Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Maitotoxin (MTX) is a polyether marine toxin produced by the dinoflagellate Gambierdiscus toxicus that can be ingested by the surgeon fish Ctenochateus striatus and induce severe human intoxication in subjects eating this fish (Yokoyama et al., 1988). On exposure to nanomolar concentrations of this extremely potent toxin, a massive increase in [Ca²⁺]_i occurs in several excitable cells, such as pheochromocytoma PC12 cells, pituitary GH₃ cells, and hypothalamic synaptosomes (Takahashi et al., 1982; Gusovsky and Daly, 1990; Taglialatela et al., 1990; Annunziato et al., 1993). Different mechanisms have been proposed to explain the [Ca²⁺]_i increase induced by MTX. Evidence suggests that this toxin activates voltage-gated Ca²⁺ channels (VGCCs; Xi et al., 1992; Fatatis et al., 1994), the plasmamembrane channel responsible for the refilling of endoplasmic reticulum Ca²⁺ stores (Soergel et al., 1992) and Na⁺ channels (Nishio et al., 1993, 1996), as well as a nonselective cationic current (Murata et al., 1992; Dietl and Völkl, 1994; Worley et al., 1994). Furthermore, it has been shown that this toxin indirectly activates phospholipase C through an increase in [Ca²⁺]_i, (Gusovsky et al., 1989). All these different MTX effects could therefore coexist in the same cell and cooperate to increase [Ca²⁺]_i, especially if elevated concentrations of this toxin are used.

It has been shown recently that concentrations higher than 0.5 ng/ml MTX induce a massive increase in [Ca²⁺]_i in a nimodipine-insensitive manner, whereas at concentrations of toxin ranging from 0.01 to 0.1 ng/ml, the toxin activates VGCCs in a specific and nimodipine-dependent way (Xi et al., 1996). However, evidence for the direct interaction of MTX with L-type VGCCs has not yet been provided; in addition, the subunit required for this interaction has yet to be characterized. Therefore, in the present study we investigated MTX action in Chinese hamster ovary (CHO) cells, a nonexcitable cell type lacking both endogenous voltage-sensitive Na⁺ channels (Scheuer et al., 1990) and VGCCs (Bosse et al., 1992), which were stably transfected with cDNAs encoding for different subunits of VGCCs (Hofmann et al., 1994; Catterall, 1995). In particular, the effect of MTX on [Ca²⁺]_i was studied with Fura-2 single-cell videoimaging in untransfected CHO cells and in CHO cells stably transfected either with the pore-forming _1c subunit of L-type VGCCs (CHOC9 cells; Bosse et al., 1992) or with the entire _1c324 complex of this channel (CHOC932/4 cells; Welling et al., 1993). The use of these transfected clones allowed us to investigate the role played by the pore-forming (_1c) and accessory (₃₂₄) subunits in MTX induction of [Ca²⁺]_i increase. In addition, patch-clamp studies were performed in CHOC932/4 cells to better characterize the electrophysiological effects exerted by the toxin. Finally, the possible effects of MTX on [Ca²⁺]_i in CHOC9 and in CHOC932/4 cells were compared with those obtained in GH₃ cells, an excitable cell line constitutively expressing L-type VGCCs (Armstrong and Mattson, 1985).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Culture. GH₃ cells were obtained from Flow Laboratories (Irvine, Scotland) and grown on plastic dishes in Ham's F-10 medium (GIBCO BRL, San Giuliano Milanese, Italy) with (in v/v) 15% horse serum (Flow Laboratories), 2.5% fetal calf serum (FCS; Hyclone, Logan, UT), 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured in a humidified 5% CO₂ atmosphere. Culture medium was changed every 2 days. The cells used belonged to cultures subjected to 34 to 60 passages.

