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NEUROPHARMACOLOGY

Flufenamic Acid Bi-Directionally Modulates the Transient Outward K⁺ Current in Rat Cerebellar Granule Cells

School of Life Sciences, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai China

Received November 24, 2006; accepted April 2, 2007.

	Abstract

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In this report, the effect of flufenamic acid on voltage-activated transient outward K⁺ current (I_A) in cultured rat cerebellar granule cells was investigated. At a concentration of 20 µMto1 mM, flufenamic acid reversibly inhibited I_A in a dose-dependent manner. However, flufenamic acid at a concentration of 0.1 to 10 µM significantly increased the current amplitude of I_A. In addition to the current amplitude of I_A, a higher concentration of flufenamic acid had a significant effect on the kinetic parameters of the steady-state activation and inactivation process, suggesting that the binding affinity of flufenamic acid to I_A channels may be state-dependent. Silencing the K_v4.2, K_v4.3, and K_v1.1 genes of I_A channels using small interfering RNA did not change the inhibitory effect of flufenamic on I_A, indicating that flufenamic acid did not act specifically on any of the subunits of the I_A-channel protein. Intracellular application of flufenamic acid could significantly increase the I_A amplitude but did not alter the inhibited effect induced by extracellular application of flufenamic acid, implying that flufenamic acid may exert its effect from both the inside and outside sites of the channel. Furthermore, the activation of current induced by intracellular application of flufenamic acid could mimic other cyclooxygenase inhibitors and arachidonic acid. Our data are the first that demonstrate how flufenamic acid is able to bidirectionally modulate I_A channels in neurons at different concentrations and by different methods of application and that two different mechanisms may be involved.

On the other hand, the effects of fenamate NSAIDs on different ion channels have been studied widely. It has been shown that local administration of voltage-dependent or Ca²⁺-activated K⁺ channel blockers could prevent the fenamate-induced peripheral antinociception, suggesting that fenamate may open several K⁺ channels at the primary afferent neurons (Alves and Duarte, 2002). In the trabecular meshwork of the eye, flufenamic acid enhances current through maxi-K channels (Stumpff et al., 2001). In addition, activation and inhibition of CLC-K channels (kidney CLC chloride channels) by distinct binding sites of niflumic acid and fenamate acid has been reported by Liantonio et al. (2006). However, the effect of fenamate NSAIDs on ion channels was considered to directly block ion channels, and few report were concerned with the main mechanism that mediates fenamate-induced inhibition of cyclooxygenase.

It has been shown that cerebellar granule cells grown in primary culture have two main voltage-activated outward K⁺ currents: rectify outward K⁺ current (I_K) and transient outward K⁺ (I_A) (Mei et al., 2004). They are distinguishable by their activation and inactivation voltage ranges and kinetics and by their pharmacological sensitivities. In general, the functional roles of I_A include influencing cell excitability and action-potential firing and controlling spike latency and repetitive firing (Shibata et al., 2000). Recently, our own data indicated that apoptosis of cerebellar granule neurons induced by low K⁺ and free serum incubation is associated with an increase of I_A (Hu et al., 2005, 2006). Therefore, finding a new I_A-channel modulator or modulated mechanism is highly useful for further investigations into neuron excitability, neuronal apoptosis, or neuronal protection.

Our recent studies demonstrated that the diphenyl structural NSAID, diclofenac, could activate I_A in rat cerebellar granule cells as a novel voltage-dependent I_A channel opener (Liu et al., 2005). In the present study, we used a whole-cell patch-clamping technique and small interfering RNA (siRNA) to investigate the effects of flufenamic acid on I_A channels, as well as on their steady-state activation and steady-state inactivation. The results first demonstrate that flufenamic acid bidirectionally modulated the I_A current of rat granule neurons at different concentrations and that two different mechanisms were involved.

	Materials and Methods

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Cell Culture. Cells were derived from cerebellum of 7- to 8-day-old Sprague-Dawley rat pups as described previously. Isolated cells then were plated onto 35-mm-diameter Petri dishes coated with poly-L-lysine (1 µg/ml) at a density of 10⁶ cells/ml. Cultured cells were incubated at 37°C with 5% CO₂ in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, glutamine (5 mM), insulin (5 µg/ml), KCl (25 mM), and 1% antibiotic-antimycotic solution. After culturing for 24 h, cytosine -D-arabinofuranoside (5 µM) was added to the culture medium to inhibit the proliferation of non-neuronal cells. All experiments were carried using cerebellar granule neurons at 5 to 7 days in culture.

