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Flufenamic Acid Affects Multiple Currents and Causes Intracellular Ca²⁺ Release in Aplysia Bag Cell Neurons

Department of Physiology and Centre for Neuroscience Studies, Queen's University, Kingston, Ontario, Canada

Submitted 12 February 2008; accepted in final form 17 April 2008

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Flufenamic acid (FFA) is a nonsteroidal antiinflammatory agent, commonly used to block nonselective cation channels. We previously reported that FFA potentiated, rather than inhibited, a cation current in Aplysia bag cell neurons. Prompted by this paradoxical result, the present study examined the effects of FFA on membrane currents and intracellular Ca²⁺ in cultured bag cell neurons. Under whole cell voltage clamp, FFA evoked either outward (I_out) or inward (I_in) currents. I_out had a rapid onset, was inhibited by the K⁺ channel blocker, tetraethylammonium, and was associated with both an increase in membrane conductance and a negative shift in the whole cell current reversal potential. I_in developed more slowly, was inhibited by the cation channel blocker, Gd³⁺, and was concomitant with both an increased conductance and positive shift in reversal potential. FFA also enhanced the use-dependent inactivation and caused a positive-shift in the activation curve of the voltage-dependent Ca²⁺ current. Furthermore, as measured by ratiometric imaging, FFA produced a rise in intracellular Ca²⁺ that persisted in the absence of extracellular Ca²⁺ and was reduced by depleting either the endoplasmic reticulum and/or mitochondrial stores. Ca²⁺ appeared to be involved in the activation of I_in, as strong intracellular Ca²⁺ buffering effectively eliminated I_in but did not alter I_out. Finally, the effects of FFA were likely not due to block of cyclooxygenase given that the general cyclooxygenase inhibitor, indomethacin, failed to evoke either current. That FFA influences a number of neuronal properties needs to be taken into consideration when employing it as a cation channel antagonist.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Flufenamic acid (FFA) was initially identified as an antiinflammatory drug by Winder et al. (1963); subsequently, Pong and Levine (1976) found it to be an inhibitor of cyclooxygenase (Cox). Beyond its effects on prostaglandin synthesis, this drug has been used more recently as a cation channel antagonist. Gogelein and Pfannmuller (1989) were the to first demonstrate that FFA inhibited nonselective cation channels, specifically in rat pancreas. Subsequently this agent has been employed as a cation channel blocker in nonneuronal preparations (Albert et al. 2006; Gogelein et al. 1990; YM Lee et al. 2003) as well as in neurons from both vertebrates and invertebrates (Bengtson et al. 2004; Cho et al. 2003; Derjean et al. 2005; Egorov et al. 2002; Ghamari-Langroudi and Bourque 2002; Green and Cottrell 1997; Haj-Dahmane and Andrade 1997; Morisset and Nagy 1999; Partridge and Valenzuela 2000; Shaw et al. 1995; Yamashita and Isa 2003).

We previously reported that FFA elicits a large outward current and potentiates a Ca²⁺-activated cation current in the bag cell neurons of the marine mollusk, Aplysia californica (Hung and Magoski 2007). These neuroendocrine cells are found in two clusters at the rostral end of the abdominal ganglion, and they initiate egg-laying behavior through a long-lasting afterdischarge and neuropeptide release (Arch 1972; Dudek et al. 1979; Kupfermann 1967; Kupfermann and Kandel 1970; Loechner et al. 1990; Pinsker and Dudek 1977; Stuart et al. 1980). At least two species of nonselective, Ca²⁺-sensitive cation channel are triggered at the onset of the afterdischarge to provide depolarizing drive for the burst. One is a voltage-dependent cation channel that is directly activated by both Ca²⁺, through closely associated calmodulin, and phosphorylation, from closely associated protein kinase C (Lupinsky and Magoski 2006; Magoski 2004; Magoski and Kaczmarek 2005; Magoski et al. 2002; Wilson et al. 1996, 1998). The other is a voltage-independent cation channel that appears to be activated by calmodulin kinase-dependent phosphorylation (Hung and Magoski 2007). This second channel is responsible for a prolonged depolarization that can be evoked by a brief train of action potentials in cultured bag cell neurons (Hung and Magoski 2007; Whim and Kaczmarek 1998).

We have found that FFA does not block either of these well-characterized cation channels in bag cell neurons (Hung and Magoski 2007; D. A. Lupinsky and N. S. Magoski, unpublished observation). Rather it activates an outward current and actually enhances the voltage-independent cation current (Hung and Magoski 2007). In the present study, we confirm that the outward current is due to opening of a K⁺ conductance. We also now show that FFA is capable of eliciting an inward current that, curiously, appears to be the result of opening a nonselective cation conductance. FFA is widely used as both a Cox inhibitor and a cation channel antagonist. Given that this drug has the potential to exert broad-spectrum effects on neuronal function, its use needs to be judicious. Alternatively, FFA may prove of value yet again as a means to activate specific conductances or cause the release of intracellular Ca²⁺ when warranted.