Intracellular Calcium Measurements

[Ca²⁺]_i was measured using a microfluorometric technique as previously reported (Cataldi et al., 1996). Briefly, the cells grown on glass coverslips were loaded with 5 µM Fura-2 acetoxymethyl ester (AM) for 1 h at room temperature in Krebs-Ringer saline solution (5.5 mM KCl, 160 mM NaCl, 1.2 mM MgCl₂, 1.5 mM CaCl₂, 10 mM d-glucose, and 10 mM HEPES-NaOH, pH 7.4). At the end of Fura-2 AM loading, the coverslip was mounted in a microscope chamber (Medical System Co., Greenvale, NY) on an inverted Nikon Diaphot fluorescence microscope. The cells were kept in Krebs-Ringer saline solution throughout the experiment. All the drugs tested were introduced into the microscope chamber by fast injection. A 100-W xenon lamp (Osram, Germany) with a computer-operated filter wheel bearing two different interference filters (340 and 380 nm) illuminated the microscopic field with UV light alternatively at the wavelength of 340 and 380 nm, with an interval of 500 ms between excitation at 340 and 380 nm. The interval between excitation by each pair of wavelengths was 4 s, and 1 s elapsed during filter movements. Consequently, a [Ca²⁺]_i determination was performed every 5 s. Emitted light was passed through a 400-nm dichroic mirror, filtered at 510 nm, and collected by a charge coupled device camera (Photonic Science, Robertsbridge, East Sussex, UK) connected to a light amplifier (Applied Imaging Ltd., Dukesway Gateshead, UK). Images were digitized and analyzed with a Magiscan image processor (Applied Imaging Ltd.). The Tardis software (Applied Imaging Ltd.) calculated the [Ca²⁺]_i corresponding to each pair of images using a calibration curve from the ratio between the intensity of the light emitted when the cells were lighted at 340 and 380 nm.

Patch-Clamp Electrophysiology. Currents from CHOC932/4 cells were recorded at room temperature using a commercially available amplifier (Axopatch 200A; Axon Instruments, Foster City, CA). The whole-cell configuration of the patch-clamp technique was adopted using glass micropipettes of 3- to 7-M resistance. No compensation was performed for pipette resistance and cell capacitance. The cells were perfused with an extracellular solution containing 10 mM BaCl₂, 125 mM NaCl, 1 mM MgCl₂, 10 mM HEPES, and 300 nM tetrodotoxin, pH 7.3. The pipettes were filled with 110 mM CsCl, 10 mM tetraethylammonium-Cl, 2 mM MgCl₂, 10 mM EGTA, 8 mM glucose, 2 mM Mg-ATP, 0.25 mM cAMP, and 10 mM HEPES, pH 7.3. Ba²⁺ currents flowing through VGCCs were activated by continuous ramp pulses from 60 to +80 mV (32 ms/pulse, 100 µs/sampling point) elicited at 0.066-Hz frequency (1 pulse every 15 s). The Ba²⁺ current through Ca²⁺ channels was obtained by subtracting the current elicited with identical protocols in the presence of 100 µM CdSO₄.

Drugs and Chemicals. All the chemicals were of analytical grade and were purchased from Sigma Italia (Milan, Italy). Fura-2 AM, MTX, nimodipine, and G-418 were from Calbiochem (La Jolla, CA). In most of the data in the literature, the toxin concentrations are expressed in nanograms per milliter. Given that the molecular weight of MTX is approximately 3500 (Yokoyama et al., 1988), the molarity of a toxin concentration of 1 ng/ml corresponds to approximately 300 pM.

