Ana Cristina do Nascimento Pinheiro

Succinate modulates Ca2+ transient and cardiomyocyte viability through PKA-dependent pathway

GPR91 is an orphan G-protein-coupled receptor (GPCR) that has been characterized as a receptor for succinate, a citric acid cycle intermediate, in several tissues. In the heart, the role of succinate is unknown. We now report that rat ventricular cardiomyocytes express GPR91. We found that succinate, through GPR91, increases the amplitude and the rate of decline of global Ca(2+) transient, by increasing the phosphorylation levels of ryanodine receptor and phospholamban, two well known Ca(2+) handling proteins. The effects of succinate on Ca(2+) transient were abolished by pre-treatment with adenylyl cyclase and cAMP-dependent protein kinase (PKA) inhibitors. Direct PKA activation by succinate was further confirmed using a FRET-based A-kinase activity reporter. Additionally, succinate decreases cardiomyocyte viability through a caspase-3 activation pathway, effect also prevented by PKA inhibition. Taken together, these observations show that succinate acts as a signaling molecule in cardiomyocytes, modulating global Ca(2+) transient and cell viability through a PKA-dependent pathway.

Mitochondrial calcium regulates rat liver regeneration through the modulation of apoptosis.

Subcellular Ca2+ signals control a variety of responses in the liver. For example, mitochondrial Ca2+ (Ca  mit2+ ) regulates apoptosis, whereas Ca2+ in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca  mit2+ also affects liver regeneration. The Ca2+‐buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin–mitochondrial targeting sequence–green fluorescent protein (PV‐MITO‐GFP). This construct properly localized to and effectively buffered Ca2+ signals in the mitochondrial matrix. Additionally, the expression of PV‐MITO‐GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl‐2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2–associated X protein (bax), apoptotic peptidase activating factor 1, and caspase‐6]. PV‐MITO‐GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca  mit2+ buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl‐2 and the decreased expression of bax. Conclusion: Together, these results reveal an essential role for Ca  mit2+ in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis. (HEPATOLOGY 2011;)

Neuroprotective effect on brain injury by neurotoxins from the spider Phoneutria nigriventer.

The role of calcium channels blockers in ischemic condition has been well documented. The PhTx3 neurotoxic fraction of the spider Phoneutria nigriventer venom is a broad-spectrum calcium channel blocker that inhibits glutamate release, calcium uptake and also glutamate uptake in synaptosomes. In the present study we describe the effect of PhTx3 (1.0 microg/mL), omega-conotoxin GVIA (1.0 micromol/L) and omega-conotoxin MVIIC (100 nmol/L) on neuroprotection of hippocampal slices and SN56 cells subjected to ischemia by oxygen deprivation and low glucose insult (ODLG). After the insult, cell viability in the slices and SN56 cells was assessed by confocal microscopy and epifluorescence, using live/dead kit containing calcein-AM and ethidium homodimer. Confocal images of CA1 region of the rat hippocampal slices subjected to ischemia insult and treated with omega-conotoxin GVIA, omega-conotoxin MVIIC and PhTx3 showed a percentage of dead cells of 68%, 54% and 18%, respectively. The SN56 cells subjected to ischemia were almost completely protected from damage by PhTx3 while with omega-conotoxin GVIA or omega-conotoxin MVIIC the cell protection was only partial. Thus, PhTx3 provided robust ischemic neuroprotection showing potential as a novel class of agents that targets multiple components and exerts neuroprotection in in vitro model of brain ischemia.

Phoneutria spider toxins block ischemia‐induced glutamate release, neuronal death, and loss of neurotransmission in hippocampus