	METHODS

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Animals and cell culture

Adult Aplysia californica weighing 150–300 g were obtained from Marinus (Long Beach, CA) and housed in an 300-l aquarium containing continuously circulating, aerated sea water (Instant Ocean; Aquarium Systems, Mentor, OH or Kent sea salt; Kent Marine, Acworth, GA) at 15°C on a 12/12 h light/dark cycle and fed Romaine lettuce five times a week.

For primary cultures of isolated bag cell neurons, animals were anesthetized by an injection of isotonic MgCl₂ (50% of body weight), and the abdominal ganglion was removed and treated with neutral protease (13.33 mg/ml; 165859; Roche Diagnostics, Indianapolis, IN) for 18 h at 20–22°C dissolved in tissue culture artificial sea water (tcASW; composition in mM: 460 NaCl, 10.4 KCl, 11 CaCl₂, 55 MgCl₂, 15 HEPES, 1 mg/ml glucose, 100 U/ml penicillin, and 0.1 mg/ml streptomycin, pH 7.8 with NaOH). The ganglion was then transferred to fresh tcASW for 1 h, after which the bag cell neuron clusters were dissected from their surrounding connective tissue. Using a fire-polished Pasteur pipette and gentle trituration, neurons were dispersed in tcASW onto 35 x 10-mm polystyrene tissue culture dishes (430165; Corning, Corning, NY). Cultures were maintained in tcASW in a 14°C incubator and used for experimentation within 1–3 days. Salts were obtained from Fisher Scientific (Ottawa, ON, Canada), ICN (Aurora, OH), or Sigma-Aldrich (St. Louis, MO).

Whole cell, voltage-clamp recordings

Voltage-clamp recordings were made using an EPC-8 amplifier (HEKA Electronics; Mahone Bay, NS, Canada) and the tight-seal, whole cell method. Microelectrodes were pulled from 1.5 mm ID, borosilicate glass capillaries (TW150F-4; World Precision Instruments, Sarasota, FL) and had a resistance of 1–2 M when filled with various intracellular salines. Pipette junction potentials were nulled immediately before seal formation. After seal formation, the pipette capacitive current was cancelled and, following break through, the whole cell capacitive current was also cancelled, while the series resistance (3–5 M) was compensated to 80% and monitored throughout the experiment. Current was filtered at 1 kHz with the EPC-8 Bessel filter and sampled at 2 kHz using an IBM-compatible personal computer, a Digidata 1322A A/D converter (Axon Instruments/Molecular Devices, Sunnyvale, CA), and the Clampex acquisition program of pCLAMP (version 8.0; Axon Instruments/Molecular Devices). Data were gathered at room temperature (20–22°C).

Most recordings were made in normal ASW (nASW; composition as per tcASW but lacking the glucose and antibiotics) with regular intracellular saline in the pipette [composition in mM: 500 K-aspartate, 70 KCl, 1.25 MgCl₂, 10 HEPES, 11 glucose, 10 glutathione, 5 ethylene glycol-bis-(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 ATP (grade 2, disodium salt; A3377; Sigma-Aldrich), and 0.1 GTP (type 3, disodium salt; G8877; Sigma-Aldrich) pH 7.3 with KOH]. The free Ca²⁺ concentration of this saline was set at 300 nM by adding an appropriate amount of CaCl₂, as calculated by WebMaxC (http://www.stanford.edu/cpatton/webmaxcS.htm). For experiments where intracellular Ca²⁺ was strongly buffered, the regular intracellular saline contained 20 mM EGTA and no added Ca²⁺. A junction potential of 15 mV was calculated for these intracellular salines versus nASW and compensated for by subtraction off-line.

Ca²⁺ currents were isolated using an ASW where Na⁺ was replaced with tetraethylammonium (TEA) and K⁺ with Cs⁺ (composition in mM: 460 TEA-Cl, 10.4 CsCl, 55 MgCl₂, 11 CaCl₂, 15 HEPES, pH 7.8 with CsOH). The protocol also employed an intracellular saline where the K⁺ was replaced with Cs⁺ (composition in mM): 70 CsCl, 10 HEPES, 11 glucose, 10 glutathione, 5 EGTA, 500 aspartic acid, 5 ATP, and 0.1 GTP, pH 7.3 with CsOH. In some instances, on-line leak subtraction was performed using a P/4 protocol from –60 mV with subpulses of opposite polarity and one-fourth the magnitude, an inter-subpulse interval of 500 ms, and 100 ms before actual test pulses. In other cases, 10 mM Ni²⁺ (NiCl₂; N6136; Sigma-Aldrich) was used to completely block the Ca²⁺ current (Hung and Magoski 2007), and this remaining, Ni²⁺-insensitive current was subtracted from the prior current to remove leak. A junction potential of 20 mV was compensated for by subtraction off-line.