Statistical Analysis. All data are expressed as mean ± S.E. The statistical analysis was performed using Student's t test for paired or unpaired data where required. The threshold for statistical significance was set at p < .05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of High K⁺ Concentrations on [Ca²⁺]_i in CHO Cells Stably Transfected with cDNAs Encoding for L-Type VGCC Subunits. When the CHO clone stably transfected with cDNA encoding for the _1c VGCC subunit was exposed to 55 mM K⁺, a rapid [Ca²⁺]_i elevation occurred. CHO cells stably transfected with the entire _1c32 subunit complex responded to high K⁺ concentration with a significantly higher [Ca²⁺]_i increase (Fig. 1). Interestingly, [Ca²⁺]_i decline after high K⁺ concentration addition was faster in CHOC9 than in CHOC932/4 cells (Fig. 1). In contrast, in untransfected CHO cells, 55 mM K⁺ did not induce any increase in [Ca²⁺]_i (Fig. 1).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Effect of 55 mM K+ on [Ca2+]i in CHO wild-type, C9, and CHOC932/4 cells. This figure shows the [Ca2+]i response to 55 mM K+ in CHO wild-type (), CHOC9 (), and CHOC932/4 () cells; traces are the mean of six, five, and four single-cell recordings, respectively, obtained during single experimental sessions representative of at least two other experimental sessions. KCl (55 mM) was added after 130 s of basal [Ca2+]i monitoring and kept in the chamber for another 90 s. Then, it was washed out by the fast injection of Krebs-Ringer saline solution. The inset reports the % of the peak of [Ca2+]i increase in response to 55 mM K+ (defined as {(peak [Ca2+]i response to 55 mM K+ minus mean baseline [Ca2+]i)/mean baseline [Ca2+]i}). The values are the mean ± S.E. of 20, 40, and 48 single-cell recordings, respectively, obtained at least in three different experimental sessions. *p < .05 versus wild-type CHO; **p < .05 versus wild-type CHO and CHOC9.

Concentration Dependence and Time Course of MTX-Induced [Ca²⁺]_i Increase in CHO Cells Stably Expressing VGCC Subunits. MTX (0.01-1 ng/ml) induced a significant concentration-dependent increase in [Ca²⁺]_i that was similar in CHOC9 and CHOC932/4 cells. The [Ca²⁺]_i was significantly higher in CHOC932/4 than in CHOC9 cells only at 0.1 ng/ml MTX (Fig. 2A). In CHO wild-type cells, which do not express VGCCs, MTX induced a much lower, yet significant, [Ca²⁺]_i increase only at concentrations of 0.1 and 0.5 ng/ml. In addition, the time course of MTX-induced [Ca²⁺]_i rise showed that in both CHOC9 and CHOC932/4 cells, the increase was much faster than that in untransfected CHO cells (Fig. 2B).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effect of different MTX concentrations (0.01-0.5 ng/ml) on [Ca2+]i in CHOwt, C9, and CHOC932/4 cells. A, concentration-effect curve of MTX-induced [Ca2+]i increase in CHO wild-type (), CHOC9 (), and CHOC932/4 () cells. MTX was added after 520 s of baseline [Ca2+]i monitoring. The response is expressed as the [Ca2+]i increase over baseline, calculated as the difference between peak [Ca2+]i detected in the presence of MTX, and mean baseline [Ca2+]i recorded before MTX or nimodipine addition. The data are the mean ± S.E. of the single-cell data (n = 14-37) recorded during at least three different experimental sessions. *p < .05 versus 0.01 ng/ml MTX; **p < .05 versus 0.01 and 0.1 ng/ml MTX; p < .05 versus 0.1 and 0.5 ng/ml MTX in CHOC9 and in CHOC932/4 cells. B, time course of 0.1 ng/ml MTX effect on [Ca2+]i in CHO wild-type, CHOC9, and CHOC932/4 cells is reported. The traces are the mean of 17, 7, and 5 single-cell recordings, respectively, obtained during a single experimental session representative of at least two other experimental sessions.

