A subfraction obtained from the venom of the tarantula Poecilotheria regalis contains inhibitor cystine knot peptides and induces relaxation of rat aorta by inhibiting L-type voltage-gated calcium channels.

Venoms from tarantulas contain low molecular weight vasodilatory compounds whose biological action is conceived as part of the envenomation strategy due to its propagative effects. However, some properties of venom-induced vasodilation do not match those described by such compounds, suggesting that other toxins may cooperate with these ones to produce the observed biological effect. Owing to the distribution and function of voltage-gated ion channels in blood vessels, disulfide-rich peptides isolated from venoms of tarantulas could be conceived into potential vasodilatory compounds. However, only two peptides isolated from spider venoms have been investigated so far. This study describes for the first time a subfraction containing inhibitor cystine knot peptides, PrFr-I, obtained from the venom of the tarantula Poecilotheria regalis. This subfraction induced sustained vasodilation in rat aortic rings independent of vascular endothelium and endothelial ion channels. Furthermore, PrFr-I decreased calcium-induced contraction of rat aortic segments and reduced extracellular calcium influx to chromaffin cells by the blockade of L-type voltage-gated calcium channels. This mechanism was unrelated to the activation of potassium channels from vascular smooth muscle, since vasodilation was not affected in the presence of TEA, and PrFr-I did not modify the conductance of the voltage-gated potassium channel Kv10.1. This work proposes a new envenomating function of peptides from venoms of tarantulas, and establishes a new mechanism for venom-induced vasodilation.

Pyridostigmine reduces mortality of patients with severe SARS-CoV-2 infection: A phase 2/3 randomized controlled trial.

BACKGROUND: Respiratory failure in severe coronavirus disease 2019 (COVID-19) is associated with a severe inflammatory response. Acetylcholine (ACh) reduces systemic inflammation in experimental bacterial and viral infections. Pyridostigmine increases the half-life of endogenous ACh, potentially reducing systemic inflammation. We aimed to determine if pyridostigmine decreases a composite outcome of invasive mechanical ventilation (IMV) and death in adult patients with severe COVID-19. METHODS: We performed a double-blinded, placebo-controlled, phase 2/3 randomized controlled trial of oral pyridostigmine (60 mg/day) or placebo as add-on therapy in adult patients admitted due to confirmed severe COVID-19 not requiring IMV at enrollment. The primary outcome was a composite of IMV or death by day 28. Secondary outcomes included reduction of inflammatory markers and circulating cytokines, and 90-day mortality. Adverse events (AEs) related to study treatment were documented and described. RESULTS: We recruited 188 participants (94 per group); 112 (59.6%) were men; the median (IQR) age was 52 (44-64) years. The study was terminated early due to a significant reduction in the primary outcome in the treatment arm and increased difficulty with recruitment. The primary outcome occurred in 22 (23.4%) participants in the placebo group vs. 11 (11.7%) in the pyridostigmine group (hazard ratio, 0.47, 95% confidence interval 0.24-0.9; P = 0.03). This effect was driven by a reduction in mortality (19 vs. 8 deaths, respectively). CONCLUSION: Our data indicate that adding pyridostigmine to standard care reduces mortality among patients hospitalized for severe COVID-19.

Study on the mechanisms of Suaeda rigida polysaccharides on the heart inhibition and skeletal muscle promotion in the frog / [Estudo sobre os mecanismos dos polissacarídeos de Suaeda rigida na inibição do coração e promoção da musculatura esquelética no sapo]

The objectives of this study were to investigate the effects of different concentrations of Suaeda rigida polysaccharides (SRPs) on the physiological characteristics of the frog heart and gastrocnemius muscle, compare their similarities and differences, and analyze the mechanisms. CaCl2 and acetylcholine (Ach) were selected respectively to be co-incubated with the high concentration SRPs to observe the effects on the heart contraction of frog. The effects of different concentrations of the SRPs on the activities of acetylcholinesterase (A-CHE), Na+-K+-ATPase and Ca2+-Mg2+-ATPase in the isolated frog heart were detected by UV-Vis spectrophotometry. The gastrocnemius muscle was immersed in the high concentration of SRPs for 10 min, and the systolic indexes were recorded. The effects of SRPs on the Ach content and A-CHE activity at the sciatic nerve-gastrocnemius junction were determined by UV-Vis spectrophotometry and enzyme-linked immunosorbent assay (ELISA). The results showed that the SRPs had significant inhibitory effects on the contractile amplitude of isolated heart and the contractile amplitude induced by CaCl2 and Ach, respectively (P<0.01). The activity of Ca2+-Mg2+-ATPase was significantly promoted, and the activity of A-CHE was significantly inhibited (P<0.01). The contraction amplitude, contraction rate, relaxation rate of gastrocnemius muscle and the Ach content at the junction of sciatic nerve-gastrocnemius muscle were significantly increased (P<0.01), and the activity of A-CHE at the junction was significantly inhibited (P<0.01) by the SRPs. All the results suggested that the SRPs could inhibit the contraction of heart and promote the contraction and relaxation of skeletal muscle. The mechanism was related to blocking the fast INa channel, inhibiting the ICa-L and activating the M receptors of myocardial membrane and then inhibiting external Ca2+ influx, increasing Ca2+-Mg2+-ATPase activity, decreasing a-che activity.