Intracellular Ca²⁺ measurements

Somatic intracellular Ca²⁺ was measured by ratiometric imaging of the dye, fura PE3 (K⁺ salt; 0110; Teflabs, Austin, TX) (Vorndran et al. 1995). Fura-PE3 was pressure injected via sharp electrodes using a PMI-100 pressure microinjector (Dagan, Minneapolis, MN), while simultaneously monitoring membrane potential with an Axoclamp 2B amplifier (Axon Instruments/Molecular Devices). Microelectrodes were pulled from 1.2 mm ID, borosilicate glass capillaries (1B120F-4; World Precision Instruments) and had a resistance of 30–50 M when the tip was filled with 10 mM fura-PE3 then backfilled with 3 M KCl. Injections usually required 10–15 300- to 900-ms pulses at 30–60 kPa to fill the neurons with an optimal amount of dye (estimated at 50–100 µM). All neurons used for imaging showed resting potentials of –50 to –60 mV and displayed action potentials that overshot 0 mV following depolarizing current injection (0.5–1 nA, directly from the amplifier). After dye injection, neurons were allowed to equilibrate for 30 min.

Ca²⁺ imaging was performed using a Nikon TS100-F inverted microscope (Nikon, Mississauga, ON, Canada) equipped with a Nikon Plan Fluor x10 objective (NA = 0.3). The light source was a 75 W Xenon arc lamp and a multi-wavelength DeltaRAM V monochromatic illuminator (Photon Technology International, London, ON, Canada) coupled to the microscope with a UV-grade liquid-light guide. Between acquisition episodes, the excitation illumination was blocked by a shutter, which along with the excitation wavelength was controlled by a IBM-compatible personal computer, a Photon Technology International computer interface, and ImageMaster Pro software (version 1.49; Photon Technology International). The emitted light passed through a 510/40-nm barrier filter prior to being detected by a Photon Technology International IC200 intensified charge coupled device camera. The camera intensifier voltage was set based on the initial fluorescence intensity of the cells at the beginning of each experiment and maintained constant thereafter. The camera black level was set prior to an experiment using the camera controller such that at a gain of 1 there was a 50:50 distribution of blue and black pixels on the image display with no light going to the camera. The ratioed image of the fluorescence intensities (converted to pixel values) from 340 and 380 nm excitation wavelengths was derived and averaged four to eight frames per acquisition, resulting in a single full-frame (520 x 480 pixels) acquisition time of 0.5–4 s. A sample of the fluorescence intensities ratio was taken typically at 1-min intervals using regions of interest (ROIs) defined over the neuronal somata prior to the start of the experiment. The ratio was recorded as 340/380 to reflect free intracellular Ca²⁺. The black level determination, image acquisition, frame averaging, emitted light ROI sampling, and ratio calculations were carried out using the ImageMaster Pro software. Ratio calculations were saved for subsequent analysis (see following text). Imaging was carried out at room temperature (20–22°C) and performed in both nASW and Ca²⁺-free ASW (cfASW; composition as per nASW but with CaCl₂ omitted and 0.5 mM EGTA added).

Drug application and reagents

The culture dish served as the bath with salines and/or drugs being applied using a gravity-driven perfusion system of 1 ml/min. In some cases, drugs were introduced directly into the bath by pipetting a small volume (<10 µl) of concentrated stock solution or a larger volume of saline (–500 µl) that was initially removed from the bath, mixed with the stock solution, and then reintroduced. Care was taken to perform all pipetting near the side of the dish and as far away as possible from the neuron(s). GdCl₃ (G-7532; Sigma-Aldrich) and tetraethylammonium-Cl (TEA; AC150905000; Fisher) were dissolved directly into nASW. N-(3-[trifluoromethyl]phenyl)anthranilic flufenamic acid (FFA; F9005; Sigma-Aldrich) was dissolved in ethanol, while dimethyl sulfoxide (DMSO; BP231-1; Fisher) was used to dissolve bafilomycin A (B1793; Sigma-Aldrich), cyclopiazonic acid (CPA) (C1530; Sigma-Aldrich or 239805; Calbiochem; San Diego, CA), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; C2920; Sigma-Aldrich), indomethacin (I7378; Sigma-Aldrich), and paxilline (P-2928; Sigma-Adrich). The maximal final concentration of DMSO or ethanol was 0.01 µM or 0.01%, respectively, which in control experiments had no effect on intracellular Ca²⁺ or membrane properties.

Analysis

The Clampfit analysis program of pCLAMP (Axon Instruments/Molecular Devices) was used to determine the amplitude and time course of currents evoked by FFA. Cursors were placed at the baseline current, prior to FFA delivery, as well as at the peak after the drug. The difference between the two cursor values was taken as the peak amplitude. Conductance was derived using Ohm's law (G = I/V) from the current during a 200-ms step from –60 to –70 mV. The percentage change was calculated from the conductance before and after FFA delivery. The current-voltage relationship of the Ca²⁺ current was determined by measuring peak current between cursors set at the start and end of the traces in Clampfit. Current was normalized to cell size by dividing by the whole cell capacitance (as determined by the EPC-8 slow capacitance compensation circuitry) and plotted against voltage using Origin (version 7.0; OriginLab, Northampton, MA). Activation curves were made by dividing the Ca²⁺ current amplitude at all voltages by that at +10 mV (the peak current voltage). These curves were fit with Boltzmann functions using Origin to derive the half-maximal voltage of activation (V_1/2; the voltage required to recruit half of the maximum current), and the slope factor (k; the amount of voltage required to shift the V_1/2 e-fold). For intracellular Ca²⁺, Origin was used to import and plot ImageMaster Pro files as line graphs. Values were derived from changes determined by eye or with adjacent-averaging from regions that had reached steady-state for 3–5 min.

