Physiologically induced restructuring of focal adhesions causes mobilization of vinculin by a vesicular endocytic recycling pathway

AbstractIn epithelial cells, vinculin is enriched in cell adhesion structures but is in equilibrium with a large cytosolic pool. It is accepted that when cells adhere to the extracellular matrix, a part of the soluble cytosolic pool of vinculin is recruited to specialized sites on the plasma membrane called focal adhesions (FAs) by binding to plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We have previously shown that bradykinin (BK) induces both a reversible dissipation of vinculin from FAs, by the phospholipase C (PLC)-mediated hydrolysis of PtdIns(4,5)P2, and the concomitant internalization of vinculin. Here, by using an immunomagnetic method, we isolated vinculin-containing vesicles induced by BK stimulation. By analyzing the presence of proteins involved in vesicle traffic, we suggest that vinculin can be delivered in the site of FA reassembly by a vesicular endocytic recycling pathway. We also observed the formation of vesicle-like structures containing vinculin in the cytosol of cells treated with lipid membrane-affecting agents, which caused dissipation of FAs due to their deleterious effect on membrane microdomains where FAs are inserted. However, these vesicles did not contain markers of the recycling endosomal compartment. Vinculin localization in vesicles has not been reported before, and this finding challenges the prevailing model of vinculin distribution in the cytosol. We conclude that the endocytic recycling pathway of vinculin could represent a physiological mechanism to reuse the internalized vinculin to reassembly new FAs, which occurs after long time of BK stimulation, but not after treatment with membrane-affecting agents

Nuevo ligando con acción alostérica sobre el receptor nicotínico de Torpedo californica

The nicotinic acetylcholine receptor belongs to a superfamily of ion channel receptors\ntriggered by ligand, also known as cystine-loop receptors. It is widely distributed in\nnature; in mammalian the neuronal subtype is present at the cholinergic synapses of the\nperipheral and central nervous systems whereas muscle subtype is located at the\nneuromuscular junction. Alterations in nicotinic receptor subtypes cause a variety of\npathologies in humans: myasthenia gravis, Alzheimer's disease, certain types of epilepsy,\netc. Currently, there are no drugs which are allosteric modulators of the receptor and\nused in therapeutics.\nIn this work we studied its interaction with a ligand called AC4-ASA, with properties\nof allosteric modulator of the nicotinic receptor, in order to establish its binding\ndeterminants. We used the photo-affinity labeling strategy, followed by a mass\nspectrometry study.\nAC4-ASA binds to two sites at the ?/? interface, one site in ?/? and two other sites\nlocated on the ? subunit. We propose binding models for the interaction sites with the\nreceptor, as well as a possible mechanism of modulation of receptor affinity for nicotine.Fil: Paván, Carlos Humberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, ArgentinaEl receptor nicotínico es un receptor ionotrópico de la superfamilia de canales iónicos\ngatillados por ligando, ampliamente distribuido en la naturaleza. En los mamíferos el\nsubtipo neuronal está presente en las sinapsis colinérgicas del sistema nervioso central y\ndel periférico mientras que el subtipo muscular se encuentra en la placa neuromuscular.\nLas alteraciones del receptor nicotínico ocasionan una amplia variedad de patologías:\nmiastenia gravis, enfermedad de Alzheimer, epilepsia, etc. Actualmente no se dispone\nde drogas con propiedades de modulación alostérica sobre este receptor útiles en\ntratamientos farmacológicos.\nEl presente trabajo estudia su interacción con un ligando que tiene propiedades de\nmodulador alostérico para el receptor nicotínico, denominado AC4-ASA; para ello, se\nutilizó la estrategia de marcación por fotoafinidad seguida de espectrometría de masas;\ncomplementariamente, se modeló su interacción mediante docking molecular.\nSe encontró que el AC4-ASA se une a un sitio en la interfase ?/?, a dos en la ?/? y a\notros dos sitios sobre ?, de modo que el ligando posee más de un sitio de interacción y se\nconfirma su capacidad de unión al sitio ortostérico. Del análisis de los sitios de unión se\npropone un mecanismo de modulación de la afinidad por la nicotina

Chalcone derivatives: synthesis, in vitro and in vivo evaluation of their anti-anxiety, anti-depression and analgesic effects

Anxiety disorders, depression and pain are highly prevalent pathologies. Their pharmacotherapy is associated with unwanted side effects; hence there is a clinical need to develop more effective drugs with fewer adverse reactions.Chalcones are one of the major classes of naturally occurring compounds. Chalcones and their derivatives have a huge importance in medicinal chemistry, displaying a wide range of pharmacological activities including anti-inflammatory, antimicrobial, antioxidant, cytotoxic and antitumor actions.The aim of this work was to evaluate chalcone effects on different targets involved in these pathologies. We have synthesized a series of simple chalcone derivatives taking common structural requirements described in literature related to their anxiolytic-like, antidepressant-like and/or antinociceptive properties into account.Furthermore, their potential in vitro effects towards different targets involved in these pathologies were evaluated. We have obtained twenty chalcones with moderate to high yields and assessed their ability to bind distinctive receptors, from rat brain homogenates, by displacement of labelled specific ligands: [3H] FNZ (binding site of benzodiazepines/GABAA), [3H] 8-OH-DPAT (serotonin 5-HT1A) and [3H] DAMGO (μ-opioid). Those compounds that showed the better in vitro activities were evaluated in mice using different behavioural tasks. In vivo results showed that 5′-methyl-2′-hydroxychalcone (9) exerted anxiolytic-like effects in mice in the plus maze test. While chalcone nuclei (1) revealed antidepressant-like activities in the tail suspension test. In addition, the novel 5′-methyl-2′-hydroxy-3′-nitrochalcone (12) exhibited antinociceptive activity in acute chemical and thermal nociception tests (writhing and hot plate tests). In conclusion, chalcones are thus promising compounds for the development of novel drugs with central nervous system (CNS) actions