Patch-Clamp Recordings. Whole-cell currents of granule neurons were recorded using a conventional patch-clamp technique. Before I_A current recording, the culture medium was replaced with a bath solution containing 125 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1 mM MgCl₂, 0.001 mM tetrodotoxin, and 20 mM tetraethylammonium (pH adjusted to 7.4 using NaOH). Soft glass recording pipettes were filled with an internal solution containing 135 mM sodium gluconate, 10 mM KCl, 10 mM HEPES, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM EGTA (pH adjusted to 7.3 using KOH). The pipette resistance is 6 to 7 M after filling with the internal solution. Flufenamic acid solutions were prepared extemporaneously and gravity ejected for 10 to 20 s from MSC-200 manual solution changer (Bio-Logic-Science Instruments, Grenoble, France). All recordings were performed at room temperature.

Data Acquisition and Analysis. All currents were recorded using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) operated in voltage-clamp mode. Data acquisition and analysis were performed with pClamp 8.01 software (Molecular Devices) and/or Origin 6.1 (Microcal analysis software; OriginLab Corp., Northampton, MA). Statistical analysis was performed using the Student's t test with nonpaired comparison or paired comparisons where it is relevant. Values were given as means ± S.E.M. with n as the number of cells tested. P value < 0.05 was used to denote the statistical difference between groups. When multiple comparisons were made, data were analyzed by a one-way ANOVA test. siRNA Vector Construction. The oligonucleotides specifying the short-hairpin RNAs were designed via the website http://katahdin.cshl.org:9331/RNAi/html/rnai.html (Table 1). For preparation of duplexes, 2 µl of sense-stranded and antisense-stranded oligonucleotides (1 µg/µl each) were mixed together in 46 µl of DNA annealing buffer (30 mM Hepes, 100 mM potassium acetate, and 2 mM magnesium acetate, adjusted to pH 7.4), heat-denatured at 90°C for 3 min, and annealed at 37°C for 1 h. Following this, 1 µl of annealed DNA was ligated into 30 to 50 ng of BamHI/EcoRI restriction-digested, gel-purified siRNA vector (RNAi-Ready pSIREN-RetroQ; BD Biosciences Clontech, San Jose, CA) using phage T4 DNA ligase in a volume of 10 µl. Several colonies were sequenced to select the correct one.

Transfection. The correct siRNA vectors were extracted using a QIAGEN (Valencia, CA) plasmid midi kit. The DNA concentration and purity were determined by measuring the absorbance at 260 and 280 nm. Ten microliters of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) then was preincubated with 250 µl of Opti-MEM I reduced serum medium (Gibco) at room temperature for 5 min. Meanwhile, the siRNA and pEGFP-F (BD Biosciences Clontech, Franklin Lakes, NJ) vectors (total, 4 µg of plasmids) were diluted to 50 µl with Opti-MEM I reduced serum medium (Invitrogen) at a ratio of 5:1 (v/v). Following this, the Lipofectamine 2000 and plasmid dilution were mixed and incubated at room temperature for 15 min. The DNA and Lipofectamine 2000 complexes were added to the 4-day-old granule cells that had been cultured in 1 ml of growth medium without antibiotics. After incubation for 5 h, the transfection medium was replaced with normal growth medium containing antibiotics. At 48 h after transfection, the cells with green fluorescence were further analyzed.

Chemicals. All drugs used were purchased from Sigma-Aldrich (St. Louis, MO), with the exception of the fetal calf serum. Dulbecco's modified Eagle's medium culture medium and antibiotic-antimycotic solution were obtained from Invitrogen. Flufenamic acid, diclofenac, mefenamic acid, indomethacin, and arachidonic acid were first dissolved in dimethyl sulfoxide (DMSO) and then diluted in extracellular or intracellular solution, with a final DMSO concentration <0.1%, which alone did not produce any modulation of I_A.