Data are presented as the means ± SE as calculated using either Origin or Instat (version 3.05; GraphPad Software; San Diego, CA). Statistical analysis was performed using Instat. The Kolmogorov-Smirnov method was used to test data sets for normality. A one-sample t-test was used to determine if the mean of a single group was different from zero. Paired and unpaired Student's t-test (standard or Welch corrected) or the Mann-Whitney test was used to test whether the mean differed between two groups. Comparisons between three or more means used a standard one-way ANOVA with Dunnett's multiple comparisons post hoc test. All tests were two-tailed. Data were considered significantly different if the P value was <0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
FFA activates a large outward current in Aplysia bag cell neurons

Initially we set out to confirm and characterize the outward current activated by FFA as previously reported by Hung and Magoski (2007). Cultured bag cell neurons were whole cell voltage-clamped using regular intracellular saline in the pipette (K⁺-aspartate based with 300 nM free Ca²⁺) and nASW in the bath. At a holding potential of –60 mV, application of 300 µM FFA elicited a prominent outward current (I_out) that was, on average, 1.75 nA with a time to peak of <1 min (n = 8; Fig. 1, A and B). Given that many outward currents pass K⁺, the effects of a common K⁺ channel blocker, TEA (Hagiwara and Saito 1959), was examined on I_out. After allowing I_out to fully develop, 50 mM TEA was perfused along with the FFA. This consistently resulted in the current completely returning to baseline - typically near zero (n = 6; Fig. 1C). When Hung and Magoski (2007) first described the effects of FFA on bag cell neurons, they showed that concentrations between 100 and 200 µM also activated I_out, although the current was larger and more reliably evoked with 300 µM. Moreover, concentrations >300 µM had a negative impact on neuronal viability, perhaps due to the effects of FFA on intracellular Ca²⁺ (see following text). As such, we have used a concentration of 300 µM throughout the present study. When used as a cation channel blocker, for both vertebrate and invertebrate cells, FFA is typically employed at 100–500 µM (Derjean et al. 2005; Ghamari-Langroudi and Bourque 2002; Green and Cottrell 1997; Morisset and Nagy 1999; Partridge and Valenzuela 2000; Shaw et al.1995).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Flufenamic acid (FFA) activates an outward current that is sensitive to TEA. A: perfusion of 300 µM FFA onto a cultured bag cell neuron, voltage-clamped at –60 mV, elicits a prominent outward current. B, left: summary amplitude data showing that the average peak outward current (Iout) is 1.5 nA. Right: summary time course data indicating that Iout develops relatively quickly and reaches peak amplitude within 1 min. C: after allowing Iout to fully activate in the presence of 300 µM FFA, simultaneous perfusion of 50 mM TEA results in a return to baseline for the current (representative of n = 6).

View larger version (13K): [in this window] [in a new window]   FIG. 2. The outward current activated by FFA is consistent with opening of a K+ channel. A: the whole cell conductance rises markedly following addition of 300 µM FFA. Sample traces, taken during control and at the peak of Iout, show that FFA increases the current flowing during a test pulse to –70 mV from a holding potential of –60 mV. Although after FFA there is a large increase in holding current, the traces have been aligned for comparison. B: in FFA, the whole cell current shifts to a more negative reversal potential and is dominated by an outward component. During a 10-s voltage ramp from –120 to 0 mV (see inset), again taken at control vs. peak Iout, the FFA current is prominently outward, roughly linear over much of the voltage range, and reverses near –90 mV. C, left: summary graph of the change in conductance shows a nearly 40-fold elevation with FFA, suggesting that channels open with the drug. Right: summary graph of the significant negative shift in reversal potential, toward EK, of the current evoked by the –120 to 0 mV ramp (paired Student's t-test).

In addition to I_out, approximately half of the neurons displayed a much smaller inward current (I_in) on exposure to 300 µM FFA during recording conditions identical to that described in the preceding text (n = 13; Fig. 3A). The two currents were not observed in the same individual neuron although a given group of neurons from a single animal could yield cells that responded to FFA with I_out or I_in. The mean amplitude of I_in was close to 300 pA and, in comparison to I_out, showed a slower time to peak at just under 4 min (Fig. 3B). Recognizing that I_in is small compared with I_out, we sought to ascertain if I_out was obscuring I_in. Using a set of neurons that consistently displayed an I_out under control conditions, TEA was applied before delivery of FFA. However, the application of 300 µM FFA in the presence of 50 mM TEA did not reveal a inward component (n = 5; data not shown).

View larger version (12K): [in this window] [in a new window]   FIG. 3. FFA activates an inward current that is sensitive to Gd3+. A: perfusion of 300 µM FFA onto a bag cell neuron, voltage-clamped at –60 mV, elicits a modest inward current. B, left: summary amplitude data showing that the average peak inward current (Iin) is nearly 300 pA. Right: summary time course data indicating that Iin develops more slowly as compared with Iout and reaches peak amplitude within 4 min. C: in the presence of 300 µM FFA, fully activated Iin is completely blocked by simultaneous perfusion of 100 µM Gd3+ (representative of n = 5).