Effect of L-type Ca²⁺ Channel Blocker Nimodipine on MTX-Induced [Ca²⁺]_i Elevation in CHOC9 and CHOC932/4 Cells. When CHOC9 and CHOC932/4 cells were preincubated for 6 min with a supramaximal concentration of nimodipine (10 µM), a significant lowering of [Ca²⁺]_i elevation elicited by increasing concentrations (0.01-1 ng/ml) of MTX occurred in both CHOC9 (Fig. 3A) and CHOC932/4 cells (Fig. 3B). However, a nimodipine-insensitive component of MTX-induced [Ca²⁺]_i increase was still present at the effective concentrations of 0.1 and 0.5 ng/ml in CHOC9 as well as in CHOC932/4 cells. This nimodipine-insensitive MTX-induced [Ca²⁺]_i increase was of the same entity as that observed in untransfected CHO cells.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effect of nimodipine on [Ca2+]i response to different MTX concentrations (0.01-0.5 ng/ml) in CHOC9 and CHOC932/4 cells. A, effect on [Ca2+]i of different concentrations of MTX in CHOC9 cells in the absence () and in the presence of 10 µM nimodipine (). The response is expressed as the [Ca2+]i increase over baseline, calculated as the difference between peak [Ca2+]i recorded in the presence of MTX, and mean [Ca2+]i before MTX or nimodipine addition. MTX was added after 520 s of baseline [Ca2+]i monitoring, whereas nimodipine was added 150 s after the beginning of [Ca2+]i monitoring and kept until the end of the experiment. The traces are the mean of seven single-cell recordings obtained during a single experimental session, representative of at least two other experimental sessions. A inset, time course of the effect of 0.1 ng/ml MTX on [Ca2+]i (absolute concentrations) in the absence (top) and in the presence (bottom) of 10 µM nimodipine. The black bar at the bottom of the inset denotes the duration of exposure to 0.1 ng/ml MTX in both experimental groups. *p < .05 versus respective 0.01 ng/ml MTX group; **p < .05 versus respective 0.1 ng/ml MTX group; p < .05 versus nimodipine-treated group. B, effect of different MTX (0.01-0.5 ng/ml) in CHOC932/4 cells in the absence () and in the presence () of 10 µM nimodipine. B inset, time course of the effect of 0.1 ng/ml MTX on [Ca2+]i (absolute concentrations) in the absence (top) and in the presence (bottom) of 10 µM nimodipine. The black bar at the bottom of the inset denotes the duration of exposure to 0.1 ng/ml MTX in both experimental groups. *p < .05 versus respective 0.01 ng/ml MTX group; **p < .05 versus respective 0.1 ng/ml MTX group; p < .05 versus nimodipine-treated group. For other experimental details see legend of A.

Concentration-Dependence of MTX-Induced [Ca²⁺]_i Increase and Its Reversal by Nimodipine in GH₃ Cells. MTX (0.01-1 ng/ml) induced a concentration-dependent increase in [Ca²⁺]_i in GH₃ cells (Fig. 4). Preincubation with nimodipine (10 µM) caused a remarkable inhibition of MTX-induced [Ca²⁺]_i elevation; however, this inhibition was not complete because a Ca²⁺ channel blocker-resistant elevation of [Ca²⁺]_i was still detected at 0.1 and 0.5 ng/ml MTX. The entity of this nimodipine-resistant MTX-induced [Ca²⁺]_i increase was similar to that observed in untransfected CHO cells.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of different MTX concentrations (0.01-0.5 ng/ml) on [Ca2+]i in GH3 cells. This figure shows the concentration-effect curve of MTX-induced [Ca2+]i increase in control () and nimodipine-treated () GH3 cells. MTX was added after 520 s of baseline [Ca2+]i monitoring. Nimodipine (10 µM) was added 150 s after the beginning of [Ca2+]i monitoring and kept until the end of the experiment. The response is expressed as [Ca2+]i increase over baseline, calculated as the difference between peak [Ca2+]i detected in the presence of MTX and mean baseline [Ca2+]i before MTX or nimodipine addition. The data are the mean ± S.E. of all the single-cell data recorded during at least three different experimental sessions (n = 14-53). *p < .05 versus respective 0.01 ng/ml MTX group; **p < .05 versus respective 0.1 ng/ml MTX group; p < .05 versus nimodipine-treated group.

