State-Dependent Blockade of Dorsal Root Ganglion Voltage-Gated Na+ Channels by Anethole.

Anethole is a phenolic compound synthesized by many aromatic plants. Anethole is a substance that humans can safely consume and has been studied for years as a biologically active molecule to treat a variety of conditions, including nerve damage, gastritis, inflammation, and nociception. Anethole is thought to carry out its biological activities through direct interaction with ion channels. Anethole is beneficial for neurodegenerative Alzheimer's and Parkinson's diseases. Nevertheless, nothing has been investigated regarding the effects of anethole on voltage-gated Na+ channels (VGSCs), which are major players in neuronal function. We used cultured dorsal root ganglion neurons from neonatal rats as a source of natively expressed VGSCs for electrophysiological studies using the whole-cell patch-clamp technique. Our data show that anethole interacts directly with VGSCs. Anethole quickly blocks and unblocks (when removed) voltage-activated Na+ currents in this preparation in a fully reversible manner. Anethole's binding affinity to these channels increases when the inactive states of these channels are populated, similar to lidocaine's effect on the same channels. Our data show that anethole inhibits neuronal activity by blocking VGSCs in a state-dependent manner. These findings relate to the putative anesthetic activity attributable to anethole, in addition to its potential benefit in neurodegenerative diseases.

Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons.

The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca(2+) and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca(2+) and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s.

Crotamine inhibits preferentially fast-twitching muscles but is inactive on sodium channels.

Crotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that induces a typical hind-limb paralysis of unknown nature. Hind limbs have a predominance of fast-twitching muscles that bear a higher density of sodium channels believed until now to be the primary target of crotamine. Hypothetically, this makes these muscles more sensitive to crotamine and would explain such hind-limb paralysis. To challenge this hypothesis, we performed concentration vs. response curves on fast (extensor digitorum longus (EDL)) and slow (soleus) muscles of adult male rats. Crotamine was tested on various human Na+ channel isoforms (Na(v)1.1-Na(v)1.6 alpha-subunits) expressed in HEK293 cells in patch-clamp experiments, as well as in acutely dissociated dorsal root ganglion (DRG) neurons. Also, the behavioral effects of crotamine intoxication were compared with those of a muscle-selective sodium channel antagonist mu-CgTx-GIIIA, and other sodium-acting toxins such as tetrodotoxin alpha- and beta-pompilidotoxins, sea anemone toxin BcIII, spider toxin Tx2-6. Results pointed out that EDL was more susceptible to crotamine than soleus under direct electrical stimulation. Surprisingly, electrophysiological experiments in human Na(v)1.1 to Na(v)1.6 Na+ channels failed to show any significant change in channel characteristics, in a clear contrast with former studies. DRG neurons did not respond to crotamine. The behavioral effects of the toxins were described in detail and showed remarkable differences. We conclude that, although differences in the physiology of fast and slow muscles may cause the typical crotamine syndrome, sodium channels are not the primary target of crotamine and therefore, the real mechanism of action of this toxin is still unknown.

Variant proteins of Plasmodium vivax are not clonally expressed in natural infections.

Plasmodium vivax is the most widely distributed human malaria parasite and responsible for 70-80 million clinical cases each year and a large socio-economical burden. The sequence of a chromosome end from P. vivax revealed the existence of a multigene superfamily, termed vir (P. vivax variant antigens), that can be subdivided into different subfamilies based on sequence similarity analysis and which represents close to 10-20% of the coding sequences of the parasite. Here we show that there is a vast repertoire of vir genes abundantly expressed in isolates obtained from human patients, that different vir gene subfamilies are transcribed in mature asexual blood stages by individual parasites, that VIR proteins are not clonally expressed and that there is no significant difference in the recognition of VIR-tags by immune sera of first-infected patients compared with sera of multiple-infected patients. These data provide to our knowledge the first comprehensive study of vir genes and their encoding variant proteins in natural infections and thus constitute a baseline for future studies of this multigene superfamily. Moreover, whereas our data are consistent with a major role of vir genes in natural infections, they are inconsistent with a predominant role in the strict sense of antigenic variation.

Activation of peripheral ATP-sensitive K+ channels mediates the antinociceptive effect of Crotalus durissus terrificus snake venom.

The role of peripheral potassium channels on the antinociceptive effect of Crotalus durissus terrificus venom, a mixed delta- and kappa-opioid receptor agonist, was investigated in hyperalgesia induced by carrageenin or prostaglandin E(2). Rat paw pressure test was applied before and 3 h after the intraplantar (i.pl.) injection of the nociceptive stimuli. Oral administration of venom 2 h after carrageenin or prostaglandin E(2) induces antinociception. Local pretreatment with 4-aminopyridine and tetraethylammonium (blockers of voltage-dependent K(+) channel) or charybdotoxin and apamin (inhibitors of large- and small-conductance Ca(2+)-activated K(+) channel, respectively) did not modify venom effect. On the other hand, glybenclamide, an inhibitor of ATP-sensitive K(+) channel abolished antinociception induced by the venom. Glybenclamide also inhibited the antinociceptive effect of [D-Pen(2.5)] enkephalin (DPDPE), a delta opioid receptor agonist, but did not modify the effect of (+)-trans-(1R,2R)-U-50488 (U50488), a kappa opioid receptor agonist. Diazoxide and pinacidil, two ATP-sensitive K(+) channel openers, injected by intraplantar route, induced a long-lasting increment of pain threshold of the animals and produced antinociception in both models of hyperalgesia. These results suggest that the antinociceptive effect of crotalid venom is mediated by activation of ATP-sensitive K(+) channels at peripheral afferent neurons.

Activation of peripheral ATP-sensitive K+ channels mediates theantinociceptive effect of Crotalus durissus terrificus snake venom

The role of peripheral potassium channels on the antinociceptive effect of Crotalus durissus terrificus venom, a mixed ä- and ê-opioid receptor agonist, was investigated in hyperalgesia induced by carrageenin or prostaglandin E2. Rat paw pressure test was applied before and 3 h after the intraplantar (i.pl.) injection of the nociceptive stimuli. Oral administration of venom 2 h after carrageenin or prostaglandin E2 induces antinociception. Local pretreatment with 4-aminopyridine and tetraethylammonium (blockers of voltage-dependent K+ channel) or charybdotoxin and apamin (inhibitors of large- and small-conductance Ca2+-activated K+ channel, respectively) did not modify venom effect. On the other hand, glybenclamide, an inhibitor of ATP-sensitive K+ channel abolished antinociception induced by the venom. Glybenclamide also inhibited the antinociceptive effect of [D-Pen2.5] enkephalin (DPDPE), a delta opioid receptor agonist, but did not modify the effect of (+)-trans-(1R,2R)-U-50488 (U50488), a kappa opioid receptor agonist. Diazoxide and pinacidil, two ATP-sensitive K+ channel openers, injected by intraplantar route, induced a long-lasting increment of pain threshold of the animals and produced antinociception in both models of hyperalgesia. These results suggest that the antinociceptive effect of crotalid venom is mediated by activation of ATP-sensitive K+ channels at peripheral afferent neurons.