	Results

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As rat cerebellar granule neurons display two main voltage-dependent outward K⁺ currents—the transient outward current (I_A) and the delayed rectifier current (I_K)—we first identified whether flufenamic acid is specific for I_A in the rat granule neurons. In the experiments carried out, the outward K⁺ currents were evoked by two sequential 200-ms depolarizing pulses to 40 mV at 1-s intervals, from holding potentials of –100 and –40 mV, respectively (Fig. 1A). When the membrane potential was held at –100 mV, depolarizing voltage pulses elicited a global outward current (I_A plus I_K) that activated rapidly (5–10 ms) and then decayed as time progressed. After 1-s intervals at a conditioning potential of –40 mV, the same depolarizing step only evoked a slight inactivating or noninactivating outward K⁺ current that had been previously described as a delayed rectifier I_K current. It is evident in Fig. 1a that flufenamic acid significantly (100 µM) inhibited the early inactivating I_A component that was elicited by depolarizing the pulse and then activating the later current that was evoked by the second depolarizing pulse. Flufenamic acid decreased the amplitude of I_A from 2107.5 ± 270 to 1859.6 ± 263.7 pA (n = 5, P < 0.05 using Student's t test), and by contrast, it increased the I_K amplitude from 791.5 ± 270 to 941.6 ± 263.7 pA (n = 5, P < 0.05 using the Student's t test). The effect of flufenamic acid on the amplitude of I_A and I_K was illustrated in Fig. 1B. In view of the fact that flufenamic acid was dissolved by 0.1% DMSO, we simultaneously examined the effect of DMSO on I_A and I_K. As shown in Fig. 1C, neither I_A nor I_K was modulated by 1% DMSO (n = 5, P > 0.05, using Student's t test). In some cases, washout of flufenamic acid from the bath solution took a long time; the I_A amplitude then recovered to a higher level than that of the control, suggesting that a lower concentration of flufenamic acid could augment I_A (Fig. 1D).

View larger version (22K): [in this window] [in a new window]   Fig. 1. Flufenamic acid (100 µM) inhibited IA current amplitudes but increased IK current amplitudes. A, the original current traces recorded in the absence and presence of 100 µM flufenamic acid (FFA). The current was evoked by two sequential 200-ms depolarizing pulses to 40 mV at 1-s intervals. The holding potentials were set to –100 mV (first pulse) for full activation of the IA, and at –40 mV (second pulse) for activation of the IK.B, IA and IK current recorded from the granule neuron; 1% DMSO was added to the bath solution. C, IA and IK current amplitude obtained from granule cells in the absence and presence of 100 µM flufenamic acid. Each value is the mean ± S.E.M. of five to six independent experiments. *, P < 0.05 compared with control group. D, a sample in which washout of 100 µM flufenamic acid from bath solution induced the augmentation of IA current amplitude. The current was elicited by a depolarizing pulse to 40 mV from the holding potentials of –100 mV.

