Crotamine Pharmacology Revisited: Novel Insights Based on the Inhibition of K-V Channels

Crotamine, a 5-kDa peptide, possesses a unique biological versatility. Not only has its cell-penetrating activity become of clinical interest but, moreover, its potential selective antitumor activity is of great pharmacological importance. In the past, several studies have attempted to elucidate the exact molecular target responsible for the crotamine-induced skeletal muscle spasm. The aim of this study was to investigate whether crotamine affects voltage-gated potassium (K-V) channels in an effort to explain its in vivo effects. Crotamine was studied on ion channel function using the two-electrode voltage clamp technique on 16 cloned ion channels (12 K-V channels and 4 Na-V channels), expressed in Xenopus laevis oocytes. Crotamine selectively inhibits K-V 1.1, K-V 1.2, and K-V 1.3 channels with an IC50 of similar to 300 nM, and the key amino acids responsible for this molecular interaction are suggested. Our results demonstrate for the first time that the symptoms, which are observed in the typical crotamine syndrome, may result from the inhibition of K-V channels. The ability of crotamine to inhibit the potassium current through K-V channels unravels it as the first snake peptide with the unique multifunctionality of cell-penetrating and antitumoral activity combined with K-V channel-inhibiting properties. This new property of crotamine might explain some experimental observations and opens new perspectives on pharmacological uses.

Ionotropic Glutamate Receptors in Hypothalamic Paraventricular and Supraoptic Nuclei Mediate Vasopressin and Oxytocin Release in Unanesthetized Rats

We report changes in plasma arginine vasopressin (AVP) and oxytocin (OT) concentrations evoked by the microinjection of L-glutamate (L-glu) into the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus(PVN) of unanesthetized rats, as well as which local mechanisms are involved in their mediation. L-Glu microinjection (10 nmol/100 nl) into the SON increased the circulating levels of both AVP and OT. The AVP increases were blocked by local pretreatment with the selective non-N-methyl-D-aspartate (NMDA) receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (2 nmol/100 nl), but it was not affected by pretreatment with the NMDA-receptor antagonist LY235959 (2 nmol/100 nl). The OT response to L-glu microinjection into the SON was blocked by local pretreatment with either NBQX or LY235959. Furthermore, the administration of either the non-NMDA receptor agonist (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) (5 nmol/100 nl) or NMDA receptor agonist NMDA (5 nmol/100 nl) into the SON had no effect on OT baseline plasma levels, but when both agonists were microinjected together these levels were increased. L-Glu microinjection into the PVN did not change circulating levels of either AVP or OT. However, after local pretreatment with LY235959, the L-glu microinjection increased plasma levels of the hormones. The L-glu microinjection into the PVN after the local treatment with NBQX did not affect the circulating AVP and OT levels. Therefore, results suggest the AVP release from the SON is mediated by activation of non-NMDA glutamate receptors, whereas the OT release from this nucleus is mediated by an interaction of NMDA and non-NMDA receptors. The present study also suggests an inhibitory role for NMDA receptors in the PVN on the release of AVP and OT. (Endocrinology 153: 2323-2331, 2012)

Neural differentiation of rat aorta pericyte cells

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins beta 3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain. (C) 2011 International Society for Advancement of Cytometry

Ether-A -go-go 1 (Eag1) Potassium Channel Expression in Dopaminergic Neurons of Basal Ganglia is Modulated by 6-Hydroxydopamine Lesion

The ether A go-go (Eag) gene encodes the voltage-gated potassium (K+) ion channel Kv10.1, whose function still remains unknown. As dopamine may directly affect K+ channels, we evaluated whether a nigrostriatal dopaminergic lesion induced by the neurotoxin 6-hydroxydopamine (6-OHDA) would alter Eag1-K+ channel expression in the rat basal ganglia and related brain regions. Male Wistar rats received a microinjection of either saline or 6-OHDA (unilaterally) into the medial forebrain bundle. The extent of the dopaminergic lesion induced by 6-OHDA was evaluated by apomorphine-induced rotational behavior and by tyrosine hydroxylase (TH) immunoreactivity. The 6-OHDA microinjection caused a partial or complete lesion of dopaminergic cells, as well as a reduction of Eag1+ cells in a manner proportional to the extent of the lesion. In addition, we observed a decrease in TH immunoreactivity in the ipsilateral striatum. In conclusion, the expression of the Eag1-K+-channel throughout the nigrostriatal pathway in the rat brain, its co-localization with dopaminergic cells and its reduction mirroring the extent of the lesion highlight a physiological circuitry where the functional role of this channel can be investigated. The Eag1-K+ channel expression in dopaminergic cells suggests that these channels are part of the diversified group of ion channels that generate and maintain the electrophysiological activity pattern of dopaminergic midbrain neurons.

