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Parecoxib, a selective blocker of cyclooxygenase-2, directly inhibits neuronal delayed-rectifier K+ current, M-type K+ current and Na+ current
European Journal of Pharmacology 844 (2019) 95–101
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Parecoxib, a selective blocker of cyclooxygenase-2, directly inhibits neuronal delayed-rectifier K+ current, M-type K+ current and Na+ current Yuan-Yuarn Liua, Hung-Tsung Hsiaob, Jeffrey Chi-Fei Wangb, Yen-Chin Liub, Sheng-Nan Wuc,d,
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a
Division of Trauma, Department of Emergency, Kaohsiung Veterans General Hospital, Kaohsiung City, Taiwan Department of Anesthesiology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Taiwan c Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan City, Taiwan d Department of Physiology, National Cheng Kung University Medical College, Tainan City, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Parecoxib Delayed rectifier K+ current M-type K+ current Sodium current Action potential Neuroblastoma cell
Parecoxib, a prodrug of valdecoxib, is a selective inhibitor of cyclooxygenase-2 and widely used for traumatic and postoperative patients to avoid opioid-induced side effects. It is a potent analgesic and has a role in multimodal analgesic and enhanced recovery after surgery. Whether parecoxib exerts any actions on these types of ionic currents remains unclear. In this study, we investigated whether it exerts any effects on ion currents in differentiated NG108-15 neuronal cells. Cell exposure to parecoxib (1–30 μM) caused a reversible reduction in the amplitude of IK(DR) with an IC50 value of 9.7 μM. The time course for the IK(DR) inactivation in response to a long-lasting pulse was changed to the biexponential process during cell exposure to 3 μM parecoxib. Other agents known to inhibit the cyclooxygenase activity have minimal effects on IK(DR). Parecoxib enhanced the degree of excessive accumulative inhibition of IK(DR) inactivation evoked by a train of brief repetitive stimuli. This compound suppressed the amplitude of M-type K+ current. It depressed the peak amplitude of voltage-gated Na+ current with no change in the current-voltage relationship of this current. However, it did not have any effect on hyperpolarization-activated cation current. No change in the expression level of KV3.1 mRNA was detected in the presence of parecoxib. The effects of parecoxib on ion currents are direct and unrelated to its inhibition of the enzymatic activity of cyclooxygenase-2. The inhibition of these ion channels by parecoxib may partly contribute to the underlying mechanisms by which it affects neuronal function in vivo.
1. Introduction Parecoxib sodium (PARE, N-[4-(5-methyl-3-phenylisoxazol-4-yl) phenylsulfonyl] propionamide) is a specific inhibitor of cyclooxygenase-2 and available for intravenous or intramuscular administration to painful traumatic or postoperative patients. This compound can rapidly be converted to active valdecoxib (Karim et al., 2001; Martinou et al., 2018; Mehta et al., 2008) and produce a more rapid onset than ketorolac (Talley et al., 2000). Parecoxib administration also significantly diminished morphine consumption (Bikhazi et al., 2004; Langford et al., 2009; Szental et al., 2015; Wheeler et al., 2002; Yuksel et al., 2018). The explanation for its effect is often thought its inhibition of cyclooxygenase-2 (Hersh et al., 2005; Talley et al., 2000). However, Urdaneta et al. reported that the effects of parecoxib may not be fully explained only by its inhibition on cyclooxygenase-2 (Urdaneta et al., 2009). Voltage-gated K+ (KV) channels play an essential role in
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determining the excitability of neurons and the delayed rectifier K+ (KDR) channels are ubiquitous in neurons. A causal relationship between KV3 (or KCNC) and the delayed rectifier K+ current (IK(DR)) has been established (Rudy and McBain, 2001). The KV3 subfamily of KV channels is characterized by positively shifted voltage dependency and fast deactivation rate. These properties can be expected to limit the Na+ channel, thereby leading to depolarization block and accommodation of repetitive firing at high frequencies (Rudy and McBain, 2001; Tateno and Robinson, 2007). KDR channels from the KV3.1-KV3.2 types are the major determinants of IK(DR) in NG108-15 neuronal cells (Huang et al., 2009). Our previous study showed that dexmedetomidine suppress the amplitude of IK(DR) in NG108-15 cells (Chen et al., 2009). If KV3-encoded K+ currents are located at nerve terminals, modulation of current amplitude might cause a profound effect on neurotransmitter release (Dodson and Forsythe, 2004; Rudy and McBain, 2001). Previous report showed that celecoxib can modify the KV1.3- or KV2.1-encoded K+ channels (Frolov et al., 2008; Frolov and Singh, 2015). However,
Correspondence to: Department of Physiology, National Cheng Kung University Medical College, No. 1, University Road, Tainan City 70101, Taiwan. E-mail address: [email protected] (S.-N. Wu).
https://doi.org/10.1016/j.ejphar.2018.12.005 Received 14 July 2018; Received in revised form 29 November 2018; Accepted 5 December 2018 Available online 06 December 2018 0014-2999/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Electrophysiological measurements
whether parecoxib can directly interact with IK(DR) or other types of ion currents in neurons remains unclear, despite its widely clinical use (Martinou et al., 2018; Szental et al., 2015; Yuksel et al., 2018). In another way, the KCNQ2, KCNQ3, and KCNQ5 genes are known to encode the core subunits of KV7.2, KV7.3 and KV7.5 channels, respectively. The increased activity of these KV channels can generate the Mtype K+ current (IK(M)) with a slowly activating and deactivating property (Brown and Yu, 2000; Shu et al., 2007). Targeting IK(M) is recognized as an adjunctive regimen for the treatment of many neurological disorders. However, whether this compound exerts any actions on these types of ionic currents remains unexplored, although celecoxib can increase the amplitude of KV7.2/7.3 currents (Du et al., 2011). Therefore, in this study, we determine the mechanism of parecoxib actions on perturbation of different ion currents in NG108-15 cells. Currents in these cells include IK(DR), IK(M), voltage-gated Na+ current (INa), and hyperpolarization-elicited cation current (Ih). Our findings provide the evidence that parecoxib can directly inhibit ion currents which is highly unlikely to be linked to its inhibitory effects on cyclooxygenase-2 enzymes.
Before the experiments, cells were dissociated with 1% trypsin/ EDTA solution and an aliquot of cell suspension was subsequently transferred to a recording chamber affixed to the stage of a DM-IL inverted microscope (Leica, Wetzlar, Germany). Cells were bathed at room temperature (20–25 °C) in normal Tyrode's solution containing 1.8 mM CaCl2. Microelectrodes were pulled from Kmax-51 capillaries (Kimble Glass, Vineland, NJ) using a PP-83 puller (Narishige, Tokyo, Japan) and had a resistance of 3–5 MΩ when filled with different intracellular solutions. Patch clamp recordings were made in the wholecell configuration with an RK-400 amplifier (Bio-Logic, Claix, France), as described previously (Wu et al., 2001). The signals were displayed on an HM-507 oscilloscope (Hameg, East Meadow, NY) and a liquid crystal projector (AV600; Delta, Taipei, Taiwan). The data were stored online in a TravelMate-6253 laptop computer (Acer, Taipei, Taiwan) at 10 kHz through a Digidata-1322A interface (Molecular Devices). The latter device was equipped with an Adaptec SlimSCSI card (Milpitas, CA) via a PCMCIA slot and was controlled by pCLAMP 9.2 (Molecular Devices). Current signals were lowpass filtered at 1 or 3 kHz. The signals were analyzed using pCLAMP 9.2 (Molecular Devices) or OriginPro 2016 (OriginLab, Northampton, MA). The pCLAMP-generated voltage-step protocols were used to examine the current-voltage (I-V) relationships for ion currents (e.g., IK(DR) and INa).
2. Materials and methods 2.1. Cell culture The clonal strain NG108-15 cell line, formed by Sendai virus-induced fusion of the mouse neuroblastoma clone N18TG-2 and the rat glioma clone C6 BV-1, was obtained from the European Collection of Cell Cultures (ECACC-88112302; Wiltshire, UK). Cells were kept in monolayer cultures at a density of 106/ml in plastic disks containing Dulbecco's modified Eagle's medium supplemented with 100 μM hypoxanthine, 1 μM aminopterin, 16 μM thymidine, and 5% fetal bovine serum as the culture medium, in a humidified incubator equilibrated with 90% air/10% CO2 at 37 °C. Media were replenished every 2–3days with fresh media. To induce neuronal differentiation, the culture medium was replaced with a medium containing 1 mM dibutyryl cyclic AMP and cells were cultured in an incubator for 1–7 days (Chen et al., 2009; Lin et al., 2008). To observe neurite growth, a Nikon Eclipse Ti-E inverted microscope (Li Trading Co., Taipei, Taiwan) equipped with a five-megapixel cooled digital camera was used. The digital camera was connected to a personal computer controlled by NIS-Elements BR3.0 software (Nikon, Kanagawa, Japan). The number of neurites and varicosities was increased as cells were pre-incubated with 1 mM dibutyryl cyclic AMP. Cell viability was evaluated using a WST-1 assay (RocheDiagnostics, Indianapolis, IN) and an ELISA reader (Dynatech, Chantilly, VA).
2.4. Data analyses To evaluate the percentage inhibition of parecoxib on IK(DR), NG108-15 cells were bathed in Ca2+-free Tyrode's solution, and each cell was depolarized from −50 to + 50 mV. The amplitude of IK(DR) at the end of depolarizing pulse during exposure to different concentrations (0.3–100 μM) of parecoxib was measured. Concentration-response data for inhibition of IK(DR) were fitted with a modified form of the Hill equation:
y=
(1 − α ) × [PARE ]−nH + α, −nH [PARE ]−nH + IC50
where y is the normalized amplitude of IK(DR); [PARE] represents the parecoxib concentration and IC50 and nH are the concentration required for 50% inhibition and Hill coefficient, respectively. This also allows the estimation of maximal inhibition (1-α). The paired or unpaired Student's t-test, or one-way analysis of variance (ANOVA) followed by post-hoc Fisher's least-significance difference for multiple-group comparisons, were used for the statistical evaluation of differences among means. Statistical analyses were performed with the use of SPSS 14.0 (SPSS Inc., Chicago, IL). Statistical significance was determined at a P value of < 0.05. Values are provided as means ± standard error of mean (S.E.M.) with sample sizes (n) indicating the number of cells from which the data were taken.
2.2. RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) To detect the expression of KV3.1 mRNA in NG108-15 cells treated with or without dibutyryl cyclic AMP, a semi-quantitative RT-PCR assay was carried out. Total RNA samples were extracted from NG108-15 cells with TRIzol reagent (Invitrogen, Grand Island, NY) and reversetranscribed into complementary DNA using Superscript II reversetranscriptase (Invitrogen, Grand Island, NY). The sequences of oligonucleotide primers used for KV3.1 (NM_008421) were 5′-CGTGCCGAC GAGTTCTTCT-3′ and 5′-GGTCATCTCCAGCTCGTCCT-3′ (Sacco et al., 2006). Amplification of Kv3.1 was done using PCR SuperMix from Invitrogen under the following conditions: 35 cycles composed of 30 s denaturation at 95 °C, 30 s primer annealing at 62 °C, a 1 min extension at 72 °C, followed by the final extension for 2 min at 72 °C. PCR products were analyzed on 1.5% (w/v) agarose gel containing ethidium bromide and then visualized under ultraviolet light. Optical densities of the DNA bands were scanned and quantified with Scion Image Software (Scion, Frederick, MD).