Os objetivos deste estudo foram investigar os efeitos de diferentes concentrações de Suaeda rigida polissacarídeos (SRPs) sobre as características fisiológicas do coração de rã e do músculo gastrocnêmio, comparar suas semelhanças e diferenças, e analisar os mecanismos. CaCl2 e acetilcolina (Ach) foram selecionados respectivamente para serem co-incubados com os SRPs de alta concentração para observar os efeitos sobre a contração do coração de rã. Os efeitos das diferentes concentrações dos SRPs sobre as atividades da acetilcolinesterase (A-CHE), Na+-K+-ATPase e Ca2+-Mg2+-ATPase no coração isolado de rã foram detectados pela espectrofotometria UV-Vis. O músculo gastrocnêmio foi imerso na alta concentração de SRP por 10 min, e os índices sistólicos foram registrados. Os efeitos das SRPs no conteúdo de Ach e na atividade de A-CHE na junção nervo-gastrocnêmio ciático foram determinados pela espectrofotometria UV-Vis e pelo ensaio de imunoabsorção enzimática (ELISA). Os resultados mostraram que os SRPs tiveram efeitos inibidores significativos sobre a amplitude contrátil do coração isolado e a amplitude contrátil induzida por CaCl2 e Ach, respectivamente (P<0,01). A atividade do Ca2+-Mg2+-ATPase foi significativamente promovida e a atividade do A-CHE foi significativamente inibida (P<0,01). A amplitude de contração, a taxa de contração, a taxa de relaxamento do músculo gastrocnêmio e o conteúdo de Ach na junção do músculo nervo ciático-gastrocnêmio foram significativamente aumentados (P<0,01), e a atividade do A-CHE na junção foi significativamente inibida (P<0,01) pelas SRPs. Todos os resultados sugeriram que os SRPs poderiam inibir a contração do coração e promover a contração e o relaxamento do músculo esquelético. O mecanismo estava relacionado ao bloqueio do canal INa rápido, inibindo a ICa-L e ativando os receptores M da membrana miocárdica e depois inibindo o influxo externo de Ca2+, aumentando a atividade de Ca2+-Mg2+-ATPase, diminuindo a atividade a-che.

Angiotensin II type 1 receptor is involved in hypertension and vascular alterations caused by environmental toxicant hexachlorobenzene.

Environmental hexachlorobenzene (HCB) increases blood pressure (BP) in female rats, causing alterations in arterial structure and function. Here we study the role of Angiotensin II receptor type 1 (AT1) in HCB-induced hypertension through the use of AT1 antagonist losartan. HCB-treated male rats showed a 22.7% increase in BP which was prevented by losartan. Losartan blocked HCB-induced changes in arterial morphology (decreased aorta cell number and increased wall thickness). Losartan also prevented HCB-induced alterations in artery relaxation by acetylcholine and nitroprusside but not the reduction in the maximum contraction by phenylephrine. Losartan rescued arterial molecular alterations caused by HCB (i.e. an increase in TGF-ß1 and AT1 expression and a decrease in eNOS expression and nitrite levels) and reduced hydrogen sulfide plasma concentration. In conclusion: in this work we demonstrate that AT1 activity is involved in HCB effects on the vascular system leading to hypertension.

