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Thioxanthenes, chlorprothixene and flupentixol inhibit proton currents in BV2 microglial cells
European Journal of Pharmacology 779 (2016) 31–37
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Thioxanthenes, chlorprothixene and flupentixol inhibit proton currents in BV2 microglial cells Jiwon Kim, Jin-Ho Song n Department of Pharmacology, College of Medicine, Chung-Ang University, 84 Heukseok-Ro, Dongjak-Gu, Seoul 06974, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 21 October 2015 Received in revised form 2 March 2016 Accepted 2 March 2016 Available online 3 March 2016
The thioxanthene antipsychotic drugs chlorprothixene and flupentixol have anti-inflammatory and antioxidant properties. The reactive oxygen species produced by NADPH oxidase during microglia-mediated inflammatory responses cause neuronal damage, thereby contributing to various neurodegenerative diseases. Voltage-gated proton channels sustain the NADPH oxidase activity, and inhibition of the channels’ activity reduces the production of reactive oxygen species. Herein, the effects of chlorprothixene and flupentixol on proton currents were investigated in BV2 microglial cells using the wholecell patch-clamp method. Both drugs inhibited the proton currents in a concentration-dependent manner (IC50 ¼ 1.7 μM and 6.6 μM, respectively). Chlorprothixene at 3 μM slightly shifted the activation voltage toward depolarization. Both the activation and the deactivation kinetics of the proton currents were slowed by chlorprothixene 1.2- and 3.5-fold, respectively. Thus, the inhibition of proton currents may be partly responsible for the antioxidant effects of thioxanthene antipsychotic drugs. & 2016 Elsevier B.V. All rights reserved.
Keywords: Chlorprothixene Flupentixol Microglia Proton channel Thioxanthenes
1. Introduction Microglia-mediated inflammatory responses and the release of reactive oxygen species contribute to the neurotoxicity observed in neurodegenerative diseases (Block et al., 2007; Sorce et al., 2012). NADPH oxidase is an important source of reactive oxygen species and transports electrons across the plasma membrane to produce superoxide and other downstream reactive oxygen species (Bedard and Krause, 2007). In a schizophrenia model, NADPH oxidase activity was up-regulated in the nucleus accumbens and the prefrontal cortex with a concomitant increase in markers of oxidative stress and immunoreactive microglia (Schiavone et al., 2009). In Alzheimer’s disease patients, cognitive performance decreased with increasing NADPH oxidase activity in the frontal and temporal cortex (Ansari and Scheff, 2011). Moreover, diphenyleneiodonium, an NADPH oxidase inhibitor, was neuroprotective against lipopolysaccharide-, 1-methyl-4-phenylpyridinium-, and rotenone-induced insults (Wang et al., 2014). Voltage-gated proton channels (HV1) are unique since they lack the pore domain found in other conventional voltage-gated ion channels (Ramsey et al., 2006; Sasaki et al., 2006). Instead, a proton channel is a dimer of voltage sensors in which each monomer contains its own pore and gate (Tombola et al., 2008). In mammals, proton channels are expressed predominantly in the n
Corresponding author. E-mail address: [email protected] (J.-H. Song).
http://dx.doi.org/10.1016/j.ejphar.2016.03.009 0014-2999/& 2016 Elsevier B.V. All rights reserved.
immune cells, where they are involved in acid extrusion (DeCoursey, 2013; Sasaki et al., 2006). Proton channels are functionally closely related to NADPH oxidase. For example, phorbol myristate acetate, a potent stimulator of the respiratory burst in neutrophils, increases the electron transport by NADPH oxidase and the proton currents simultaneously (DeCoursey et al., 2000). The charge displacement across the plasma membrane and the intracellular acid accumulation during NADPH oxidase activity must be compensated to sustain NADPH oxidase’s high rate of reactive oxygen species production (DeCoursey et al., 2003; Morgan et al., 2005). Proton channels are the most suitable for this purpose (Murphy and DeCoursey, 2006). When proton channels are inhibited by Zn2 þ , or are absent, such as in HV1 / mice, the intracellular pH regulation is severely impaired and the production of reactive oxygen species decreases (El Chemaly et al., 2010; Morgan et al., 2009). Neuroinflammation and oxidative stresses are believed to contribute to the pathophysiological mechanisms of schizophrenia, and several antipsychotics show an anti-inflammatory effect (Leza et al., 2015). Chlorprothixene, a thioxanthene antipsychotic drug, suppressed the production of reactive oxygen species from activated macrophages, because of its immunomodulatory effect on macrophages, rather than through a direct radical-scavenging effect (Hadjimitova et al., 2004). Flupentixol, another thioxanthene antipsychotic drug, inhibited lymphocyte proliferation, interleukin-2 production, and immune responses, both in vivo and in vitro. These effects were not mediated via specific dopamine receptors (Boukhris et al., 1988;
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Panajotova, 1997). In addition, flupentixol inhibited the release of pro-inflammatory cytokines, tumor necrosis factor-α, interleukin1β, interleukin-2, and nitric oxide by lipopolysaccharide-stimulated rat microglia (Kowalski et al., 2003, 2004). In the present study, we investigated the effects of chlorprothixene and flupentixol on the proton currents in microglial BV2 cells in relation to their anti-inflammatory and antioxidant potential.