2.5. Chemicals and solutions Parecoxib (Dynastat®, N-[4-(5-methyl-3-phenylisoxazol-4-yl) phenylsulfonyl]propionamide) was obtained from Pfizer Inc. (New York, NY). 4-Aminopyridine, dibutyryl cyclic AMP, diazoxide, indomethacin, flupirtine, linopirdine, tetraethylammonium chloride and tetrodotoxin were purchased from Sigma-Aldrich Chemicals (St. Louis, MO), curcumin, dibutyryl cyclic AMP, ketoprofen, meclofenamate and meloxicam were from Biomol (Plymouth Meeting, PA), and pinacidil, piroxicam and ZD7288 were from Tocris (Bristol, UK). All other chemicals, including CsCl, CsOH and CdCl2, were commercially available and of reagent grade. Deionized water purified by a Millipore-Q system (Millipore, Bedford, MA) was used in all experiments. The composition of the bath solution (i.e., normal Tyrode's solution) was 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 96
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mM glucose, and 5.5 mM HEPES-NaOH buffer, with a solution pH of 7.4. To measure IK(DR), a patch pipette was filled with a solution consisting of 140 mM KCl, 1 mM MgCl2, 3 mM Na2ATP, 0.1 mM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES-KOH buffer at a solution pH of 7.2. To measure M-type K+ current (IM), cells were bathed in high-K+, Ca2+free solution consisting of 145 mM KCl 145, 0.53 mM MgCl2, and 5 mM HEPES-KOH buffer, with a solution pH of 7.4. To record voltage-gated sodium current (INa) or hyperpolarization-elicited cation current (Ih), KCl inside the pipette solution was replaced with equimolar CsCl, and the pH was adjusted to 7.2 with CsOH. 3. Results 3.1. Effect of parecoxib on IK(DR) in differentiated NG108-15 neuronal cells Fig. 2. Effect of parecoxib on the inactivation time course of IK(DR) in differentiated NG108-15 cells. (A) Superimposed current traces obtained in the absence and presence of parecoxib. a: control; b: 10 μM parecoxib. The cell was depolarized from a holding potential of −50 mV to + 50 mV with a duration of 10 s. The smooth gray curves shown in (A) were nonlinear least squares fits to either one or two exponentials to the data. The inset in (A) indicates the voltage protocol used. (B) Summary of the data showing the effect of parecoxib on the fast component of IK(DR) inactivation (mean ± S.E.M.; n = 5–8 for each bar). *Significantly different from parecoxib (1 μM) alone group (P < 0.05).
In an initial set of experiments, a whole-cell configuration of the patch-clamp technique was used to evaluate the effects of parecoxib on ion currents in NG108-15 cells differentiated with dibutyryl cyclic AMP (1 mM). To measure IK(DR), cells were bathed in Ca2+-free Tyrode's solution which contained tetrodotoxin (1 μM) and CdCl2 (0.5 mM), and the recording pipette was filled with a K+-containing solution. Fig. 1 shows that parecoxib can diminish the amplitude of IK(DR) in a concentration-dependent manner. In these experiments, each cell was depolarized from −50 to + 50 mV with a duration of 300 ms, and current amplitudes of IK(DR) were measured at the end of depolarizing pulses. The relationship between the parecoxib concentration (0.3–100 μM) and the relative amplitude of IK(DR) was also constructed and is plotted in Fig. 1B. Fitting the concentration-response curve with the modified Hill equation as described in Section 2 yielded a half-maximal concentration (i.e., IC50) of 9.7 μM and a slope coefficient of 1.1. Parecoxib at a concentration of 100 μM nearly abolished current amplitude. Fig. 1C illustrates the averaged I-V relationships of IK(DR) obtained in the
control and during cell exposure to 10 μM parecoxib. These results demonstrate that parecoxib has a significant depressant action on IK(DR) functionally expressed in differentiated NG108-15 cells. During cell exposure to parecoxib, in addition to the decreased amplitude of IK(DR), the time to the peak of IK(DR) tended to be shortened, and the time course of current inactivation was progressively changed to the biexponential process (Fig. 2). For example, as the depolarizing pulse from −50 to + 50 mV with a duration of 10 s was evoked, the inactivation of IK(DR) was well fitted to a single exponential process with a mean time constant of 2012 ± 32 ms (n = 12); however, in the presence of parecoxib (30 μM), the fast and slow constants of IK(DR) inactivation were 815 ± 13 and 3231 ± 34 ms (n = 11), respectively. The fast, rather than the slow component was found to become significantly concentration-dependent (Fig. 2B). In other words, the fast component of inactivation time constant decreased when the parecoxib concentration was elevated. After parecoxib was removed, IK(DR) returned almost to the control level. 3.2. Effect of parecoxib on the accumulative inhibition of IK(DR) inactivation The IK(DR) inactivation is capable of accumulating during repetitive short pulses as reported previously (Lin et al., 2008). For this reason, in another set of experiments, we sought to determine whether it affects the degree of IK(DR) inactivation elicited by brief repetitive depolarizations. Under control conditions, a single 20-s depolarizing step to + 70 mV from a holding potential of −50 mV produced a decline with a time constant of 5.7 ± 0.2 s (n = 7). However, the time constant for 20-s repetitive pulses to + 60 mV, each of which lasted 40 ms with 20-ms intervals at the level of −50 mV between the depolarizing pulses, was significantly reduced to 2.3 ± 0.2 s (n = 7, P < 0.05). During exposure to 1 μM parecoxib, the value of the time constant obtained in response to such a train of short repetitive pulses was further diminished to 1.5 ± 0.1 s (n = 6, P < 0.05). A representative example of the cumulative inhibition of IK(DR) inactivation in control is illustrated in Fig. 3A. As shown in Fig. 3B, application of parecoxib was also able to increase the cumulative inhibition of IK(DR) inactivation in NG108-15 cells. For example, when cells were exposed to 1 μM parecoxib, the amplitude of IK(DR) measured at the end of a train of short repetitive stimuli was
Fig. 1. Inhibitory effect of parecoxib on IK(DR) in NG108-15 cells differentiated with dibutyryl cyclic AMP. Cells were bathed in Ca2+-free Tyrode's solution in which tetrodotoxin (1 μM) and CdCl2 (0.5 mM) were included. (A) Superimposed current traces obtained in the absence and presence of parecoxib. The inset in (A) indicates the voltage protocol used. a: control; b: 1 μM parecoxib; c: 3 μM parecoxib; d: 10 μM parecoxib. (B) Concentration-response relationship for parecoxib-induced inhibition of IK(DR). Each cell was depolarized from −50 to + 50 mV. Each point represents the mean ± S.E.M. (n = 6–12). The smooth line represents the best fit to the modified Hill equation as described in Section 2. The IC50 value was estimated with the non-linear curve fit. The IC50 value, maximally inhibited the percentage of IK(DR), and the Hill coefficient (nH) were 9.7 μM, 74% (i.e., α = 0.26), and 1.1, respectively. (C) Averaged I-V relationships of IK(DR) obtained in the absence (•) and presence (◯) of 10 μM parecoxib (mean ± S.E.M.; n = 8–12 for each point). 97
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Fig. 3. Excessive accumulative inactivation of IK(DR) during repetitive stimuli in the absence and presence of parecoxib in differentiated NG108-15 cells. Currents in (B) were obtained during repetitive depolarizations to + 70 mV with a return to −50 mV. The individual depolarizing pulse used to elicit outward K+ current lasted 40 ms (indicated in (A)). Current trace with a single exponential of 2.3 s was obtained in the control. (B) Summary of the data showing the effect of parecoxib (1 and 3 μM) on the amplitude of IK(DR) during repetitive depolarization (mean ± S.E.M.; n = 6–8 for each bar). Current amplitude was measured at the end of repetitive depolarizing pulses (i.e., 20 s). *Significantly different from control (P < 0.05).
effects on IK(DR) in these cells.
significantly decreased by 35 ± 2%, from 2903 ± 285–1887 ± 203 pA (n = 8, P < 0.05). It is thus clear from these results that exposure to this compound can increase the accumulative inhibition of IK(DR) inactivation in NG108-15 cells.
3.4. Effect of parecoxib on M-type K+ current (IK(M)) in differentiated NG108-15 cells Celexcoxib, another inhibitor of cyclooxygenase-2, was previously used to stimulate IK(M) (Du et al., 2011). As such, in another set of experiments, we explored the possible effect of parecoxib on IK(M) observed in these cells. The examined cells were bathed in high-K+, Ca2+free solution and the recording pipette was filled with K+-containing solution. As shown in Fig. 5, when the cell was depolarized from −50 to −10 mV with a duration of 1 s, K+ inward current with the slowly activating and deactivating properties was readily evoked. This K+ current elicited by long-lasting membrane depolarization was sensitive to inhibition by linopirdine (10 μM), yet not by either 4-aminopyridine (1 mM) or tetraethylammonium (10 mM), and it was hence identified as an IK(M) (Hsu et al., 2014; Huang et al., 2008; Selyanko et al., 1999). Linopirdine is recognized as a selective blocker of IK(M) (Huang et al.,
3.3. Comparison between the effects of indomethacin, curcumin, ketoprofen, meclofenamate, meloxicam, piroxicam, and 4-aminopyridine on IK(DR) Effects of other compounds which are the inhibitors of cyclooxygenase were also examined and compared. In these experiments, each cell was depolarized from −50 to + 50 mV with a duration of 300 ms, and the amplitude of IK(DR) was measured at the end of the depolarizing pulse. The results showed that neither indomethacin (10 μM), curcumin (10 μM), ketoprofen (10 μM), nor meclofenamate (10 μM) could be effective in suppressing IK(DR) (Fig. 4) (Kuo et al., 2018). However, meloxicam (10 μM) and piroxicam (10 μM) slightly diminished this current, while 4-aminopyridine (1 mM) decreased it by about 80% (Fig. 4). Indomethacin, curcumin, ketoprofen, and meclofenamate are known to be blockers of cyclooxygenases, while meloxicam is a selective inhibitor of cyclooxygenase-2. Therefore, these results indicate that, unlike parecoxib, these inhibitors of cyclooxygenase had minimal
Fig. 5. Effect of parecoxib on the amplitude of M-type K+ current (IK(M)) in differentiated NG108-15 cells. These experiments were conducted in cells bathed in high K+, Ca2+-free solution and the recording pipette was filled with K+-containing solution. (A) Superimposed IK(M) traces obtained in the absence (a) and presence of 10 μM (b) and 30 μM parecoxib (c). Arrowhead indicates the zero current level. The upper part indicates the voltage protocol applied. (B) Bar graph showing the effect of parecoxib, linopirdine and parecoxib plus flupirtine on IK(M) amplitude (mean ± S.E.M.; n = 10–11 for each bar). The IK(M) amplitude elicited by long-lasting membrane depolarization from −50 to −10 mV was measured at the end of depolarizing pulse. a: control; b: 10 μM parecoxib; c: 30 μM parecoxib; d: 10 μM linopirdine; e: 30 μM parecoxib plus 10 μM flupirtine. *Significantly different from control (P < 0.05) and **significantly different from parecoxib (10 μM) alone group (P < 0.05).
