (1999) Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells. J Physiol 516:421–432.
    OpenUrlCrossRefPubMed
  57. ↵
    1. von Rüden L and
    2. Neher E
    (1993) A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells. Science 262:1061–1065.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Weihe E,
    2. Schäfer MK,
    3. Erickson JD, and
    4. Eiden LE
    (1994) Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat. J Mol Neurosci 5:149–164.
    OpenUrlCrossRefPubMed
  59. ↵
    1. Yang Y,
    2. Udayasankar S,
    3. Dunning J,
    4. Chen P, and
    5. Gillis KD
    (2002) A highly Ca2+-sensitive pool of vesicles is regulated by protein kinase C in adrenal chromaffin cells. Proc Natl Acad Sci USA 99:17060–17065.
    OpenUrlAbstract/FREE Full Text
  60. ↵
    1. Yero T and
    2. Rey JA
    (2008) Tetrabenazine (xenazine), an FDA-approved treatment option for Huntington’s disease-related chorea. P&T 33:690–694.
    OpenUrl
  61. ↵
    1. Yuchi Z,
    2. Lau K, and
    3. Van Petegem F
    (2012) Disease mutations in the ryanodine receptor central region: crystal structures of a phosphorylation hot spot domain. Structure 20:1201–1211.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleNeuropharmacology

Tetrabenazine Facilitates Exocytosis through Ryanodine Receptor

Ricardo de Pascual, Nuria Álvarez-Ortego, Cristóbal de los Ríos, Gema Jacob-Mazariego and Antonio G. García
Journal of Pharmacology and Experimental Therapeutics October 1, 2019, 371 (1) 219-230; DOI: https://doi.org/10.1124/jpet.119.256560
del.icio.us logo Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo

Jump to section

Advertisement