2. Materials and methods 2.1. Cell culture A mouse microglial BV2 cell culture was routinely maintained in Dulbecco’s modified Eagle medium supplemented with fetal bovine serum (10%) (WELGENE, Daegu, Republic of Korea), penicillin (100 units/ml) and streptomycin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO) at 37°C in 5% CO2/humidified air. For the experiments, the cells were plated onto 12-mm glass coverslips coated with poly-L-lysine in 6-well cell culture plates. The experiments were performed for a period of 2–4 days following cell plating. 2.2. Solutions The cells along with the coverslip were bathed in the external solution containing 85 mM N-methyl-D-glucamine aspartate, 100 mM HEPES, 1 mM CaCl2, and 1 mM MgCl2 (pH 7.3 with CsOH). A patch pipette was filled with the internal solution containing 85 mM N-methyl-D-glucamine aspartate, 120 mM 2-(N-morpholino)ethanesulfonic acid (MES), 1 mM EGTA, and 3 mM MgCl2 (pH 5.5 with CsOH). Chlorprothixene and flupentixol were dissolved in dimethylsulfoxide to obtain a 1000-fold stock solution. The stock solution was stored at 20°C in small aliquots, and was diluted to the desired concentrations in the external solution immediately before the experiments. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). 2.3. Whole-cell patch-clamp recordings The whole-cell patch-clamp recordings were performed at room temperature (22–24°C). The patch pipette electrode was pulled from a borosilicate glass capillary tube (G150TF-4, Warner Instrument, Hamden, CT) using a pipette puller (PP83, Narishige, Tokyo, Japan), and the tip of the pipette was heat-polished using a microforge (MF83, Narishige) to a final resistance of 6–8 MΩ when filled with the internal solution. The cells were voltage-clamped in the conventional whole-cell configuration using an Axopatch 200B amplifier, Digidata 1322A interface, and pCLAMP8 acquisition software (Molecular Devices, Sunnyvale, CA). The analog data were filtered and digitized at 1 kHz. An Ag-AgCl pellet/3 M KCl-agar bridge was used as the reference electrode. The liquid junction potential of þ1.3 mV between the internal and external solutions was corrected before gigaohm-seal formation. The data were expressed as the mean 7S.E.M. of at least 7 determinations, with n referring to the number of cells examined. The statistical significance was assessed by the Student’s t-test, and was considered significant at P o0.05.
3. Results 3.1. Chlorprothixene and flupentixol inhibit proton currents The cells were voltage-clamped at a holding potential of 70 mV in the standard whole-cell recording configuration, and
Fig. 1. Chlorprothixene-induced inhibition of the amplitude of voltage-gated proton currents in BV2 microglial cells. The proton currents were evoked by a 2-s depolarizing step from a holding potential of 70 mV to þ20 mV, every 15 s. (A) Representative current traces in the control (a), in the presence of chlorprothixene at 3 μM for 15 min (b), and after washout for 30 min (c) are shown. (B) Time-course showing the inhibition of the proton current amplitude by chlorprothixene. Points (a), (b), and (c) indicate the times at which the current traces in (A) were acquired.
the proton currents were activated by 2-s steps to þ20 mV. The resultant proton currents showed slow kinetics with both activation and deactivation developing in hundreds of milliseconds (Fig. 1A). The effect of chlorprothixene on the proton current amplitude was examined. The application of chlorprothixene at 3 μM decreased the proton current amplitude slowly to 4373% (n ¼10) of the control in 15 min (Fig. 1). The inhibition was partially recovered to 72 74% of the control upon washout with the external solution for a period of 30 min. The effects of chlorprothixene were concentration-dependent (Fig. 2). Chlorprothixene was applied at several concentrations and the reduction in the current amplitude was assessed with the following Hill equation:
Ichlorprothixene/Icontrol = 1/(1 + ([chlorprothixene]/IC50)h) where Ichlorprothixene is the current amplitude measured after treatment with chlorprothixene for 15 min, Icontrol is the control current amplitude, [chlorprothixene] is the concentration of chlorprothixene, IC50 is the half-maximal inhibitory concentration, and h is the Hill coefficient. The IC50 and the Hill coefficient of chlorprothixene were estimated to be 1.7 μM and 0.93 (n ¼7–10), respectively. Flupentixol also inhibited the proton currents in a concentration-dependent manner, with an IC50 and Hill coefficient of 6.6 μM and 0.80 (n ¼ 7–9), respectively. At higher concentrations (10 μM and 30 μM), flupentixol was toxic and induced cell membrane instability within a short period. Since it was difficult to maintain the electrical stability of the membrane for a period of 15 min, the effect was measured at 5 min after treatment when a complete effect had not yet developed. Thus, the potency of flupentixol was underestimated and the actual IC50 value should have been less than the one estimated.
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maximal conductance to 487 2% of the control. G-V data for flupentixol could not be determined due to its toxicity. Instead, the steady-state conductance at þ20 mV, at which the conductance was near maximum, was measured. Flupentixol, under the same conditions as chlorprothixene (at 3 μM for 15 min), reduced the conductance to 58 77% (n ¼8) of the control. 3.3. The effects of chlorprothixene on the reversal potential of proton currents
Fig. 2. Concentration-dependent inhibition of proton currents by chlorprothixene and flupentixol. The proton currents were evoked by a 2-s depolarizing step from a holding potential of 70 mV to þ20 mV. The cells were perfused with an external solution containing different concentrations of chlorprothixene (filled circles) or flupentixol (open circles) for 15 min, except for flupentixol at concentrations of 10 μM and 30 μM (5-min perfusion). The current amplitude after treatment with the respective drug was normalized to that of the control. The data points represent the mean 7 S.E.M. of 7–10 cells. The solid lines show the fit with a standard doseresponse relationship, which yielded an estimated IC50 of 1.7 μM and 6.6 μM and a Hill coefficient of 0.93 and 0.80 for chlorprothixene and flupentixol, respectively.