Effect of Depolarizing Concentrations of Extracellular K⁺ Ions on MTX-Induced [Ca²⁺]_i Increase in CHOC932/4 Cells. In CHOC932/4 cells, the perfusion with depolarizing concentrations of extracellular K⁺ ions (55 mM), which are widely used to enhance the activity of VGCCs, caused an immediate increase in [Ca²⁺]_i (Fig. 5A). In the continuous presence of elevated K⁺ concentrations, the peak in the [Ca²⁺]_i was followed by a plateau phase (Fig. 5A). On the other hand, MTX (0.1 ng/ml) caused an increase in [Ca²⁺]_i that was characterized by a slower onset and a progressive elevation (Fig. 5B). The [Ca²⁺]_i increase induced by 0.1 ng/ml MTX reached a value of about 1000 nM after 10 min of exposure (Figs. 5B and 2A). When MTX was simultaneously superfused with depolarizing concentrations of extracellular K⁺ (55 mM), the [Ca²⁺]_i showed a considerably faster rise and reached a value at least twice higher (>2000 nM) than that induced by the toxin in the presence of 5 mM extracellular K⁺ (Fig. 5C). In addition, this enhanced MTX-induced increase of [Ca²⁺]_i observed in the presence of depolarizing concentrations of extracellular K⁺ was almost completely prevented by the preincubation with 10 µM nimodipine.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effect of 55 mM extracellular K+ on MTX-induced [Ca2+]i increase in CHOC932/4 cells. A, time course of [Ca2+]i response to 55 mM K+ in CHOC932/4 cells. The bar represents the time of exposure to 55 mM K+. B, time course of [Ca2+]i response to 0.1 ng/ml MTX in the presence of 5 mM extracellular K+ in CHOC932/4 cells. The bar represents the time of exposure to 0.1 ng/ml MTX. C, [Ca2+]i response to 0.1 ng/ml MTX in the presence of 55 mM extracellular K+ in CHOC932/4, in both the absence (Controls) and presence of 10 µM nimodipine. The bar represents the time of exposure to 0.1 ng/ml MTX plus 55 mM K+. The arrow after 150 s of basal [Ca2+]i monitoring denotes the time of exposure to 10 µM nimodipine only in the nimodipine-treated group. After this time, the dihydropyridine was present throughout the experiment in the nimodipine group.

Effect MTX on Ba²⁺ Currents Recorded in CHOC932/4 Cells. CHOC932/4 cells expressing VGCCs showed voltage-dependent currents that activated around 20 mV, peaked at +30 mV, and reversed at potentials more positive than +60 mV. These currents were inhibited by approximately 90% by 10 µM nimodipine (Fig. 6A); furthermore, the dihydropyridine VGCC activator Bay K 8644 (1 µM) potentiated ~2-fold these Ba²⁺ currents (Fig. 6B). This pharmacological profile is similar to that previously reported (Bosse et al., 1992; Welling et al., 1993). In the same cells, low concentrations of MTX (0.03 ng/ml) induced the appearance of an inward current at 60 mV, which progressively increased with time and was entirely prevented by the subsequent addition of 100 µM Cd²⁺ (Fig. 6C). In addition, 10 µM nimodipine was able to prevent the appearance of the MTX-induced inward current; this MTX-induced current reappeared on the removal of the dihydropyridine antagonist (Fig. 6D). By contrast, the same concentration of nimodipine failed to prevent the increase in inward currents induced by higher MTX concentrations (3 ng/ml; Fig. 6E).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Effect of nimodipine, Bay K 8644, and MTX on Ba2+ currents in CHOC932/4 cells. The effects of nimodipine (10 µM) and Bay K 8644 (1 µM) on high voltage-activated Ca2+ channel currents in CHOC932/4 cells are shown in A and B, respectively. Ba2+ currents flowing through VGCCs were activated by ramp pulses from 60 to +80 mV (32 ms/pulse, 100 µs/sampling point) elicited at 0.066-Hz frequency (1 pulse every 15 s). The traces shown in A and B represent control and drug effect after 2-min perfusion with each drug. Each trace was obtained by subtracting the current elicited with identical protocols in the presence of 100 µM Cd2+. Shown the effects of 0.03 ng/ml MTX on Ba2+ currents in CHOC932/4 cells at the holding potential of 60 mV and their reversal by 100 µM Cd2+ (C), the inhibition of MTX-induced currents by 10 µM nimodipine (D), and the inability of the same dihydropyridine concentration to block the effects of 3 ng/ml MTX (E). Each panel is representative of at least four cells exposed to the same experimental protocol.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results of the present study show that the transfection of a nonexcitable cell type like CHO cells with cDNA encoding only for the pore-forming _1c subunit of L-type VGCCs (CHOC9 cells) is sufficient to induce a remarkable increase in [Ca²⁺]_i in response to different concentrations of MTX. The transfection of CHO cells with the entire _1c324 complex of VGCCs resulted in an MTX-induced [Ca²⁺]_i elevation that was similar to that observed in CHOC9 cells. The specificity of low MTX concentrations on the Ca²⁺ channel subunits expressed in transfected CHO cells is further supported by the capacity of the L-type Ca²⁺ channel blocker nimodipine to inhibit the [Ca²⁺]_i increase elicited by this toxin. These results provided evidence that the _1c subunit of L-type VGCCs represents an important target mediating the MTX effect on [Ca²⁺]_i.