Fig. 4. Summary of the data showing the effects of indomethacin (10 μM), curcumin (10 μM), ketoprofen (10 μM), meclofenamate (10 μM), meloxicam (10 μM), piroxicam (10 μM) and 4-aminopyridine (1 mM) on IK(DR). In these experiments, cells were bathed in Ca2+-free Tyrode’s solution and the recording pipettes were filled with a K+-containing solution. Each cell was depolarized from −50 to + 50 mV with a duration of 300 ms. At the end of the depolarizing pulse, the current amplitude in the control was considered to be 1.0, and the relative amplitude of IK(DR) after application of each agent was plotted. 4-AP: 1 mM 4-aminopyridine. *Significantly different from control (P < 0.05). Each bar represents the mean ± S.E.M. (n = 6–9). 98
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Fig. 6. Effects of parecoxib on INa and Ih in differentiated NG108-15 cells. Cells were bathed in Ca2+-free Tyrode's solution, and the recording pipette was filled with a Cs+-containing solution. (A) Original INa currents obtained in the absence (trace a) and presence of 3 μM (trace b) and 10 μM (trace c) parecoxib. The examined cell was depolarized from −80 to −20 mV. The inset indicates expanded time scale of current traces (indicated by dashed box). (B) Averaged I-V relationships of peak INa obtained in the absence (•) or presence (◯) of 10 μM parecoxib (mean ± S.E.M.; n = 6–10 for each point). Notably, parecoxib diminishes the peak amplitude of INa without any change in the overall I-V configuration of this current. *Significantly different from controls measured at the same level of membrane potential (P < 0.05). In these experiments on recording Ih, cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl2. The pipette was filled with a K+-containing solution. In each cell, a long-step hyperpolarizing stimulus was applied from a holding potential of −50 mV. In (C), traces a (black) and b (gray) were, respectively, obtained in the absence and presence of 30 μM parecoxib. The inset shown in (A) indicates the voltage protocol used. (D) Averaged I-V relationships of Ih in the absence and presence of 30 μM parecoxib (mean ± S.E.M.; n = 4–8 for each point). The amplitude of Ih was measured at the end of each hyperpolarizing pulse.
further studied whether this compound could have any influence on Ih. The results showed its inability to affect the amplitude or gating of Ih in these cells. When cells were hyperpolarized from −50 to the different voltages ranging from −150 to −50 mV in 10-mV increments, the Ih measured at the end of the hyperpolarizing pulse in the control did not significantly differ from that measured during the exposure to 30 μM parecoxib (Fig. 6C and D). Moreover, similar to the findings observed in that of INa (Fig. 6A and B), no discernible change in the overall I-V relationships of Ih was seen in the presence of parecoxib (Fig. 6D). However, at the same voltage protocol, 30 μM ZD7288, an inhibitor of Ih (Liu et al., 2009), could be effective in suppressing Ih, as evidenced by a significant reduction of Ih from 2012 ± 15–986 ± 11 pA during the exposure to 30 μM ZD7288 (n = 11, P < 0.05). Therefore, it is clear that, unlike IK(DR), IK(M) or INa, Ih is not subject to inhibition by parecoxib.
2008). Notably, as cells were exposed to parecoxib, the IK(M) amplitude evoked in response to depolarizing pulse from −50 to −10 mV was progressively diminished, as evidenced by the data showing that addition of 30 μM parecoxib caused a significant reduction in current amplitude from 41.2 ± 7.9–7.4 ± 1.1 pA (n = 11, P < 0.05). Moreover, in continued presence of 30 μM parecoxib, subsequent addition of 10 μM flupirtine, an activator of IK(M) (Wu et al., 2012), was found to reverse its inhibition of IK(M) significantly. Therefore, distinguishable from previous observations showing the ability of celecoxib to increase IK(M) amplitude (Du et al., 2011), it is possible that the presence of parecoxib suppressed the amplitude of IK(M) effectively in these cells. 3.5. Inhibitory effect of parecoxib on voltage-gated Na+ current (INa) in differentiated NG108-15 cells The functional defects of the SCN9A gene were recently associated with inherited erythromelagia, a painful neuropathic disorder (Waxman, 2007). Therefore, the effect of parecoxib on INa was further investigated in these cells. The experiments were conducted with a Cs+containing pipette solution. As shown in Fig. 6A, when the cell was depolarized from −80 to −10 mV, the application of parecoxib (10 μM) significantly decreased the peak amplitude of INa by 21 ± 2%, from 1122 ± 87–898 ± 98 pA (n = 8, P < 0.05). After washout of parecoxib, the amplitude of INa was partially returned to 1108 ± 91 pA (n = 5). However, no change in the overall I-V relationships of INa could be demonstrated in the presence of parecoxib (Fig. 6B). Furthermore, neither the activation nor inactivation time course of INa elicited by rapid membrane depolarization was modified by application of parecoxib. Likewise, the non-inactivating INa in response to a long-lasting ramp pulse was unaffected in the presence of this agent (data not shown). Taken together, these results indicate that in differentiated NG108-15 cells, parecoxib produces a depressant action on the peak amplitude of INa.
3.7. The mRNA expression for KV3.1 in differentiated NG108-15 cells Previous studies showed that the inhibitors of cyclooxygenase enzymes were able to alter the expression level of cyclooxygenase-2 mRNA increased by carrageenan (Pham-Marcou et al., 2008; Urdaneta et al., 2009). The IK(DR) in these cells is recognized to be carried by the Shaw-related K+ channels, which are the products of the KV3.1 (or KCNC1) gene. Therefore, we further examined whether the mRNA levels of KV3.1 in differentiated NG108-15 cells can be altered in the presence of parecoxib. In parecoxib-treated cells, cells were pre-incubated with 30 μM parecoxib for 2 days. Our RT-PCR analysis demonstrated that the extent of mRNA expression for KV3.1 in differentiated cells was found to be significantly increased. However, the mRNA level of KV3.1 subunit in cells did not differ between the presence and absence of parecoxib (Fig. 7). 4. Discussion In this study, we provide the direct evidence to show that parecoxib, which is the first cyclooxygenase-2 to be administered parenterally, has a depressant action on IK(DR) in differentiated NG108-15 cells. The halfblocking concentration of this compound required for inhibition of IK(DR) with an IC50 value of 9.7 μM appears to be of the same order of magnitude as the concentrations of celecoxib used to suppress KV1.3- or KV2.1-encoded K+ currents (Frolov et al., 2008; Frolov and Singh,
3.6. No effect of parecoxib on hyperpolatization-activated cation current (Ih) in differentiated NG108-15 cells In recent studies done at our laboratory, we demonstrated that when undergoing differentiation with a non-degradable analogue of cyclic AMP (i.e., dibutyryl cyclic AMP), NG108-15 cells could also express the activity of hyperpolarization-evoked cation channels. We 99
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block of IK(DR) (data not shown). Diazoxide or pinacidil are the openers of ATP-sensitive K+ channels (Wu et al., 2000). Therefore, parecoxibinduced inhibition of IK(DR) observed in NG108-15 cells is unlikely to be connected with the activity of either large-conductance Ca2+-activated K+ channels or ATP-sensitive K+ channels. Voltage-gated Na+ (NaV) channels are generally involved in the generation and propagation of APs in most excitable cells. Our study showed the ability of parecoxib to suppress the peak amplitude of INa in NG108-15 cells. The tetrodotoxin-sensitive INa identified in differentiated NG108-15 cells is reported to resemble NaV1.7 encoded by SCN9A gene (Kawaguchi et al., 2007). However, neither the activation nor the inactivation rate of INa was modified in the presence of parecoxib. In addition, the application of parecoxib failed to alter the noninactivating component of INa elicited by a slow ramp pulse. Parecoxib was also not found to have any effects on Ih. Taken together, the electrophysiological effects of parecoxib and other coxibs on ion currents are presumably not limited to their modification of KDR channels. Recent studies have demonstrated that NaV1.7 can be expressed both in peripheral sensory and sympathetic neurons as well as olfactory epithelia (Momin and Wood, 2008). Gain-of-function mutations in NaV1.7 (e.g., the L858H mutation) are recognized to cause primary erythromelagia and paroxysmal extreme pain disorder, while loss-offunction mutations in NaV1.7 cause a channelopathy accompanied by insensitivity to pain (Momin and Wood, 2008; Waxman, 2007). In our experimental conditions, the effects of parecoxib on ion currents were generally observed within only 2 min of application of this compound. The inhibitory effect of parecoxib on INa is thus direct and could be an important mechanism by which it depresses the amplitude of neuronal action potentials. Parecoxib-induced blockade of IK(DR), IK(M) and INa may synergistically affect the functional activity of neurons. In conclusion, this present study clearly demonstrates that parecoxib can directly inhibit IK(DR), IK(M) and INa in differentiated NG10815 cells and strongly indicates that this compound is apparently not an inactive prodrug. The suppression of these ionic currents is responsible for its effects on functional activities in these cells. Following intravenous or intramuscular administration, parecoxib can easily pass across the blood-brain barriers, although it may rapidly be hydrolyzed to its active moiety, valedoxib. Therefore, the electrophysiological effects of parecoxib presented herein could lead to changes in firing behavior of neurons, hence altering neuronal function (Kim et al., 2011; Polascheck et al., 2010). However, to what extent parecoxib-induced inhibition of ion channels contributes to its anti-inflammatory and antinociceptive actions has yet to be further delineated.
Fig. 7. Results of RT-PCR for the KV3.1 mRNA in differentiated NG108-15 cells obtained in the presence (lane A) and absence (lane B) of 10 μM parecoxib for 48 h. Total RNA was isolated, and RT-PCR analysis was performed. Amplified RT-PCR products were obtained for use as a marker lane of DNA (leftmost lane; M.W.) and KV3.1 subunit (306 bp) indicated by arrow. The mRNA level of KV3.1 subunit obtained in the presence of parecoxib (30 μM) did not differ from that in the control (i.e., undifferentiated NG108-15 cells).
2015). Notably, immediately following a single intramuscular injection with 20 and 40 mg parecoxib, the peak plasma concentration of approximately 2.7 and 4.5 μM can be respectively reached within 15 min (Karim et al., 2001). Other compounds known to inhibit the activity of cyclooxygenases were found to have minimal effects on IK(DR), while 4aminopyridine suppressed this current effectively. 4-Aminopyridine was previously reported to modulate amyloid β1–42-induced intracellular signaling in human microglia (Franciosi et al., 2006). Consistent with previous observations (Frolov et al., 2008; Frolov and Singh, 2015), the KDR and KM channels may thus be an important target for the action of parecoxib, despite its ability to suppress activity of cyclooxygenase-2. Both the alteration in the channel gating and the reduction of the currents caused by parecoxib or other coxibs are also likely to be clinically relevant if similar results are found in intact neurons in vivo. Indeed, it has been recently shown that the anti-inflammatory and anti-nociceptive actions of parecoxib cannot be fully explained by its inhibition of cyclooxygenase-2 activity (Urdaneta et al., 2009). In our study, the mechanism of parecoxib actions on IK(DR) apparently differed from those on INa or Ih. In differentiated NG108-15 neuronal cells, the effect exerted by parecoxib is a time-, and concentration-dependent increase of the inactivation process of IK(DR), along with no change in the activation kinetics of this current. No significant change in the mRNA expression of KV3.1 detected in NG10815 cells was detected in the presence of parecoxib. However, its presence was also found to increase the accumulative inhibition of IK(DR) inactivation elicited by long trains of brief repetitive depolarizations in these cells. Therefore, as action potentials in neurons in vivo fire more frequently, the inhibitory action of parecoxib on IK(DR) would become enhanced. Parecoxib-induced increase of IK(DR) inactivation may enhance a process of long-lasting facilitation or potentiation as described previously (Huang et al., 2009; Shu et al., 2007; Wu et al., 2008). In our study, as the recording pipettes were filled with parecoxib (30 μM) and whole-cell currents were established, no change in the amplitude of IK(DR) was clearly seen. However, when cells were exposed to parecoxib, the time course of IK(DR) inactivation was changed to the biexponential process. The fast component of IK(DR) inactivation became concentration-dependent. Therefore, with respect to its effects on IK(DR), parecoxib tends to have a mechanism that is a combination of open channel block and tight binding to inactivated states of the channel. During cell exposure to it, the conformation of the external pore mouth appears to be similar to the characteristics of C-type inactivation because C-type inactivation is thought to involve conformational changes at the external mouth of the pore (Baukrowitz and Yellen, 1995). All recordings of IK(DR) presented herein were conducted in a Ca2+free Tyrode's solution containing 0.5 mM CdCl2. Rottlerin, an activator of large-conductance Ca2+-activated K+ channels (Wu et al., 2007), did not reverse the inhibition of IK(DR) caused by parecoxib. Furthermore, neither diazoxide nor pinacidil had any effects on parecoxib-induced
CRediT authorship contribution statement Yuan-Yuarn Liu: Conceptualization, Resources, Methodology, Funding acquisition. Hung-Tsung Hsiao: Conceptualization, Methodology. Jeffrey Chi-Fei Wang: Conceptualization, Resources, Funding acquisition. Yen-Chin Liu: Writing - original draft, Visualization, Project administration. Sheng-Nan Wu: Methodology, Software, Writing - review & editing, Supervision.