3.2. The effects of chlorprothixene on the voltage-dependent activation of proton currents The voltage-dependence of the proton current activation was assessed by measuring the current amplitude following a series of depolarizing steps, and the current-voltage (I-V) relationship was determined. Since the currents did not reach the steady-state during 2-s depolarizing pulses, the steady-state current amplitude was measured by a fit with an exponential function. Fig. 3 shows the effects of chlorprothixene on the I-V curve. The activation threshold at which the proton channels start to open was around 40 mV, which did not change after the treatment with chlorprothixene (3 μM) for 15 min. Chlorprothixene inhibited the amplitude of proton currents across a range of test voltages, with no obvious shift in the I-V curve. For the quantitative assessment of the voltage-dependence of proton currents, the conductance-voltage (G-V) relationship was calculated from the I-V data. The conductance (G) was obtained by dividing the current amplitude (I) by the driving force (Vm Vrev), where Vm is the test voltage and Vrev is the reversal potential determined from the individual cells. G-V plots were fitted by the following Boltzmann equation:
G/Gmax = 1/(1 + exp((V0.5 − Vm)/k )) where Gmax is the maximal conductance at þ40 mV, V0.5 is the potential at half-maximal activation, and k is the slope factor. In the absence of chlorprothixene, V0.5 and k were estimated to be 21.971.4 mV and 8.9 70.3 mV (n¼ 8), respectively. After treatment with chlorprothixene, they were 19.7 71.5 mV (P o0.05) and 7.870.4 mV (Po0.01), respectively. Despite the minimal change in the values, a statistically significant difference was observed in both parameters before and after treatment with chlorprothixene. The steady-state conductance reached a maximum around þ30 mV (Fig. 3C). Chlorprothixene reduced the
The reversal potential at which the direction of proton currents changes from inward to outward reflects the pH gradient between the intra- and extracellular environment. The pH gradient affects the gating of the proton channels as well as the driving force for protons (Cherny et al., 1995). It is postulated that chlorprothixene – being a weak base – transverses the plasma membrane and binds to protons, thereby increasing the intracellular pH. The effects of chlorprothixene on the reversal potential were determined (Fig. 4). The cells were stimulated with a 2-s depolarizing step from a holding potential of 70 mV to þ40 mV, and were then repolarized to potentials between 70 mV and 100 mV in 10mV decrements. The tail current at the repolarizing potential was fitted with an exponential function, and the current amplitude at the start of repolarization was measured by extrapolating the fitted curve. The voltage at which the tail current crossed the baseline was considered as the reversal potential. In the absence of chlorprothixene, the reversal potential was estimated to be 87.071.7 mV (n ¼ 8). However, after treatment with chlorprothixene (3 μM) for 15 min, a statistically significant difference was observed in the reversal potential, which had shifted toward hyperpolarization at 90 71.0 mV (P o0.01). For flupentixol, the reversal potential was estimated by the base line intercept of a line connecting the current at the end of a 2-s depolarizing pulse to þ20 mV and the tail current. In control conditions, it was 83.4 71.3 mV (n ¼8), which had shifted toward hyperpolarization at 84.1 71.2 mV (P o0.05) after treatment with flupentixol (3 μM) for 15 min. 3.4. The effects of chlorprothixene on the proton current kinetics As shown in Figs. 1, 3, and 4, a retardation of the time course of both the peak proton current and the tail current was observed after the treatment with chlorprothixene. To quantify the change in kinetics, the activation and deactivation phases of the proton currents recorded at a depolarizing and repolarizing step to þ20 mV and 70 mV, respectively, were fitted with a mono-exponential function (Fig. 5). In the absence of chlorprothixene, the activation and deactivation time constants were estimated to be 721 749 ms and 315 715 ms (n ¼50), respectively. However, after treatment with chlorprothixene (3 μM) for 15 min, the activation and deactivation time constants increased significantly to 829 760 ms (P o0.001) and 1085 760 ms (P o0.001), respectively. Flupentixol, under the same conditions as chlorprothixene (at 3 μM for 15 min), increased the activation time constant from 602 759 ms to 7767 105 ms (n¼ 8, P o0.05), but had no effect on the deactivation constant (211 714 ms and 215 734 ms, before and after treatment with flupentixol, respectively).
4. Discussion Previously, we have shown that certain antipsychotic drugs strongly inhibit proton currents (Shin et al., 2015; Shin and Song, 2014). These drugs, including chlorpromazine (a phenothiazine), haloperidol (a butyrophenone), clozapine (a dibenzodiazepine), and olanzapine (a thienobenzodiazepine), have different chemical
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Fig. 3. The effects of chlorprothixene on the voltage-dependent activation of proton currents. The cells were stimulated from a holding potential of 70 mV to test potentials ranging from 50 mV to þ40 mV in a series of steps of 10-mV increments, first under control conditions and then in the presence of chlorprothixene at 3 μM for 15 min. (A) Representative current traces recorded with this protocol. (B) Current-voltage (I-V) relationship plot for the data obtained from (A). The steady-state current amplitude was estimated by a fit with an exponential function. (C) Conductance-voltage (G-V) relationship plot under control conditions (open circles) and in the presence of chlorprothixene (filled circles and triangles) (n¼ 8). The conductance was normalized to that of the test potential of þ40 mV (circles) or to a maximal control conductance (triangles). The solid line is the fit with a Boltzmann function.