A parallel-group, multicenter randomized, double-blinded, placebo-controlled, phase 2/3, clinical trial to test the efficacy of pyridostigmine bromide at low doses to reduce mortality or invasive mechanical ventilation in adults with severe SARS-CoV-2 infection: the Pyridostigmine In Severe COvid-19 (PISCO) trial protocol.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the causative agent of coronavirus disease 2019 (COVID-19), may lead to severe systemic inflammatory response, pulmonary damage, and even acute respiratory distress syndrome (ARDS). This in turn may result in respiratory failure and in death. Experimentally, acetylcholine (ACh) modulates the acute inflammatory response, a neuro-immune mechanism known as the inflammatory reflex. Recent clinical evidence suggest that electrical and chemical stimulation of the inflammatory reflex may reduce the burden of inflammation in chronic inflammatory diseases. Pyridostigmine (PDG), an ACh-esterase inhibitor (i-ACh-e), increases the half-life of endogenous ACh, therefore mimicking the inflammatory reflex. This clinical trial is aimed at evaluating if add-on of PDG leads to a decrease of invasive mechanical ventilation and death among patients with severe COVID-19. METHODS: A parallel-group, multicenter, randomized, double-blinded, placebo-controlled, phase 2/3 clinical trial to test the efficacy of pyridostigmine bromide 60 mg/day P.O. to reduce the need for invasive mechanical ventilation and mortality in hospitalized patients with severe COVID-19. DISCUSSION: This study will provide preliminary evidence of whether or not -by decreasing systemic inflammation- add-on PDG can improve clinical outcomes in patients with severe COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov NCT04343963 (registered on April 14, 2020).

Determinants of Copper Resistance in Acidithiobacillus Ferrivorans ACH Isolated from the Chilean Altiplano.

The use of microorganisms in mining processes is a technology widely employed around the world. Leaching bacteria are characterized by having resistance mechanisms for several metals found in their acidic environments, some of which have been partially described in the Acidithiobacillus genus (mainly on ferrooxidans species). However, the response to copper has not been studied in the psychrotolerant Acidithiobacillus ferrivorans strains. Therefore, we propose to elucidate the response mechanisms of A. ferrivorans ACH to high copper concentrations (0-800 mM), describing its genetic repertoire and transcriptional regulation. Our results show that A. ferrivorans ACH can grow in up to 400 mM of copper. Moreover, we found the presence of several copper-related makers, belonging to cop and cus systems, as well as rusticyanins and periplasmatic acop protein in the genome. Interestingly, the ACH strain is the only one in which we find three copies of copB and copZ genes. Moreover, transcriptional expression showed an up-regulation response (acop, copZ, cusA, rusA, and rusB) to high copper concentrations. Finally, our results support the important role of these genes in A. ferrivorans copper stress resistance, promoting the use of the ACH strain in industrial leaching under low temperatures, which could decrease the activation times of oxidation processes and the energy costs.

Brain-wide mapping of c-fos expression in the single prolonged stress model and the effects of pretreatment with ACH-000029 or prazosin.

Post-traumatic stress disorder (PTSD) is a mental health condition that is triggered by a stressful event, with symptoms including exaggerated startle response, intrusive traumatic memories and nightmares. The single prolonged stress (SPS) is a multimodal stress protocol that comprises a sequential exposure to physical restraint, forced swimming, predator scent and ether anesthesia. This procedure generates behavioral and neurobiological alterations that resemble clinical findings of PTSD, and thus it is commonly used to model the disease in rodents. Here, we applied c-fos mapping to produce a comprehensive view of stress-activated brain regions in mice exposed to SPS alone or to SPS after oral pretreatment with the serotonin-noradrenaline receptor dual modulator ACH-000029 or the α1-adrenergic blocker prazosin. The SPS protocol evoked c-fos expression in several brain regions that control the stress-anxiety response, including the central and medial amygdala, the bed nucleus of the stria terminalis, the pallidum, the paraventricular hypothalamus, the intermediodorsal, paraventricular and central medial thalamic nuclei, the periaqueductal gray, the lateral habenula and the cuneiform nucleus. These effects were partially blocked by pretreatment with prazosin but completely prevented by ACH-000029. Collectively, these findings contribute to the brain-wide characterization of neural circuits involved in PTSD-related stress responses. Furthermore, the identification of brain areas regulated by ACH-000029 and prazosin revealed regions in which SPS-induced activation may depend on the combined or isolated action of the noradrenergic and serotonergic systems. Finally, the dual regulation of serotonin and α1 receptors by ACH-000029 might represent a potential pharmacotherapy that can be applied in the peri-trauma or early post-trauma period to mitigate the development of symptoms in PTSD patients.

Role of Muscarinic Acetylcholine Receptors in Breast Cancer: Design of Metronomic Chemotherapy.

