Gustavo J.S. Pereira

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Regulates Autophagy in Cultured Astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing messenger that in many cells releases Ca2+ from the endolysosomal system. Recent studies have shown that NAADP-induced Ca2+ mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca2+ signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca2+ signals mediated by TPCs regulate autophagy.

Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney and cultured renal tubular epithelia

Bites from snake (Bothrops genus) cause local tissue damage and systemic complications, which include alterations such as hemostatic system and acute renal failure (ARF). Recent studies suggest that ARF pathogenesis in snakebite envenomation is multifactorial and involves hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. The aim of the work was to investigate the effects of the Bothrops leucurus venom (BlV) in the renal perfusion system and in cultured renal tubular cells of the type MDCK (Madin-Darby Canine kidney). BlV (10 μg/mL) reduced the perfusion pressure at 90 and 120 min. The renal vascular resistance (RVR) decreased at 120 min of perfusion. The effect on urinary flow (UF) and glomerular filtration rate (GFR) started 30 min after BlV infusion, was transient and returned to normal at 120 min of perfusion. It was also observed a decrease on percentual tubular transport of sodium (%TNa(+)) at 120 min and of chloride (%TCl(-)) at 60 and 90 min. The treatment with BlV caused decrease in cell viability to the lowest concentration tested with an IC(50) of 1.25 μg/mL. Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by necrosis. However, a cell death process may involve apoptosis in lower concentrations. BlV treatment (1.25 μg/mL) led to significant depolarization of the mitochondrial membrane potential and, indeed, we found an increase in the expression of cell death genes in the lower concentrations tested. The venom also evoked an increase in the cytosolic Ca(2+) in a concentration dependent manner, indicating that Ca(2+) may participate in the venom of B. leucurus effect. The characterization of the effects in the isolated kidney and renal tubular cells gives strong evidences that the acute renal failure induced by this venom is a result of the direct nephrotoxicity which may involve the cell death mechanism.

Apoptosis induced by Aβ25-35 peptide is Ca(2+) -IP3 signaling-dependent in murine astrocytes.

Although the accumulation of the neurotoxic peptide β‐amyloid (Aβ) in the central nervous system is a hallmark of Alzheimers disease, whether Aβ acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca2+ dysregulation, induced by a neurotoxic fragment (Aβ25–35), caused apoptosis in a concentration‐dependent manner, leading to cytoplasmic Ca2+ 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 Ca2+ chelator, indicating that an increase in intracellular Ca2+ was involved in cell death. Interestingly, the Bax translocation was dependent on Ca2+ 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 Ca2+ homeostasis induces ER depletion of Ca2+ 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.

Autophagy inhibited Ehrlich ascitic tumor cells apoptosis induced by the nitrostyrene derivative compounds: Relationship with cytosolic calcium mobilization

Apoptosis induction is often associated with increased autophagy, indicating interplay between these two important cellular events in cell death and survival. In this study, the programmed cell death and autophagy induced by two nitrostyrene derivative compounds (NTS1 and NTS2) was studied using the tumorigenic Ehrlich ascitic tumor (EAT) cells. EAT cells were highly sensitive to NTS1 and NTS2 cytotoxicity in a dose-dependent manner. NTS1 and NTS2 IC(50) was less than 15.0μM post 12h incubation. Apoptosis was primarily induced by both compounds, as demonstrated by an increase in Annexin-V positive cells, concurrently with cytochrome c release from mitochondria to cytosol and caspase-3 activation. Although cytosolic Ca(2+) mobilization is involved in autophagy as well as apoptosis in response to cellular stress in many cancer cell types, from the two nitrostyrene derivative compounds studied, mainly NTS1 mobilized this ion and disparate autophagy in EAT cells. These results suggest that EAT induced cell death by NTS1 and NTS2 involved a Ca(2+)-dependent and a Ca(2+)-independent pathways, respectively. In accordance with these results, the treatment of EAT cells with 3 methyladenine (3-MA), an autophagy inhibitor; significantly increased the number of apoptotic cells after NTS1 treatment, suggesting that pharmacological modulation of autophagy augments the NTS1 efficacy. Thus, we denote the importance of studies involving autophagy and apoptosis during pre-clinical studies of new drugs with anticancer properties.

L-Aminoacid Oxidase from Bothrops leucurus Venom Induces Nephrotoxicity via Apoptosis and Necrosis

Acute renal failure is a common complication caused by Bothrops viper envenomation. In this study, the nefrotoxicity of a main component of B. leucurus venom called L-aminoacid oxidase (LAAO-Bl) was evaluated by using tubular epithelial cell lines MDCK and HK-2 and perfused kidney from rats. LAAO-Bl exhibited cytotoxicity, inducing apoptosis and necrosis in MDCK and HK-2 cell lines in a concentration-dependent manner. MDCK apoptosis induction was accompanied by Ca2+ release from the endoplasmic reticulum, reactive oxygen species (ROS) generation and mitochondrial dysfunction with enhanced expression of Bax protein levels. LAAO-Bl induced caspase-3 and caspase-7 activation in both cell lines. LAAO-Bl (10 μg/mL) exerts significant effects on the isolated kidney perfusion increasing perfusion pressure and urinary flow and decreasing the glomerular filtration rate and sodium, potassium and chloride tubular transport. Taken together our results suggest that LAAO-Bl is responsible for the nephrotoxicity observed in the envenomation by snakebites. Moreover, the cytotoxic of LAAO-Bl to renal epithelial cells might be responsible, at least in part, for the nephrotoxicity observed in isolated kidney.