structures. The drugs’ proton current-inhibiting potency was unrelated to their affinity for dopamine or serotonin receptors. Moreover, risperidone (a benzisoxazole) had no substantial effects on proton currents. These observations suggest that the pharmacological profiles of these antipsychotic drugs may not be directly related to the inhibition of proton currents. Instead, their specific chemical structure may be an important determinant of such activity. Thioxanthenes are structurally similar to phenothiazines, in that the nitrogen atom at position 10 in phenothiazines is substituted by a carbon atom that is flanked by a double bond to the side chain. In this study, we report that the two thioxanthenes chlorprothixene and flupentixol inhibit proton currents with high potency. The IC50 values for chlorprothixene and flupentixol were 1.7 μM and 6.6 μM, respectively (Fig. 2). The potency of chlorprothixene is comparable to that of chlorpromazine (IC50 ¼ 2.2 μM) (Shin and Song, 2014). Chlorprothixene, containing a tricyclic ring structure, resembles chlorpromazine but has a more rigid structure (Bergmann et al., 1974). Nevertheless, herein, the rigidity of the structure or the substitution of the nitrogen atom by the carbon atom did not seem to affect the proton current inhibition. Although flupentixol had a slightly less potent proton current-inhibiting capacity compared to that of chlorprothixene, it inhibited dopamine-sensitive responses more potently and showed higher clinical antipsychotic efficacy than were observed for
chlorprothixene (Feinberg and Snyder, 1975). The bulkier piperazineethanol and trifluoromethyl side chains in flupentixol may hinder the drug’s interaction with proton channels. The structure-dependent effect of thioxanthenes can also be observed in their interaction with other membrane proteins. Permeability glycoprotein (P-glycoprotein) encoded by the multidrug resistant (MDR) gene resembles an ion channel both in structure and function, and several ion channel blockers, such as verapamil and quinidine, are also permeability glycoprotein modulators (Rodriguez et al., 1999). Thioxanthenes sensitize cancer cells that are multidrug-resistant to chemotherapeutic drugs in a structuredependent manner, which is not related to their antipsychotic potencies (Ford et al., 1990). For example, trans-flupentixol has a greater anti-multidrug-resistance activity than that of cis-flupentixol, but lacks binding affinity for dopamine and serotonin receptors. Such stereospecificity is related to the different orientation of the molecules in the membrane’s lipid environment. Indeed, trans-flupentixol interacts more strongly with phospholipids than cis-flupentixol (Wiese and Pajeva, 1997). Eventually, flupentixol interacts with the protein allosterically at the lipidprotein interface (Mandal et al., 2012). Chlorprothixene slowed both the activation and the deactivation kinetics of proton currents; the latter was more markedly affected (Fig. 5). This effect suggests that chlorprothixene interferes with the gating of proton channels. Hanatoxin, a tarantula
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Fig. 4. The effects of chlorprothixene on the reversal potential of proton currents. The cells were stimulated with a 2-s depolarizing step from a holding potential of 70 mV to þ40 mV, and were then repolarized to potentials between 70 mV and 100 mV in 10-mV decrements. (A) Representative current families before and after treatment with chlorprothixene 3 μM for 15 min. The insets show enlarged views of the tail currents at the repolarizing potential. The dotted line denotes the zero current level. (B) The amplitude of the tail current measured at the repolarizing potential, for which the data were obtained from (A). (C) A comparison of the reversal potentials before and after treatment with chlorprothixene (n¼ 8, **P o 0.01).
Fig. 5. The effects of chlorprothixene on the proton current kinetics. The proton currents were evoked by a 2-s depolarizing step from a holding potential of 70 mV to þ20 mV, before and after treatment with chlorprothixene (3 μM) for 15 min. The peak current and the tail current were fitted with a mono-exponential function. The activation and the deactivation time constants and the fitted traces before (dashed lines) and after (dotted lines) treatment with chlorprothixene are shown (n¼ 50, ***P o0.001).
toxin, inhibited the proton currents expressed in HEK cells. The toxin was presumed to move into the lipid membrane and interact with the voltage-sensor paddle motif at the lipid-protein interface, stabilizing its resting state (Alabi et al., 2007). As a result, hanatoxin shifted the activation voltage in the positive direction and a strong depolarization could relieve the current inhibition. Another gating modifier, NH17, a diphenylamine carboxylate derivative, also inhibited the proton currents with a positive shift in the activation voltage (Kornilov et al., 2014). In the present study, chlorprothixene at 3 μM, shifted the activation voltage of the proton currents only marginally (þ2.2 mV) (Fig. 3). Thus, it appears that chlorprothixene slows the opening transition of the proton channel rather than stabilizing its resting/closed state. Upon repolarization, the open channel is deactivated, which is reflected by the tail current decay. The deactivation time constant was markedly increased by chlorprothixene compared to the activation time constant. Thus, chlorpromazine hinders the closing transition of the proton channel more effectively. Although flupentixol increased the activation constant similarly to chlorprothixene, it had almost no effect on the deactivation constant, suggesting somewhat different modes of action between two drugs.
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The gating of the proton channel is regulated by the pH gradient across the plasma membrane (Cherny et al., 1995). Local anesthetics, such as lidocaine and bupivacaine, inhibit proton currents by increasing the intracellular pH, an observation that is supported by the large positive shift in both the reversal potential and the activation voltage (Matsuura et al., 2012). Chlorprothixene, however, produced a small but statistically significant shift in the reversal potential to the opposite direction (Fig. 4). Considering its negligible effect on the activation voltage, this result negates the assumption that chlorprothixene inhibits proton currents by altering the intracellular pH. This notion holds true for flupentixol, since it also shifted the reversal potential slightly to the hyperpolarizing direction. A small shift in the activation voltage could not have accounted for more than half the reduction of the proton currents by chlorprothixene at 3 μM (Fig. 3). It was suggested that extracellularly applied imipramine inhibited proton currents from the interior of the cell after crossing the plasma membrane (Song et al., 2012). Having a similar tricyclic ring structure, chlorprothixene is assumed to act similarly. Likewise, 2-guanidinobenzimidazole (2GBI), a guanidine derivative, blocks proton channels only when applied intracellularly, without altering the activation voltage (Hong et al., 2013). In consistent with this notion, increasing the intracellular pH from 5.5 to 6.0, which would decrease its accumulation inside the cell, chlorprothixene (3 μM) inhibited the proton currents at þ40 mV by 447 1% (n¼ 3), which was less compared to that attained at pH 5.5 (57 7 3%, n ¼10). Both chlorprothixene and flupentixol reduced the steady-state conductance, suggesting that they hinder the conducting pathway of the proton channels. Deletion of HV1 reduced NADPH oxidase-dependent reactive oxygen species production, and prevented neuronal death and brain damage after ischemic stroke (Wu et al., 2012). Thus, inhibition of the proton channels may be a therapeutic strategy in the treatment of ischemic stroke and other neurodegenerative diseases associated with oxidative stresses. HV1 is a more preferable target than NADPH oxidase since it is expressed in microglia but not in neurons; hence, inhibition of proton channels will not interfere with neuronal NADPH oxidase activity. Inflammation and oxidative stress are contributing factors in the pathogenesis of schizophrenia (Leza et al., 2015). Chlorprothixene and flupentixol have been shown to possess immunosuppressive and antioxidant properties (Hadjimitova et al., 2004; Kowalski et al., 2003, 2004). The drug’s proton channel inhibition may be partly responsible for these effects.
Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2010-0022213 and NRF2014R1A1A2054977).
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Thioxanthenes, chlorprothixene and flupentixol inhibit proton currents in BV2 microglial cells Jiwon Kim, Jin-Ho Song n Department of Pharmacology, College of Medicine, Chung-Ang University, 84 Heukseok-Ro, Dongjak-Gu, Seoul 06974, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 21 October 2015 Received in revised form 2 March 2016 Accepted 2 March 2016 Available online 3 March 2016
The thioxanthene antipsychotic drugs chlorprothixene and flupentixol have anti-inflammatory and antioxidant properties. The reactive oxygen species produced by NADPH oxidase during microglia-mediated inflammatory responses cause neuronal damage, thereby contributing to various neurodegenerative diseases. Voltage-gated proton channels sustain the NADPH oxidase activity, and inhibition of the channels’ activity reduces the production of reactive oxygen species. Herein, the effects of chlorprothixene and flupentixol on proton currents were investigated in BV2 microglial cells using the wholecell patch-clamp method. Both drugs inhibited the proton currents in a concentration-dependent manner (IC50 ¼ 1.7 μM and 6.6 μM, respectively). Chlorprothixene at 3 μM slightly shifted the activation voltage toward depolarization. Both the activation and the deactivation kinetics of the proton currents were slowed by chlorprothixene 1.2- and 3.5-fold, respectively. Thus, the inhibition of proton currents may be partly responsible for the antioxidant effects of thioxanthene antipsychotic drugs. & 2016 Elsevier B.V. All rights reserved.
Keywords: Chlorprothixene Flupentixol Microglia Proton channel Thioxanthenes
1. Introduction Microglia-mediated inflammatory responses and the release of reactive oxygen species contribute to the neurotoxicity observed in neurodegenerative diseases (Block et al., 2007; Sorce et al., 2012). NADPH oxidase is an important source of reactive oxygen species and transports electrons across the plasma membrane to produce superoxide and other downstream reactive oxygen species (Bedard and Krause, 2007). In a schizophrenia model, NADPH oxidase activity was up-regulated in the nucleus accumbens and the prefrontal cortex with a concomitant increase in markers of oxidative stress and immunoreactive microglia (Schiavone et al., 2009). In Alzheimer’s disease patients, cognitive performance decreased with increasing NADPH oxidase activity in the frontal and temporal cortex (Ansari and Scheff, 2011). Moreover, diphenyleneiodonium, an NADPH oxidase inhibitor, was neuroprotective against lipopolysaccharide-, 1-methyl-4-phenylpyridinium-, and rotenone-induced insults (Wang et al., 2014). Voltage-gated proton channels (HV1) are unique since they lack the pore domain found in other conventional voltage-gated ion channels (Ramsey et al., 2006; Sasaki et al., 2006). Instead, a proton channel is a dimer of voltage sensors in which each monomer contains its own pore and gate (Tombola et al., 2008). In mammals, proton channels are expressed predominantly in the n
Corresponding author. E-mail address: [email protected] (J.-H. Song).
http://dx.doi.org/10.1016/j.ejphar.2016.03.009 0014-2999/& 2016 Elsevier B.V. All rights reserved.
immune cells, where they are involved in acid extrusion (DeCoursey, 2013; Sasaki et al., 2006). Proton channels are functionally closely related to NADPH oxidase. For example, phorbol myristate acetate, a potent stimulator of the respiratory burst in neutrophils, increases the electron transport by NADPH oxidase and the proton currents simultaneously (DeCoursey et al., 2000). The charge displacement across the plasma membrane and the intracellular acid accumulation during NADPH oxidase activity must be compensated to sustain NADPH oxidase’s high rate of reactive oxygen species production (DeCoursey et al., 2003; Morgan et al., 2005). Proton channels are the most suitable for this purpose (Murphy and DeCoursey, 2006). When proton channels are inhibited by Zn2 þ , or are absent, such as in HV1 / mice, the intracellular pH regulation is severely impaired and the production of reactive oxygen species decreases (El Chemaly et al., 2010; Morgan et al., 2009). Neuroinflammation and oxidative stresses are believed to contribute to the pathophysiological mechanisms of schizophrenia, and several antipsychotics show an anti-inflammatory effect (Leza et al., 2015). Chlorprothixene, a thioxanthene antipsychotic drug, suppressed the production of reactive oxygen species from activated macrophages, because of its immunomodulatory effect on macrophages, rather than through a direct radical-scavenging effect (Hadjimitova et al., 2004). Flupentixol, another thioxanthene antipsychotic drug, inhibited lymphocyte proliferation, interleukin-2 production, and immune responses, both in vivo and in vitro. These effects were not mediated via specific dopamine receptors (Boukhris et al., 1988;
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Panajotova, 1997). In addition, flupentixol inhibited the release of pro-inflammatory cytokines, tumor necrosis factor-α, interleukin1β, interleukin-2, and nitric oxide by lipopolysaccharide-stimulated rat microglia (Kowalski et al., 2003, 2004). In the present study, we investigated the effects of chlorprothixene and flupentixol on the proton currents in microglial BV2 cells in relation to their anti-inflammatory and antioxidant potential.