BACKGROUND: muscarinic acetylcholine receptors (mAChRs) have attracted interest as targets for therapeutic interventions in different illnesses like Alzheimer´s disease, viral infections and different tumors. Regarding the latter, many authors have studied each subtype of mAChRs, which seem to be involved in the progression of distinct types of malignancies. METHODS: We carefully revised research literature focused on mAChRs expression and signaling as well as in their involvement in cancer progression and treatment. The characteristics of screened papers were described using the mentioned conceptual framework. RESULTS: Muscarinic antagonists and agonists have been assayed for the treatment of tumors established in lung, brain and breast with beneficial effects. We described an up-regulation of mAChRs in mammary tumors and the lack of expression in non-tumorigenic breast cells and normal mammary tissues. We and others demonstrated that muscarinic agonists can trigger anti-tumor actions in a dose-dependent manner on tumors originated in different organs like brain or breast. At pharmacological concentrations, they exert similar effects to traditional chemotherapeutic agents. Metronomic chemotherapy refers to the administration of anti-cancer drugs at low doses with short intervals among them, and it is a different regimen applied in cancer treatment reducing malignant growth and angiogenesis, and very low incidence of adverse effects. CONCLUSION: The usage of subthreshold concentrations of muscarinic agonists combined with conventional chemotherapeutic agents could be a promising tool for breast cancer therapy.

Methanolic extract of Rhinella schneideri (Anura: Bufonidae) poison causes ultrastructural changes in nerve terminals of phrenic nerve-diaphragm preparations in mice / Extracto metanólico de Rhinella schneideri (Anura: Bufonidae) causa cambios ultraestructurales en las terminaciones nerviosas de preparaciones de nervio frénico de ratón

Abstract Rhinella schneideri (or Bufo paracnemis), popularly known in Brazil as cururu toad, is also found in other countries in South America. The cardiovascular effects of this poison are largely known and recently was shown that it is capable to affect the neuromuscular junction on avian and mice isolated preparation. In this work, we used transmission electron microscopy to investigate the ultrastructure of the motor nerve terminal and postsynaptic junctional folds of phrenic nerve-hemidiaphragm preparations incubated for either 5 or 60 min with the methanolic extract of R. schneideri (50 µg/mL). In addition, the status of the acetylcholine receptors (AChR) was examined by TRITC-α-bungarotoxin immunofluorescence location at the endplate membrane. The results show that 5 min of incubation with the gland secretion extract significantly decreased (32 %) the number of synaptic vesicles into the motor nerve terminal, but did not decrease the electron density on the top of the junctional folds where nicotinic receptors are concentrated; however, 60 min of incubation led to significant nerve terminal reloading in synaptic vesicles whereas the AChR immunoreactivity was not as marked as in control and after 5 min incubation. Muscle fibers were well-preserved but intramuscular motor axons were not. The findings corroborated pharmacological data since the decrease in the number of synaptic vesicles (5 min) followed by recovery (60 min) is in accordance with the transient increase of MEPPs frequency meaning increased neurotransmitter release. These data support the predominant presynaptic mode of action of the R. schneideri, but do not exclude the possibility of a secondary postsynaptic action depending on the time the preparation is exposed to poison. Rev. Biol. Trop. 66(3): 1290-1297. Epub 2018 September 01.

Resumen Rhinella schneideri (o Bufo paracnemis), conocido popularmente en Brasil como sapo cururu, también se encuentra en otros países de América del Sur. Los efectos cardiovasculares de este veneno son ampliamente conocidos y recientemente se demostró que es capaz de afectar la unión neuromuscular en la preparación aislada de aves y ratones. En este trabajo, utilizamos microscopía electrónica de transmisión para investigar la ultraestructura de la terminación nerviosa motora y pliegues de unión postsináptica de preparaciones de nervio frénico-hemidiafragma incubadas durante 5 o 60 min con el extracto metanólico de R. schneideri (50 μg/mL). Además, se examinó el estado de los receptores de acetilcolina (AChR) mediante la ubicación de inmunofluorescencia de TRITC-α-bungarotoxina en la membrana de la placa terminal. Los resultados muestran que 5 min de incubación con el extracto de secreción de glándula disminuyeron significativamente (32 %) el número de vesículas sinápticas en el terminal del nervio motor, pero no disminuyeron la densidad electrónica en la parte superior de los pliegues de unión donde se concentran los receptores nicotínicos. Sin embargo, 60 min de incubación condujeron a una recarga significativa de los terminales nerviosos en las vesículas sinápticas, mientras que la inmunorreactividad del AChR no fue tan marcada como en el control y después de 5 min de incubación. Las fibras musculares estaban bien conservadas, pero los axones motores intramusculares no. Los hallazgos corroboraron los datos farmacológicos ya que la disminución en el número de vesículas sinápticas (5 min) seguida de recuperación (60 min) está de acuerdo con el aumento transitorio de la frecuencia de MEPPs, lo que significa una mayor liberación de neurotransmisores. Estos datos apoyan el modo de acción presináptico predominante de R. schneideri, pero no excluyen la posibilidad de una acción postsináptica secundaria dependiendo del tiempo en que la preparación esté expuesta al veneno.