The aim of this study was to investigate the effect of spider toxins on brain injury induced by oxygen deprivation and low glucose (ODLG) insult on slices of rat hippocampus. After ODLG insult cell viabilility in hippocampal slices was assessed by confocal microscopy and epifluorescence using the live/dead kit containing calcein‐AM and ethidium homodimer and CA1 population spike amplitude recording during stimulation of Schaffer collateral fibers. Spider toxins Tx3–3 or Tx3–4 and conus toxins, ω‐conotoxin GVIA or ω‐conotoxin MVIIC are calcium channel blockers and protected against neuronal damage in slices subjected to ODLG insult. Confocal imaging of CA1 region of rat hippocampal slices subject to ischemic insult treated with Tx3–3, Tx3–4, ω‐conotoxin GVIA or ω‐conotoxin MVIIC showed a decrease in cell death that amounted to 68 ± 4.2%, 77 ± 3.8%, 32 ± 2.3%, and 46 ± 2.9%, respectively. This neuroprotective effect of Tx3–4 was corroborated by eletrophysiological recordings of population spikes amplitudes in CA1. The neuroprotection promoted on hippocampal slices by Tx3–3 or Tx3–4 was also observed when the toxins were applied 10, 20, 30, 60, 90, or 120 min after induction of the ODLG injury. During the ischemic insult, glutamate release from slices was increased by 71% (from 7.0 ± 0.3 nM/mg of protein control slices not subjected to ischemia to 12 ± 0.4 nM/mg of protein in slices exposed to ischemia). Tx3–3, Tx3–4, ω‐conotoxin GVIA or ω‐conotoxin MVIIC inhibited the ischemia‐induced increase on glutamate release by 54, 72, 60, and 70%, respectively. Thus Tx3–3 and Tx3–4 provided robust ischemic neuroprotection showing potential as a novel class of agent that exerts neuroprotection in an in vitro model of brain ischemia.

Phoneutria spider toxins block ischemia-induced glutamate release and neuronal death of cell layers of the retina.

Purpose: To investigate the effect of calcium channel blockers, spider toxins, on cell viability and the glutamate content of ischemic retinal slices. Methods: Rat retinal slices were subjected to ischemia via exposure to oxygen-deprived low-glucose medium for 45 minutes. Slices were either treated or not treated with the toxins PhTx3, Tx3-3, and Tx3-4. After oxygen-deprived low-glucose insult, glutamate content and cell viability were assessed in the slices by confocal and optical microscopy. Results: In the retinal ischemic slices that were treated with PhTx3, Tx3-3, and Tx3-4, confocal imaging showed a decrease in cell death of 79.5 ± 3.1%, 75.5 ± 5.8%, and 61 ± 3.8%, respectively. Neuroprotective effects were also observed 15, 30, 60, and 90 minutes after the onset of the retinal ischemic injury. As a result of the ischemia, glutamate increased from 6.2 ± 1.0 nMol/mg protein to 13.2 ± 1.0 nMol/mg protein and was inhibited by PhTx3, Tx3-3, and Tx3-4 to 8.6 ± 0.7, 8.8 ± 0.9, and 7.4 ± 0.8 nMol/mg protein, respectively. Histologic analysis of the live cells in the outer, inner, and ganglion cell layers of the ischemic slices showed a considerable reduction in cell death by the toxin treatment. Conclusion: Spider toxins reduced glutamate content and cell death of retinal ischemic slices.

Tx3-4 a toxin from the venom of spider Phoneutria nigriventer blocks calcium channels associated with exocytosis

The purpose of the present work was to investigate the pharmacological action of a calcium channel-blocking toxin from the venom of the spider Phonetic nigriventer, Tx3-4 on calcium channels coupled to exocytosis of synaptic vesicles. Tx3-4 blocked KCl-induced exocytosis of synaptic vesicles with an IC50 of 1.1 nM. To investigate whether the target of Tx3-4 overlaps with known calcium channels that mediate calcium entry and exocytosis, we used omega-toxins that interact selectively with neuronal calcium channels. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-4 is P/Q calcium channels. In conclusion, Tx3-4 is a potent inhibitor of calcium channels involved in the KCl-induced exocytosis of synaptic vesicles in brain cortical synaptosomes.

Effects of a progressive resistance exercise program with high-speed component on the physical function of older women with sarcopenic obesity: a randomized controlled trial

ABSTRACT Background Sarcopenic obesity is associated with disability in older people, especially in women. Resistance exercises are recommended for this population, but their efficacy is not clear. Objective To evaluate the effects of a progressive resistance exercise program with high-speed component on the physical function of older women with sarcopenic obesity. Method Twenty-eight women 65 to 80 years old, with a body mass index ≥30kg/m2 and handgrip strength ≤21kg were randomly allocated to two groups. The experimental group underwent a 10-week resistance exercise program designed to improve strength, power, and endurance of lower-limb muscles, with open chain and closed chain exercises. The control group had their health status monitored through telephone calls. The primary outcomes were lower limb muscle performance measured by knee extensor strength, power and fatigue by isokinetic dynamometry, and mobility measured by the Short Physical Performance Battery and by gait velocity. The secondary outcome was health-related quality of life assessed by the SF-36 Questionnaire. Results The average rate of adherence was 85%, with few mild adverse effects. There were no significant between-group differences for any of the outcomes. Conclusion In this study, a progressive resistance exercise program with high-speed component was not effective for improving the physical function of older women with sarcopenic obesity.