Acknowledgments The work in this laboratory was supported by a grant from National Cheng Kung University (D106-35A13), Tainan City, Taiwan. The authors are grateful to Dr. Bing-Shuo Chen for the earlier work.
Conflict of interest None of the authors in this study have any potential conflict of interest or financial interests to disclose. 100
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References
Voros, D., Fragulidis, G.P., 2018. Parecoxib's effects on anastomotic and abdominal wound healing: a randomized Controlled trial. J. Surg. Res. 223, 165–173. Mehta, V., Johnston, A., Cheung, R., Bello, A., Langford, R.M., 2008. Intravenous parecoxib rapidly leads to COX-2 inhibitory concentration of valdecoxib in the central nervous system. Clin. Pharmacol. Ther. 83, 430–435. Momin, A., Wood, J.N., 2008. Sensory neuron voltage-gated sodium channels as analgesic drug targets. Curr. Opin. Neurobiol. 18, 383–388. Pham-Marcou, T.A., Beloeil, H., Sun, X., Gentili, M., Yaici, D., Benoit, G., Benhamou, D., Mazoit, J.X., 2008. Antinociceptive effect of resveratrol in carrageenan-evoked hyperalgesia in rats: prolonged effect related to COX-2 expression impairment. Pain 140, 274–283. Polascheck, N., Bankstahl, M., Loscher, W., 2010. The COX-2 inhibitor parecoxib is neuroprotective but not antiepileptogenic in the pilocarpine model of temporal lobe epilepsy. Exp. Neurol. 224, 219–233. Rudy, B., McBain, C.J., 2001. Kv3 channels: voltage-gated K+ channels designed for highfrequency repetitive firing. Trends Neurosci. 24, 517–526. Sacco, T., De Luca, A., Tempia, F., 2006. Properties and expression of Kv3 channels in cerebellar Purkinje cells. Mol. Cell Neurosci. 33, 170–179. Selyanko, A.A., Hadley, J.K., Wood, I.C., Abogadie, F.C., Delmas, P., Buckley, N.J., London, B., Brown, D.A., 1999. Two types of K(+) channel subunit, Erg1 and KCNQ2/3, contribute to the M-like current in a mammalian neuronal cell. J. Neurosci. 19, 7742–7756. Shu, Y., Yu, Y., Yang, J., McCormick, D.A., 2007. Selective control of cortical axonal spikes by a slowly inactivating K+ current. Proc. Natl. Acad. Sci. USA 104, 11453–11458. Szental, J.A., Webb, A., Weeraratne, C., Campbell, A., Sivakumar, H., Leong, S., 2015. Postoperative pain after laparoscopic cholecystectomy is not reduced by intraoperative analgesia guided by analgesia nociception index (ANI(R)) monitoring: a randomized clinical trial. Br. J. Anaesth. 114, 640–645. Talley, J.J., Bertenshaw, S.R., Brown, D.L., Carter, J.S., Graneto, M.J., Kellogg, M.S., Koboldt, C.M., Yuan, J., Zhang, Y.Y., Seibert, K., 2000. N-[[(5-methyl-3-phenylisoxazol-4-yl)-phenyl]sulfonyl]propanamide, sodium salt, parecoxib sodium: a potent and selective inhibitor of COX-2 for parenteral administration. J. Med. Chem. 43, 1661–1663. Tateno, T., Robinson, H.P., 2007. Quantifying noise-induced stability of a cortical fastspiking cell model with Kv3-channel-like current. Biosystems 89, 110–116. Urdaneta, A., Siso, A., Urdaneta, B., Cardenas, R., Quintero, L., Avila, R., Suarez-Roca, H., 2009. Lack of correlation between the central anti-nociceptive and peripheral antiinflammatory effects of selective COX-2 inhibitor parecoxib. Brain Res. Bull. 80, 56–61. Waxman, S.G., 2007. Channel, neuronal and clinical function in sodium channelopathies: from genotype to phenotype. Nat. Neurosci. 10, 405–409. Wheeler, M., Oderda, G.M., Ashburn, M.A., Lipman, A.G., 2002. Adverse events associated with postoperative opioid analgesia: a systematic review. J. Pain 3, 159–180. Wu, S.N., Chen, B.S., Lin, M.W., Liu, Y.C., 2008. Contribution of slowly inactivating potassium current to delayed firing of action potentials in NG108-15 neuronal cells: experimental and theoretical studies. J. Theor. Biol. 252, 711–721. Wu, S.N., Hsu, M.C., Liao, Y.K., Wu, F.T., Jong, Y.J., Lo, Y.C., 2012. Evidence for inhibitory effects of flupirtine, a centrally acting analgesic, on delayed rectifier K+ currents in motor neuron-like cells. Evid. Based Complement Altern. Med. 2012, 148403. Wu, S.N., Li, H.F., Chiang, H.T., 2000. Characterization of ATP-sensitive potassium channels functionally expressed in pituitary GH3 cells. J. Membr. Biol. 178, 205–214. Wu, S.N., Lo, Y.K., Chen, H., Li, H.F., Chiang, H.T., 2001. Rutaecarpine-induced block of delayed rectifier K+ current in NG108-15 neuronal cells. Neuropharmacology 41, 834–843. Wu, S.N., Wang, Y.J., Lin, M.W., 2007. Potent stimulation of large-conductance Ca2+activated K+ channels by rottlerin, an inhibitor of protein kinase C-δ, in pituitary tumor (GH3) cells and in cortical neuronal (HCN-1A) cells. J. Cell Physiol. 210, 655–666. Yuksel, U., Bakar, B., Dincel, G.C., Budak Yildiran, F.A., Ogden, M., Kisa, U., 2018. The investigation of the Cox-2 selective inhibitor parecoxib effects in spinal cord injury in rat. J. Investig. Surg. 1–12.
Baukrowitz, T., Yellen, G., 1995. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron 15, 951–960. Bikhazi, G.B., Snabes, M.C., Bajwa, Z.H., Davis, D.J., LeComte, D., Traylor, L., Hubbard, R.C., 2004. A clinical trial demonstrates the analgesic activity of intravenous parecoxib sodium compared with ketorolac or morphine after gynecologic surgery with laparotomy. Am. J. Obstet. Gynecol. 191, 1183–1191. Brown, B.S., Yu, S.P., 2000. Modulation and genetic identification of the M channel. Prog. Biophys. Mol. Biol. 73, 135–166. Chen, B.S., Peng, H., Wu, S.N., 2009. Dexmedetomidine, an α2-adrenergic agonist, inhibits neuronal delayed-rectifier potassium current and sodium current. Br. J. Anaesth. 103, 244–254. Dodson, P.D., Forsythe, I.D., 2004. Presynaptic K+ channels: electrifying regulators of synaptic terminal excitability. Trends Neurosci. 27, 210–217. Du, X.N., Zhang, X., Qi, J.L., An, H.L., Li, J.W., Wan, Y.M., Fu, Y., Gao, H.X., Gao, Z.B., Zhan, Y., Zhang, H.L., 2011. Characteristics and molecular basis of celecoxib modulation on K(v)7 potassium channels. Br. J. Pharmacol. 164, 1722–1737. Franciosi, S., Ryu, J.K., Choi, H.B., Radov, L., Kim, S.U., McLarnon, J.G., 2006. Broadspectrum effects of 4-aminopyridine to modulate amyloid beta1-42-induced cell signaling and functional responses in human microglia. J. Neurosci. 26, 11652–11664. Frolov, R.V., Berim, I.G., Singh, S., 2008. Inhibition of delayed rectifier potassium channels and induction of arrhythmia: a novel effect of celecoxib and the mechanism underlying it. J. Biol. Chem. 283, 1518–1524. Frolov, R.V., Singh, S., 2015. Evidence of more ion channels inhibited by celecoxib: KV1.3 and L-type Ca2+ channels. BMC Res. Notes 8, 62. Hersh, E.V., Lally, E.T., Moore, P.A., 2005. Update on cyclooxygenase inhibitors: has a third COX isoform entered the fray? Curr. Med. Res. Opin. 21, 1217–1226. Hsu, H.T., Tseng, Y.T., Lo, Y.C., Wu, S.N., 2014. Ability of naringenin, a bioflavonoid, to activate M-type potassium current in motor neuron-like cells and to increase BKCachannel activity in HEK293T cells transfected with alpha-hSlo subunit. BMC Neurosci. 15, 135. Huang, C.W., Huang, C.C., Lin, M.W., Tsai, J.J., Wu, S.N., 2008. The synergistic inhibitory actions of oxcarbazepine on voltage-gated sodium and potassium currents in differentiated NG108-15 neuronal cells and model neurons. Int. J. Neuropsychopharmacol. 11, 597–610. Huang, C.W., Tsai, J.J., Huang, C.C., Wu, S.N., 2009. Experimental and simulation studies on the mechanisms of levetiracetam-mediated inhibition of delayed-rectifier potassium current (KV3.1): contribution to the firing of action potentials. J. Physiol. Pharmacol. 60, 37–47. Karim, A., Laurent, A., Slater, M.E., Kuss, M.E., Qian, J., Crosby-Sessoms, S.L., Hubbard, R.C., 2001. A pharmacokinetic study of intramuscular (i.m.) parecoxib sodium in normal subjects. J. Clin. Pharmacol. 41, 1111–1119. Kawaguchi, A., Asano, H., Matsushima, K., Wada, T., Yoshida, S., Ichida, S., 2007. Enhancement of sodium current in NG108-15 cells during neural differentiation is mainly due to an increase in NaV1.7 expression. Neurochem. Res. 32, 1469–1475. Kim, Y.H., Lee, P.B., Park, J., Lim, Y.J., Kim, Y.C., Lee, S.C., Ahn, W., 2011. The neurological safety of epidural parecoxib in rats. Neurotoxicology 32, 864–870. Kuo, P.C., Yang, C.J., Lee, Y.C., Chen, P.C., Liu, Y.C., Wu, S.N., 2018. The comprehensive electrophysiological study of curcuminoids on delayed-rectifier K+ currents in insulin-secreting cells. Eur. J. Pharmacol. 819, 233–241. Langford, R.M., Joshi, G.P., Gan, T.J., Mattera, M.S., Chen, W.H., Revicki, D.A., Chen, C., Zlateva, G., 2009. Reduction in opioid-related adverse events and improvement in function with parecoxib followed by valdecoxib treatment after non-cardiac surgery: a randomized, double-blind, placebo-controlled, parallel-group trial. Clin. Drug Investig. 29, 577–590. Lin, M.W., Wang, Y.J., Liu, S.I., Lin, A.A., Lo, Y.C., Wu, S.N., 2008. Characterization of aconitine-induced block of delayed rectifier K+ current in differentiated NG108-15 neuronal cells. Neuropharmacology 54, 912–923. Liu, Y.C., Wang, Y.J., Wu, P.Y., Wu, S.N., 2009. Tramadol-induced block of hyperpolarization-activated cation current in rat pituitary lactotrophs. Naunyn Schmiede. Arch. Pharmacol. 379, 127–135. Martinou, E., Drakopoulou, S., Aravidou, E., Sergentanis, T., Kondi-Pafiti, A., Argyra, E.,
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Parecoxib, a selective blocker of cyclooxygenase-2, directly inhibits neuronal delayed-rectifier K+ current, M-type K+ current and Na+ current Yuan-Yuarn Liua, Hung-Tsung Hsiaob, Jeffrey Chi-Fei Wangb, Yen-Chin Liub, Sheng-Nan Wuc,d,
T
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a
Division of Trauma, Department of Emergency, Kaohsiung Veterans General Hospital, Kaohsiung City, Taiwan Department of Anesthesiology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Taiwan c Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan City, Taiwan d Department of Physiology, National Cheng Kung University Medical College, Tainan City, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Parecoxib Delayed rectifier K+ current M-type K+ current Sodium current Action potential Neuroblastoma cell
Parecoxib, a prodrug of valdecoxib, is a selective inhibitor of cyclooxygenase-2 and widely used for traumatic and postoperative patients to avoid opioid-induced side effects. It is a potent analgesic and has a role in multimodal analgesic and enhanced recovery after surgery. Whether parecoxib exerts any actions on these types of ionic currents remains unclear. In this study, we investigated whether it exerts any effects on ion currents in differentiated NG108-15 neuronal cells. Cell exposure to parecoxib (1–30 μM) caused a reversible reduction in the amplitude of IK(DR) with an IC50 value of 9.7 μM. The time course for the IK(DR) inactivation in response to a long-lasting pulse was changed to the biexponential process during cell exposure to 3 μM parecoxib. Other agents known to inhibit the cyclooxygenase activity have minimal effects on IK(DR). Parecoxib enhanced the degree of excessive accumulative inhibition of IK(DR) inactivation evoked by a train of brief repetitive stimuli. This compound suppressed the amplitude of M-type K+ current. It depressed the peak amplitude of voltage-gated Na+ current with no change in the current-voltage relationship of this current. However, it did not have any effect on hyperpolarization-activated cation current. No change in the expression level of KV3.1 mRNA was detected in the presence of parecoxib. The effects of parecoxib on ion currents are direct and unrelated to its inhibition of the enzymatic activity of cyclooxygenase-2. The inhibition of these ion channels by parecoxib may partly contribute to the underlying mechanisms by which it affects neuronal function in vivo.
