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S100B, a homodimeric Ca 2+ -binding protein, is produced and secreted by astrocytes, and its extracellular levels have been used as a glial marker in brain damage and neurodegenerative and psychiatric diseases; however, its mechanism of secretion is elusive. We used primary astrocyte cultures and calcium measurements from real-time fluorescence microscopy to investigate the role of intracellular calcium in S100B secretion. In addition, the dimethyl sulfoxide (DMSO) effect on S100B was investigated in vitro and in vivo using Wistar rats. We found that DMSO, a widely used vehicle in biological assays, is a powerful S100B secretagogue, which caused a biphasic response of Ca 2+ mobilization. Our data show that astroglial S100B secretion is triggered by the increase in intracellular Ca 2+ and indicate that this increase is due to Ca 2+ mobilization from the endoplasmic reticulum. Also, blocking plasma membrane Ca 2+ channels involved in the Ca 2+ replenishment of internal stores decreased S100B secretion. The DMSO-induced S100B secretion was confirmed in vivo and in ex vivo hippocampal slices. Our data support a nonclassic vesicular export of S100B modulated by Ca 2+ , and the results might contribute to understanding the mechanism underlying the astroglial release of S100B.

α-Synuclein is the major component of neuronal cytoplasmic aggregates called Lewy bodies, the main pathological hallmark of Parkinson disease. Although neurons are the predominant cells expressing α-synuclein in the brain, recent studies have demonstrated that primary astrocytes in culture also express α-synuclein and regulate α-synuclein trafficking. Astrocytes have a neuroprotective role in several detrimental brain conditions; we therefore analyzed the effects of the overexpression of wild-type α-synuclein and its A30P and A53T mutants on autophagy and apoptosis. We observed that in immortalized astrocyte cell lines, overexpression of α-synuclein proteins promotes the decrease of LC3-II and the increase of p62 protein levels, suggesting the inhibition of autophagy. When these cells were treated with rotenone, there was a loss of mitochondrial membrane potential, especially in cells expressing mutant α-synuclein. The level of this decrease was related to the toxicity of the mutants because they show a more intense and sustained effect. The decrease in autophagy and the mitochondrial changes in conjunction with parkin expression levels may sensitize astrocytes to apoptosis., (© 2017 Wiley Periodicals, Inc.)

Although the accumulation of the neurotoxic peptide β-amyloid (Aβ) in the central nervous system is a hallmark of Alzheimer's disease, whether Aβ acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca(2+) dysregulation, induced by a neurotoxic fragment (Aβ25-35), caused apoptosis in a concentration-dependent manner, leading to cytoplasmic Ca(2+) mobilization from extra- and intracellular sources, mainly from the endoplasmic reticulum (ER) via IP3 receptor activation. This mechanism was related to Aβ-mediated apoptosis by the intrinsic pathway because the expression of pro-apoptotic Bax was accompanied by its translocation in cells transfected with GFP-Bax. Aβ-mediated apoptosis was reduced by BAPTA-AM, a fast Ca(2+) chelator, indicating that an increase in intracellular Ca(2+) was involved in cell death. Interestingly, the Bax translocation was dependent on Ca(2+) mobilization from IP3 receptors because pre-incubation with xestospongin C, a selective IP3 receptor inhibitor, abolished this response. Taken together, these results provide evidence that Aβ dysregulation of Ca(2+) homeostasis induces ER depletion of Ca(2+) stores and leads to apoptosis; this mechanism plays a significant role in Aβ apoptotic cell death and might be a new target for neurodegeneration treatments., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Autophagy is a mechanism of protection against various forms of human diseases, such as cancer, in which autophagy seems to have an extremely complex role. In cancer, there is evidence that autophagy may be oncogenic in some contexts, whereas in others it clearly contributes to tumor suppression. In addition, studies have demonstrated the existence of a complex relationship between autophagy and cell death, determining whether a cell will live or die in response to anticancer therapies. Nevertheless, we still need to complete the autophagy-apoptosis puzzle in the tumor context to better address appropriate chemotherapy protocols with autophagy modulators. Generally, tumor cell resistance to anticancer induced-apoptosis can be overcome by autophagy inhibition. However, when an extensive autophagic stimulus is activated, autophagic cell death is observed. In this review, we discuss some details of autophagy and its relationship with tumor progression or suppression, as well as role of autophagy-apoptosis in cancer treatments., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

