ARTICLE pubs.acs.org/crt
A Comparative Study of the Effect of Ciguatoxins on Voltage-Dependent Naþ and Kþ Channels in Cerebellar Neurons† Sheila Perez,‡ Carmen Vale,‡ Eva Alonso,‡ Carmen Alfonso,‡ Paula Rodríguez,‡ Paz Otero,‡ Amparo Alfonso,‡ Paulo Vale,§ Masahiro Hirama,|| Mercedes R. Vieytes,^ and Luis M. Botana*,‡ ‡
Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain. IPIMAR, Av. Brasilia, Lisboa 1449-006, Portugal Department of Chemistry, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan ^ Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain
)
§
ABSTRACT: Ciguatera is a global disease caused by the consumption of certain warm-water fish (ciguateric fish) that have accumulated orally effective levels of sodium channel activator toxins (ciguatoxins) through the marine food chain. The effect of ciguatoxin standards and contaminated ciguatoxin samples was evaluated by electrophysiological recordings in cultured cerebellar neurons. The toxins affected both voltagegated sodium (Nav) and potassium channels (Kv) although with different potencies. CTX 3C was the most active toxin blocking the peak inward sodium currents, followed by P-CTX 1B and 51-OH CTX 3C. In contrast, P-CTX 1B was more effective in blocking potassium currents. The analysis of six different samples of contaminated fish, in which a ciguatoxin analogue of mass 1040.6, not identical with the standard 51-OH CTX 3C, was the most prevalent compound, indicated an additive effect of the different ciguatoxins present in the samples. The results presented here constitute the first comparison of the potencies of three different purified ciguatoxins on sodium and potassium channels in the same neuronal preparation and indicate that electrophysiological recordings from cultured cerebellar neurons may provide a valuable tool to detect and quantify ciguatoxins in the very low nanomolar range.
’ INTRODUCTION Ciguatera fish poisoning (CFP) is a major economic and social problem worldwide, with more than 25000 persons poisoned annually.1 The causative toxins, ciguatoxins (CTX), are produced by the dinoflagellate Gambierdiscus toxicus.2,3 CTX are highly lipophilic, polycyclic ether compounds3 which bind competitively to the receptor-site 5 of voltage-gated sodium channels.4,5 In humans, the onset of ciguatera commonly begins with gastrointestinal problems such as nausea, vomiting, diarrhea, and abdominal pain within 12 h of eating a toxic fish. Moreover, a combination of bradycardia and hypotension may be present during this acute period. From a few hours to two weeks after exposure, subjective neurological complaints may emerge. Paresthesias (numbness and tingling of perioral region and extremities) and disturbance of temperature sensation are considered pathognomic symptoms of CFP.6 Ciguatoxins in finfish comprise an assemblage of principal ciguatoxins and numerous closely related structural isomers and congeners. The first CTX structure elucidated was the principal CTX from the Pacific (P-CTX 1), which exhibits extreme lethality in mouse models with a median lethal dose of 0.25 μg/kg after intraperitoneal injection.6 Nowadays, 29 ciguatoxin congeners have been identified among the pacific ciguatoxins r 2011 American Chemical Society
(P-CTX) that comprise products of epimerization, hydroxylation, and oxidation of P-CTX 1 and CTX 4B (see ref 6 for a review). Like P-CTX 1 in the Pacific area, the main CTX involved in CFP in the Caribbean area is C-CTX 1. This toxin is also a potent sodium channel activator with a mouse LD50 of 3.6 μg/ kg.7 Neurological symptoms of CFP are believed to be the direct consequence of the interaction of CTX with voltage-gated sodium channels.1 Notwithstanding, the small quantities of pure ciguatoxins available, the physiological effects of several CTX have been evaluated in a wide variety of preparations. In this sense, pharmacological studies have revealed that pacific CTX activate voltage-sensitive sodium channels at low nanomolar concentrations to cause cell depolarization, spontaneous nerve firing, elevation of the intracellular free Ca2þ concentration, and edema of Schwann cell and axons present on excitable membranes.4,8-11 Research on other ciguatoxins is less extensive, mainly due to difficulties in obtaining purified toxin, and as a consequence, there is scarce information on the biological activity of different ciguatoxins in the same cellular model. Thus, P-CTX 1 had no effect on the kinetics of Nav currents, but it Received: January 27, 2011 Published: February 25, 2011 587
dx.doi.org/10.1021/tx200043j | Chem. Res. Toxicol. 2011, 24, 587–596
Chemical Research in Toxicology
ARTICLE
decreased the peak current amplitude in mammalian sensory neurons and caused a hyperpolarizing shift in the voltagedependence of sodium channel activation12 in cultured rat myotubes.13 Moreover, different potencies of P-CTX 1B and P-CTX 4B to affect Nav and Kv channels have also been recently reported on myelinated axons.14 Although, the scarcity of CTX has hampered quantitative evaluations of their effects on voltagegated sodium and potassium channels, the recent accomplishment of the chemical synthesis of the ciguatoxin congener CTX 3C15,16 has allowed detailed patch-clamp analysis of the effects of this toxin. Thus, CTX 3C markedly affected the operation of Nav but was ineffective on Kv channels in mouse taste cells.17 Moreover, CTX 3C at high concentrations (1 μM) has been shown to activate tetrodotoxin-sensitive sodium channels (Nav1.2, Nav1.4, and Nav1.5) by accelerating their activation kinetics and shifting the activation curve toward hyperpolarization;18 however, the toxin at 100 nM preferentially affected the activation process of the tetrodotoxin-resistant Nav1.8 channel compared with those of Nav1.2 and