Age-, Gender-, and in Vivo Different Doses of Isoproterenol Modify in Vitro Aortic Vasoreactivity and Circulating VCAM-1.

Different human-like cardiomyopathies associated to ß-adrenergic stimulation are experimentally modeled in animals through variations in dose, route, and duration of administration of different cardiotoxic drugs. However, associated changes in the vasculature and their relation to systemic inflammation, and the influence of cardiovascular diseases risk factors (gender and age) upon them are seldom analyzed. Here we studied the effect of age and gender on the vasoreactivity of aortas from mice subjected to in vivo repeated ß-adrenergic stimulation with different doses of isoproterenol (ISO) in association with circulating inflammatory cytokines. Young (2 months) and old (18 months) male and female mice received 0 (control), 5, 40, 80 or 160 µg/g/d of ISO (7 days, s.c.). IL-1α, IL-4 and vascular cell adhesion molecule-1 (VCAM-1) were quantified in plasma. In vitro, norepinephrine-induced vasoconstriction and acetylcholine-induced relaxation were measured in aortas. No differences in contraction, relaxation, IL-1α, and IL-4 were found between control young males and females. Age decreased contraction in males and relaxation was lower in females and abolished in males. VCAM-1 was higher in young males than in females and increased in old mice. Vasoconstriction in ISO-treated mice results as a bell-shaped curve on contraction in young and old males, with lower values in the latter. In females, ISO-160 increased contraction in young females but decreased it in old females. Vasorelaxation was reduced in ISO-treated young males and females. ISO-80 and 160 reduced vasorelaxation in old females, and intermediate doses relaxed aortas from old males. VCAM-1 was higher in young and old males with ISO-80 and 160; while VCAM-1 was higher only with ISO-160 in old females. Our results demonstrate that repeated ß-adrenergic stimulation modifies vascular reactivity depending on gender, age, and dose. Females were less sensitive to alterations in vasoreactivity, and young females required a higher amount of the adrenergic stimuli than old females to show vascular alterations. Changes were independent of IL-1α and IL-4. VCAM-1 only changed in old females stimulated with ISO 160. Our results highlight the relevance of considering and comparing in the same study females and aged organisms to improve the accuracy of applications to clinical studies.

Mitochondrial involvement in memory impairment induced by scopolamine in rats.

OBJECTIVE: Scopolamine (SCO) administration to rats induces molecular features of AD and other dementias, including impaired cognition, increased oxidative stress, and imbalanced cholinergic transmission. Although mitochondrial dysfunction is involved in different types of dementias, its role in cognitive impairment induced by SCO has not been well elucidated. The aim of this work was to evaluate the in vivo effect of SCO on different brain mitochondrial parameters in rats to explore its neurotoxic mechanisms of action. METHODS: Saline (Control) or SCO (1 mg/kg) was administered intraperitoneally 30 min prior to neurobehavioral and biochemical evaluations. Novel object recognition and Y-maze paradigms were used to evaluate the impact on memory, while redox profiles in different brain regions and the acetylcholinesterase (AChE) activity of the whole brain were assessed to elucidate the amnesic mechanism of SCO. Finally, the effects of SCO on brain mitochondria were evaluated both ex vivo and in vitro, the latter to determine whether SCO could directly interfere with mitochondrial function. RESULTS: SCO administration induced memory deficit, increased oxidative stress, and increased AChE activities in the hippocampus and prefrontal cortex. Isolated brain mitochondria from rats administered with SCO were more vulnerable to mitochondrial swelling, membrane potential dissipation, H2O2 generation and calcium efflux, all likely resulting from oxidative damage. The in vitro mitochondrial assays suggest that SCO did not affect the organelle function directly. CONCLUSION: In conclusion, the present results indicate that SCO induced cognitive dysfunction and oxidative stress may involve brain mitochondrial impairment, an important target for new neuroprotective compounds against AD and other dementias.

Supplementation with Brazil nuts and green tea extract regulates targeted biomarkers related to colorectal cancer risk in humans.

