A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes

Producción CientíficaSecretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2- EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange, neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH3 cells. We find that all the acidic dyes labelled the same population of vesicles. However, that population was largely different from the one labelled by the targeted proteins, with very little colocalization among them, in all the cell types studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles are not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide (GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting that they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle dynamics is studied using these techniques

Ca2+ Dynamics in the Secretory Vesicles of Neurosecretory PC12 and INS1 Cells

Producción CientíficaWe have investigated the dynamics of the free [Ca2+] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca2+-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca2+] ([Ca2+]SG] was around 20–40 lM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca2+ pumps with thapsigargin largely blocked Ca2+ uptake by the granules in Ca2+-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca2+ pump inhibitor benzohydroquinone induced a rapid release of Ca2+ from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca2+ pumps is essential to maintain the steady-state [Ca2+]SG. Both inositol 1,4, 5-trisphosphate (InsP3) and caffeine produced a rapid Ca2+ release from the granules, suggesting the presence of InsP3 and ryanodine receptors in the granules. The response to high-K+ depolarization was different in both cell types, a decrease in [Ca2+]SG in PC12 cells and an increase in [Ca2+]SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca2+]SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca2+ through SERCA-type Ca2+ pumps and can release it through InsP3 and ryanodine receptors, supporting the hypothesis that secretory granule Ca2+ may be released during cell stimulation and contribute to secretion

Plan de seguimiento y mejora de la rúbrica de evaluación de los Trabajos Fin de Grado del área de mecánica de medios continuos y teoría de estructuras

Memoria ID-0231. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2014-2015

Repositorio de material docente para el desarrollo de trabajos de fin de grado en el área de mecánica de medios continuos y teoría de estructuras

Memoria ID-0061. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2016-2017

The mitochondrial Na+/Ca2+ exchanger plays a key role in the control of cytosolic Ca2+ oscillations

Producción CientíficaThere is increasing evidence that mitochondria play an important role in the control of cytosolic Ca2+ signaling. We show here that the main mitochondrial Ca2+-exit pathway, the mitochondrial Na+/Ca2+ exchanger, controls the pattern of cytosolic Ca2+ oscillations in nonexcitable cells. In HeLa cells, the inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca2+] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na+/Ca2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca2+ release and in that way controls cytosolic Ca2+ oscillations

Calcium dynamics in catecholamine-containing secretory vesicles

Producción CientíficaWe have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca2+] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca2+] around 40 M. Cell stimulation with either ATP, caffeine or high-K+ depolarization increased cytosolic [Ca2+] and decreased secretory granule [Ca2+] ([Ca2+]SG). Inositol-(1,4,5)- trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca2+ from the granules. Changes in cytosolic [Na+] (0–140 mM) or [Ca2+] (0–10 M) did not modify either ([Ca2+]SG). Instead, [Ca2+]SG was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca2+]SG, while cytosolic alcalinization reversibly increased [Ca2+]SG. These results are consistent with the operation of a H+/Ca2+ antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K+ on [Ca2+]SG, because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca2+]SG was reversible in 10–15 min even in the absence of cytosolic Ca2+ or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca2+ buffer. This large store buffers [Ca2+]SG changes in the long-term but allows highly dynamic free [Ca2+]SG changes to occur in seconds or minutes

Diseño coordinado de guiones de prácticas para el laboratorio de Mecánica de Medios Continuos y Teoría de Estructuras de la Escuela Politécnica Superior de Zamora

Memoria ID12-0057. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2012-2013

SERCA inhibition improves lifespan and healthspan in a chemical model of Parkinson disease in Caenorhabditis elegans

Introduction: The high prevalence of neurodegenerative diseases in our population and the lack of effective treatments encourage the search for new therapeutic targets for these pathologies. We have recently described that submaximal inhibition of the Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA), the main responsible for ER calcium storage, is able to increase lifespan in Caenorhabditis elegans worms by mechanisms involving mitochondrial metabolism and nutrient-sensitive pathways.Methods: We have studied here the effects of submaximal SERCA inhibition in a chemical model of Parkinson’s disease (PD) induced in C. elegans worms by treatment with the mitochondrial complex I inhibitor rotenone. For specific SERCA inhibition, we treated worms with RNAi against sca-1, the sole orthologue of SERCA in C. elegans.Results and Discussion: Our results show that rotenone produces alterations in worms that include decreased lifespan, smaller size, reduced fertility, decreased motility, defecation and pumping rate, increased mitochondrial ROS production, reduced mitochondrial membrane potential and oxygen consumption rate, altered mitochondrial structure, and altered ethanol preference in behavioral studies. Most of these alterations were either fully or partially reversed in worms treated with sca-1 RNAi, suggesting that SERCA inhibition could be a novel pharmacological target in the prevention or treatment of neurodegeneration

Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules

Producción CientíficaThe secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50–100 lm Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular [Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+ during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.2015-09-1

Gut-derived short-chain fatty acids modulate skin barrier integrity by promoting keratinocyte metabolism and differentiation

Barrier integrity is central to the maintenance of healthy immunological homeostasis. Impaired skin barrier function is linked with enhanced allergen sensitization and the development of diseases such as atopic dermatitis (AD), which can precede the development of other allergic disorders, for example, food allergies and asthma. Epidemiological evidence indicates that children suffering from allergies have lower levels of dietary fibre-derived short-chain fatty acids (SCFA). Using an experimental model of AD-like skin inflammation, we report that a fermentable fibre-rich diet alleviates systemic allergen sensitization and disease severity. The gut-skin axis underpins this phenomenon through SCFA production, particularly butyrate, which strengthens skin barrier function by altering mitochondrial metabolism of epidermal keratinocytes and the production of key structural components. Our results demonstrate that dietary fibre and SCFA improve epidermal barrier integrity, ultimately limiting early allergen sensitization and disease development. The Graphical Abstract was designed using Servier Medical Art images (https://smart.servier.com). [Image: see text