Recent in vitro evidence indicates that astrocytes can modulate synaptic plasticity by releasing neuroactive substances (gliotransmitters). However, whether gliotransmitter release from astrocytes is necessary for higher brain function in vivo , particularly for memory, as well as the contribution of connexin (Cx) hemichannels to gliotransmitter release, remain elusive. Here, we microinfused into the rat basolateral amygdala (BLA) TAT‐Cx43L2, a peptide that selectively inhibits Cx43‐hemichannel opening while maintaining synaptic transmission or interastrocyte gap junctional communication. In vivo blockade of Cx43 hemichannels during memory consolidation induced amnesia for auditory fear conditioning, as assessed 24 h after training, without affecting short‐term memory, locomotion, or shock reactivity. The amnesic effect was transitory, specific for memory consolidation, and was confirmed after microinfusion of Gap27, another Cx43‐hemichannel blocker. Learning capacity was recovered after coinfusion of TAT‐Cx43L2 and a mixture of putative gliotransmitters (glutamate, glutamine, lactate, d ‐serine, glycine, and ATP). We propose that gliotransmitter release from astrocytes through Cx43 hemichannels is necessary for fear memory consolidation at the BLA. Thus, the present study is the first to demonstrate a physiological role for astroglial Cx43 hemichannels in brain function, making these channels a novel pharmacological target for the treatment of psychiatric disorders, including post‐traumatic stress disorder.—Stehberg, J., Moraga‐Amaro, R., Salazar, C., Becerra, A., Echeverría, C., Orellana, J. A., Bultynck, G., Ponsaerts, R., Leybaert, L., Simon, F., Sáez, J. C., Retamal, M. A. Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala. FASEB J. 26, 3649–3657 (2012). www.fasebj.org

Background Endothelial dysfunction is a crucial step in the pathogenesis of cardiovascular diseases. Reactive oxygen species (ROS) generated in response to lipopolysaccharide (LPS) during sepsis promotes progressive endothelial failure. Typically, LPS-stimulated leukocytes produce pro-inflammatory cytokines, which trigger endothelial ROS production through NAD(P)H oxidase (Nox) activation, in a process that takes hours. Noteworthy, endothelial cells exposed to LPS may also generate ROS in just a few minutes. However, the mechanisms underlying this early event and its deleterious effect in endothelial function are unknown. Here, we investigated the mechanisms of early LPS-induced ROS generation and its effect in endothelial cell viability. Methods Human umbilical vein endothelial cells were exposed to LPS for 1–40 min to study ROS generation, cytokines expression, and signaling transduction by confocal microscopy, real-time PCR (RT-PCR), western blot, and immunoprecipation. Fourty-eight hour treatments were used to determine cell death by MTT assay, cell counting, and flow cytometry. Contribution of specific Nox isoform was evaluated using a siRNAs approach. Results LPS rapidly evoked a cytokine-independent ROS production, eliciting a rapid increase in p47^phox phosphorylation by a phospholipase C/conventional protein kinase C and PI3-K signaling. It is noteworthy that the early LPS-induced ROS production triggered significant endothelial necrosis, which was prevented by a previous, but not a posterior, antioxidant treatment. The early LPS-induced ROS production as well as endothelial necrosis was totally dependent of Nox2 and Nox4 activity. Conclusion Endothelial cells exposure to LPS triggers an early ROS production. Remarkably, this single early ROS production is enough to generate extensive endothelial cell death by necrosis dependent on the activity of Nox2 and Nox4. Because, in sepsis, ROS production can cause endothelial dysfunction, results here provided may be relevant when considering the development of strategies for sepsis therapy.

Skeletal muscle atrophy is a pathological condition mainly characterized by a loss of muscular mass and the contractile capacity of the skeletal muscle as a consequence of muscular weakness and decreased force generation. Cachexia is defined as a pathological condition secondary to illness characterized by the progressive loss of muscle mass with or without loss of fat mass and with concomitant diminution of muscle strength. The molecular mechanisms involved in cachexia include oxidative stress, protein synthesis/degradation imbalance, autophagy deregulation, increased myonuclear apoptosis, and mitochondrial dysfunction. Oxidative stress is one of the most common mechanisms of cachexia caused by different factors. It results in increased ROS levels, increased oxidation‐dependent protein modification, and decreased antioxidant system functions. In this review, we will describe the importance of oxidative stress in skeletal muscles, its sources, and how it can regulate protein synthesis/degradation imbalance, autophagy deregulation, increased myonuclear apoptosis, and mitochondrial dysfunction involved in cachexia.

Endothelial dysfunction is decisive in the progression of cardiovascular diseases. Lipopolysaccharide (LPS)-induced reactive oxygen species (ROS)-mediated endothelial cell death is a main feature observed in inflammation secondary to endotoxaemia, emerging as a leading cause of death among critically ill patients in intensive care units. However, the molecular mechanism underlying LPS-induced endothelial cell death is not well understood. Transient receptor protein melastatin 4 (TRPM4) is an ion channel associated with cell death that is expressed in endothelium and modulated by ROS. Here, we investigate the role of TRPM4 in LPS-induced endothelial cell death, testing whether suppression of the expression of TRPM4 confers endothelial cell resistance to LPS challenge. Using primary cultures of human umbilical vein endothelial cells (HUVEC), we demonstrate that TRPM4 is critically involved in LPS-induced endothelial cell death. HUVEC exposed to LPS results in Na⁺-dependent cell death. Pharmacological inhibition of TRPM4 with 9-phenanthrol or glibenclamide protects endothelium against LPS exposure for 48 h. Furthermore, TRPM4-like currents increase in cells pre-treated with LPS and inhibited with glibenclamide. Of note, suppression of TRPM4 expression by siRNA or suppression of its activity in a dominant negative mutant is effective in decreasing LPS-induced endothelial cell death when cells are exposed to LPS for 24–30 h. TRPM4 is critically involved in LPS-induced endothelial cell death. These results demonstrate that either pharmacological inhibition of TRPM4, suppression of TRPM4 expression, or inhibition of TRPM4 activity are able to protect endothelium against LPS injury. These results are useful in sepsis drug design and development of new strategies for sepsis therapy.

Endothelial dysfunction is crucial in endotoxaemia‐derived sepsis syndrome pathogenesis. It is well accepted that lipopolysaccharide (LPS) induces endothelial dysfunction through immune system activation. However, LPS can also directly generate actions in endothelial cells (ECs) in the absence of participation by immune cells. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge. However, the mechanism that confers endothelial resistance to LPS is not known. LPS‐resistant ECs exhibit a fibroblast‐like morphology, suggesting that these ECs enter a fibrotic programme in response to LPS. Thus, our aim was to investigate whether LPS is able to induce endothelial fibrosis in the absence of immune cells and explore the underlying mechanism. Using primary cultures of ECs and culturing intact blood vessels, we demonstrated that LPS is a crucial factor to induce endothelial fibrosis. We demonstrated that LPS was able and sufficient to promote endothelial fibrosis, in the absence of immune cells through an activin receptor–like kinase 5 (ALK5) activity–dependent mechanism. LPS‐challenged ECs showed an up‐regulation of both fibroblast‐specific protein expression and extracellular matrix proteins secretion, as well as a down‐regulation of endothelial markers. These results demonstrate that LPS is a crucial factor in inducing endothelial fibrosis in the absence of immune cells through an ALK5‐dependent mechanism. It is noteworthy that LPS‐induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxaemia‐derived sepsis syndrome and other inflammatory diseases.