2. Materials and methods 2.1. Cell culture A mouse microglial BV2 cell culture was routinely maintained in Dulbecco’s modified Eagle medium supplemented with fetal bovine serum (10%) (WELGENE, Daegu, Republic of Korea), penicillin (100 units/ml) and streptomycin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO) at 37°C in 5% CO2/humidified air. For the experiments, the cells were plated onto 12-mm glass coverslips coated with poly-L-lysine in 6-well cell culture plates. The experiments were performed for a period of 2–4 days following cell plating. 2.2. Solutions The cells along with the coverslip were bathed in the external solution containing 85 mM N-methyl-D-glucamine aspartate, 100 mM HEPES, 1 mM CaCl2, and 1 mM MgCl2 (pH 7.3 with CsOH). A patch pipette was filled with the internal solution containing 85 mM N-methyl-D-glucamine aspartate, 120 mM 2-(N-morpholino)ethanesulfonic acid (MES), 1 mM EGTA, and 3 mM MgCl2 (pH 5.5 with CsOH). Chlorprothixene and flupentixol were dissolved in dimethylsulfoxide to obtain a 1000-fold stock solution. The stock solution was stored at 20°C in small aliquots, and was diluted to the desired concentrations in the external solution immediately before the experiments. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). 2.3. Whole-cell patch-clamp recordings The whole-cell patch-clamp recordings were performed at room temperature (22–24°C). The patch pipette electrode was pulled from a borosilicate glass capillary tube (G150TF-4, Warner Instrument, Hamden, CT) using a pipette puller (PP83, Narishige, Tokyo, Japan), and the tip of the pipette was heat-polished using a microforge (MF83, Narishige) to a final resistance of 6–8 MΩ when filled with the internal solution. The cells were voltage-clamped in the conventional whole-cell configuration using an Axopatch 200B amplifier, Digidata 1322A interface, and pCLAMP8 acquisition software (Molecular Devices, Sunnyvale, CA). The analog data were filtered and digitized at 1 kHz. An Ag-AgCl pellet/3 M KCl-agar bridge was used as the reference electrode. The liquid junction potential of þ1.3 mV between the internal and external solutions was corrected before gigaohm-seal formation. The data were expressed as the mean 7S.E.M. of at least 7 determinations, with n referring to the number of cells examined. The statistical significance was assessed by the Student’s t-test, and was considered significant at P o0.05.
3. Results 3.1. Chlorprothixene and flupentixol inhibit proton currents The cells were voltage-clamped at a holding potential of 70 mV in the standard whole-cell recording configuration, and
Fig. 1. Chlorprothixene-induced inhibition of the amplitude of voltage-gated proton currents in BV2 microglial cells. The proton currents were evoked by a 2-s depolarizing step from a holding potential of 70 mV to þ20 mV, every 15 s. (A) Representative current traces in the control (a), in the presence of chlorprothixene at 3 μM for 15 min (b), and after washout for 30 min (c) are shown. (B) Time-course showing the inhibition of the proton current amplitude by chlorprothixene. Points (a), (b), and (c) indicate the times at which the current traces in (A) were acquired.
the proton currents were activated by 2-s steps to þ20 mV. The resultant proton currents showed slow kinetics with both activation and deactivation developing in hundreds of milliseconds (Fig. 1A). The effect of chlorprothixene on the proton current amplitude was examined. The application of chlorprothixene at 3 μM decreased the proton current amplitude slowly to 4373% (n ¼10) of the control in 15 min (Fig. 1). The inhibition was partially recovered to 72 74% of the control upon washout with the external solution for a period of 30 min. The effects of chlorprothixene were concentration-dependent (Fig. 2). Chlorprothixene was applied at several concentrations and the reduction in the current amplitude was assessed with the following Hill equation:
Ichlorprothixene/Icontrol = 1/(1 + ([chlorprothixene]/IC50)h) where Ichlorprothixene is the current amplitude measured after treatment with chlorprothixene for 15 min, Icontrol is the control current amplitude, [chlorprothixene] is the concentration of chlorprothixene, IC50 is the half-maximal inhibitory concentration, and h is the Hill coefficient. The IC50 and the Hill coefficient of chlorprothixene were estimated to be 1.7 μM and 0.93 (n ¼7–10), respectively. Flupentixol also inhibited the proton currents in a concentration-dependent manner, with an IC50 and Hill coefficient of 6.6 μM and 0.80 (n ¼ 7–9), respectively. At higher concentrations (10 μM and 30 μM), flupentixol was toxic and induced cell membrane instability within a short period. Since it was difficult to maintain the electrical stability of the membrane for a period of 15 min, the effect was measured at 5 min after treatment when a complete effect had not yet developed. Thus, the potency of flupentixol was underestimated and the actual IC50 value should have been less than the one estimated.
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maximal conductance to 487 2% of the control. G-V data for flupentixol could not be determined due to its toxicity. Instead, the steady-state conductance at þ20 mV, at which the conductance was near maximum, was measured. Flupentixol, under the same conditions as chlorprothixene (at 3 μM for 15 min), reduced the conductance to 58 77% (n ¼8) of the control. 3.3. The effects of chlorprothixene on the reversal potential of proton currents
Fig. 2. Concentration-dependent inhibition of proton currents by chlorprothixene and flupentixol. The proton currents were evoked by a 2-s depolarizing step from a holding potential of 70 mV to þ20 mV. The cells were perfused with an external solution containing different concentrations of chlorprothixene (filled circles) or flupentixol (open circles) for 15 min, except for flupentixol at concentrations of 10 μM and 30 μM (5-min perfusion). The current amplitude after treatment with the respective drug was normalized to that of the control. The data points represent the mean 7 S.E.M. of 7–10 cells. The solid lines show the fit with a standard doseresponse relationship, which yielded an estimated IC50 of 1.7 μM and 6.6 μM and a Hill coefficient of 0.93 and 0.80 for chlorprothixene and flupentixol, respectively.