1. Introduction Parecoxib sodium (PARE, N-[4-(5-methyl-3-phenylisoxazol-4-yl) phenylsulfonyl] propionamide) is a specific inhibitor of cyclooxygenase-2 and available for intravenous or intramuscular administration to painful traumatic or postoperative patients. This compound can rapidly be converted to active valdecoxib (Karim et al., 2001; Martinou et al., 2018; Mehta et al., 2008) and produce a more rapid onset than ketorolac (Talley et al., 2000). Parecoxib administration also significantly diminished morphine consumption (Bikhazi et al., 2004; Langford et al., 2009; Szental et al., 2015; Wheeler et al., 2002; Yuksel et al., 2018). The explanation for its effect is often thought its inhibition of cyclooxygenase-2 (Hersh et al., 2005; Talley et al., 2000). However, Urdaneta et al. reported that the effects of parecoxib may not be fully explained only by its inhibition on cyclooxygenase-2 (Urdaneta et al., 2009). Voltage-gated K+ (KV) channels play an essential role in
⁎
determining the excitability of neurons and the delayed rectifier K+ (KDR) channels are ubiquitous in neurons. A causal relationship between KV3 (or KCNC) and the delayed rectifier K+ current (IK(DR)) has been established (Rudy and McBain, 2001). The KV3 subfamily of KV channels is characterized by positively shifted voltage dependency and fast deactivation rate. These properties can be expected to limit the Na+ channel, thereby leading to depolarization block and accommodation of repetitive firing at high frequencies (Rudy and McBain, 2001; Tateno and Robinson, 2007). KDR channels from the KV3.1-KV3.2 types are the major determinants of IK(DR) in NG108-15 neuronal cells (Huang et al., 2009). Our previous study showed that dexmedetomidine suppress the amplitude of IK(DR) in NG108-15 cells (Chen et al., 2009). If KV3-encoded K+ currents are located at nerve terminals, modulation of current amplitude might cause a profound effect on neurotransmitter release (Dodson and Forsythe, 2004; Rudy and McBain, 2001). Previous report showed that celecoxib can modify the KV1.3- or KV2.1-encoded K+ channels (Frolov et al., 2008; Frolov and Singh, 2015). However,
Correspondence to: Department of Physiology, National Cheng Kung University Medical College, No. 1, University Road, Tainan City 70101, Taiwan. E-mail address: [email protected] (S.-N. Wu).
https://doi.org/10.1016/j.ejphar.2018.12.005 Received 14 July 2018; Received in revised form 29 November 2018; Accepted 5 December 2018 Available online 06 December 2018 0014-2999/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Electrophysiological measurements
whether parecoxib can directly interact with IK(DR) or other types of ion currents in neurons remains unclear, despite its widely clinical use (Martinou et al., 2018; Szental et al., 2015; Yuksel et al., 2018). In another way, the KCNQ2, KCNQ3, and KCNQ5 genes are known to encode the core subunits of KV7.2, KV7.3 and KV7.5 channels, respectively. The increased activity of these KV channels can generate the Mtype K+ current (IK(M)) with a slowly activating and deactivating property (Brown and Yu, 2000; Shu et al., 2007). Targeting IK(M) is recognized as an adjunctive regimen for the treatment of many neurological disorders. However, whether this compound exerts any actions on these types of ionic currents remains unexplored, although celecoxib can increase the amplitude of KV7.2/7.3 currents (Du et al., 2011). Therefore, in this study, we determine the mechanism of parecoxib actions on perturbation of different ion currents in NG108-15 cells. Currents in these cells include IK(DR), IK(M), voltage-gated Na+ current (INa), and hyperpolarization-elicited cation current (Ih). Our findings provide the evidence that parecoxib can directly inhibit ion currents which is highly unlikely to be linked to its inhibitory effects on cyclooxygenase-2 enzymes.
Before the experiments, cells were dissociated with 1% trypsin/ EDTA solution and an aliquot of cell suspension was subsequently transferred to a recording chamber affixed to the stage of a DM-IL inverted microscope (Leica, Wetzlar, Germany). Cells were bathed at room temperature (20–25 °C) in normal Tyrode's solution containing 1.8 mM CaCl2. Microelectrodes were pulled from Kmax-51 capillaries (Kimble Glass, Vineland, NJ) using a PP-83 puller (Narishige, Tokyo, Japan) and had a resistance of 3–5 MΩ when filled with different intracellular solutions. Patch clamp recordings were made in the wholecell configuration with an RK-400 amplifier (Bio-Logic, Claix, France), as described previously (Wu et al., 2001). The signals were displayed on an HM-507 oscilloscope (Hameg, East Meadow, NY) and a liquid crystal projector (AV600; Delta, Taipei, Taiwan). The data were stored online in a TravelMate-6253 laptop computer (Acer, Taipei, Taiwan) at 10 kHz through a Digidata-1322A interface (Molecular Devices). The latter device was equipped with an Adaptec SlimSCSI card (Milpitas, CA) via a PCMCIA slot and was controlled by pCLAMP 9.2 (Molecular Devices). Current signals were lowpass filtered at 1 or 3 kHz. The signals were analyzed using pCLAMP 9.2 (Molecular Devices) or OriginPro 2016 (OriginLab, Northampton, MA). The pCLAMP-generated voltage-step protocols were used to examine the current-voltage (I-V) relationships for ion currents (e.g., IK(DR) and INa).
2. Materials and methods 2.1. Cell culture The clonal strain NG108-15 cell line, formed by Sendai virus-induced fusion of the mouse neuroblastoma clone N18TG-2 and the rat glioma clone C6 BV-1, was obtained from the European Collection of Cell Cultures (ECACC-88112302; Wiltshire, UK). Cells were kept in monolayer cultures at a density of 106/ml in plastic disks containing Dulbecco's modified Eagle's medium supplemented with 100 μM hypoxanthine, 1 μM aminopterin, 16 μM thymidine, and 5% fetal bovine serum as the culture medium, in a humidified incubator equilibrated with 90% air/10% CO2 at 37 °C. Media were replenished every 2–3days with fresh media. To induce neuronal differentiation, the culture medium was replaced with a medium containing 1 mM dibutyryl cyclic AMP and cells were cultured in an incubator for 1–7 days (Chen et al., 2009; Lin et al., 2008). To observe neurite growth, a Nikon Eclipse Ti-E inverted microscope (Li Trading Co., Taipei, Taiwan) equipped with a five-megapixel cooled digital camera was used. The digital camera was connected to a personal computer controlled by NIS-Elements BR3.0 software (Nikon, Kanagawa, Japan). The number of neurites and varicosities was increased as cells were pre-incubated with 1 mM dibutyryl cyclic AMP. Cell viability was evaluated using a WST-1 assay (RocheDiagnostics, Indianapolis, IN) and an ELISA reader (Dynatech, Chantilly, VA).
2.4. Data analyses To evaluate the percentage inhibition of parecoxib on IK(DR), NG108-15 cells were bathed in Ca2+-free Tyrode's solution, and each cell was depolarized from −50 to + 50 mV. The amplitude of IK(DR) at the end of depolarizing pulse during exposure to different concentrations (0.3–100 μM) of parecoxib was measured. Concentration-response data for inhibition of IK(DR) were fitted with a modified form of the Hill equation:
y=
(1 − α ) × [PARE ]−nH + α, −nH [PARE ]−nH + IC50
where y is the normalized amplitude of IK(DR); [PARE] represents the parecoxib concentration and IC50 and nH are the concentration required for 50% inhibition and Hill coefficient, respectively. This also allows the estimation of maximal inhibition (1-α). The paired or unpaired Student's t-test, or one-way analysis of variance (ANOVA) followed by post-hoc Fisher's least-significance difference for multiple-group comparisons, were used for the statistical evaluation of differences among means. Statistical analyses were performed with the use of SPSS 14.0 (SPSS Inc., Chicago, IL). Statistical significance was determined at a P value of < 0.05. Values are provided as means ± standard error of mean (S.E.M.) with sample sizes (n) indicating the number of cells from which the data were taken.
2.2. RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) To detect the expression of KV3.1 mRNA in NG108-15 cells treated with or without dibutyryl cyclic AMP, a semi-quantitative RT-PCR assay was carried out. Total RNA samples were extracted from NG108-15 cells with TRIzol reagent (Invitrogen, Grand Island, NY) and reversetranscribed into complementary DNA using Superscript II reversetranscriptase (Invitrogen, Grand Island, NY). The sequences of oligonucleotide primers used for KV3.1 (NM_008421) were 5′-CGTGCCGAC GAGTTCTTCT-3′ and 5′-GGTCATCTCCAGCTCGTCCT-3′ (Sacco et al., 2006). Amplification of Kv3.1 was done using PCR SuperMix from Invitrogen under the following conditions: 35 cycles composed of 30 s denaturation at 95 °C, 30 s primer annealing at 62 °C, a 1 min extension at 72 °C, followed by the final extension for 2 min at 72 °C. PCR products were analyzed on 1.5% (w/v) agarose gel containing ethidium bromide and then visualized under ultraviolet light. Optical densities of the DNA bands were scanned and quantified with Scion Image Software (Scion, Frederick, MD).
