Aedes aegypti salivary gland extract alleviates acute itching by blocking TRPA1 channels.

Aedes aegypti (Ae. aegypti) saliva induces a variety of anti-inflammatory and immunomodulatory activities. Interestingly, although it is known that mosquito bites cause allergic reactions in sensitised hosts, the primary exposure of humans to Ae. aegypti does not evoke significant itching. Whether active components in the saliva of Ae. aegypti can counteract the normal itch reaction to injury produced by a histaminergic or non-histaminergic pathway in vertebrate hosts is unknown. This study investigated the effects of Ae. aegypti mosquito salivary gland extract (SGE) on sensitive reactions such as itching and associated skin inflammation. Acute pruritus and plasma extravasation were induced in mice by the intradermal injection of either compound 48/80 (C48/80), the Mas-related G protein-coupled receptor (Mrgpr) agonist chloroquine (CQ), or the transient receptor potential ankyrin 1 (TRPA1) agonist allyl isothiocyanate (AITC). The i.d. co-injection of Ae. aegypti SGE inhibited itching, plasma extravasation, and neutrophil influx evoked by C48/80, but it did not significantly affect mast cell degranulation in situ or in vitro. Additionally, SGE partially reduced CQ- and AITC-induced pruritus in vivo, suggesting that SGE affects pruriceptive nerve firing independently of the histaminergic pathway. Activation of TRPA1 significantly increased intracellular Ca2+ in TRPA-1-transfected HEK293t lineage, which was attenuated by SGE addition. We showed for the first time that Ae. aegypti SGE exerts anti-pruriceptive effects, which are partially regulated by the histamine-independent itch TRPA1 pathway. Thus, SGE may possess bioactive molecules with therapeutic potential for treating nonhistaminergic itch.

Long-Term Culture of Mouse EmbryonicStem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neuronsfunctional properties and features, have been developed. Most of these protocols are short lasting, which,therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describehere a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during3 months under several splitting...

Long-term culture of mouse embryonic stem cell-derived adherent neurospheres and functional neurons.

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

Effects of the blockade of high voltage-activated calcium channels on in vitro pineal melatonin synthesis.

The presence of high voltage-activated calcium channels in the rat pineal gland is well known. However, their role in pineal metabolism is not completely understood and is even controversial. Better to understand this matter, we investigated the effects of L-, N- or P/Q-type calcium channel blockers (nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, respectively) on melatonin content and arylalkylamine-N-acetyltransferase activity of denervated rat pineal glands kept for 48 h in culture and stimulated with norepinephrine. Melatonin was measured by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase activity was quantified by radiometric assay. Pre-incubation with any of these high voltage-activated calcium channel blockers reduced the melatonin production induced by norepinephrine although arylalkylamine-N-acetyltransferase activity was reduced only by the N-type calcium channel antagonist, omega-conotoxin GVIA. The results indicate that calcium influx through L-, N- or P/Q-type of high voltage-activated calcium channels is necessary for the full expression of the metabolic process leading to melatonin synthesis in the rat pineal glands. However, the mechanisms involved in this process are different for the L- or P/Q- and N-type calcium channels.

Electrophysiology of cardiac myocytes of Aplysia brasiliana.

The electrical properties of Aplysia brasiliana myogenic heart were evaluated. Two distinct types of action potentials (APs) were recorded from intact hearts, an AP with a slow rising phase followed by a slow repolarizing phase and an AP with a 'fast' depolarizing phase followed by a plateau. Although these two APs differ in their rates of depolarization (2.2 x 0.3 V/s), both APs were abolished by the addition of Co2+, Mn2+ and nifedipine or by omitting Ca2+ from the external solution. These data suggest that a Ca2+ inward current is responsible for the generation of both types of APs. Two outward currents activated at -40 mV membrane potential were prominent in isolated cardiac myocytes: a fast activating, fast inactivating outward current similar to the A-type K+ current and a slow activating outward current with kinetics similar to the delayed rectifier K+ current were recorded under voltage clamp conditions. Based on the effects of 4-AP and TEA on the electrical properties of ventricular myocytes, we suggest that the fast kinetic outward current substantially attenuates the peak values of the APs and that the slow activating outward current is involved on membrane repolarization.

Filtered load of buffer and renal H-ion secretion: mechanism of proximal tubule load dependence

Cuando la carga filtrada de "buffers" como bicarbonato o fosfato es incrementada por elevación del FG o de la concentración plasmática de "buffer", la reabsorción global de bicarbonato o la formación de acidez titulable son estimuladas. Lo mismo ocurre con la reabsorción proximal de bicarbonato durante la perfusión con concentraciones crecientes de este ion o cuando se eleva el flujo de fluido. En los últimos años, hemos estudiado los mecanismos de esta dependencia funcional. Observamos que el ritmo de reabsorción de bicarbonato es siempre proporcional a la concentración luminal de "buffer" cuando se inyecta una columna estacionaria de fluido en la luz tubular. La secreción de H**+ es tambiénn proporcional a nivels luminales de otros "buffers" distintos al bicarbonato. Empleando la técnica de pH-stat adaptada a túbulos renales, demostramos que la secreción proxim de H**+ depende del pH luminal y es independiente del sistema de "buffer" utilizado. Un análisis cinético de estos datos demuestra una relación no linear entre pH luminal y secreción de H**+, compatible con transporte mediado por "carrier"

Filtered load of buffer and renal H-ion secretion: mechanism of proximal tubule load dependence

Cuando la carga filtrada de "buffers" como bicarbonato o fosfato es incrementada por elevación del FG o de la concentración plasmática de "buffer", la reabsorción global de bicarbonato o la formación de acidez titulable son estimuladas. Lo mismo ocurre con la reabsorción proximal de bicarbonato durante la perfusión con concentraciones crecientes de este ion o cuando se eleva el flujo de fluido. En los últimos años, hemos estudiado los mecanismos de esta dependencia funcional. Observamos que el ritmo de reabsorción de bicarbonato es siempre proporcional a la concentración luminal de "buffer" cuando se inyecta una columna estacionaria de fluido en la luz tubular. La secreción de H**+ es tambiénn proporcional a nivels luminales de otros "buffers" distintos al bicarbonato. Empleando la técnica de pH-stat adaptada a túbulos renales, demostramos que la secreción proxim de H**+ depende del pH luminal y es independiente del sistema de "buffer" utilizado. Un análisis cinético de estos datos demuestra una relación no linear entre pH luminal y secreción de H**+, compatible con transporte mediado por "carrier" (AU)