The hypothesis that the MTX-induced [Ca²⁺]_i increase is mainly exerted through VGCCs is also supported by the results showing that the toxin-induced [Ca²⁺]_i increase is potentiated under experimental conditions in which the activity of VGCCs is enhanced by depolarizing concentrations of extracellular K⁺, suggesting that the depolarization of VGCCs facilitates the onset and the entity of MTX action. Furthermore, the involvement of VGCCs in MTX action is also supported by its reversal by the dihydropyridine VGCC blocker nimodipine.

The action of MTX on the _1c subunit is not the only mechanism responsible for [Ca²⁺]_i elevation. In fact, an increase in [Ca²⁺]_i, although of a much lower entity, was also observed in wild-type CHO cells that lack VGCCs. Evidence that the [Ca²⁺]_i-increasing effect of MTX not only is due to an action on _1c subunit but also recognizes another mechanism is further suggested by the results showing that the Ca²⁺ channel blocker nimodipine, which binds to _1c subunit, used in a supramaximal concentration did not completely counteract MTX-induced [Ca²⁺]_i elevation in CHOC9 and CHOC932/4 cells. This nimodipine-insensitive [Ca²⁺]_i elevation was also observed in GH₃ cells, an excitable cell line constitutively expressing L-type VGCCs. The mechanism by which MTX increases [Ca²⁺]_i in a VGCC-independent way has been widely investigated (Gusovsky and Daly, 1990; Annunziato et al., 1993). In fact, it has been shown that MTX can promote Ca²⁺ entrance through an interaction with the channels responsible for the refilling of endoplasmic reticulum Ca²⁺ stores (Soergel et al., 1992). In addition, it has been reported that MTX can induce phosphoinositide breakdown through the indirect activation of phospholipase C (Gusovsky et al., 1989; Gusovsky and Daly, 1990). Finally, it has been electrophysiologically found by means of the patch-clamp technique that MTX might activate a nonselective Na⁺/Ca²⁺ current (Dietl and Völkl, 1994). This nimodipine-insensitive mechanism shown in this study seems to be particularly present in the higher range of active concentrations of MTX. In accordance, Xi et al. (1996) found that in GH₄C₁ cells, this non-Ca²⁺ channel-mediated component of MTX action is present only at concentrations higher than 0.5 ng/ml.

Evidence that MTX can exert different pharmacological actions depending on its concentration is provided by the results of the present electrophysiological experiments. In fact, in CHOC932/4 cells, low concentrations of MTX (0.03 ng/ml) induced an inward current at 60 mV that was entirely blocked by Cd²⁺ and nimodipine, whereas the same concentration of the dihydropyridine failed to prevent the membrane currents induced by a higher MTX concentration (3 ng/ml; Kobayashi et al., 1987). The fact that MTX activates an inward Ba²⁺ current at a membrane potential of 60 mV, whereas VGCCs are typically activated around 20 mV, could be due to the removal of the channel inactivation process caused by the toxin, thus allowing the modified channel to open at resting membrane potential, as also suggested by Yoshii et al. (1987). This hypothesis seems to be supported by the ability of the selective L-type VGCC blocker nimodipine to prevent the current induced by low MTX concentrations at 60 mV.

In conclusion, the results of the present study show that MTX-induced [Ca²⁺]_i elevation recognizes two components: 1) an action on L-type VGCCs at the pore-forming _1c subunit level that is responsible for the greatest rise of [Ca²⁺]_i, occurring mainly at lower toxin concentrations; and 2) a VGCC-independent mechanism that is present in both excitable and nonexcitable cells is responsible for a lower elevation of [Ca²⁺]_i, and mainly occurs at higher MTX concentrations.