3.2. The effects of chlorprothixene on the voltage-dependent activation of proton currents The voltage-dependence of the proton current activation was assessed by measuring the current amplitude following a series of depolarizing steps, and the current-voltage (I-V) relationship was determined. Since the currents did not reach the steady-state during 2-s depolarizing pulses, the steady-state current amplitude was measured by a fit with an exponential function. Fig. 3 shows the effects of chlorprothixene on the I-V curve. The activation threshold at which the proton channels start to open was around 40 mV, which did not change after the treatment with chlorprothixene (3 μM) for 15 min. Chlorprothixene inhibited the amplitude of proton currents across a range of test voltages, with no obvious shift in the I-V curve. For the quantitative assessment of the voltage-dependence of proton currents, the conductance-voltage (G-V) relationship was calculated from the I-V data. The conductance (G) was obtained by dividing the current amplitude (I) by the driving force (Vm Vrev), where Vm is the test voltage and Vrev is the reversal potential determined from the individual cells. G-V plots were fitted by the following Boltzmann equation:
G/Gmax = 1/(1 + exp((V0.5 − Vm)/k )) where Gmax is the maximal conductance at þ40 mV, V0.5 is the potential at half-maximal activation, and k is the slope factor. In the absence of chlorprothixene, V0.5 and k were estimated to be 21.971.4 mV and 8.9 70.3 mV (n¼ 8), respectively. After treatment with chlorprothixene, they were 19.7 71.5 mV (P o0.05) and 7.870.4 mV (Po0.01), respectively. Despite the minimal change in the values, a statistically significant difference was observed in both parameters before and after treatment with chlorprothixene. The steady-state conductance reached a maximum around þ30 mV (Fig. 3C). Chlorprothixene reduced the
The reversal potential at which the direction of proton currents changes from inward to outward reflects the pH gradient between the intra- and extracellular environment. The pH gradient affects the gating of the proton channels as well as the driving force for protons (Cherny et al., 1995). It is postulated that chlorprothixene – being a weak base – transverses the plasma membrane and binds to protons, thereby increasing the intracellular pH. The effects of chlorprothixene on the reversal potential were determined (Fig. 4). The cells were stimulated with a 2-s depolarizing step from a holding potential of 70 mV to þ40 mV, and were then repolarized to potentials between 70 mV and 100 mV in 10mV decrements. The tail current at the repolarizing potential was fitted with an exponential function, and the current amplitude at the start of repolarization was measured by extrapolating the fitted curve. The voltage at which the tail current crossed the baseline was considered as the reversal potential. In the absence of chlorprothixene, the reversal potential was estimated to be 87.071.7 mV (n ¼ 8). However, after treatment with chlorprothixene (3 μM) for 15 min, a statistically significant difference was observed in the reversal potential, which had shifted toward hyperpolarization at 90 71.0 mV (P o0.01). For flupentixol, the reversal potential was estimated by the base line intercept of a line connecting the current at the end of a 2-s depolarizing pulse to þ20 mV and the tail current. In control conditions, it was 83.4 71.3 mV (n ¼8), which had shifted toward hyperpolarization at 84.1 71.2 mV (P o0.05) after treatment with flupentixol (3 μM) for 15 min. 3.4. The effects of chlorprothixene on the proton current kinetics As shown in Figs. 1, 3, and 4, a retardation of the time course of both the peak proton current and the tail current was observed after the treatment with chlorprothixene. To quantify the change in kinetics, the activation and deactivation phases of the proton currents recorded at a depolarizing and repolarizing step to þ20 mV and 70 mV, respectively, were fitted with a mono-exponential function (Fig. 5). In the absence of chlorprothixene, the activation and deactivation time constants were estimated to be 721 749 ms and 315 715 ms (n ¼50), respectively. However, after treatment with chlorprothixene (3 μM) for 15 min, the activation and deactivation time constants increased significantly to 829 760 ms (P o0.001) and 1085 760 ms (P o0.001), respectively. Flupentixol, under the same conditions as chlorprothixene (at 3 μM for 15 min), increased the activation time constant from 602 759 ms to 7767 105 ms (n¼ 8, P o0.05), but had no effect on the deactivation constant (211 714 ms and 215 734 ms, before and after treatment with flupentixol, respectively).
4. Discussion Previously, we have shown that certain antipsychotic drugs strongly inhibit proton currents (Shin et al., 2015; Shin and Song, 2014). These drugs, including chlorpromazine (a phenothiazine), haloperidol (a butyrophenone), clozapine (a dibenzodiazepine), and olanzapine (a thienobenzodiazepine), have different chemical
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Fig. 3. The effects of chlorprothixene on the voltage-dependent activation of proton currents. The cells were stimulated from a holding potential of 70 mV to test potentials ranging from 50 mV to þ40 mV in a series of steps of 10-mV increments, first under control conditions and then in the presence of chlorprothixene at 3 μM for 15 min. (A) Representative current traces recorded with this protocol. (B) Current-voltage (I-V) relationship plot for the data obtained from (A). The steady-state current amplitude was estimated by a fit with an exponential function. (C) Conductance-voltage (G-V) relationship plot under control conditions (open circles) and in the presence of chlorprothixene (filled circles and triangles) (n¼ 8). The conductance was normalized to that of the test potential of þ40 mV (circles) or to a maximal control conductance (triangles). The solid line is the fit with a Boltzmann function.