2.5. Chemicals and solutions Parecoxib (Dynastat®, N-[4-(5-methyl-3-phenylisoxazol-4-yl) phenylsulfonyl]propionamide) was obtained from Pfizer Inc. (New York, NY). 4-Aminopyridine, dibutyryl cyclic AMP, diazoxide, indomethacin, flupirtine, linopirdine, tetraethylammonium chloride and tetrodotoxin were purchased from Sigma-Aldrich Chemicals (St. Louis, MO), curcumin, dibutyryl cyclic AMP, ketoprofen, meclofenamate and meloxicam were from Biomol (Plymouth Meeting, PA), and pinacidil, piroxicam and ZD7288 were from Tocris (Bristol, UK). All other chemicals, including CsCl, CsOH and CdCl2, were commercially available and of reagent grade. Deionized water purified by a Millipore-Q system (Millipore, Bedford, MA) was used in all experiments. The composition of the bath solution (i.e., normal Tyrode's solution) was 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 96
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mM glucose, and 5.5 mM HEPES-NaOH buffer, with a solution pH of 7.4. To measure IK(DR), a patch pipette was filled with a solution consisting of 140 mM KCl, 1 mM MgCl2, 3 mM Na2ATP, 0.1 mM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES-KOH buffer at a solution pH of 7.2. To measure M-type K+ current (IM), cells were bathed in high-K+, Ca2+free solution consisting of 145 mM KCl 145, 0.53 mM MgCl2, and 5 mM HEPES-KOH buffer, with a solution pH of 7.4. To record voltage-gated sodium current (INa) or hyperpolarization-elicited cation current (Ih), KCl inside the pipette solution was replaced with equimolar CsCl, and the pH was adjusted to 7.2 with CsOH. 3. Results 3.1. Effect of parecoxib on IK(DR) in differentiated NG108-15 neuronal cells Fig. 2. Effect of parecoxib on the inactivation time course of IK(DR) in differentiated NG108-15 cells. (A) Superimposed current traces obtained in the absence and presence of parecoxib. a: control; b: 10 μM parecoxib. The cell was depolarized from a holding potential of −50 mV to + 50 mV with a duration of 10 s. The smooth gray curves shown in (A) were nonlinear least squares fits to either one or two exponentials to the data. The inset in (A) indicates the voltage protocol used. (B) Summary of the data showing the effect of parecoxib on the fast component of IK(DR) inactivation (mean ± S.E.M.; n = 5–8 for each bar). *Significantly different from parecoxib (1 μM) alone group (P < 0.05).
In an initial set of experiments, a whole-cell configuration of the patch-clamp technique was used to evaluate the effects of parecoxib on ion currents in NG108-15 cells differentiated with dibutyryl cyclic AMP (1 mM). To measure IK(DR), cells were bathed in Ca2+-free Tyrode's solution which contained tetrodotoxin (1 μM) and CdCl2 (0.5 mM), and the recording pipette was filled with a K+-containing solution. Fig. 1 shows that parecoxib can diminish the amplitude of IK(DR) in a concentration-dependent manner. In these experiments, each cell was depolarized from −50 to + 50 mV with a duration of 300 ms, and current amplitudes of IK(DR) were measured at the end of depolarizing pulses. The relationship between the parecoxib concentration (0.3–100 μM) and the relative amplitude of IK(DR) was also constructed and is plotted in Fig. 1B. Fitting the concentration-response curve with the modified Hill equation as described in Section 2 yielded a half-maximal concentration (i.e., IC50) of 9.7 μM and a slope coefficient of 1.1. Parecoxib at a concentration of 100 μM nearly abolished current amplitude. Fig. 1C illustrates the averaged I-V relationships of IK(DR) obtained in the
control and during cell exposure to 10 μM parecoxib. These results demonstrate that parecoxib has a significant depressant action on IK(DR) functionally expressed in differentiated NG108-15 cells. During cell exposure to parecoxib, in addition to the decreased amplitude of IK(DR), the time to the peak of IK(DR) tended to be shortened, and the time course of current inactivation was progressively changed to the biexponential process (Fig. 2). For example, as the depolarizing pulse from −50 to + 50 mV with a duration of 10 s was evoked, the inactivation of IK(DR) was well fitted to a single exponential process with a mean time constant of 2012 ± 32 ms (n = 12); however, in the presence of parecoxib (30 μM), the fast and slow constants of IK(DR) inactivation were 815 ± 13 and 3231 ± 34 ms (n = 11), respectively. The fast, rather than the slow component was found to become significantly concentration-dependent (Fig. 2B). In other words, the fast component of inactivation time constant decreased when the parecoxib concentration was elevated. After parecoxib was removed, IK(DR) returned almost to the control level. 3.2. Effect of parecoxib on the accumulative inhibition of IK(DR) inactivation The IK(DR) inactivation is capable of accumulating during repetitive short pulses as reported previously (Lin et al., 2008). For this reason, in another set of experiments, we sought to determine whether it affects the degree of IK(DR) inactivation elicited by brief repetitive depolarizations. Under control conditions, a single 20-s depolarizing step to + 70 mV from a holding potential of −50 mV produced a decline with a time constant of 5.7 ± 0.2 s (n = 7). However, the time constant for 20-s repetitive pulses to + 60 mV, each of which lasted 40 ms with 20-ms intervals at the level of −50 mV between the depolarizing pulses, was significantly reduced to 2.3 ± 0.2 s (n = 7, P < 0.05). During exposure to 1 μM parecoxib, the value of the time constant obtained in response to such a train of short repetitive pulses was further diminished to 1.5 ± 0.1 s (n = 6, P < 0.05). A representative example of the cumulative inhibition of IK(DR) inactivation in control is illustrated in Fig. 3A. As shown in Fig. 3B, application of parecoxib was also able to increase the cumulative inhibition of IK(DR) inactivation in NG108-15 cells. For example, when cells were exposed to 1 μM parecoxib, the amplitude of IK(DR) measured at the end of a train of short repetitive stimuli was
Fig. 1. Inhibitory effect of parecoxib on IK(DR) in NG108-15 cells differentiated with dibutyryl cyclic AMP. Cells were bathed in Ca2+-free Tyrode's solution in which tetrodotoxin (1 μM) and CdCl2 (0.5 mM) were included. (A) Superimposed current traces obtained in the absence and presence of parecoxib. The inset in (A) indicates the voltage protocol used. a: control; b: 1 μM parecoxib; c: 3 μM parecoxib; d: 10 μM parecoxib. (B) Concentration-response relationship for parecoxib-induced inhibition of IK(DR). Each cell was depolarized from −50 to + 50 mV. Each point represents the mean ± S.E.M. (n = 6–12). The smooth line represents the best fit to the modified Hill equation as described in Section 2. The IC50 value was estimated with the non-linear curve fit. The IC50 value, maximally inhibited the percentage of IK(DR), and the Hill coefficient (nH) were 9.7 μM, 74% (i.e., α = 0.26), and 1.1, respectively. (C) Averaged I-V relationships of IK(DR) obtained in the absence (•) and presence (◯) of 10 μM parecoxib (mean ± S.E.M.; n = 8–12 for each point). 97
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Fig. 3. Excessive accumulative inactivation of IK(DR) during repetitive stimuli in the absence and presence of parecoxib in differentiated NG108-15 cells. Currents in (B) were obtained during repetitive depolarizations to + 70 mV with a return to −50 mV. The individual depolarizing pulse used to elicit outward K+ current lasted 40 ms (indicated in (A)). Current trace with a single exponential of 2.3 s was obtained in the control. (B) Summary of the data showing the effect of parecoxib (1 and 3 μM) on the amplitude of IK(DR) during repetitive depolarization (mean ± S.E.M.; n = 6–8 for each bar). Current amplitude was measured at the end of repetitive depolarizing pulses (i.e., 20 s). *Significantly different from control (P < 0.05).
effects on IK(DR) in these cells.
significantly decreased by 35 ± 2%, from 2903 ± 285–1887 ± 203 pA (n = 8, P < 0.05). It is thus clear from these results that exposure to this compound can increase the accumulative inhibition of IK(DR) inactivation in NG108-15 cells.
3.4. Effect of parecoxib on M-type K+ current (IK(M)) in differentiated NG108-15 cells Celexcoxib, another inhibitor of cyclooxygenase-2, was previously used to stimulate IK(M) (Du et al., 2011). As such, in another set of experiments, we explored the possible effect of parecoxib on IK(M) observed in these cells. The examined cells were bathed in high-K+, Ca2+free solution and the recording pipette was filled with K+-containing solution. As shown in Fig. 5, when the cell was depolarized from −50 to −10 mV with a duration of 1 s, K+ inward current with the slowly activating and deactivating properties was readily evoked. This K+ current elicited by long-lasting membrane depolarization was sensitive to inhibition by linopirdine (10 μM), yet not by either 4-aminopyridine (1 mM) or tetraethylammonium (10 mM), and it was hence identified as an IK(M) (Hsu et al., 2014; Huang et al., 2008; Selyanko et al., 1999). Linopirdine is recognized as a selective blocker of IK(M) (Huang et al.,
3.3. Comparison between the effects of indomethacin, curcumin, ketoprofen, meclofenamate, meloxicam, piroxicam, and 4-aminopyridine on IK(DR) Effects of other compounds which are the inhibitors of cyclooxygenase were also examined and compared. In these experiments, each cell was depolarized from −50 to + 50 mV with a duration of 300 ms, and the amplitude of IK(DR) was measured at the end of the depolarizing pulse. The results showed that neither indomethacin (10 μM), curcumin (10 μM), ketoprofen (10 μM), nor meclofenamate (10 μM) could be effective in suppressing IK(DR) (Fig. 4) (Kuo et al., 2018). However, meloxicam (10 μM) and piroxicam (10 μM) slightly diminished this current, while 4-aminopyridine (1 mM) decreased it by about 80% (Fig. 4). Indomethacin, curcumin, ketoprofen, and meclofenamate are known to be blockers of cyclooxygenases, while meloxicam is a selective inhibitor of cyclooxygenase-2. Therefore, these results indicate that, unlike parecoxib, these inhibitors of cyclooxygenase had minimal
Fig. 5. Effect of parecoxib on the amplitude of M-type K+ current (IK(M)) in differentiated NG108-15 cells. These experiments were conducted in cells bathed in high K+, Ca2+-free solution and the recording pipette was filled with K+-containing solution. (A) Superimposed IK(M) traces obtained in the absence (a) and presence of 10 μM (b) and 30 μM parecoxib (c). Arrowhead indicates the zero current level. The upper part indicates the voltage protocol applied. (B) Bar graph showing the effect of parecoxib, linopirdine and parecoxib plus flupirtine on IK(M) amplitude (mean ± S.E.M.; n = 10–11 for each bar). The IK(M) amplitude elicited by long-lasting membrane depolarization from −50 to −10 mV was measured at the end of depolarizing pulse. a: control; b: 10 μM parecoxib; c: 30 μM parecoxib; d: 10 μM linopirdine; e: 30 μM parecoxib plus 10 μM flupirtine. *Significantly different from control (P < 0.05) and **significantly different from parecoxib (10 μM) alone group (P < 0.05).