View larger version (13K): [in this window] [in a new window]   FIG. 4. The inward current activated by FFA is consistent with opening of a nonselective cation channel. A: there is an increase in whole cell conductance after perfusion of 300 µM FFA. Sample traces, taken during control and at the peak of Iin, show that FFA increases the current flowing during a test pulse to –70 mV from a holding potential of - 60 mV. Again, the traces have been aligned for comparison. B: in FFA, the whole cell current shifts to a more positive reversal potential and displays a very prominent inward component. During a 10-s voltage ramp from –120 to 0 mV (see inset), again taken at control vs. peak Iout, the FFA current is largely inward, essentially linear over the majority of the voltage range, and reverses near –30 mV. C, left: summary graph of the change in conductance shows greater than a sixfold increase with FFA, suggesting that channels open with the drug. Right: summary graph of the significant positive shift in reversal potential, toward 0 mV, of the current evoked by the –120 to 0 mV ramp (paired Student's t-test).

The prior observation that FFA potentiated a Ca²⁺-activated cation current in bag cell neurons (Hung and Magoski 2007) led us to consider that FFA could be exerting an affect on voltage-gated Ca²⁺ influx. This is to say, the cation current could have been enhanced indirectly by upregulating Ca²⁺ channels. Under conditions where cultured bag cell neuron voltage-gated Ca²⁺ current was isolated (see METHODS) (DeRiemer et al. 1985; Hung and Magoski 2007), we observed a strongly voltage-dependent Ca²⁺ current that activated between –30 and –20 mV, peaked near +10 mV, and showed moderate inactivation over 200-ms test pulses (Fig. 5A). Delivery of 100 µM FFA did not alter Ca²⁺ current amplitude or activation characteristics (n = 4; data not shown). However, compared with ethanol controls (n = 3), addition of 300 µM FFA (n = 4) markedly decreased the Ca²⁺ current during a 5-Hz train of 100-ms voltage steps from –60 to +10 mV (Fig. 5B). Thus over the course of the voltage train, FFA appeared to block voltage-gated Ca²⁺ channels in a use-dependent manner. This voltage train was the same as that used by Hung and Magoski (2007) to evoke the Ca²⁺-activated cation current first observed to be potentiated by FFA. Parenthetically, the Ca²⁺ current activation curve, following the FFA-induced use-dependent block, displayed a rightward shift in half activation (V_1/2; from –8.5 to –6.3 mV) with little change in sensitivity (k; from 4.5 to 5.1; Fig. 5C).

View larger version (13K): [in this window] [in a new window]   FIG. 5. The voltage-gated Ca2+ current is altered by FFA. A, left: typical whole cell, voltage-gated Ca2+ currents evoked by 200-ms steps from a holding potential of –60 up to +60 mV in 10-mV increments. Right: current-voltage relationship for 7 neurons plotting peak current vs. test potential shows that the maximum Ca2+ current occurs at +10 mV. B: use-dependent inactivation of the Ca2+ current is enhanced with perfusion of 300 µM FFA. Left: summary graph of peak current evoked during a 5-Hz, 10-s train of 100-ms test pulses to +10 mV from a holding potential of –60 mV. Compared with control, the Ca2+ current in FFA undergoes more rapid use-dependent inactivation, particularly early in the train. This is best illustrated by the current flowing during pulses 2–4, which in FFA show prominent, successive reductions. In both cases, data are normalized to the current during the first pulse. Right: sample current traces evoked by the first and last test pulse in control and the presence of FFA. The amount of reduction in the peak of the first compared with the last current is greater with FFA. C: activation curves for Ca2+ current in control and the presence of FFA. Current is normalized by dividing peak current at each test voltage by the peak current at +10 mV. Voltage protocol as per A. A Boltzmann function fit of the points shows very similar voltage-dependent activation properties, with FFA producing a small, positive shift in the half-activation (V1/2) without an appreciable change in sensitivity (k).

An inhibition of voltage-gated Ca²⁺ current does not explain how FFA is able to both evoke I_out and I_in, as well potentiate the Ca²⁺-activated cation current reported by Hung and Magoski (2007). Alternatively, a mechanism may be found in prior work showing that FFA can cause release of intracellular Ca²⁺ in Helix and hippocampal neurons as well as a mandibular cell line (Lee et al. 1996; Partridge and Valenzuela 2000; Poronnik et al. 1992; Shaw et al. 1995). We used ratiometric imaging of fura PE3-loaded cultured bag cell neurons to examine if 300 µM FFA altered intracellular Ca²⁺. In Ca²⁺- containing nASW, FFA produced a clear elevation of intracellular Ca²⁺ that reached a stable peak in <10 min (n = 11; Fig. 6A). To determine if the Ca²⁺ increase was due to release of intracellular Ca²⁺ or influx of extracellular Ca²⁺ (possibly through I_in or voltage-gated Ca²⁺ current activated by depolarization), FFA was introduced in cfASW. Under those conditions, the FFA-induced elevation of intracellular Ca²⁺ was unaltered although in some instances, it was slower to reach peak amplitude (n = 11; Fig. 6B).

