Carvacrol modulates voltage-gated sodium channels kinetics in dorsal root ganglia.

Recent studies have shown that many of plant-derived compounds interact with specific ion channels and thereby modulate many sensing mechanisms, such as nociception. The monoterpenoid carvacrol (5-isopropyl-2-methylphenol) has an anti-nociceptive effect related to a reduction in neuronal excitability and voltage-gated Na(+) channels (NaV) inhibition in peripheral neurons. However, the detailed mechanisms of carvacrol-induced inhibition of neuronal NaV remain elusive. This study explores the interaction between carvacrol and NaV in isolated dorsal root ganglia neurons. Carvacrol reduced the total voltage-gated Na(+) current and tetrodotoxin-resistant (TTX-R) Na(+) current component in a concentration-dependent manner. Carvacrol accelerates current inactivation and induced a negative-shift in voltage-dependence of steady-state fast inactivation in total and TTX-R Na(+) current. Furthermore, carvacrol slowed the recovery from inactivation. Carvacrol provoked a leftward shift in both the voltage-dependence of steady-state inactivation and activation of the TTX-R Na(+) current component. In addition, carvacrol-induced inhibition of TTX-R Na(+) current was enhanced by an increase in stimulation frequency and when neurons were pre-conditioned with long depolarization pulse (5s at -50 mV). Taken all results together, we herein demonstrated that carvacrol affects NaV gating properties. The present findings would help to explain the mechanisms underlying the analgesic activity of carvacrol.

The effects of volatile anesthetics on the extracellular accumulation of [(3)H]GABA in rat brain cortical slices.

GABA is an inhibitory neurotransmitter that appears to be associated with the action of volatile anesthetics. These anesthetics potentiate GABA-induced postsynaptic currents by synaptic GABAA receptors, although recent evidence suggests that these agents also significantly affect extrasynaptic GABA receptors. However, the effect of volatile anesthetics on the extracellular concentration of GABA in the central nervous system has not been fully established. In the present study, rat brain cortical slices loaded with [(3)H]GABA were used to investigate the effect of halothane and sevoflurane on the extracellular accumulation of this neurotransmitter. The accumulation of [(3)H]GABA was significantly increased by sevoflurane (0.058, 0.11, 0.23, 0.46, and 0.93 mM) and halothane (0.006, 0.012, 0.024, 0.048, 0072, and 0.096 mM) with an EC50 of 0.26 mM and 35 µM, respectively. TTX (blocker of voltage-dependent Na(+) channels), EGTA (an extracellular Ca(2+) chelator) and BAPTA-AM (an intracellular Ca(2+) chelator) did not interfere with the accumulation of [(3)H]GABA induced by 0.23 mM sevoflurane and 0.048 mM halothane. SKF 89976A, a GABA transporter type 1 (GAT-1) inhibitor, reduced the sevoflurane- and halothane-induced increase in the accumulation of GABA by 57 and 63 %, respectively. Incubation of brain cortical slices at low temperature (17 °C), a condition that inhibits GAT function and reduces GABA release through reverse transport, reduced the sevoflurane- and halothane-induced increase in the accumulation of [(3)H]GABA by 82 and 75 %, respectively, relative to that at normal temperature (37 °C). Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce GABA release through reverse transport, abolished the sevoflurane and halothane effects on the accumulation of [(3)H]GABA. The effect of sevoflurane and halothane did not involve glial transporters because ß-alanine, a blocker of GAT-2 and GAT-3, did not inhibit the effect of the anesthetics. In conclusion, the present study suggests that sevoflurane and halothane increase the accumulation of GABA by inducing the reverse transport of this neurotransmitter. Therefore, volatile anesthetics could interfere with neuronal excitability by increasing the action of GABA on synaptic and extrasynaptic GABA receptors.

Toxicity of cultured bullseye puffer fish Sphoeroides annulatus.