Nav1.4.19 In this article, we took advantage of the chemical identification of the ciguatoxin profile in a recent CFP case in Europe,20 to perform a detailed evaluation of the biological activity of the combination of six different ciguatoxins in different proportions and to analyze in the same preparation the biological effects of synthetic CTX 3C, 51-OH CTX 3C, and purified P-CTX 1B. In this study, we analyzed six samples extracted from two species of fish Seriola dumerili (S.Dumerili) and Seriola fasciata (S. Fasciata), caught at Salvagen Islands, a part of Madeira Arquipielago (Portugal). In this ciguatera outbreak, a ciguatoxin analogue of 51-OH CTX 3C with a mass of 1040.6 was the most abundant toxin in all samples, followed by C/I-CTX 1/2 and CTX 4A/B. CTX 1B and CTX 3C were present at very low levels.20 The biological activity of different fish extracts containing this ciguatoxin combination in varying amounts and its effect on voltagegated sodium and potassium channels are described in this article and compared with the biological activity of synthetic 51-OH CTX 3C, CTX 3C, and purified P-CTX 1B in cerebellar granule cells (CGC) using the perforated patch-clamp technique. Cultured cerebellar granule cells were chosen to perform this analysis since primary cultures of CGC constitute one of the most reliable neuronal models for the study of neural function and pathology.21,22 CGCs in cultures express voltage-gated sodium currents, blocked by TTX and at least two types of potassium currents: a delayed rectifier current (IK) and an inactivating current (IA).23 Thus, in our knowledge, this report constitutes the first comparison of the activities of different ciguatoxins and their associations in the same neuronal system.
laboratory. All other chemicals were reagent grade and purchased from Sigma-Aldrich (Madrid, Spain). Cell Cultures. Primary cultures of cerebellar granule cells were obtained from cerebella of 7-day-old mice as previously described.22,24 In brief, cells were dissociated by mild trypsinization at 37 C, followed by trituration in a DNase solution (0.004% w/v) containing a soybean trypsin inhibitor (0.05% w/v). The cells were suspended in DMEM containing 25 mM KCl, 31 mM glucose, and 0.2 mM glutamine supplemented with p-amino benzoate, insulin, penicillin, and 10% fetal calf serum. The cell suspension was seeded in 18 mm glass coverslips precoated with poly-D-lysine and incubated in 12 multiwell plates, for 6-11 days in culture, in a humidified 5% CO2/95% air atmosphere at 37 C. Cytosine arabinoside, 20 μM, was added before 48 h in culture to prevent glial proliferation. Electrophysiology. Membrane currents from single cells were studied at room temperature (22-25 C) by gramicidin-perforatedpatch recordings in voltage-clamp mode in order to minimize the dialysis of intracellular components25,26 using a computer-controlled current and voltage clamp amplifier (Multiclamp 700B, Molecular Devices). Signals were recorded and analyzed using a Pentium computer equipped with a Digidata 1440 data acquisition system and pClamp10 software (Molecular Devices, Sunnyvale, CA). pClamp10 was used to generate current and voltage-clamp commands and to record the resulting data. Signals were prefiltered at 10 kHz and digitized at 20 μs intervals. Recording electrodes were fabricated from borosilicate glass microcapillaries (outer diameter, 1.5 mm), and the tip resistance was 5-10 MΩ. Gramicidin (10-20 μg/mL) (Sigma, St. Louis, MO) was used as the membrane-perforating agent. The internal pipet solution contained (in mM): 108 Cs gluconate, 1.7 NaCl, 0.9 EGTA, 9 HEPES, 1.8 MgCl2, 4 Na2ATP, and 0.3 NaGTP at pH 7.2.27 The progress of perforation was evaluated by monitoring the decrease in membrane resistance. After the membrane resistance had stabilized (usually between 5 and 20 min after obtaining the GΩ seal), data were obtained. For the perforated patch-clamp, the extracellular medium contained (in mM): 154 NaCl, 5.6 KCl, 3.6 NaHCO3, 1.3 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES (pH 7.4). For voltage-dependent sodium channels, voltage-gated ion currents were elicited in CGCs by applying a series of 25 ms depolarizing pulses (voltage steps), in 5 mV increments, from a holding potential of -100 mV.28 The current-voltage (I-V) relationship for transient voltagegated sodium currents were obtained by measuring the peak amplitude of the current for each given membrane potential during the voltage steps. As granule cell neurons display two main voltage-dependent outward Kþ currents, transient outward IA current, and delayed rectifier IK currents, the effect of ciguatoxin on these currents was also examined. To evaluate the effect of ciguatoxins on Kþ channels, cesium was replaced by Kþ in the intracellular pipet solution, and 50 nM saxitoxin was added to the bathing solution. In these experiments, transient outward IA and delayed rectifier IK currents were elicited 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 activation of the global Kþ current (IA plus IK current), and at -40 mV (second pulse) for the activation of IK currents. IA currents were obtained by subtraction of the outward Kþ currents elicited at a holding potential of -40 mV from the global Kþ current evoked at a holding potential of -100 mV.29 Concentration-response curves for each ciguatoxin standard were obtained by serial dilutions of the stocks in a final volume of 500 μL of extracellular solution in the recording chamber. No washing attempts were performed. The same amount of toxin solvent, in this case methanol, was used for control measurements. Statistical Method. All data are expressed as means ( SEM of n experiments (each performed in duplicate). Statistical comparison was by nonpaired Student’s t test. P values