View larger version (16K): [in this window] [in a new window]   FIG. 6. FFA increases intracellular Ca2+. A: in Ca2+-containing normal artificial seawater (nASW), bath application 300 µM FFA causes a relatively rapid rise in intracellular Ca2+ as monitored by ratiometric imaging of fura PE3. Inset: summary time course data shows the FFA-induced Ca2+ increase reaches peak amplitude in 8 min. B: when extracellular Ca2+ is removed and FFA applied while bathing the neurons in Ca2+-free ASW (cfASW), the rise in intracellular Ca2+ persists. Although more variable, the time course is similar to that seen in nASW. C: pretreatment with 20 µM of the endoplasmic reticulum Ca2+-ATPase blocker, cyclopiazonic acid (CPA), attenuates the FFA-induced Ca2+ increase in cfASW. D: similarly, prior application of 20 µM of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an agent that collapses the mitochondrial membrane potential, reduces the FFA-induced Ca2+ increase in cfASW. E: if both the ER and the mitochondria are depleted by simultaneous application of CPA and FCCP, the ability of FFA to elevate Ca2+ in cfASW is largely abolished. Note that while the absolute values on the ordinate for B–E are not necessarily the same, the range is identical in all cases. F: summary graph of the FFA-induced Ca2+ increase in cfASW alone as well as cfASW with CPA, FCCP, CPA plus FCCP, or bafilomycin A (Baf A; 100 nM). The latter is a vesicular H+-ATPase inhibitor that depletes Ca2+ from acidic stores. The response to FFA is significantly reduced by pretreatment with CPA, FCCP, or CPA plus FCCP but not Baf A (standard ANOVA followed by Dunnett's multiple comparisons test of cfASW vs. cfASW with CPA, FCCP, CPA plus FCCP, or Baf A).

FFA-evoked inward current, but not the outward current, depends on intracellular Ca²⁺

The release of intracellular Ca²⁺ by FFA raises the possibility that I_out and/or I_in may be gated by cytosolic Ca²⁺. For I_out, we tested this by recording the FFA-induced current while dialyzing cultured bag cell neurons with either regular intracellular saline (5 mM EGTA) or a high EGTA (20 mM) intracellular saline in the whole cell pipette. The prediction being that if I_out was Ca²⁺ sensitive, the high EGTA would buffer the Ca²⁺ released by FFA and prevent activation. However, there was no difference between the magnitude of I_out recorded with the two internal salines (n = 5 and 5; Fig. 7, A, B, and D). Moreover, delivery of 10 µM paxilline, a Ca²⁺-activated K⁺ channel blocker (Knaus et al. 1994) known to be effective in bag cell neurons (Zhang et al. 2002) did not alter I_out (n = 5; Fig. 7, C and D). With respect to I_in, when FFA was used to evoked the current in a different group of neurons, it proved sensitive to high EGTA intracellular saline. While control neurons dialyzed with regular intracellular saline all displayed an I_in (n = 9; Fig. 7E), the cells recorded using high EGTA in the pipette failed to display any current change (n = 7; Fig. 7F; see inset for quantification).

View larger version (14K): [in this window] [in a new window]   FIG. 7. Iout appears Ca2+-independent, whereas Iin appears Ca2+-dependent. A: while voltage-clamping at –60 mV and dialyzing with regular intracellular saline, perfusion of 300 µM FFA elicits a prominent Iout. B: in a parallel experiment, dialysis with intracellular saline containing high (20 mM) EGTA does not occlude Iout. C: following activation of Iout by FFA (regular intracellular saline dialysis), introduction of 10 µM paxilline does not alter the steady-state current. D: summary graph showing no significant difference between Iout in neurons dialyzed with regular intracellular saline and those dialyzed with high EGTA intracellular saline (unpaired Student's t-test, Welch corrected). Similarly, application of 10 µM paxilline, a Ca2+-activated K+ channel blocker, does not significantly decrease FFA-induced Iout (paired Student's t-test). E: in a different group of neurons from those used in A–D, FFA elicits a typical Iin when the cell is dialyzed with regular intracellular saline (representative of n = 9). F: attempting to evoke Iin during a parallel experiment, involving dialysis with high EGTA intracellular saline, fails to elicit any change in current (representative of n = 7). Inset: summary graph shows a significant difference between control and high EGTA intracellular conditions (unpaired Student's t-test, Welch corrected).

A final option for a mechanism that could generate I_out and/or I_in is the inhibitory effect of FFA on Cox (Pong and Levine 1976). Potentially, a decrease in the resting prostaglandin level could remove some steady-state inhibition and open I_out and/or I_in. The inhibitory action of FFA does not distinguish between Cox-1 and -2 isoforms (Ouellet and Percival 1995). As such, we employed indomethacin, a general Cox antagonist (Laneuville et al. 1994) that is known to inhibit prostaglandin synthesis in Aplysia nervous tissue (Piomelli et al. 1987a). When 10 µM indomethacin was applied to cultured bag cell neurons voltage-clamped at –60 mV in nASW, it produced no change in the holding current (n = 7; Fig. 8A). However, following washout of the indomethacin, delivery of 300 µM FFA to those same neurons elicited either I_out or I_in (n = 4 and 3; Fig. 8B).