The Effect of Spider Toxin PhTx3-4, ω-Conotoxins MVIIA and MVIIC on Glutamate Uptake and on Capsaicin-Induced Glutamate Release and [Ca2+]i in Spinal cord Synaptosomes

In spinal cord synaptosomes, the spider toxin PhTx3-4 inhibited capsaicin-stimulated release of glutamate in both calcium-dependent and -independent manners. In contrast, the conus toxins, ω-conotoxin MVIIA and ω-conotoxin MVIIC, only inhibited calcium-dependent glutamate release. PhTx3-4, but not ω-conotoxin MVIIA or ω-conotoxin MVIIC, is able to inhibit the uptake of glutamate by synaptosomes, and this inhibition in turn leads to a decrease in the Ca2+-independent release of glutamate. No other polypeptide toxin so far described has this effect. PhTx3-4 and ω-conotoxins MVIIC and MVIIA are blockers of voltage-dependent calcium channels, and they significantly inhibited the capsaicin-induced rise of intracellular calcium [Ca2+]i in spinal cord synaptosomes, which likely reflects calcium entry through voltage-gated calcium channels. The inhibition of the calcium-independent glutamate release by PhTx3-4 suggests a potential use of the toxin to block abnormal glutamate release in pathological conditions such as pain.

The effect of sevoflurane on intracellular calcium concentration from cholinergic cells

The mechanism of action of volatile anesthetics is not completely understood. Calcium release from internal stores may alter signaling pathways that influence neurotransmission. Abnormalities of the regulation of intracellular calcium concentration ([Ca2+]i) from patients with malignant hyperthermia is a hallmark of this syndrome indicating the potential of these agents to interact with proteins involved in Ca2+ signaling. In the present study, a cholinergic cell line (SN56) was used to examine whether the release of calcium from intracellular stores occurs in the presence of sevoflurane. Changes in [Ca2+]i were measured using fluo-4, a fluorescent calcium sensitive dye and laser scanning confocal microscopy. Sevoflurane induced an increase on [Ca2+]i from SN56 cells. The sevoflurane-induced increase on [Ca2+]i remained even when the cells were perfused with medium lacking extracellular calcium. However, this effect was abolished by BAPTA-AM, a chelator of intracellular calcium, suggesting the involvement of intracellular Ca2+ stores. Using cyclopiazonic acid, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, we investigated whether the depletion of intracellular Ca2+ stores interfered with the effect of sevoflurane. In the presence of this agent, sevoflurane caused a small but not significant rise on [Ca2+]i of the SN56 cells. Dantrolene, an inhibitor of ryanodine-sensitive calcium stores did not modify the sevoflurane increase on [Ca2+]i. Carbachol, a drug that releases Ca2+ from the IP3 pool, abolished the effect of sevoflurane. In addition, xestospongin D, a cell-permeant IP3 receptor antagonist, decreased significantly the sevoflurane increase on [Ca2+]i. Our data suggest that the sevoflurane-induced increase on [Ca2+]i from SN56 cells occurs through the release of calcium from IP3-sensitive calcium stores.

PhTx3-4, a Spider Toxin Calcium Channel Blocker, Reduces NMDA-Induced Injury of the Retina

The in vivo neuroprotective effect of PhTx3-4, a spider toxin N-P/Q calcium channel blocker, was studied in a rat model of NMDA-induced injury of the retina. NMDA (N-Methyl-d-Aspartate)-induced retinal injury in rats reduced the b-wave amplitude by 62% ± 3.6%, indicating the severity of the insult. PhTx3-4 treatment increased the amplitude of the b-wave, which was almost equivalent to the control retinas that were not submitted to injury. The PhTx3-4 functional protection of the retinas recorded on the ERG also was observed in the neuroprotection of retinal cells. NMDA-induced injury reduced live cells in the retina layers and the highest reduction, 84%, was in the ganglion cell layer. Notably, PhTx3-4 treatment caused a remarkable reduction of dead cells in the retina layers, and the highest neuroprotective effect was in the ganglion cells layer. NMDA-induced cytotoxicity of the retina increased the release of glutamate, reactive oxygen species (ROS) production and oxidative stress. PhTx3-4 treatment reduced glutamate release, ROS production and oxidative stress measured by malondialdehyde. Thus, we presented for the first time evidence of in vivo neuroprotection from NMDA-induced retinal injury by PhTx3-4 (-ctenitoxin-Pn3a), a spider toxin that blocks N-P/Q calcium channels.