The toxin content in various life cycle stages of tank-cultivated bullseye puffer (Sphoeroides annulatus) were analyzed by mouse bioassay and ESI-MS spectrometry analysis. The presence of toxin content was determined in extracts of sperm, eggs, embryo, larvae, post-larvae, juvenile, pre-adult, and adult fish, as well as in food items used during the cultivation of the species. Our findings show that only the muscle of juveniles, the viscera of pre-adults, and muscle, liver, and gonad of adult specimens were slightly toxic (<1 mouse unit). Thus, cultivated S. annulatus, as occurs with other cultivated puffer fish species, does not represent a food safety risk to consumers. This is the first report of toxin analysis covering the complete life stages of a puffer fish under controlled conditions.

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects.

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freeze-thaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A(2) were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 ± 34.7% of control twitch height, which were blocked with either 100 µ m of phentolamine or 1 m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-N(G) -Nitroarginine methyl ester (0.5 m m) or 1 m m of lidocaine. PHY-N (1, 3 and 10 µg ml(-1) ) induced an increase in tonus of the biventer-cervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 µ m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release.

First report on toxins in the Panamanian toads Atelopus limosus, A. glyphus and A. certus.

Major toxins from skin extracts of 18 specimens of six Atelopus toad species collected in Panama were analyzed. Chiriquitoxin was identified using (1)H NMR in A. limosus and A. glyphus for the first time. Zetekitoxin in A. zeteki and tetrodotoxin in A. varius, A. chiriquiensis and A. zeteki were identified again. Furthermore, A. certus was suggested to contain a water-soluble toxin other than tetrodotoxin.

Biomedical and pharmacological potential of tetrodotoxin-producing bacteria isolated from marine pufferfish Arothron hispidus (Muller, 1841)

Specimens of the pufferfish Arothron hispidus collected at Parangipettai, on the southeast coast of India, were subjected to bacterial isolation and identification. Three species were identified, namely Bacillus sp., Kytococcus sedentarius and Cellulomonas fimi. Partially-purified microbial filtrates exhibited hemolytic activity on chicken and human erythrocytes of O, B and AB blood groups, with maximum activity of 32 HU. The microbial filtrates also presented ATPase, Mg2+-ATPase, Na+K+-ATPase and AchE enzymatic activities of positive neuromodulation in Kytococcus sedentarius with 1300, 300.1, 1549.98 and 140.55%, in Cellulomonas fimi with 620, 300, 10 and 128.42%, and in Bacillus species with 40, 200, 849.98 and 158.69%, respectively. Toxicity symptoms were observed when the bacterial filtrate was intraperitoneally injected into mice. The bacterial filtrate caused adverse effects on viability of the mouse muscle cell line (L929) and leukemia cell line (P388). Maximum level of inhibition was observed on the growth of L929 cell line. Bacillus lentimorbus inhibited the cell line from 84.03 to 94.43% whereas Bacillus species inhibited the growth in a range between 77.25 and 86.16% at the lowest dilution.(AU)

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish.

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques--mouse bioassay, ELISA, HPLC and mass spectrometry-we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6+/-12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6+/-0 5MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry.

Further report of the occurrence of tetrodotoxin and new analogues in the Anuran family Brachycephalidae.

Tetrodotoxin (TTX) is one of the most potent toxin already isolated, which occurs in a wide range of marine as well as terrestrial animals such as in newts and anurans. In this work, the occurrence of TTX and analogues was examined in three brachycephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix using LC-FLD and LC-MS/MS. In toxicity assay (intra-peritonial injection in mice) B. nodoterga extracts were non-toxic, while B. pernix extract exhibit the highest toxicity among the studied species. Skin showed the highest toxic, followed by the liver. Retention time data in the LC-FLD system indicated the presence of TTX, 4-epiTTX, 4,9-anhydroTTX and TDA, SIM data confirmed the presence of these compounds and revealed other analogs such as 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX, 6-epiTTX. Two new components were also identified by mass spectrometry (348 and 330Da). These unknown compounds have daughter ions similar to TTX, suggesting new putative TTX analogues.