Therefore, we tested whether flufenamic acid inhibited I_A in a concentration-dependent manner. To investigate merely the effect of flufenamic acid on I_A, all the results described thereafter were obtained using a bath solution containing 20 mM tetraethylammonium to eliminate the I_K currents. I_A currents were then evoked by 200-ms constant depolarizing pulses, ranging from –100 mV to 40 mV at 10-s intervals. We unexpectedly found that application of flufenamic acid to the bath solution produced bidirectional modulation of current amplitude depending on the concentration used (Fig. 2A). At concentrations from 20 µM to 1 mM, flufenamic acid significantly inhibited I_A amplitude. The inhibitory effect of flufenamic acid on I_A was fast and reversible following washing; application of flufenamic acid to the bath solution resulted in a clear, rapid decrease of current amplitude that reached its maximal effect within 30 to 60 s and returned to control levels after 1 to 2 min of washout. Moreover, the inhibition of I_A is concentration-dependent. The inhibition of I_A current by flufenamic acid at 50, 250, and 500 µM and 1 mM was 24 ± 5% (n = 8), 27.3 ± 3.8% (n = 5), 44.6 ± 5.8% (n = 5), and 69.3 ± 6.3% (n = 3), respectively (P < 0.05, using a one-way ANOVA test). Interestingly, when reducing the concentration to 10, 1, and 0.1 µM, flufenamic acid significantly and reversibly increased I_A current amplitude to 143 ± 8% (n = 9), 119.5 ± 3.7% (n = 5), and 106.7 ± 1.7% (n = 4) of control, respectively (P < 0.05, using a one-way ANOVA test). Moreover, flufenamic acid activated I_A current gradually and reached its maximal activation of I_A within 2 to 3 min of drug application. Figure 2A illustrates typical experiments in which flufenamic acid was applied at different concentrations. The sum of bidirectional modulation on I_A amplitude was demonstrated in Fig. 2B, which revealed that stimulatory or inhibitory effect of FA on I_A current was dose-dependent.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Flufenamic acid bimodulated IA current at different concentrations with dose-dependent. A, superimposed K+ current evoked by 200-ms depolarizing pulses from –100 to 40 mV. The current traces were obtained in the absence and presence of flufenamic acid (FFA) at a concentration of 0.1 µM to 1 mM, respectively. B, statistical analysis of the effect of various flufenamic acid concentrations on IA. The data represent the mean values ± S.E.M. obtained from five to seven cells. *, P < 0.05 compared with the control; #, P < 0.05 compared with the data obtained from the different concentration, using a one-way ANOVA test.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Flufenamic acid externally applied altered the steady-state activation property of IA.A, IA recordings using an activation voltage protocol in the absence (top) and presence (bottom) of flufenamic acid (FFA, 100 µM). The cells were held at –100 mV and depolarized in 10-mV steps from –80 to 20 mV at 10-s intervals. B, the voltage-dependent activation curve of IA obtained in the absence or presence of flufenamic acid. C, plot of the normalized conductance as a function of the command potential in the absence or presence of flufenamic acid. The data points were fitted with a Boltzmann function. A leftward shift of the voltage-dependent activation curve was observed in the presence of flufenamic acid. The data represent the mean values ± S.E.M. obtained from six cells.

We then studied the effect of flufenamic acid on voltage-dependent steady-state inactivation of I_A. Currents were elicited using 1-s conditioning prepulses from –110 mV to various membrane potentials before a 200-ms test pulse of +20 mV (Fig. 4, A and B). The data obtained from Fig. 4A showed that, when conditioning prepulse applying at –90 mV, the flufenamic acid-induced inhibitory effect on I_A was 26 ± 5.5%. By contrast, when conditioning prepulse was at –60 mV, the inhibitory effect of flufenamic acid reduced to 3 ± 3%. After normalizing each current peak to the maximal current amplitude obtained from the –110 mV prepulses as a function of the conditioning prepulse potential, we found that flufenamic acid modified the steady-state inactivation curve of I_A. In five cells studied, the half-maximal inactivation voltage was –58.5 ± 1.8 and –49.7 ± 1.1 mV in the absence or presence of flufenamic acid, respectively (n = 6, P < 0.05). Figure 4C shows the statistical results of an inactivation curve that has been shifted approximately 9 mV toward the depolarizing potential by the addition of flufenamic acid. These results suggest that flufenamic acid decreased the I_A current by modifying the steady-state I_A-channel activation and inactivation properties.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Flufenamic acid externally applied altered the steady-state inactivation property of IA.A, IA currents recorded in the absence (top) and presence of flufenamic acid (FFA, 100 µM, bottom). One-second conditioning prepulses from –110 to 0 mV in 10-mV increments were applied before the test pulse to 20 mV. The voltage protocol is shown below the current records. B, steady-state inactivation curves of IA plot in the absence or presence of flufenamic acid. The abscissa indicates conditioning prepulse potentials. C, the peak current amplitude normalized to the maximal current was plotted against the prepulse potential. Normalized current points were fitted with a Boltzmann function, showing that the application of flufenamic acid shifted the steady-state inactivation curve toward positive potential. The data represent the mean values ± S.E.M. obtained from six cells.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Silencing three main -subunits of IA channel expressed in rat cerebellar granule cells did not modulate the flufenamic acid-induced inhibitory effect. A, the main -subunits of IA channel detected in primary cultured rat cerebellar granule cells using RT-PCR. B, the IA current recorded after silencing the Kv4.2, Kv4.3, and Kv1.1 genes, respectively, by siRNA in the absence and presence of flufenamic acid (FFA). Three types of siRNA vector of Kv gene, Kv4.2, Kv4.3, and Kv1.1, respectively, were cotransfected with pEGFP-F to label the transfected cells. The K+ current was evoked by the 200-ms depolarizing pulse to 40 mV at 1-s intervals from holding potentials of –100 mV. C, the inhibitory effect of flufenamic acid on IA amplitude when silencing Kv4.2, Kv4.3, and Kv1.1 genes. Each value is the mean ± S.E.M. of 5 to 15 independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 6. The IA current amplitude could be bimodulated by intracellular and extracellular application of flufenamic acid. A, the time course of changed IA amplitudes induced by the internal application of flufenamic acid (FFA, 10 µM). The insets in the graphs show the superimposed IA traces taken from the initial control levels (after establishment of the whole-cell configuration), after internal infusion of flufenamic acid, respectively. The time points (a, b, and c) noted on the curves correspond to the superimposed IA current traces illustrated by insets. B, time course of the changed current amplitudes obtained from the cell with intracellular and external flufenamic acid. The insets in the graphs show the superimposed IA traces taken from the same cell. C, statistical analysis of the effect induced by internal flufenamic acid alone and without internal flufenamic acid. The data represent the mean values ± S.E.M. obtained from five to seven cells. *, P < 0.05 compared with the control group without internal flufenamic acid; #, P < 0.05 compared with the internal flufenamic acid.