View larger version (8K): [in this window] [in a new window]   FIG. 8. Inhibition of Cox does not alter steady-state membrane current. A: perfusion of 10 µM indomethacin, a general Cox inhibitor, results in no change to the holding current under voltage-clamp at –60 mV in nASW (representative of n = 7). B: in the same neuron, subsequent application of 300 µM FFA results in a robust Iout.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

The marked increase in steady-state membrane conductance associated with the FFA-induced I_out in cultured bag cell neurons points to channel opening. The candidate ions that could cause I_out are K⁺ or Cl^–. The Nernst potential for Cl^– in our recording conditions is approximately –55 mV. As such, Cl^– channel opening at a holding potential of –60 mV would result in Cl^– efflux, a small inward current, and a slight, positive shift in the reversal potential of the whole cell current. On the contrary, I_out implicates a K⁺ channel, as it is associated with a negative shift, from roughly –65 mV to nearly –80 mV, in the reversal potential. Presumably, this shift only approaches the K⁺ Nernst potential (calculated to be around –100 mV) because other steady-state channels comprising the resting conductance are still open. Thus I_out strongly influences, but does not completely dominate, whole cell current reversal potential.

I_out is also sensitive to TEA, a well-recognized K⁺ channel blocker (Hagiwara and Saito 1959) known to inhibit both voltage-sensitive and Ca²⁺-activated K⁺ conductances in bag cell neurons (Fink et al. 1988; Quattrocki et al. 1994). I_out is similar to a weakly voltage-dependent K⁺ current that is activated by FMRFamide and perhaps inositol triphosphate in bag cell neurons (Fink et al. 1988; Fisher et al. 1993). I_out also resembles the serotonin-sensitive S-channel found in Aplysia sensory neurons (Shuster et al. 1991). Thus I_out is likely part of the resting conductance, and its gating would have profound consequences for the resting potential and excitability.

In canine jejunal smooth muscle, FFA activates a weakly voltage-sensitive outward current characterized as a K⁺ channel (Farrugia et al. 1993). Similarly, FFA stimulates opening of a two-pore leak K⁺ channel expressed in cell lines (Takahira et al. 2005). Furthermore, Helix neurons display a slow, outward current triggered by FFA (Lee et al. 1996; Shaw et al. 1995) although no information is available regarding the reversal potential, pharmacology, or voltage dependence of that current nor if it is associated with an increase in membrane conductance. Interestingly, the outward current in Helix is reduced by depleting the endoplasmic reticulum of Ca²⁺. This suggests that, unlike I_out in bag cell neurons, the FFA-induced current in Helix may depend on intracellular Ca²⁺ release. The rapid onset and lack of an effect of high intracellular EGTA suggests that I_out is not Ca²⁺ dependent.

The FFA-evoked I_in in cultured bag cell neurons was also accompanied by a conductance increase and a positive shift (from approximately –70 to nearly –15 mV) in the reversal potential of the whole cell current. The current-voltage relationship during activation of I_in was voltage independent up to the point where it reversed, after which some outward rectification was apparent. The reversal potential is consistent with the opening of a channel that is nonselective for cations. Specifically, reversal between –40 and +20 mV is typical for channels that pass cations with a varying degree of selectivity and no overwhelming preference (Colquhoun et al. 1981; Kass et al. 1978; Partridge and Swandulla 1988; Partridge et al. 1994). Further support for I_in being a cation channel comes from the fact that it is blocked by Gd³⁺. This trivalent cation is a well-established nonspecific cation channel blocker with relatively few side-effects (Chakfe and Bourque 2000; Franco and Lansman 1990; Popp et al. 1993; Yang and Sachs 1989). Finally, it appears that I_in may be activated in part by FFA-induced Ca²⁺ release. Both the Ca²⁺ elevation and I_in required several min to fully develop; furthermore, FFA failed to evoke I_in when intracellular Ca²⁺ was strongly buffered with high EGTA.

If Ca²⁺-activation is the gating mechanism for I_in, it is possible that one or more bag cell neuron Ca²⁺-activated channels contributes to the conductance as a whole. While the voltage-dependent cation channel which reverses well above 0 mV is likely not I_in (Lupinsky and Magoski 2006; Magoski 2004; Wilson et al. 1996), the voltage-independent cation channel triggered by Ca²⁺ influx, with a reversal potential near –40 mV and a sensitivity to Gd³⁺, may be a component (Hung and Magoski 2007). That FFA potentiated this current (Hung and Magoski 2007), despite actually inhibiting the voltage-gated Ca²⁺ current, could be due to a synergistic effect of Ca²⁺ liberation from intracellular stores and subsequent activation of I_in. In addition, Knox et al. (1996) reported that depletion of Ca²⁺ from the endoplasmic reticulum by thapsigargin activated a cation channel which reversed near –20 mV, was voltage-independent, and was blocked by pretreatment with BAPTA-AM. This third Ca²⁺-activated cation channel may also contribute to I_in. Incidentally, it is unlikely that a possible inhibitory effect of FFA on gap junctions, as electrical synapses or hemi-channels (Harks et al. 2001; Srinivas and Spray 2003; Ye et al. 2003), is the cause of I_in. All of the neurons used in the present study were single cells that did not touch other neurons and had no opportunity to make electrical synapses. Regarding hemi-channels, they certainly could be present, but their block would result in a decreased whole cell conductance, rather than the increase seen with both I_in and I_out.