Se and green tea have been shown in epidemiological, observational and preclinical studies to be inversely related to the risk of developing colorectal cancer (CRC). However, there are limited studies to evaluate their regulatory effects on genes/proteins that relate to CRC oncogenesis in human subjects, such as selenoproteins, WNT signalling pathway, inflammation and methylation. This study examined the effects of supplementation of Se using Brazil nuts and green tea extract (GTE) capsules, alone and in combination, on targeted biomarkers. In total, thirty-two volunteers (>50 years of age) with plasma Se≤1·36 µmol/l were randomised to one of three treatment groups: nine to Se (approximately 48 µg/d) as six Brazil nuts, eleven to four GTE capsules (800 mg (-)-epigallocatechin-3-gallate) and twelve to a combination of Brazil nuts and GTE. Blood and rectal biopsies were obtained before and after each intervention. Plasma Se levels, rectal selenoprotein P (SePP) and ß-catenin mRNA increased significantly in subjects consuming Brazil nuts alone or in combination, whereas rectal DNA methyltransferase (DNMT1) and NF-κB mRNA were reduced significantly in subjects consuming GTE alone or in combination. None of the interventions significantly affected rectal acetylated histone H3 or Ki-67 expression at the protein level or plasma C-reactive protein. Effects of the combination of Brazil nuts and GTE did not differ from what would be expected from either agent alone. In conclusion, supplementation of Brazil nuts and/or GTE regulates targeted biomarkers related to CRC oncogenesis, specifically genes associated with selenoproteins (SePP), WNT signalling (ß-catenin), inflammation (NF-κB) and methylation (DNMT1). Their combination does not appear to provide additional effects compared with either agent alone.

The Role of Acetylcholine in the Inflammatory Response in Animals Surviving Sepsis Induced by Cecal Ligation and Puncture.

The cholinergic anti-inflammatory pathway controls the inflammatory response and nonreflexive consciousness through bidirectional communication between the brain and immune system. Moreover, brain acetylcholinesterase activity may have a role in regulating the vagus nerve in this pathway. Thus, we analyzed the role of acetylcholine (ACh) in the inflammatory response 15 days after induction of sepsis by cecal ligation and puncture (CLP). Balb/c mice were pretreated with or without donepezil (5 mg/kg/day, orally) 7 days before CLP, and mice homozygous for vesicular ACh transporter (VAChT) knockdown (KD) were subjected to CLP. All animals were sacrificed 15 days after CLP, and the plasma, spleen, and hippocampus were collected. Characterization of splenic lymphocytes and cytokine levels in the plasma, spleen, and hippocampus was determined. Our results showed a splenomegaly in group CLP. The numbers of cytotoxic T cells, helper T cells, regulatory T cells, B cells, and Th17 cells differed between mice subjected to CLP and to sham operation in both untreated and donepezil-treated groups. In VAChT-KD mice, CLP resulted in decreased cytotoxic and helper T cells and increased in Th17 cells compared with the sham. Additionally, in VAChT-KD mice, the levels of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α, were increased following CLP. Thus, we concluded that ACh affected the inflammatory response at 15 days after CLP since stimulation of cholinergic transmission increased the proliferation of lymphocytes, including regulatory T cells, in association with a lower inflammatory profile and VAChT-KD decreased the number of lymphocytes and increased inflammation.

Sulfamethoxazole-trimethoprim associated with resveratrol for the treatment of toxoplasmosis in mice: Influence on the activity of enzymes involved in brain neurotransmission.

This study aimed to investigate the influence of sulfamethoxazole-trimethoprim (ST) associated with resveratrol on the enzymatic activities of acetylcholinesterase (AChE), adenylate kinase (AK), pyruvate kinase (PK), and creatine kinase (CK) in the brain of mice experimentally infected by Toxoplasma gondii. For that, 60 mice were divided into ten groups with 6 animals each: groups A to D composed by healthy mice and groups E to J consisting of animals infected by T. gondii (VEG strain). Animals started treatment 20 days post-infection for 10 consecutive days with oral doses of 0.5 mg kg(-1) of ST (groups B and F), 100 mg kg(-1) of free resveratrol (groups C and G) and inclusion complex of resveratrol (nanoparticles containing resveratrol) (groups D and H), as well as with an association of both drugs (groups I and J). The results showed increased (P < 0.001) AChE activity on infected animals (groups E-J) when compared to not-infected (A) animals, and also uninfected animals treated with ST (group B) had increased AChE activity. AK activity decreased (P < 0.001) in the infected and untreated (group E), differently from the other groups that did not differ. PK activity did not differ between groups (P > 0.05). When comparing control groups (uninfected (A) and infected (E)), we verified a significant (P < 0.001) increase in CK activity in the brain, and it is noteworthy that the animals treated with resveratrol associated with ST (group I and J) had similar CK activity to those animals from the group A. Treatment with the combination of ST and resveratrol was able to reduce (P < 0.05) the number of parasitic cysts in the brain, thus reduced inflammatory infiltrates in the liver, and prevented the occurrence of hepatocytes lesions due to toxoplasmosis in mice. Based on these results, it is possible to conclude that increased AChE and CK activities after T. gondii infection did not change with the treatment of ST-resveratrol association. In addition, decreased AK activity caused by T. gondii infection was normalized by ST-resveratrol treatment. T. gondii infection and treatment does not affect PK activity in brain.