As fenamates are the inhibitors of the cyclooxygenase, like all NSAIDs (Ouellet and Percival, 1995), we tested whether I_A activation was common to all NSAIDs by using three NSAIDs of different chemical groups (diclofenac, mefenamic acid, and indomethacin). All of the three NSAIDs can mimic the flufenamic acid-induced activatory effect on I_A. The internal application of mefenamic acid (Fig. 7A), diclofenac (Fig. 7B), and indomethacin (Fig. 7C) was performed by pipette solution, and the current amplitude increased after establishment of the whole-cell configuration and reached its maximum within 2 to 4 min. The percentage of current amplitude increased by intracellular methflunamic, indomethacin, and diclofenac was 145 ± 7% (n = 6), 130.2 ± 3.6% (n = 6), and 124.5 ± 1.3% (n = 6), respectively (P < 0.05, using Student's t test). Statistical analyses of the above data are shown in Fig. 7D.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Intracellular application of three NSAIDs of different chemical groups could mimic the internal flufenamic acid-induced effect on IA current. A to C, the time course of increased IA amplitudes induced by the internal application of 10 µM mefenamic acid (MA, A), 10 µM diclofenac (DIC, B), and 10 µM indomethacin (INDO, C). The insets in the graphs show the superimposed IA traces taken from the same cells. D, statistical analysis of the effect on IA current amplitudes induced by internal flufenamic acid, mefenamic acid, diclofenac, and indomethacin. The data represent the mean values ± S.E.M. obtained from five to seven cells.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Intracellular application of ETYA and arachidonic acid could mimic the internal flufenamic acid-induced effect on IA current. A and B, the time course of increased IA current amplitudes induced by the internal application of ETYA (10 µM, A) and AA (10 µM, B). C, time course of the changed current amplitudes obtained from the cell with intracellular AA (10 µM) and external flufenamic acid (10 µM, FFA). The insets in the graphs show the superimposed IA traces taken from the same cells. D, statistical analysis of the effect on IA current amplitudes induced by internal arachidonic acid, ETYA, and 1% DMSO, which is a solvent of ETYA and arachidonic acid. And the effect on IA current amplitudes induced by extracellular FFA while internal arachidonic acid was used. The data represent the mean values ± S.E.M. obtained from five to seven cells. *, P < 0.05 compared with the control; #, P < 0.05 compared with intracellular arachidonic acid alone.

	Discussion

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Our results first demonstrate that flufenamic acid bidirectionally modulated the I_A current of rat granule neurons at different concentrations and via different methods of application; external application at concentrations beyond 10 µM resulted in inhibiting I_A, whereas external application or intracellular application of 10 µM flufenamic acid augmented I_A. Stimulatory or inhibitory effect of flufenamic acid on I_A current was dose-dependent.

