Ischemia-reperfusion damage is attenuated by GQ-11, a peroxisome proliferator-activated receptor (PPAR)-α/γ agonist, after aorta clamping in rats.

INTRODUCTION: Ischemia-Reperfusion (I/R) damage is one of the major challenges in cardiothoracic surgeries and in a pathological manner, is identified by exacerbated damage signals resulted from blood supply restriction and subsequent flow restoration and re­oxygenation. I/R damage includes cellular dysfunction and death, impairing tissue and organ function. Inflammation and oxidative stress are known to underlie either ischemia or reperfusion, leaded by HIF, TNF-α, NF-κB, IL-6 and ROS formation. However, the available approaches to prevent I/R damage has been unsuccessful so far. As agonists of peroxisome-proliferation activation receptor (PPAR) are described as transcription factors related to anti-inflammatory factors, we proposed to observe the effects of novel dual agonist, GQ-11, in I/R-related damage. METHODS: Male, Wistar rats, 60 days age and 305 g body weight average were treated with vehicle, pioglitazone or GQ-11 (20 mg/kg) for 7 consecutive days and were submitted to aorta clamping for 30 min followed by 3 h of reperfusion. 18F-fluorodeoxyglucose (18F-FDG), an analog of glucose associated with inflammation when accumulated, was observed in liver and bowel by positron emission tomography (PET). RESULTS: GQ-11 decreased 18F-FDG uptake in liver and bowel when compared to vehicle and pioglitazone. The treatment also modulated inflammatory markers IL-10, TGF-ß, IL-6, IL1-ß, TNFα, and CCL-2, besides antioxidant enzymes such as catalase, GPx and SOD. CONCLUSION: Inflammation and oxidative stress showed to be important processes to be regulated in I/R in order to prevent exacerbated responses that leads to cell/tissue dysfunction and death. PPAR agonists - including GQ-11 - might be promising agents in a strategy to avoid tissue dysfunction and death after cardiothoracic surgeries.

Abnormal activation of calpain and protein kinase Cα promotes a constitutive release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients.

Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion.

Physiological Roles of Calpain 1 Associated to Multiprotein NMDA Receptor Complex.

We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

Interaction between calpain-1 and HSP90: new insights into the regulation of localization and activity of the protease.

Here we demonstrate that heat shock protein 90 (HSP90) interacts with calpain-1, but not with calpain-2, and forms a discrete complex in which the protease maintains its catalytic activity, although with a lower affinity for Ca2+. Equilibrium gel distribution experiments show that this complex is composed by an equal number of molecules of each protein partner. Moreover, in resting cells, cytosolic calpain-1 is completely associated with HSP90. Since calpain-1, in association with HSP90, retains its proteolytic activity, and the chaperone is displaced by calpastatin also in the absence of Ca2+, the catalytic cleft of the protease is not involved in this association. Thus, calpain-1 can form two distinct complexes depending on the availability of calpastatin in the cytosol. The occurrence of a complex between HSP90 and calpain-1, in which the protease is still activable, can prevent the complete inhibition of the protease even in the presence of high calpastatin levels. We also demonstrate that in basal cell conditions HSP90 and calpain-1, but not calpain-2, are inserted in the multi-protein N-Methyl-D-Aspartate receptor (NMDAR) complex. The amount of calpain-1 at the NMDAR cluster is not modified in conditions of increased [Ca2+]i, and this resident protease is involved in the processing of NMDAR components. Finally, the amount of calpain-1 associated with NMDAR cluster is independent from Ca2+-mediated translocation. Our findings show that HSP90 plays an important role in maintaining a given and proper amount of calpain-1 at the functional sites.

Vibrio cholerae interactions with Mytilus galloprovincialis hemocytes mediated by serum components.

Edible bivalves (e.g., mussels, oysters) can accumulate large amount of bacteria in their tissues and act as passive carriers of pathogens to humans. Bacterial persistence inside bivalves depends, at least in part, on hemolymph anti-bacterial activity that is exerted by both serum soluble factors and phagocytic cells (i.e., the hemocytes). It was previously shown that Mytilus galloprovincialis hemolymph serum contains opsonins that mediate D-mannose-sensitive interactions between hemocytes and Vibrio cholerae O1 El Tor bacteria that carry the mannose-sensitive hemagglutinin (MSHA). These opsonins enhance phagocytosis and killing of vibrios by facilitating their binding to hemocytes. Since V. cholerae strains not carrying the MSHA ligand (O1 classical, non-O1/O139) are present in coastal water and can be entrapped by mussels, we studied whether in mussel serum, in addition to opsonins directed toward MSHA, other components can mediate opsonization of these bacteria. By comparing interactions of O1 classical and non-O1/O139 strains with hemocytes in artificial sea water and serum, it was found that M. galloprovincialis serum contains components that increase by at approximately twofold their adhesion to, association with, and killing by hemocytes. Experiments conducted with high and low molecular mass fractions obtained by serum ultrafiltration indicated that these compounds have molecular mass higher than 5000 Da. Serum exposure to high temperature (80°C) abolished its opsonizing capability suggesting that the involved serum active components are of protein nature. Further studies are needed to define the chemical properties and specificity of both the involved bacterial ligands and hemolymph opsonins. This information will be central not only to better understand V. cholerae ecology, but also to improve current bivalve depuration practices and properly protect human health.