The occurrence of 11-oxotetrodotoxin, a rare tetrodotoxin analogue, in the brachycephalidae frog Brachycephalus ephippium.

11-oxoTTX is an analogue 4-5 times more toxic than TTX itself, been rare even in marine animals. Two ions at m/z 320 and 336 corresponding to TTX and 11-oxoTTX (M+H(+)), respectively, were detected in the Brachycephalidae frog Brachycephalus ephippium extracts. The fragment ion pattern of 11-oxoTTX is similar to that TTX, although its possible to verify some specific fragments.

Occurrence of tetrodotoxin and its analogues in the Brazilian frog Brachycephalus ephippium (Anura: Brachycephalidae).

Brachycephalus ephippium is a diurnal frog, that shows aposematic colouration and inhabits Atlantic forest leaf litter in south-eastern Brazil. The presence of tetrodotoxin (TTX) in the skin, liver and ovaries of B. ephippium was demonstrated. The skin (260 M.U./g) exhibited the highest toxicity followed by liver (177 M.U./g). TTX and its analogues, tetrodonic acid, 4-epitetrodotoxin and 4,9 anhydrotetrodotoxin were isolated and identified by HPLC followed by fluorimetric analysis. TTX and 11-nortetrodotoxin-6(S)-ol had their presence confirmed by mass spectrometry (MALDI-TOF). The results confirm Brachycephalidae as a fourth family of anurans containing TTX.

Histoenzymological and ultrastructural changes in lateral muscle fibers of Oreochromis niloticus (Teleostei: Cichlidae) after local injection of veratrine.

The effects of veratrine have been investigated in mammalian, amphibian, and crustacean muscle, but not in fish. In this work, the action of veratrine was studied in the lateral muscle of the freshwater teleost Oreochromis niloticus after intramuscular injection. Histoenzymological typing and electron microscopy of muscle fibers before and 15, 30, and 60 min after veratrine injection (10 ng/kg fish) were used to indirectly assess the morphological changes and the oxidative and m-ATPase activities. In some cases, muscles were pretreated with tetrodotoxin to determine whether the ultrastructural changes were the result of Na(+) channel activation by veratrine. Veratrine altered the metabolism of fibers mainly after 30 min. Oxidative fibers showed decreased NADH-TR activity, whereas that of glycolytic and oxidative-glycolytic type fibers increased. There was no change in the m-ATPase activity of the three fiber types, except at 60 min postveratrine, when a novel fiber type, which showed no reversal after acidic and alkaline preincubations, appeared. Ultrastructural damage involved sarcomeres, myofibrils, and mitochondria, but the T-tubules remained intact. Pretreatment with tetrodotoxin (1 ng/ml) prevented the ultrastructural changes caused by veratrine. These results show that in fish skeletal muscle veratrine produces some effects that are not seen in mammalian muscle.

Toxicities and distribution of tetrodotoxin in the tissues of puffer fish found in the coast of the Baja California Peninsula, Mexico.

Toxicities and tetrodotoxin distribution in tissues of five puffer fish species commonly found in the littoral of Baja California Peninsula, Mexico (Sphoeroides annulatus, S. lobatus, S. lispus, Arothron meleagris and Canthigaster punctatissima) were evaluated by bioassay and HPLC. The toxicities estimated as tetrodotoxin-equivalents of all species were more than 0.42 microg/g in at least one of the tissues tested, and the highest was found in S. lispus liver (130 microg/g).

TsTX-VII, a new toxin from Tityus serrulatus scorpion venom able to induce the release of neurotransmitters from rat brain synaptosomes not blocked by tetrodotoxin.

A procedure for the isolation of the toxin Tityustoxin VII (TsTX-VII) from Tityus serrulatus scorpion venom and its biochemical characterization is reported. This protein has a M(r) = 6,700-6,800, eight half-cystine residues accounting for four disulfide bridges and no His. Its N-terminal sequence GHZGYGS ... characterizes it as a new toxin, able to release glutamic acid and gamma aminobutyric acid from rat brain synaptosomes "in vitro". This release was also induced by the whole venom. Tetrodotoxin however blocked the effect of the whole venom but not that of TsTX-VII, thus suggesting that the releasing mechanism by TsTX-VII does not involve Na+ but perhaps K+ or Ca++ channels.