structures. The drugs’ proton current-inhibiting potency was unrelated to their affinity for dopamine or serotonin receptors. Moreover, risperidone (a benzisoxazole) had no substantial effects on proton currents. These observations suggest that the pharmacological profiles of these antipsychotic drugs may not be directly related to the inhibition of proton currents. Instead, their specific chemical structure may be an important determinant of such activity. Thioxanthenes are structurally similar to phenothiazines, in that the nitrogen atom at position 10 in phenothiazines is substituted by a carbon atom that is flanked by a double bond to the side chain. In this study, we report that the two thioxanthenes chlorprothixene and flupentixol inhibit proton currents with high potency. The IC50 values for chlorprothixene and flupentixol were 1.7 μM and 6.6 μM, respectively (Fig. 2). The potency of chlorprothixene is comparable to that of chlorpromazine (IC50 ¼ 2.2 μM) (Shin and Song, 2014). Chlorprothixene, containing a tricyclic ring structure, resembles chlorpromazine but has a more rigid structure (Bergmann et al., 1974). Nevertheless, herein, the rigidity of the structure or the substitution of the nitrogen atom by the carbon atom did not seem to affect the proton current inhibition. Although flupentixol had a slightly less potent proton current-inhibiting capacity compared to that of chlorprothixene, it inhibited dopamine-sensitive responses more potently and showed higher clinical antipsychotic efficacy than were observed for
chlorprothixene (Feinberg and Snyder, 1975). The bulkier piperazineethanol and trifluoromethyl side chains in flupentixol may hinder the drug’s interaction with proton channels. The structure-dependent effect of thioxanthenes can also be observed in their interaction with other membrane proteins. Permeability glycoprotein (P-glycoprotein) encoded by the multidrug resistant (MDR) gene resembles an ion channel both in structure and function, and several ion channel blockers, such as verapamil and quinidine, are also permeability glycoprotein modulators (Rodriguez et al., 1999). Thioxanthenes sensitize cancer cells that are multidrug-resistant to chemotherapeutic drugs in a structuredependent manner, which is not related to their antipsychotic potencies (Ford et al., 1990). For example, trans-flupentixol has a greater anti-multidrug-resistance activity than that of cis-flupentixol, but lacks binding affinity for dopamine and serotonin receptors. Such stereospecificity is related to the different orientation of the molecules in the membrane’s lipid environment. Indeed, trans-flupentixol interacts more strongly with phospholipids than cis-flupentixol (Wiese and Pajeva, 1997). Eventually, flupentixol interacts with the protein allosterically at the lipidprotein interface (Mandal et al., 2012). Chlorprothixene slowed both the activation and the deactivation kinetics of proton currents; the latter was more markedly affected (Fig. 5). This effect suggests that chlorprothixene interferes with the gating of proton channels. Hanatoxin, a tarantula
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Fig. 4. The effects of chlorprothixene on the reversal potential of proton currents. The cells were stimulated with a 2-s depolarizing step from a holding potential of 70 mV to þ40 mV, and were then repolarized to potentials between 70 mV and 100 mV in 10-mV decrements. (A) Representative current families before and after treatment with chlorprothixene 3 μM for 15 min. The insets show enlarged views of the tail currents at the repolarizing potential. The dotted line denotes the zero current level. (B) The amplitude of the tail current measured at the repolarizing potential, for which the data were obtained from (A). (C) A comparison of the reversal potentials before and after treatment with chlorprothixene (n¼ 8, **P o 0.01).
Fig. 5. The effects of chlorprothixene on the proton current kinetics. The proton currents were evoked by a 2-s depolarizing step from a holding potential of 70 mV to þ20 mV, before and after treatment with chlorprothixene (3 μM) for 15 min. The peak current and the tail current were fitted with a mono-exponential function. The activation and the deactivation time constants and the fitted traces before (dashed lines) and after (dotted lines) treatment with chlorprothixene are shown (n¼ 50, ***P o0.001).
toxin, inhibited the proton currents expressed in HEK cells. The toxin was presumed to move into the lipid membrane and interact with the voltage-sensor paddle motif at the lipid-protein interface, stabilizing its resting state (Alabi et al., 2007). As a result, hanatoxin shifted the activation voltage in the positive direction and a strong depolarization could relieve the current inhibition. Another gating modifier, NH17, a diphenylamine carboxylate derivative, also inhibited the proton currents with a positive shift in the activation voltage (Kornilov et al., 2014). In the present study, chlorprothixene at 3 μM, shifted the activation voltage of the proton currents only marginally (þ2.2 mV) (Fig. 3). Thus, it appears that chlorprothixene slows the opening transition of the proton channel rather than stabilizing its resting/closed state. Upon repolarization, the open channel is deactivated, which is reflected by the tail current decay. The deactivation time constant was markedly increased by chlorprothixene compared to the activation time constant. Thus, chlorpromazine hinders the closing transition of the proton channel more effectively. Although flupentixol increased the activation constant similarly to chlorprothixene, it had almost no effect on the deactivation constant, suggesting somewhat different modes of action between two drugs.
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The gating of the proton channel is regulated by the pH gradient across the plasma membrane (Cherny et al., 1995). Local anesthetics, such as lidocaine and bupivacaine, inhibit proton currents by increasing the intracellular pH, an observation that is supported by the large positive shift in both the reversal potential and the activation voltage (Matsuura et al., 2012). Chlorprothixene, however, produced a small but statistically significant shift in the reversal potential to the opposite direction (Fig. 4). Considering its negligible effect on the activation voltage, this result negates the assumption that chlorprothixene inhibits proton currents by altering the intracellular pH. This notion holds true for flupentixol, since it also shifted the reversal potential slightly to the hyperpolarizing direction. A small shift in the activation voltage could not have accounted for more than half the reduction of the proton currents by chlorprothixene at 3 μM (Fig. 3). It was suggested that extracellularly applied imipramine inhibited proton currents from the interior of the cell after crossing the plasma membrane (Song et al., 2012). Having a similar tricyclic ring structure, chlorprothixene is assumed to act similarly. Likewise, 2-guanidinobenzimidazole (2GBI), a guanidine derivative, blocks proton channels only when applied intracellularly, without altering the activation voltage (Hong et al., 2013). In consistent with this notion, increasing the intracellular pH from 5.5 to 6.0, which would decrease its accumulation inside the cell, chlorprothixene (3 μM) inhibited the proton currents at þ40 mV by 447 1% (n¼ 3), which was less compared to that attained at pH 5.5 (57 7 3%, n ¼10). Both chlorprothixene and flupentixol reduced the steady-state conductance, suggesting that they hinder the conducting pathway of the proton channels. Deletion of HV1 reduced NADPH oxidase-dependent reactive oxygen species production, and prevented neuronal death and brain damage after ischemic stroke (Wu et al., 2012). Thus, inhibition of the proton channels may be a therapeutic strategy in the treatment of ischemic stroke and other neurodegenerative diseases associated with oxidative stresses. HV1 is a more preferable target than NADPH oxidase since it is expressed in microglia but not in neurons; hence, inhibition of proton channels will not interfere with neuronal NADPH oxidase activity. Inflammation and oxidative stress are contributing factors in the pathogenesis of schizophrenia (Leza et al., 2015). Chlorprothixene and flupentixol have been shown to possess immunosuppressive and antioxidant properties (Hadjimitova et al., 2004; Kowalski et al., 2003, 2004). The drug’s proton channel inhibition may be partly responsible for these effects.
Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2010-0022213 and NRF2014R1A1A2054977).
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