Fig. 4. Summary of the data showing the effects of indomethacin (10 μM), curcumin (10 μM), ketoprofen (10 μM), meclofenamate (10 μM), meloxicam (10 μM), piroxicam (10 μM) and 4-aminopyridine (1 mM) on IK(DR). In these experiments, cells were bathed in Ca2+-free Tyrode’s solution and the recording pipettes were filled with a K+-containing solution. Each cell was depolarized from −50 to + 50 mV with a duration of 300 ms. At the end of the depolarizing pulse, the current amplitude in the control was considered to be 1.0, and the relative amplitude of IK(DR) after application of each agent was plotted. 4-AP: 1 mM 4-aminopyridine. *Significantly different from control (P < 0.05). Each bar represents the mean ± S.E.M. (n = 6–9). 98
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Fig. 6. Effects of parecoxib on INa and Ih in differentiated NG108-15 cells. Cells were bathed in Ca2+-free Tyrode's solution, and the recording pipette was filled with a Cs+-containing solution. (A) Original INa currents obtained in the absence (trace a) and presence of 3 μM (trace b) and 10 μM (trace c) parecoxib. The examined cell was depolarized from −80 to −20 mV. The inset indicates expanded time scale of current traces (indicated by dashed box). (B) Averaged I-V relationships of peak INa obtained in the absence (•) or presence (◯) of 10 μM parecoxib (mean ± S.E.M.; n = 6–10 for each point). Notably, parecoxib diminishes the peak amplitude of INa without any change in the overall I-V configuration of this current. *Significantly different from controls measured at the same level of membrane potential (P < 0.05). In these experiments on recording Ih, cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl2. The pipette was filled with a K+-containing solution. In each cell, a long-step hyperpolarizing stimulus was applied from a holding potential of −50 mV. In (C), traces a (black) and b (gray) were, respectively, obtained in the absence and presence of 30 μM parecoxib. The inset shown in (A) indicates the voltage protocol used. (D) Averaged I-V relationships of Ih in the absence and presence of 30 μM parecoxib (mean ± S.E.M.; n = 4–8 for each point). The amplitude of Ih was measured at the end of each hyperpolarizing pulse.
further studied whether this compound could have any influence on Ih. The results showed its inability to affect the amplitude or gating of Ih in these cells. When cells were hyperpolarized from −50 to the different voltages ranging from −150 to −50 mV in 10-mV increments, the Ih measured at the end of the hyperpolarizing pulse in the control did not significantly differ from that measured during the exposure to 30 μM parecoxib (Fig. 6C and D). Moreover, similar to the findings observed in that of INa (Fig. 6A and B), no discernible change in the overall I-V relationships of Ih was seen in the presence of parecoxib (Fig. 6D). However, at the same voltage protocol, 30 μM ZD7288, an inhibitor of Ih (Liu et al., 2009), could be effective in suppressing Ih, as evidenced by a significant reduction of Ih from 2012 ± 15–986 ± 11 pA during the exposure to 30 μM ZD7288 (n = 11, P < 0.05). Therefore, it is clear that, unlike IK(DR), IK(M) or INa, Ih is not subject to inhibition by parecoxib.
2008). Notably, as cells were exposed to parecoxib, the IK(M) amplitude evoked in response to depolarizing pulse from −50 to −10 mV was progressively diminished, as evidenced by the data showing that addition of 30 μM parecoxib caused a significant reduction in current amplitude from 41.2 ± 7.9–7.4 ± 1.1 pA (n = 11, P < 0.05). Moreover, in continued presence of 30 μM parecoxib, subsequent addition of 10 μM flupirtine, an activator of IK(M) (Wu et al., 2012), was found to reverse its inhibition of IK(M) significantly. Therefore, distinguishable from previous observations showing the ability of celecoxib to increase IK(M) amplitude (Du et al., 2011), it is possible that the presence of parecoxib suppressed the amplitude of IK(M) effectively in these cells. 3.5. Inhibitory effect of parecoxib on voltage-gated Na+ current (INa) in differentiated NG108-15 cells The functional defects of the SCN9A gene were recently associated with inherited erythromelagia, a painful neuropathic disorder (Waxman, 2007). Therefore, the effect of parecoxib on INa was further investigated in these cells. The experiments were conducted with a Cs+containing pipette solution. As shown in Fig. 6A, when the cell was depolarized from −80 to −10 mV, the application of parecoxib (10 μM) significantly decreased the peak amplitude of INa by 21 ± 2%, from 1122 ± 87–898 ± 98 pA (n = 8, P < 0.05). After washout of parecoxib, the amplitude of INa was partially returned to 1108 ± 91 pA (n = 5). However, no change in the overall I-V relationships of INa could be demonstrated in the presence of parecoxib (Fig. 6B). Furthermore, neither the activation nor inactivation time course of INa elicited by rapid membrane depolarization was modified by application of parecoxib. Likewise, the non-inactivating INa in response to a long-lasting ramp pulse was unaffected in the presence of this agent (data not shown). Taken together, these results indicate that in differentiated NG108-15 cells, parecoxib produces a depressant action on the peak amplitude of INa.
3.7. The mRNA expression for KV3.1 in differentiated NG108-15 cells Previous studies showed that the inhibitors of cyclooxygenase enzymes were able to alter the expression level of cyclooxygenase-2 mRNA increased by carrageenan (Pham-Marcou et al., 2008; Urdaneta et al., 2009). The IK(DR) in these cells is recognized to be carried by the Shaw-related K+ channels, which are the products of the KV3.1 (or KCNC1) gene. Therefore, we further examined whether the mRNA levels of KV3.1 in differentiated NG108-15 cells can be altered in the presence of parecoxib. In parecoxib-treated cells, cells were pre-incubated with 30 μM parecoxib for 2 days. Our RT-PCR analysis demonstrated that the extent of mRNA expression for KV3.1 in differentiated cells was found to be significantly increased. However, the mRNA level of KV3.1 subunit in cells did not differ between the presence and absence of parecoxib (Fig. 7). 4. Discussion In this study, we provide the direct evidence to show that parecoxib, which is the first cyclooxygenase-2 to be administered parenterally, has a depressant action on IK(DR) in differentiated NG108-15 cells. The halfblocking concentration of this compound required for inhibition of IK(DR) with an IC50 value of 9.7 μM appears to be of the same order of magnitude as the concentrations of celecoxib used to suppress KV1.3- or KV2.1-encoded K+ currents (Frolov et al., 2008; Frolov and Singh,
3.6. No effect of parecoxib on hyperpolatization-activated cation current (Ih) in differentiated NG108-15 cells In recent studies done at our laboratory, we demonstrated that when undergoing differentiation with a non-degradable analogue of cyclic AMP (i.e., dibutyryl cyclic AMP), NG108-15 cells could also express the activity of hyperpolarization-evoked cation channels. We 99
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block of IK(DR) (data not shown). Diazoxide or pinacidil are the openers of ATP-sensitive K+ channels (Wu et al., 2000). Therefore, parecoxibinduced inhibition of IK(DR) observed in NG108-15 cells is unlikely to be connected with the activity of either large-conductance Ca2+-activated K+ channels or ATP-sensitive K+ channels. Voltage-gated Na+ (NaV) channels are generally involved in the generation and propagation of APs in most excitable cells. Our study showed the ability of parecoxib to suppress the peak amplitude of INa in NG108-15 cells. The tetrodotoxin-sensitive INa identified in differentiated NG108-15 cells is reported to resemble NaV1.7 encoded by SCN9A gene (Kawaguchi et al., 2007). However, neither the activation nor the inactivation rate of INa was modified in the presence of parecoxib. In addition, the application of parecoxib failed to alter the noninactivating component of INa elicited by a slow ramp pulse. Parecoxib was also not found to have any effects on Ih. Taken together, the electrophysiological effects of parecoxib and other coxibs on ion currents are presumably not limited to their modification of KDR channels. Recent studies have demonstrated that NaV1.7 can be expressed both in peripheral sensory and sympathetic neurons as well as olfactory epithelia (Momin and Wood, 2008). Gain-of-function mutations in NaV1.7 (e.g., the L858H mutation) are recognized to cause primary erythromelagia and paroxysmal extreme pain disorder, while loss-offunction mutations in NaV1.7 cause a channelopathy accompanied by insensitivity to pain (Momin and Wood, 2008; Waxman, 2007). In our experimental conditions, the effects of parecoxib on ion currents were generally observed within only 2 min of application of this compound. The inhibitory effect of parecoxib on INa is thus direct and could be an important mechanism by which it depresses the amplitude of neuronal action potentials. Parecoxib-induced blockade of IK(DR), IK(M) and INa may synergistically affect the functional activity of neurons. In conclusion, this present study clearly demonstrates that parecoxib can directly inhibit IK(DR), IK(M) and INa in differentiated NG10815 cells and strongly indicates that this compound is apparently not an inactive prodrug. The suppression of these ionic currents is responsible for its effects on functional activities in these cells. Following intravenous or intramuscular administration, parecoxib can easily pass across the blood-brain barriers, although it may rapidly be hydrolyzed to its active moiety, valedoxib. Therefore, the electrophysiological effects of parecoxib presented herein could lead to changes in firing behavior of neurons, hence altering neuronal function (Kim et al., 2011; Polascheck et al., 2010). However, to what extent parecoxib-induced inhibition of ion channels contributes to its anti-inflammatory and antinociceptive actions has yet to be further delineated.
Fig. 7. Results of RT-PCR for the KV3.1 mRNA in differentiated NG108-15 cells obtained in the presence (lane A) and absence (lane B) of 10 μM parecoxib for 48 h. Total RNA was isolated, and RT-PCR analysis was performed. Amplified RT-PCR products were obtained for use as a marker lane of DNA (leftmost lane; M.W.) and KV3.1 subunit (306 bp) indicated by arrow. The mRNA level of KV3.1 subunit obtained in the presence of parecoxib (30 μM) did not differ from that in the control (i.e., undifferentiated NG108-15 cells).
2015). Notably, immediately following a single intramuscular injection with 20 and 40 mg parecoxib, the peak plasma concentration of approximately 2.7 and 4.5 μM can be respectively reached within 15 min (Karim et al., 2001). Other compounds known to inhibit the activity of cyclooxygenases were found to have minimal effects on IK(DR), while 4aminopyridine suppressed this current effectively. 4-Aminopyridine was previously reported to modulate amyloid β1–42-induced intracellular signaling in human microglia (Franciosi et al., 2006). Consistent with previous observations (Frolov et al., 2008; Frolov and Singh, 2015), the KDR and KM channels may thus be an important target for the action of parecoxib, despite its ability to suppress activity of cyclooxygenase-2. Both the alteration in the channel gating and the reduction of the currents caused by parecoxib or other coxibs are also likely to be clinically relevant if similar results are found in intact neurons in vivo. Indeed, it has been recently shown that the anti-inflammatory and anti-nociceptive actions of parecoxib cannot be fully explained by its inhibition of cyclooxygenase-2 activity (Urdaneta et al., 2009). In our study, the mechanism of parecoxib actions on IK(DR) apparently differed from those on INa or Ih. In differentiated NG108-15 neuronal cells, the effect exerted by parecoxib is a time-, and concentration-dependent increase of the inactivation process of IK(DR), along with no change in the activation kinetics of this current. No significant change in the mRNA expression of KV3.1 detected in NG10815 cells was detected in the presence of parecoxib. However, its presence was also found to increase the accumulative inhibition of IK(DR) inactivation elicited by long trains of brief repetitive depolarizations in these cells. Therefore, as action potentials in neurons in vivo fire more frequently, the inhibitory action of parecoxib on IK(DR) would become enhanced. Parecoxib-induced increase of IK(DR) inactivation may enhance a process of long-lasting facilitation or potentiation as described previously (Huang et al., 2009; Shu et al., 2007; Wu et al., 2008). In our study, as the recording pipettes were filled with parecoxib (30 μM) and whole-cell currents were established, no change in the amplitude of IK(DR) was clearly seen. However, when cells were exposed to parecoxib, the time course of IK(DR) inactivation was changed to the biexponential process. The fast component of IK(DR) inactivation became concentration-dependent. Therefore, with respect to its effects on IK(DR), parecoxib tends to have a mechanism that is a combination of open channel block and tight binding to inactivated states of the channel. During cell exposure to it, the conformation of the external pore mouth appears to be similar to the characteristics of C-type inactivation because C-type inactivation is thought to involve conformational changes at the external mouth of the pore (Baukrowitz and Yellen, 1995). All recordings of IK(DR) presented herein were conducted in a Ca2+free Tyrode's solution containing 0.5 mM CdCl2. Rottlerin, an activator of large-conductance Ca2+-activated K+ channels (Wu et al., 2007), did not reverse the inhibition of IK(DR) caused by parecoxib. Furthermore, neither diazoxide nor pinacidil had any effects on parecoxib-induced
CRediT authorship contribution statement Yuan-Yuarn Liu: Conceptualization, Resources, Methodology, Funding acquisition. Hung-Tsung Hsiao: Conceptualization, Methodology. Jeffrey Chi-Fei Wang: Conceptualization, Resources, Funding acquisition. Yen-Chin Liu: Writing - original draft, Visualization, Project administration. Sheng-Nan Wu: Methodology, Software, Writing - review & editing, Supervision.