Tetrodotoxin blockade of amygdala overlapping with poisoning impairs acquisition of conditioned taste aversion in rats.

The role of several forebrain structures in the association of the short-term gustatory memory (GSTM) of the conditioned stimulus (CS; 0.1% sodium saccharin) with the visceral unconditioned stimulus (US; 0.15 M LiCl, 2% b.wt.) in acquisition of conditioned taste aversion (CTA) was investigated. Experiment 1 examined the effects of bilateral reversible inactivation of amygdala (Amy), hippocampus (Hipp), gustatory cortex (GC), bed nucleus of stria terminalis (BNST), lateral hypothalamic area (LHA), ventral thalamus (VT) or LHA+VT, induced by intracerebral injection of tetrodotoxin (TTX; 10 ng/microliters per site) applied before i.p. injection of LiCl to rats anesthetized by pentobarbital (50 mg/kg) immediately after saccharin drinking. Amy blockade resulted in a complete disruption of learning, while the inactivation of the remaining areas examined produced mild or no impairments. The dose-related effects of TTX injection into Amy were investigated in Experiment 2. Doses of 3 and 1 ng TTX were as effective as the 10 ng dose used in Expt. 1. However, 0.3 ng or saline did not interfere with CTA acquisition. Analysis of the retrograde amnesic effect produced by transient amygdalectomy (Experiment 3), showed that TTX (10 ng) injected immediately or 1.5 h after LiCl application induced a marked learning disruption, whereas no amnesia was elicited at 6 and 24 h post-acquisition intervals. It is suggested that Amy plays an essential role in the associative phase of acquisition, but not in the consolidation of the permanent taste aversion engram.

Rat cortical synaptosomes have more than one mechanism for Ca2+ entry linked to rapid glutamate release: studies using the Phoneutria nigriventer toxin PhTX2 and potassium depolarization.

PhTX2, one of the components of the venom of the South American spider Phoneutria nigriventer, inhibits the closure of voltage-sensitive Na+ channels. Incubation of cerebral-cortical synaptosomes with PhTX2 causes a rapid increase in the intrasynaptosomal free Ca2+ concentration and a dose-dependent release of glutamate. This release is made up of a slow component, which appears to be due to reversal of Na(+)-dependent glutamate uptake, and more rapid component that is dependent on the entry of extrasynaptosomal Ca2+. It has previously been shown that membrane depolarization using KCl can cause rapid Ca(2+)-dependent release of glutamate from synaptosomes. This requires Ca2+ entry through a specific type of Ca2+ channel that is sensitive to Aga-GI, a toxic component of the venom of the spider Agelenopsis aperta. We have compared the effects of PhTX2 and KCl on elevation of intrasynaptosomal free Ca2+ and glutamate release, and a number of differences have emerged. Firstly, PhTX2-mediated Ca2+ influx and glutamate release, but not those caused by KCl, are inhibited by tetrodotoxin. Secondly, KCl produces a clear additional increase in Ca2+ and glutamate release following those elicited by PhTX2. Finally, 500 microM MnCl2 abolishes PhTX2-mediated, but not KCl-mediated, glutamate release. These findings suggest that more than one mechanism of Ca2+ entry may be coupled to glutamate release from nerve endings.

Occurrence of tetrodotoxin in the frog Atelopus oxyrhynchus.

Alcohol extracts from the frog Atelopus oxyrhynchus were toxic to mice when injected intraperitoneally. The toxin was purified by gel filtration on a Sephadex G-15 column, and was identified as tetrodotoxin by thin-layer chromatography and GC-MS analysis of the alkali-hydrolyzed and trimethylsilylated derivative giving the same pattern as the C9-base of tetrodotoxin.