Search

The role of liver X receptors (LXR) in rheumatoid arthritis (RA) remains controversial. We studied the effect of LXR agonists on fibroblast‐like synoviocytes (FLS) from RA patients and the K/BxN arthritis model in LXRα and β double‐deficient (Nr1h2/3−/−) mice. Two synthetic LXR agonists, GW3965 and T0901317, were used to activate LXRs and investigate their effects on cell growth, proliferation and matrix metalloproteinases, and chemokine production in cultured FLS from RA patients. The murine model K/BxN serum transfer of inflammatory arthritis in Nr1h2/3−/− animals was used to investigate the role of LXRs on joint inflammation in vivo. LXR agonists inhibited the FLS proliferative capacity in response to TNF, the chemokine‐induced migration, the collagenase activity in FLS supernatant and FLS CXCL12 production. In the K/BxN mouse model, Nr1h2/3−/− animals showed aggravated arthritis, histological inflammation, and joint destruction, as well as an increase in synovial metalloproteases and expression of proinflammatory mediators such as IL‐1β and CCL2 in joints compared with wild type animals. Taken together, these data underscore the importance of LXRs in modulating the joint inflammatory response and highlight them as potential therapeutic targets in RA. [ABSTRACT FROM AUTHOR]

Adrenal chromaffin cells release epinephrine (EPI) and norepinephrine (NE) into the bloodstream as part of the homeostatic response to situations like stress. Here we utilized EPI‐deficient mice generated by knocking out (KO) the phenylethanolamine N‐methyltransferase (Pnmt) gene. These Pnmt‐KO mice were bred to homozygosis but displayed no major phenotype. The lack of EPI was partially compensated by an increase in NE, suggesting that EPI storage was optimized in adrenergic cells. Electron microscopy showed that despite the lack of EPI, chromaffin granules retain their shape and general appearance. This indicate that granules from adrenergic or noradrenergic cells preserve their characteristics even though they contain only NE. Acute insulin injection largely reduced the EPI content in wild‐type animals, with a minimal reduction in NE, whereas there was only a partial reduction in NE content in Pnmt‐KO mice. The analysis of exocytosis by amperometry revealed a reduction in the quantum size (−30%) and Imax (−21%) of granules in KO cells relative to the wild‐type granules, indicating a lower affinity of NE for the granule matrix of adrenergic cells. As amperometry cannot distinguish between adrenergic or noradrenergic cells, it would suggest even a larger reduction in the affinity for the matrix. Therefore, our results demonstrate that adrenergic cells retain their structural characteristics despite the almost complete absence of EPI. Furthermore, the chromaffin granule matrix from adrenergic cells is optimized to accumulate EPI, with NE being a poor substitute. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/ [ABSTRACT FROM AUTHOR]

Abstract: Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca2+, although the mechanisms mediating Ca2+ uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca2+, are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca2+, dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca2+. We will also address the potential role of vesicular Ca2+ in two major processes in secretory cells, vesicle movement and exocytosis. [Copyright &y& Elsevier]

Chromogranins are the main soluble proteins in the large dense core secretory vesicles (LDCVs) found in aminergic neurons and chromaffin cells. We recently demonstrated that chromogranins A and B each regulate the concentration of adrenaline in chromaffin granules and its exocytosis. Here we have further studied the role played by these proteins by generating mice lacking both chromogranins. Surprisingly, these animals are both viable and fertile. Although chromogranins are thought to be essential for their biogenesis, LDCVs were evident in these mice. These vesicles do have a somewhat atypical appearance and larger size. Despite their increased size, single-cell amperometry recordings from chromaffin cells showed that the amine content in these vesicles is reduced by half. These data demonstrate that although chromogranins regulate the amine concentration in LDCVs, they are not completely essential, and other proteins unrelated to neurosecretion, such as fibrinogen, might compensate for their loss to ensure that vesicles are generated and the secretory pathway conserved. [ABSTRACT FROM AUTHOR]

Single-cell amperometry is a powerful technique that permits the detection of electrochemically active transmitters, such as catecholamines, histamine, or serotonin, released by exocytosis from secretory cells.Amperometry has two main characteristics that make it ideal for the study of exocytosis at the single-cell level with single-vesicle resolution quantal release. (i) It is noninvasive. The carbon fiber microelectrode can be carefully positioned on plasma membrane of a single cell, allowing the detection of the oxidation current of the secreted molecules. (ii) High temporal resolution and sensitivity. Exocytosis can be monitored with a real-time resolution that allows the determination of the kinetics release with an attomol detection sensitivity, which ensures an accurate calculation of the amount of transmitter released.Here, we compile some recommendations and advices to perform amperometry quantal analysis., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Viruses have developed different survival strategies in host cells by crossing cell-membrane compartments, during different steps of their viral life cycle. In fact, the non-regenerative viral membrane of enveloped viruses needs to encounter the dynamic cell-host membrane, during early steps of the infection process, in which both membranes fuse, either at cell-surface or in an endocytic compartment, to promote viral entry and infection. Once inside the cell, many viruses accomplish their replication process through exploiting or modulating membrane traffic, and generating specialized compartments to assure viral replication, viral budding and spreading, which also serve to evade the immune responses against the pathogen. In this review, we have attempted to present some data that highlight the importance of membrane dynamics during viral entry and replicative processes, in order to understand how viruses use and move through different complex and dynamic cell-membrane structures and how they use them to persist.