Characterization of placental cholinesterases and activity induction associated to environmental organophosphate exposure.

Although non-innervated, the placenta contains both cholinesterases (ChEs), butyrylcholinesterase (BChE) and acetylcholinesterase (AChE). These enzymes are well-known for their multiple molecular forms. In a first approach, we used recognized specific inhibitors, substrate preferences and non-denaturating gel electrophoresis in order to characterize the ChE profile of term placenta from uncomplicated pregnancy. Results strongly suggest that the predominant cholinesterasic form present was tetrameric BChE. It is well established that both ChEs are targets of cholinesterase-inhibiting organophosphates (OP), one of the most important classes of chemicals actively applied to the environment. However, we have previously reported increased ChEs activity in placenta of rural residents exposed to OP. In the present work, we have studied: 1) whether this finding was reproducible and, 2) whether AChE or BChE up regulation is behind the increase of placental ChE activity. The population studied included forty healthy women who live in an agricultural area. Samples were collected during both the OP pulverization period (PP) and the recess period (RP). The placental ChEs activity increased in PP, evidencing reproducibility of previous results. The analysis of non-denaturating gels revealed that increased activity of total ChE activity in placenta from women exposed to OP may be attributable to tetrameric BChE up-regulation.

Altered acetylcholine release in the hippocampus of dystrophin-deficient mice.

Mild cognitive impairments have been described in one-third of patients with Duchenne muscle dystrophy (DMD). DMD is characterized by progressive and irreversible muscle degeneration caused by mutations in the dystrophin gene and lack of the protein expression. Previously, we have reported altered concentrations of α7- and ß2-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal membranes of dystrophic (mdx) mice. This suggests that alterations in the central cholinergic synapses are associated with dystrophin deficiency. In this study, we examined the release of acetylcholine (ACh) and the level of the vesicular ACh transporter (VAChT) using synaptosomes isolated from brain regions that normally have a high density of dystrophin (cortex, hippocampus and cerebellum), in control and mdx mice at 4 and 12months of age. ACh release evoked by nicotinic stimulation or K(+) depolarization was measured as the tritium outflow from superfused synaptosomes preloaded with [(3)H]-choline. The results showed that the evoked tritium release was Ca(2+)-dependent and mostly formed by [(3)H]-ACh. ß2-containing nAChRs were involved in agonist-evoked [(3)H]-ACh release in control and mdx preparations. In hippocampal synaptosomes from 12-month-old mdx mice, nAChR-evoked [(3)H]-ACh release increased by 57% compared to age-matched controls. Moreover, there was a 98% increase in [(3)H]-ACh release compared to 4-month-old mdx mice. [(3)H]-ACh release evoked by K(+) depolarization was not altered, while the VAChT protein level was decreased (19%) compared to that of age-matched controls. In cortical and cerebellar preparations, there was no difference in nAChR-evoked [(3)H]-ACh release and VAChT levels between mdx and age-matched control groups. Our previous findings and the presynaptic alterations observed in the hippocampi of 12-month-old mdx mice indicate possible dysfunction of nicotinic cholinergic synapses associated with dystrophin deficiency. These changes may contribute to the cognitive and behavioral abnormalities described in dystrophic mice and patients with DMD.

Etomidate evokes synaptic vesicle exocytosis without increasing miniature endplate potentials frequency at the mice neuromuscular junction.

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na(+) channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca(2+) because its effect was abolished in Ca(2+)-free medium and also inhibited by omega-Agatoxin IVA (30 and 200nM) suggesting the participation of P/Q-subtype Ca(2+) channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca(2+) channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent.

Long-term depression of inhibitory synaptic transmission induced by spike-timing dependent plasticity requires coactivation of endocannabinoid and muscarinic receptors.

The precise timing of pre-postsynaptic activity is vital for the induction of long-term potentiation (LTP) or depression (LTD) at many central synapses. We show in synapses of rat CA1 pyramidal neurons in vitro that spike timing dependent plasticity (STDP) protocols that induce LTP at glutamatergic synapses can evoke LTD of inhibitory postsynaptic currents or STDP-iLTD. The STDP-iLTD requires a postsynaptic Ca(2+) increase, a release of endocannabinoids (eCBs), the activation of type-1 endocananabinoid receptors and presynaptic muscarinic receptors that mediate a decreased probability of GABA release. In contrast, the STDP-iLTD is independent of the activation of nicotinic receptors, GABAB Rs and G protein-coupled postsynaptic receptors at pyramidal neurons. We determine that the downregulation of presynaptic Cyclic adenosine monophosphate/protein Kinase A pathways is essential for the induction of STDP-iLTD. These results suggest a novel mechanism by which the activation of cholinergic neurons and retrograde signaling by eCBs can modulate the efficacy of GABAergic synaptic transmission in ways that may contribute to information processing and storage in the hippocampus.