Acknowledgments The work in this laboratory was supported by a grant from National Cheng Kung University (D106-35A13), Tainan City, Taiwan. The authors are grateful to Dr. Bing-Shuo Chen for the earlier work.
Conflict of interest None of the authors in this study have any potential conflict of interest or financial interests to disclose. 100
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References
Voros, D., Fragulidis, G.P., 2018. Parecoxib's effects on anastomotic and abdominal wound healing: a randomized Controlled trial. J. Surg. Res. 223, 165–173. Mehta, V., Johnston, A., Cheung, R., Bello, A., Langford, R.M., 2008. Intravenous parecoxib rapidly leads to COX-2 inhibitory concentration of valdecoxib in the central nervous system. Clin. Pharmacol. Ther. 83, 430–435. Momin, A., Wood, J.N., 2008. Sensory neuron voltage-gated sodium channels as analgesic drug targets. Curr. Opin. Neurobiol. 18, 383–388. Pham-Marcou, T.A., Beloeil, H., Sun, X., Gentili, M., Yaici, D., Benoit, G., Benhamou, D., Mazoit, J.X., 2008. Antinociceptive effect of resveratrol in carrageenan-evoked hyperalgesia in rats: prolonged effect related to COX-2 expression impairment. Pain 140, 274–283. Polascheck, N., Bankstahl, M., Loscher, W., 2010. The COX-2 inhibitor parecoxib is neuroprotective but not antiepileptogenic in the pilocarpine model of temporal lobe epilepsy. Exp. Neurol. 224, 219–233. Rudy, B., McBain, C.J., 2001. Kv3 channels: voltage-gated K+ channels designed for highfrequency repetitive firing. Trends Neurosci. 24, 517–526. Sacco, T., De Luca, A., Tempia, F., 2006. Properties and expression of Kv3 channels in cerebellar Purkinje cells. Mol. Cell Neurosci. 33, 170–179. Selyanko, A.A., Hadley, J.K., Wood, I.C., Abogadie, F.C., Delmas, P., Buckley, N.J., London, B., Brown, D.A., 1999. Two types of K(+) channel subunit, Erg1 and KCNQ2/3, contribute to the M-like current in a mammalian neuronal cell. J. Neurosci. 19, 7742–7756. Shu, Y., Yu, Y., Yang, J., McCormick, D.A., 2007. Selective control of cortical axonal spikes by a slowly inactivating K+ current. Proc. Natl. Acad. Sci. USA 104, 11453–11458. Szental, J.A., Webb, A., Weeraratne, C., Campbell, A., Sivakumar, H., Leong, S., 2015. Postoperative pain after laparoscopic cholecystectomy is not reduced by intraoperative analgesia guided by analgesia nociception index (ANI(R)) monitoring: a randomized clinical trial. Br. J. Anaesth. 114, 640–645. Talley, J.J., Bertenshaw, S.R., Brown, D.L., Carter, J.S., Graneto, M.J., Kellogg, M.S., Koboldt, C.M., Yuan, J., Zhang, Y.Y., Seibert, K., 2000. N-[[(5-methyl-3-phenylisoxazol-4-yl)-phenyl]sulfonyl]propanamide, sodium salt, parecoxib sodium: a potent and selective inhibitor of COX-2 for parenteral administration. J. Med. Chem. 43, 1661–1663. Tateno, T., Robinson, H.P., 2007. Quantifying noise-induced stability of a cortical fastspiking cell model with Kv3-channel-like current. Biosystems 89, 110–116. Urdaneta, A., Siso, A., Urdaneta, B., Cardenas, R., Quintero, L., Avila, R., Suarez-Roca, H., 2009. Lack of correlation between the central anti-nociceptive and peripheral antiinflammatory effects of selective COX-2 inhibitor parecoxib. Brain Res. Bull. 80, 56–61. Waxman, S.G., 2007. Channel, neuronal and clinical function in sodium channelopathies: from genotype to phenotype. Nat. Neurosci. 10, 405–409. Wheeler, M., Oderda, G.M., Ashburn, M.A., Lipman, A.G., 2002. Adverse events associated with postoperative opioid analgesia: a systematic review. J. Pain 3, 159–180. Wu, S.N., Chen, B.S., Lin, M.W., Liu, Y.C., 2008. Contribution of slowly inactivating potassium current to delayed firing of action potentials in NG108-15 neuronal cells: experimental and theoretical studies. J. Theor. Biol. 252, 711–721. Wu, S.N., Hsu, M.C., Liao, Y.K., Wu, F.T., Jong, Y.J., Lo, Y.C., 2012. Evidence for inhibitory effects of flupirtine, a centrally acting analgesic, on delayed rectifier K+ currents in motor neuron-like cells. Evid. Based Complement Altern. Med. 2012, 148403. Wu, S.N., Li, H.F., Chiang, H.T., 2000. Characterization of ATP-sensitive potassium channels functionally expressed in pituitary GH3 cells. J. Membr. Biol. 178, 205–214. Wu, S.N., Lo, Y.K., Chen, H., Li, H.F., Chiang, H.T., 2001. Rutaecarpine-induced block of delayed rectifier K+ current in NG108-15 neuronal cells. Neuropharmacology 41, 834–843. Wu, S.N., Wang, Y.J., Lin, M.W., 2007. Potent stimulation of large-conductance Ca2+activated K+ channels by rottlerin, an inhibitor of protein kinase C-δ, in pituitary tumor (GH3) cells and in cortical neuronal (HCN-1A) cells. J. Cell Physiol. 210, 655–666. Yuksel, U., Bakar, B., Dincel, G.C., Budak Yildiran, F.A., Ogden, M., Kisa, U., 2018. The investigation of the Cox-2 selective inhibitor parecoxib effects in spinal cord injury in rat. J. Investig. Surg. 1–12.
Baukrowitz, T., Yellen, G., 1995. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron 15, 951–960. Bikhazi, G.B., Snabes, M.C., Bajwa, Z.H., Davis, D.J., LeComte, D., Traylor, L., Hubbard, R.C., 2004. A clinical trial demonstrates the analgesic activity of intravenous parecoxib sodium compared with ketorolac or morphine after gynecologic surgery with laparotomy. Am. J. Obstet. Gynecol. 191, 1183–1191. Brown, B.S., Yu, S.P., 2000. Modulation and genetic identification of the M channel. Prog. Biophys. Mol. Biol. 73, 135–166. Chen, B.S., Peng, H., Wu, S.N., 2009. Dexmedetomidine, an α2-adrenergic agonist, inhibits neuronal delayed-rectifier potassium current and sodium current. Br. J. Anaesth. 103, 244–254. Dodson, P.D., Forsythe, I.D., 2004. Presynaptic K+ channels: electrifying regulators of synaptic terminal excitability. Trends Neurosci. 27, 210–217. Du, X.N., Zhang, X., Qi, J.L., An, H.L., Li, J.W., Wan, Y.M., Fu, Y., Gao, H.X., Gao, Z.B., Zhan, Y., Zhang, H.L., 2011. Characteristics and molecular basis of celecoxib modulation on K(v)7 potassium channels. Br. J. Pharmacol. 164, 1722–1737. Franciosi, S., Ryu, J.K., Choi, H.B., Radov, L., Kim, S.U., McLarnon, J.G., 2006. Broadspectrum effects of 4-aminopyridine to modulate amyloid beta1-42-induced cell signaling and functional responses in human microglia. J. Neurosci. 26, 11652–11664. Frolov, R.V., Berim, I.G., Singh, S., 2008. Inhibition of delayed rectifier potassium channels and induction of arrhythmia: a novel effect of celecoxib and the mechanism underlying it. J. Biol. Chem. 283, 1518–1524. Frolov, R.V., Singh, S., 2015. Evidence of more ion channels inhibited by celecoxib: KV1.3 and L-type Ca2+ channels. BMC Res. Notes 8, 62. Hersh, E.V., Lally, E.T., Moore, P.A., 2005. Update on cyclooxygenase inhibitors: has a third COX isoform entered the fray? Curr. Med. Res. Opin. 21, 1217–1226. Hsu, H.T., Tseng, Y.T., Lo, Y.C., Wu, S.N., 2014. Ability of naringenin, a bioflavonoid, to activate M-type potassium current in motor neuron-like cells and to increase BKCachannel activity in HEK293T cells transfected with alpha-hSlo subunit. BMC Neurosci. 15, 135. Huang, C.W., Huang, C.C., Lin, M.W., Tsai, J.J., Wu, S.N., 2008. The synergistic inhibitory actions of oxcarbazepine on voltage-gated sodium and potassium currents in differentiated NG108-15 neuronal cells and model neurons. Int. J. Neuropsychopharmacol. 11, 597–610. Huang, C.W., Tsai, J.J., Huang, C.C., Wu, S.N., 2009. Experimental and simulation studies on the mechanisms of levetiracetam-mediated inhibition of delayed-rectifier potassium current (KV3.1): contribution to the firing of action potentials. J. Physiol. Pharmacol. 60, 37–47. Karim, A., Laurent, A., Slater, M.E., Kuss, M.E., Qian, J., Crosby-Sessoms, S.L., Hubbard, R.C., 2001. A pharmacokinetic study of intramuscular (i.m.) parecoxib sodium in normal subjects. J. Clin. Pharmacol. 41, 1111–1119. Kawaguchi, A., Asano, H., Matsushima, K., Wada, T., Yoshida, S., Ichida, S., 2007. Enhancement of sodium current in NG108-15 cells during neural differentiation is mainly due to an increase in NaV1.7 expression. Neurochem. Res. 32, 1469–1475. Kim, Y.H., Lee, P.B., Park, J., Lim, Y.J., Kim, Y.C., Lee, S.C., Ahn, W., 2011. The neurological safety of epidural parecoxib in rats. Neurotoxicology 32, 864–870. Kuo, P.C., Yang, C.J., Lee, Y.C., Chen, P.C., Liu, Y.C., Wu, S.N., 2018. The comprehensive electrophysiological study of curcuminoids on delayed-rectifier K+ currents in insulin-secreting cells. Eur. J. Pharmacol. 819, 233–241. Langford, R.M., Joshi, G.P., Gan, T.J., Mattera, M.S., Chen, W.H., Revicki, D.A., Chen, C., Zlateva, G., 2009. Reduction in opioid-related adverse events and improvement in function with parecoxib followed by valdecoxib treatment after non-cardiac surgery: a randomized, double-blind, placebo-controlled, parallel-group trial. Clin. Drug Investig. 29, 577–590. Lin, M.W., Wang, Y.J., Liu, S.I., Lin, A.A., Lo, Y.C., Wu, S.N., 2008. Characterization of aconitine-induced block of delayed rectifier K+ current in differentiated NG108-15 neuronal cells. Neuropharmacology 54, 912–923. Liu, Y.C., Wang, Y.J., Wu, P.Y., Wu, S.N., 2009. Tramadol-induced block of hyperpolarization-activated cation current in rat pituitary lactotrophs. Naunyn Schmiede. Arch. Pharmacol. 379, 127–135. Martinou, E., Drakopoulou, S., Aravidou, E., Sergentanis, T., Kondi-Pafiti, A., Argyra, E.,
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