Ca²⁺-activated and receptor-operated TRP cation channels from heart and arterial smooth muscle are blocked by Gd³⁺ but not FFA (Hill et al. 2006; Ohki et al. 2000). FFA also initially enhances both Ca²⁺-activated and ligand-gated cation channels in Helix and hippocampal neurons (Green and Cottrell 1997; Partridge and Valenzuela 2000; Shaw et al. 1995); although once enhancement reaches a peak, a slow block then follows. Those Ca²⁺-activated cation currents were elicited by depolarizing steps or action potentials with the enhancement thought to be due to FFA-induced release of more Ca²⁺ (Partridge and Valenzuela 2000; Shaw et al. 1995). This is similar to the bag cell neurons in that FFA can enhance cation channels by releasing Ca²⁺. However, prior to the present study there were no reports that FFA could trigger cation channels to open at rest without depolarization-evoked Ca²⁺ influx. The bag cell neuron I_in and the prolonged depolarization cation current also do not show any slow block by FFA. I_in would influence the resting potential and, if activated by Ca²⁺ released during the afterdischarge (Fisher et al. 1994), contribute depolarizing drive to the burst.

As suggested, some of the effect of FFA on cultured bag cell neurons appears to be due to intracellular Ca²⁺ release. Our experiments involving depleting endoplasmic reticulum Ca²⁺ with CPA suggest that this Ca²⁺ may in part come from the endoplasmic reticulum. CPA blocks the Ca²⁺-ATPase and causes the endoplasmic reticulum to lose Ca²⁺ through leak channels (Seidler et al. 1989; Tu et al. 2006). Lee et al. (1996) found that the ability of FFA to raise intracellular Ca²⁺ levels in Helix neurons could be largely eliminated by thapsigargin, which is functionally analogous to CPA (Thastrup et al. 1990). However, the FFA-induced Ca²⁺ increase in both a mandibular cell line and hippocampal neurons was not prevented by thapsigargin (Partridge and Valenzuela 2000; Poronnik et al. 1992). Thus as in the bag cell neurons, FFA may target other stores. Data from the present study show that the FFA Ca²⁺ response is also depressed by pretreatment with FCCP, which collapses the mitochondrial membrane potential that normally drives Ca²⁺ into the mitochondria (Collins et al. 2000; Heytler and Prichard 1962; Simpson and Russell 1996). FFA has been shown to both prevent Ca²⁺ uptake and release from liver mitochondria (Jordani et al. 2000; McDougall et al. 1988). This may be achieved through either a protonophore-like effect, similar to FCCP itself, or direct activation of the mitochondrial permeability transition pore (Jordani et al. 2000). Not surprisingly, removal of Ca²⁺ from both the endoplasmic reticulum and mitochondria, by depleting with CPA and FCCP at the same time, substantially reduced the Ca²⁺ response of the bag cell neurons to FFA.

In summary, we have provided evidence that FFA directly opens a K⁺ conductance and indirectly activates a cation conductance by releasing intracellular Ca²⁺ in cultured bag cell neurons. The effect of FFA on voltage-gated Ca²⁺ current may be due to the drug acting on the channel itself or again by some Ca²⁺-dependent process. Clearly FFA can alter the function of numerous membrane proteins; as such, the mechanism of FFA cation channel block in other systems may be related to how it alters plasma membrane, and perhaps intracellular, ion channels. In some ways, FFA has been seen as a gold-standard for cation channel blockers. However, recognizing that this drug may set off other intracellular or biophysical events, its use needs to be tempered with appropriate controls. Despite this, FFA could be employed as a tool for intentionally releasing intracellular Ca²⁺ or triggering certain currents.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
K. E. Gardam holds a RS McLaughlin Fellowship, C. M. Hickey holds an Ontario Graduate Scholarship in Science and Technology, A. Y. Hung held a Canadian Institutes of Health Research (CIHR) Canada Graduate Scholarship Master's Award, and N. S. Magoski holds a CIHR New Investigator Award. This work was supported by CIHR operating grant to N. S. Magoski.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The authors thank S. L. Smith for technical assistance and N. M. Magoski for critical evaluation of previous drafts of the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: N. S. Magoski, Dept. of Physiology, Queen's University, 4^thFloor, Botterell Hall, 18 Stuart St., Kingston, ON K7L 3N6, Canada (E-mail: magoski{at}post.queensu.ca)

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