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Abstract: Previous studies have shown that nitric oxide (NO) inhibits apoptosis of retinal neurons in culture through the canonical cyclic GMP/protein kinase G (PKG)-dependent pathway, but also involving multiple kinase pathways, such as phosphatidylinositol 3′ kinase (PI3k) and AKT. NO and AKT exhibit survival-promoting properties and display important roles in both CNS development and plasticity. The purpose of this study was to evaluate the effects of exogenous NO, derived from the NO donor S-nitroso-N-acetylpenicillamin (SNAP), or endogenous NO, produced from l-arginine, on AKT phosphorylation in cultured chick retinal neurons. Our results demonstrate that SNAP or l-arginine enhances AKT phosphorylation on both serine-473 and threonine-308 residues in a concentration and time-dependent manner. This effect was mediated by the activation of soluble guanylyl cyclase and PKG, since it was blocked by the respective enzyme inhibitors ODQ or LY83583 and KT5823, as well as by transduction with shRNA lentiviruses coding PKGII shRNA, and mimicked by the respective enzyme activators YC-1 and 8-Bromo cyclic GMP, and also by the cyclic GMP phosphodiesterase inhibitor zaprinast. In addition, LY294002 or wortmannin suppressed the SNAP effect, indicating the involvement of phosphoinositide 3′ kinase. Moreover, the mTOR inhibitor KU0063794 blocked SNAP-induced AKT phosphorylation at both residues, suggesting the participation of the mTORC2 complex in the process. Glutamate and NMDA also promoted AKT phosphorylation and a nitric oxide synthase inhibitor abrogated these effects, revealing a mechanism involving the activation of NMDA receptors and NO production. We have also found that SNAP and l-arginine induced AKT translocation into the nucleus of retinal neurons as well as other neuronal cell lines. SNAP also protects retinal cells from death induced by hydrogen peroxide and this effect was blocked by the phosphoinositide 3′ kinase inhibitor LY294002. We therefore conclude that NO produced from endogenous or exogenous sources promotes AKT activation and its shuttling to the nucleus, probably participating in neuronal survival pathways important during CNS development. [Copyright &y& Elsevier]

Abstract: In this study, we evaluated the effects of PhKv, a 4584 Da peptide isolated from the spider Phoneutria nigriventer venom, in the isolated rat heart and in isolated ventricular myocytes. Ventricular arrhythmias were induced by occlusion of the left anterior descending coronary artery for 15 min followed by 30 min of reperfusion. Administration of native PhKv (240 nM) 1 min before or after reperfusion markedly reduced the duration of arrhythmias. This effect was blocked by atropine, thereby indicating the participation of muscarinic receptors in the antiarrhythmogenic effect of PhKv. Notably, recombinant PhKv (240 nM) was also efficient to attenuate the arrhythmias (3.8 ± 0.9 vs. 8.0 ± 1.2 arbitrary units in control group). Furthermore, PhKv induced a significant reduction in heart rate. This bradycardia was partially blunted by atropine and potentiated by pyridostigmine. To further evaluate the participation of acetylcholine on the PhKv effects, we examined the release of this neurotransmitter from neuromuscular junctions. It was found that Phkv (200 nM) significantly increased the release of acetylcholine in this preparation. Moreover, PhKv (250 nM) did not cause any significant change in action potential or Ca2+ transient parameters in isolated cardiomyocytes. Altogether, these findings show an important acetylcholine-mediated antiarrhythmogenic effect of the spider PhKv toxin in isolated hearts. [Copyright &y& Elsevier]

Abstract: The purpose of the present work was to investigate the pharmacological action of a calcium channel-blocking toxin from the venom of the spider Phonetic nigriventer, Tx3-4 on calcium channels coupled to exocytosis of synaptic vesicles. Tx3-4 blocked KCl-induced exocytosis of synaptic vesicles with an IC50 of 1.1nM. To investigate whether the target of Tx3-4 overlaps with known calcium channels that mediate calcium entry and exocytosis, we used ω-toxins that interact selectively with neuronal calcium channels. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-4 is P/Q calcium channels. In conclusion, Tx3-4 is a potent inhibitor of calcium channels involved in the KCl-induced exocytosis of synaptic vesicles in brain cortical synaptosomes. [Copyright &y& Elsevier]

The cholinergic anti-inflammatory pathway has been shown to regulate lung inflammation and cytokine release in acute models of inflammation, mainly via α7 nicotinic receptor (α7nAChR). We aimed to evaluate the role of endogenous acetylcholine in chronic allergic airway inflammation in mice and the effects of therapeutic nAChR stimulation in this model. We first evaluated lung inflammation and remodeling on knock-down mice with 65% of vesicular acetylcholine transport (VAChT) gene reduction (KDVAChT) and wild-type(WT) controls that were subcutaneously sensitized and then inhaled with ovalbumin(OVA). We then evaluated the effects of PNU-282987(0.5-to-2mg/kg),(α7nAChR agonist) treatment in BALB/c male mice intraperitoneal sensitized and then inhaled with OVA. Another OVA-sensitized-group was treated with PNU-282987 plus Methyllycaconitine (MLA,1 mg/kg, α7nAChR antagonist) to confirm that the effects observed by PNU were due to α7nAChR. We showed that KDVAChT-OVA mice exhibit exacerbated airway inflammation when compared to WT-OVA mice. In BALB/c, PNU-282987 treatment reduced the number of eosinophils in the blood, BAL fluid, and around airways, and also decreased pulmonary levels of IL-4,IL-13,IL-17, and IgE in the serum of OVA-exposed mice. MLA pre-treatment abolished all the effects of PNU-282987. Additionally, we showed that PNU-282987 inhibited STAT3-phosphorylation and reduced SOCS3 expression in the lung. These data indicate that endogenous cholinergic tone is important to control allergic airway inflammation in a murine model. Moreover, α7nAChR is involved in the control of eosinophilic inflammation and airway remodeling, possibly via inhibition of STAT3/SOCS3 pathways. Together these data suggest that cholinergic anti-inflammatory system mainly α7nAChR should be further considered as a therapeutic target in asthma. [ABSTRACT FROM AUTHOR]