Glutamate induces autophagy via the two-pore channels in neural cells

NAADP (nicotinic acid adenine dinucleotide phosphate) has been proposed as a second messenger for glutamate in neuronal and glial cells via the activation of the lysosomal Ca2+ channels TPC1 and TPC2. However, the activities of glutamate that are mediated by NAADP remain unclear. In this study, we evaluated the effect of glutamate on autophagy in astrocytes at physiological, non-toxic concentration. We found that glutamate induces autophagy at similar extent as NAADP. By contrast, the NAADP antagonist NED-19 or SiRNA-mediated inhibition of TPC1/2 decreases autophagy induced by glutamate, confirming a role for NAADP in this pathway. The involvement of TPC1/2 in glutamate-induced autophagy was also confirmed in SHSY5Y neuroblastoma cells. Finally, we show that glutamate leads to a NAADP-dependent activation of AMPK, which is required for autophagy induction, while mTOR activity is not affected by this treatment. Taken together, our results indicate that glutamate stimulates autophagy via NAADP/TPC/AMPK axis, providing new insights of how Ca2+ signalling glutamate-mediated can control the cell metabolism in the central nervous system.

Mitochondrial involvement in carbachol-induced intracellular Ca2+ mobilization and contraction in rat gastric smooth muscle.

AIMS Mitochondria are important modulators of Ca2+ homeostasis. However, it is not clear if they modulate and participate in smooth muscle signaling and contraction. The aim of the present work was to investigate the role of mitochondria in Ca2+ transients and contraction induced by metabotropic muscarinic receptor activation in rat gastric smooth muscle. MAIN METHODS Carbachol (CCh)-induced contraction was investigated in the absence or presence of increasing concentration of mitochondrial protonophore, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone (FCCP), in gastric fundus strips. Ca2+ and mitochondrial membrane potential (ΔΨm) measurements were performed in primarily cultured gastric smooth muscle cells loaded with FURA-2 or TMRE dyes. KEY FINDINGS Results show that CCh (1 μM)-induced contraction was inhibited by FCCP in a concentration-dependent manner. In cultured smooth muscle cells CCh (1 μM) caused a cytosolic Ca2+ rise. Preincubation with FCCP strongly inhibited CCh-evoked Ca2+ transients indicating that mitochondria shape intracellular Ca2+ signals. CCh induced elevations of ∆Ψm in 60% of the individual mitochondrion analyzed. SIGNIFICANCE Taken together our results indicate that CCh induces release of Ca2+ from intracellular stores, which may be modulated by mitochondria. Thus, mitochondria participate of the intracellular Ca2+ homeostasis in muscarinic contraction in gastric fundus smooth muscle.

Bothropoides pauloensis venom effects on isolated perfused kidney and cultured renal tubular epithelial cells

Snake envenomation (Bothrops genus) is common in tropical countries and acute kidney injury is one of the complications observed in Bothrops snakebite with relevant morbidity and mortality. Here, we showed that Bothropoides pauloensis venom (BpV) decreased cell viability (IC50 of 7.5 μg/mL). Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by apoptosis and late apoptosis, through caspases 3 and 7 activation, mitochondrial membrane potential collapse and ROS overproduction. BpV reduced perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, percentage of sodium, chloride or potassium tubular transportation. These findings demonstrated that BpV cytotoxicity on renal epithelial cells might be responsible for the nephrotoxicity observed in isolated kidney.

γ-Rays-generated ROS induce apoptosis via mitochondrial and cell cycle alteration in smooth muscle cells.

Abstract Purpose: γ-rays (IR) cause an increase in intracellular calcium [Ca2+], alters contractility and triggers apoptosis via the activation of protein kinase C in intestinal guinea pig smooth muscle cells. The present study investigated the role of the mitochondria in these processes and characterized proteins involved in IR-induced apoptosis. Materials and methods: Intestinal smooth muscle cells were exposed to 10–50 Gy from a 60Co γ-source. Reactive oxygen species (ROS) levels were measured by colourimetry with a fluorescente probe. Protein expression was analyzed by immunoblotting and immunofluorescence. Results: Apoptosis was inhibited by glutathione, possible by inhibiting the generation or scavenging ROS. Apoptosis was mediated by the mitochondria releasing cytochrome c leading to caspase 3 activation. IR increased the expression of the cyclins A, B2 and E and led to unbalanced cellular growth in an absorption dose-dependent manner. However, radiation did not induce alterations in the mitochondrial ultrastructure or in transmembrane electric potential. In contrast, IR increased the nuclear expression of cytoplasmic proteins and cyclins A and E. Conclusion: Smooth muscle cells subjected to IR undergo mitochondrial-mediated apoptosis that involves oncoproteins activation and preserves mitochondrial structure. IR also cause alterations in the expression and localization of both pro- and anti-apoptotic proteins.

Overexpression of α‐synuclein in an astrocyte cell line promotes autophagy inhibition and apoptosis

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.