The mode by which fenamatic acids inhibited ion channels is uncertain. As diphenyl carboxylate and its derivates, including flufenamic acid, are highly lipophilic molecules; modifying membrane ion channels by them can result in alterations of the permeability of cell membranes (Ottolia and Toro, 1994) or a nonspecific lipid effect on the channels (Li et al., 1999; Bock et al., 2003). In our study, the rapid onset and reversible inhibition induced by flufenamic acid suggested that its effects are probably mediated by a direct action on the channel and are not caused by indirect effects, such as lowering and/or increasing intracellular factors. The direct blocking effect on ion channels was similar to that in previous studies on rat supraoptic neurons and expressed systems in which flufenamic acid blocked the Ca²⁺-dependent nonselective cation channels and human K_v2.1 subunits stably expressed in the Chinese hamster ovary cells (Lee and Wang, 1999; Partridge and Valenzuela, 2000). These results, together with our observations on I_A channels, suggest that flufenamic acid, like other members of the fenamate acid class, have a broad spectrum of nonspecific actions on ionchannel proteins.

Transient potassium currents (I_A) have been described in neurons from many regions of the central nervous system and also in cardiac and vascular cells (Hoffman and Johnston, 1998; Song et al., 1998). Comparisons with other voltage-gated K⁺ channels showed that I_A significantly operates at subthreshold membrane potentials and transiently inactivates during depolarizing pulses. In the rat cerebellar granule cells, fenamate acid significantly shifted both the steady-state activation and steady-state inactivation curves toward a more depolarized potential. Moreover, a flufenamic acid-induced inhibitory effect on I_A channels was seen at a hyperpolarizing potential at which few I_A channels were inactivated. Thus, these results indicated that flufenamic acid might have a much more higher affinity for activated I_A channels than for inactivated channels. Therefore, these results suggested that flufenamic acid not only decreased the current amplitude by blockading the I_A channels but also modified the channel properties in terms of activation or inactivation gating. The latter function might involve modulation of neuronal excitability.

Recent reports have suggested that members of the shal (K_v4) family form the major components of the I_A channels in the central nervous system (Serôdio and Rudy, 1998; An et al., 2000). Shibata et al. (2000) reported that I_A channels encoded by the K_v4 family were found in cerebellar neurons; moreover, our own results revealed that the majority of the I_A channels were not only conducted by the K_v4 family but also by K_v1.1 in rat granule cells. As the previous study by Wang et al. (1997) showed, by using a heterologous expression system, fenamates (niflumic and flufenamic acid) more strongly inhibited K_v4.3 than K_v4.2. To identify whether the inhibitory effect induced by flufenamic acid on I_A was associated with the unitary subunit of I_A-channel protein, we tested the effect of flufenamic acid on the remaining current by silencing K_v4.2, K_v4.3, and K_v1.1, respectively. However, our results indicated that silencing the expression of any of the above subunits of the I_A channel could not eliminate the effect of flufenamic acid on the I_A current. Because there is a high degree of structural similarity between the K_v4.3 and K_v4.2 channels and the K_v4.3 channel is 75% identical to the K_v4.2 channel at the amino acid level (Dixon al., 1996), these results suggested that the effects of flufenamic acid were probably not isoform-selective. The discrepancy between our observation and Wang et al. (1997) may result from the differences between the reconstituted K_v4.2 or K_v4.3 channel complex and the native I_A channel of granule neurons; the latter consisted of K_v4 family and K_v1.1 subunits simultaneously.

It was surprising to find in our studies that when a lower concentration of flufenamic acid was used, it induced the activator effect on I_A. This was the opposite of what happened at higher concentrations but similar to what happened when the drug was administered internally via patch pipettes. Moreover, intracellular application of flufenamic acid did not alter the inhibitory effect induced by extracellular application of flufenamic acid. Therefore, it is conceivable that flufenamic acid augmented I_A via another mechanism other than the channel block. One possibility could be the inhibition of cyclooxygenase, as it has been well established that fenamates are also inhibitors of cyclooxygenase, as well as other NSAIDs (Wu, 1998). Therefore, we tested whether I_A activation is common to all NSAIDs; three NSAIDs of different chemical groups (diclofenac, mefenamic acid, and indomethacin) were randomly chosen. All of the three NSAIDs could have mimicked the flufenamic acid-induced stimulatory effect on I_A when they were applied internally by pipette solution. One possibility could be that the enzyme cyclooxygenase was inhibited, as it has been well established that fenamates are also inhibitors of the enzyme cyclooxygenase (Ouellet and Percival, 1995). Thus, it is conceivable that a lower concentration or intracellular flufenamic acid-induced augmentation of I_A was due to the modulatory effect of cyclooxygenase inhibition, whereas external application at higher concentrations of flufenamic acid decreased I_A by the channel-blocking mechanism.