Antispasmodic effects and composition of the essential oils from two South American chemotypes of Lippia alba.

ETHOPHARMACOLOGY RELEVANCE: Lippia alba (Mill.) N. E. Brown (Verbenaceae) is an aromatic species used in Central and South America as eupeptic for indigestion. In Argentina, it is used by the "criollos" from the Chaco province. There are several chemotypes which differ in the chemical composition of the essential oils. Nowadays, it is experimentally cultivated in some countries of the region, including Argentina. AIM OF THE STUDY: To compare the chemical composition and pharmacology of the essential oils from two chemotypes: "citral" (CEO) and "linalool" (LEO), in isolated rat duodenum and ileum. METHODS: Contractile concentration-response curves (CRC) of acetylcholine (ACh) and calcium in 40mM K(+)-medium (Ca(2+)-CRC) were done in isolated intestine portions, in the absence and presence of CEO or LEO at different concentrations. RESULTS: Likewise verapamil, CEO and LEO induced a non-competitive inhibition of the ACh-CRC, with IC50 of 7.0±0.3mg CEO/mL and 37.2±4.2mg LEO/mL. l-NAME, a NO-synthase blocker, increased the IC50 of CEO to 26.1±8.7mg CEO/mL. Likewise verapamil, CEO and LEO non-competitively inhibited the Ca(2+)-CRC, with IC50 of 6.3±1.7mg CEO/mL, 7.0±2.5mg LEO/mL and 0.24±0.04mg verapamil/mL (pIC50: 6.28). CEO was proved to possess limonene, neral, geranial and (-)-carvone as the major components, while LEO was rich in linalool. CONCLUSIONS: Results suggest that CEO has five times more potency than LEO to inhibit muscarinic contractions. The essential oils of both chemotypes interfered with the Ca(2+)-influx, but with an IC50 about 28 times higher than that of verapamil. Moreover, CEO partially stimulated the NO production. These results show the medicinal usefulness of both Lippia alba chemotypes, thus validating its traditional use, potency and mechanism of action.

Phytochemical study guided by the myorelaxant activity of the crude extract, fractions and constituent from stem bark of Hymenaea courbaril L.

ETHNOPHARMACOLOGICAL RELEVANCE: Hymenaea courbaril L. (Caesalpinoideae) is used in Brazilian folk medicine to treat anemia, kidney problems, sore throat and other dysfunctions of the respiratory system, such as bronchitis and asthma, although such properties are yet to be scientifically validated. AIM OF THE STUDY: In order to give a scientific basis to support the traditional use of Hymenaea courbaril, this study was designed to evaluate antioxidant, myorelaxant and anti-inflammatory properties of the ethanol extract from stem bark and its fractions. The myorelaxant effect of astilbin, a flavonoid isolated from the bioactive ethyl acetate fraction (EAF), has also been evaluated. MATERIAL AND METHODS: In the present study ethanol extract from stem bark (EEHC) and fractions were analyzed using bioassay-guided fractionation. The following activities were investigated: antioxidant by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, myorelaxant on rat tracheal smooth muscle, and anti-inflammatory using ovalbumin-induced leukocytosis and airway hyperresponsiveness in rats. RESULTS: The results of the present investigation show that the whole extract of Hymenaea courbaril and some of its fractions strongly scavenged DPPH radical. The extract showed myorelaxant activity on rat trachea, being EAF its highest efficient fraction. Bio-guided study allowed the isolation of astilbin, a well-known flavonoid. The activity induced by this compound indicates that it may be partly responsible for the myorelaxant effect of EAF. EAF reduced contractions that depended on divalent cation inflow through voltage-operated Ca(2+) channels (VOCCs) or receptor-operated Ca(2+) channels (ROCCs), but it was more potent to inhibit VOCC- than ROCC-dependent contraction induced by Ca(2+) addition in ACh-enriched Ca(2+)-free medium. Oral pretreatment of antigen-challenged animals with EAF prevented airway hyperresponsiveness on KCl-induced contraction and reduced the number of total white cells, particularly eosinophils and neutrophils in bronchoalveolar lavage. CONCLUSIONS: This study provided scientific basis that Hymenaea courbaril presents potential antioxidant, myorelaxant and anti-inflammatory actions, which support its use in folk medicine to treat inflammatory airway diseases.