Successful Improvement of Frequency and Symptoms of Premature Complexes after Oral Magnesium Administration

Background: Premature ventricular and supraventricular complexes (PVC and PsVC) are frequent and often symptomatic. The magnesium (Mg) ion plays a role in the physiology of cell membranes and cardiac rhythm. Objective: We evaluated whether the administration of Mg Pidolate (MgP) in patients with PVC and PsVC is superior to placebo (P) in improving symptoms and arrhythmia frequency. Methods: Randomized double-blind study with 60 consecutive symptomatic patients with more than 240 PVC or PsVC/h on 24-hour Holter monitoring who were selected to receive placebo or MgP. To evaluate symptom improvement, a categorical and a specific questionnaire for symptoms related to PVC and PsVC was made. Improvement in premature complex density (PCD) per hour was considered significant if percentage reduction was >= 70% after treatment. The dose of MgP was 3.0 g/day for 30 days, equivalent to 260mg of Mg element. None of the patients had structural heart disease or renal failure. Results: Of the 60 patients, 33 were female (55%). Ages ranged from 16 to 70 years old. In the MgP group, 76.6% of patients had a PCD reduction >70%, 10% of them >50% and only 13.4% <50%. In the P group, 40% showed slight improvement, <30%, in the premature complexes frequency (p < 0.001). Symptom improvement was achieved in 93.3% of patients in the MgP group, compared with only 16.7% in the P group (p < 0.001). Conclusion: Oral Mg supplementation decreases PCD, resulting in symptom improvement. (Arq Bras Cardiol 2012;98(6):480-487)

Redução da densidade de extrassístoles e dos sintomas relacionados após administração de magnésio por via oral

FUNDAMENTO: As extrassístoles ventriculares e supraventriculares (EV e ESSV) são frequentes e muitas vezes sintomáticas. O íon magnésio (Mg) desempenha um papel importante na fisiologia do potencial de ação transmembrana celular e do ritmo cardíaco. OBJETIVO: Avaliar se a administração do pidolato de magnésio (PMg) em pacientes com EV e ESSV tem desempenho superior ao uso do placebo (P) na melhora dos sintomas e densidade das extrassístoles (DES). MÉTODOS: Estudo duplo-cego, randomizado, com 60 pacientes sintomáticos consecutivos, com mais de 240/EV ou ESSV ao Holter de 24 horas e selecionados para receber P ou PMg. Para avaliar a melhora da sintomatologia, foi feito um questionário categórico e específico de sintomas relacionados às extrassístoles. Após o tratamento, foi considerada significante uma redução de mais de 70% na DES por hora. A dose do PMg foi de 3,0 g/dia por 30 dias, equivalente a 260 mg do elemento Mg. Nenhum paciente tinha cardiopatia estrutural ou insuficiência renal. RESULTADOS: Dos 60 pacientes estudados, 33 eram do sexo feminino (55%). A faixa etária variou de 16 a 70 anos. No grupo PMg, 76,6% dos pacientes tiveram redução maior que 70%, 10% deles maior que 50% e somente 13,4% tiveram redução menor que 50% na DES. No grupo P, 40% dos pacientes tiveram melhora de apenas 30% na frequência de extrassístoles (p < 0,001). A melhora dos sintomas foi alcançada em 93,3% dos pacientes do grupo PMg, comparada com somente 16,7% do grupo P (p < 0,001). CONCLUSÃO: A suplementação de Mg via oral reduziu a DES, resultando em melhora dos sintomas.