Inhibitors of the cyclooxygenase pathway in the arachidonic acid (AA) cascade can cause the buildup of AA and/or reduce its metabolic products (for a review, see Bazan, 2003). By intercellular application of AA, we determined whether the fenamate acid-induced activation effect on I_A was associated with accumulation of AA or its metabolic products. The observation that internal application of AA mimicked the effect of fenamate acid on I_A is consistent with our hypothesis. To avoid the effect of DMSO on I_A, which is a solvent of AA, we also tested the effect of AA dissolved in 0.1% ethanol and obtained the same results (data not shown). Thus, it indicated that AA could act as a mediator of I_A channels, as reported first in smooth muscle (Ordway et al., 1989). In addition, ETYA, which blocks the formation of active AA metabolites, mimicked the effect of AA activation on I_A, indicating that AA activation was direct and not mediated by its oxygenated metabolites, as reported by Danthi et al. (2003) in bovine adrenal zona fasciculata cells. However, whether AA has a direct mediatory effect on I_A channels or indirectly activates the downstream signal in granule neurons needs to be further explored.

Arachidonic acid and its metabolic products have been shown to modulate a large number of ligand- and voltagegated ion channels in a variety of systems (Ordway et al., 1991). The modulator effects of AA and its metabolites either activate or inactivate ion channels depending on the cell types studied (Patel and Honore, 2001; Takahira et al., 2001; Wei et al., 2004). However, it is noticeable that, in rat granule neurons, our study and Holmqvist et al. (2001) have revealed that extracellular application of AA remarkably inhibited I_A amplitude and modulated its kinetics (Wang et al., 2005). Moreover, molecular cloning showed that the AA-induced inhibition effect on I_A occurs by acting directly on the K_v4-K⁺ channel interacting protein complex (Holmqvist et al., 2001). Here, our results have revealed that, although AA was applied intracellularly, it evoked an opposite stimulatory effect on I_A. To date, few investigations have used AA intracellularly, although the study in excised membrane patches (Kim et al., 1995) has shown that AA applied to the cytoplasmic or extracellular side of the membrane caused opening of three types of channels. We reason that the conflicting function of AA may result from its difference in binding location in cytoplasmic or extracellular sides of the membrane. Further work will be necessary to examine this possibility.

The overall properties of I_A make it an excellent target for any modulatory mechanism influencing cell excitability and action-potential firing (Shibata et al., 2000; Kiss et al., 2002). Although the reliable mechanism of fenamate acid-induced biodirected modulation of I_A remains unknown, our results may partially account for the unwanted side effects of fenamate acid and might be a valuable new avenue to investigate in terms of therapeutic and/or basic research applications of NSAIDs.

	Acknowledgements

 
We thank Drs. H. C. Chen and H Zhu (Chinese University of Hong Kong) for technical help on setting up RNA interference method.

	Footnotes

 
This work is supported by The Ministry of Education Foundation of China (20060246018). Z.-G.Z. and M.Z. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.117556.

ABBREVIATIONS: NSAIDs, nonsteroidal anti-inflammatory drugs; ANOVA, analysis of variance; siRNA, small interfering RNA; eGFP, enhanced green fluorescent protein; DMSO, dimethyl sulfoxide; RT-PCR, reverse transcription-polymerase chain reaction; EYTA, eicosatetraynoic acid; AA, arachidonic acid.

Address correspondence to: Dr. Mei Yan-Ai, School of Life Sciences, Institute of Brain Science, Fudan University, Shanghai 200433, P.R. China. E-mail: yamei{at}fudan.edu.cn

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