BbKI is a kallikrein inhibitor with a reactive site sequence similar to that of kinins, the vasoactive peptides inserted in kininogen moieties. This structural similarity probably contributes to the strong interaction with plasma kallikrein, the enzyme that releases, from high-molecular weight kininogen (HMWK), the proinflammatory peptide bradykinin, which acts on B(2) receptors (B(2)R). BbKI was examined on smooth muscle contraction and Ca(2+) mobilization, in which the kallikrein-kinin system is involved. Contrary to expectations, BbKI (1.8 μm) increased [Ca(2+)](c) and contraction, as observed with BK (2.0 μm). Not blocked by B(1) receptors (B(1)R), the BbKI agonistic effect was blocked by the B(2)R antagonist, HOE-140 (6 μm), and the involvement of B(2)R was confirmed in B(2)R-knockout mice intestine. The same tissue response was obtained using a synthetic peptide derived from the BbKI reactive site structure, more resistant than BK to angiotensin I-converting enzyme (ACE) hydrolysis. Depending on the concentration, BbKI has a dual effect. At a low concentration, BbKI acts as a potent kallikrein inhibitor; however, due to the similarity to BK, in high concentrations, BbKI greatly increases Ca(2+) release from internal storages, as a consequence of its interaction with B(2)R. Therefore, the antagonistic and agonistic effects of BbKI may be considered in conditions of B(2)R involvement.

Background: Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd(2) [S((-))C(2), N-dmpa](2) (μ-dppe)Cl(2)} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies., Methods: B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a., Results: Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells., Conclusions: The cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.

Apoptosis is a highly complex form of cell death that can be triggered by alterations in Ca(2+) homeostasis. Members of the Bcl-2 family may regulate apoptosis and modulate Ca(2+) distribution within intracellular compartments. Bax, a proapoptotic member of the family, is constitutively expressed and soluble in the cytosol and, under apoptotic induction, translocates to mitochondrial membranes. However, it is not clear if the intracellular Ca(2+) stores and selective Ca(2+) releases can modulate or control Bax translocation. The aim of this study was to investigate the relation of intracellular Ca(2+) stores with Bax translocation in rat cortical astrocytes. Results show that the classical apoptotic inducer, staurosporine, caused high elevations of cytosolic Ca(2+) that precede Bax translocation. On the other hand, agents that mobilize Ca(2+) from endoplasmic reticulum such as noradrenaline or thapsigargin, induced Bax translocation, while mitochondrial Ca(2+) release evoked by carbonyl cyanide-p-(trifluoromethoxyphenyl) hydrazone was not able to cause Bax punctation. In addition, microinjection of inositol 1,4,5- trisphosphate induced Bax translocation. Taken together, our results show that in Bax overexpressing cortical astrocytes, endoplasmic reticulum-Ca(2+) release may induce Bax transactivation and specifically control apoptosis.

Transient increase in cytosolic (Cac2+) and mitochondrial Ca2+ (Ca m2+) are essential elements in the control of many physiological processes. However, sustained increases in Ca c2+ and Ca m2+ may contribute to oxidative stress and cell death. Several events are related to the increase in Ca m2+, including regulation and activation of a number of Ca2+ dependent enzymes, such as phospholipases, proteases and nucleases. Mitochondria and endoplasmic reticulum (ER) play pivotal roles in the maintenance of intracellular Ca2+ homeostasis and regulation of cell death. Several lines of evidence have shown that, in the presence of some apoptotic stimuli, the activation of mitochondrial processes may lead to the release of cytochrome c followed by the activation of caspases, nuclear fragmentation and apoptotic cell death. The aim of this review was to show how changes in calcium signaling can be related to the apoptotic cell death induction. Calcium homeostasis was also shown to be an important mechanism involved in neurodegenerative and aging processes.

Background and Purpose: Androgens cause non-genomic relaxation in several smooth muscle preparations. However, such an effect has not been investigated in rat vas deferens yet. Our purpose was to study the effect of testosterone and derivatives in this tissue., Experimental Approach: The influence of androgens was tested on contraction and translocation of intracellular Ca(2+) induced by KCl in rat vas deferens in vitro., Key Results: The testosterone derivative 5alpha-dihydrotestosterone produced a rapid and reversible concentration-dependent relaxation of KCl-induced contractions. Other androgens were also effective, showing the following rank order of potency: androsterone >5beta-dihydrotestosterone >androstenedione >5alpha-dihydrotestosterone >testosterone. Calcium-induced contractions were also inhibited (about 45%) by 5alpha-dihydrotestosterone (30 microM). Moreover 5alpha-dihydrotestosterone blocked the increase of intracellular Ca(2+) induced by KCl, measured by the fluorescent dye fura-2. Relaxation to 5alpha-dihydrotestosterone was resistant to the K(+) channel antagonists glibenclamide, 4-aminopyridine and charybdotoxin. It was not affected by removal of epithelium or by L-NNA (300 microM), an inhibitor of nitric oxide biosynthesis, nor by selective inhibitors of soluble guanylate cyclase, ODQ or LY 83583, indicating that nitrergic or cGMP mediated mechanisms were not involved. The androgen-induced relaxation was also not blocked by the protein synthesis inhibitor cycloheximide (300 microM) or by the classical androgen receptor flutamide (up to 100 microM), corroborating that the effect is non-genomic., Conclusions and Implications: Testosterone derivatives caused relaxation of the rat vas deferens, that did not involve epithelial tissue, K(+) channels, or nitric oxide-dependent mechanisms, but was related to a partial blockade of Ca(2+) influx.