Hypophosphorylation of ribosomal protein S6 is a molecular mechanism underlying ischemic tolerance induced by either hibernation or preconditioning.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) have an extraordinary capacity to withstand prolonged and profound reductions in blood flow and oxygen delivery to the brain without incurring any cellular damage. As such, the hibernation torpor of I. tridecemlineatus provides a valuable model of tolerance to ischemic stress. Herein, we report that during hibernation torpor, a marked reduction in the phosphorylation of the ribosomal protein S6 (rpS6) occurs within the brains of I. tridecemlineatus. Of note, rpS6 phosphorylation was shown to increase in the brains of rats that underwent an occlusion of the middle cerebral artery. However, such an increase was attenuated after the implementation of an ischemic preconditioning paradigm. In addition, cultured cortical neurons treated with the rpS6 kinase (S6K) inhibitors, D-glucosamine or PF4708671, displayed a decrease in rpS6 phosphorylation and a subsequent increase in tolerance to oxygen/glucose deprivation, an in vitro model of ischemic stroke. Collectively, such evidence suggests that the down-regulation of rpS6 signal transduction may account for a substantial part of the observed increase in cellular tolerance to brain ischemia that occurs during hibernation torpor and after ischemic preconditioning. Further identification and characterization of the mechanisms used by hibernating species to increase ischemic tolerance may eventually clarify how the loss of homeostatic control that occurs during and after cerebral ischemia in the clinic can ultimately be minimized and/or prevented. Mammalian hibernation provides a valuable model of tolerance to ischemic stress. Herein, we demonstrate that marked reductions in the phosphorylation of ribosomal protein S6 (rpS6), extracellular signal-regulated kinase family of mitogen-activated protein (MAP) kinase p44/42 (p44/42MAPK) and ribosomal protein S6 kinase (S6K) occur within the brains of both hibernating squirrels and rats, which have undergone an ischemic preconditioning paradigm. We therefore propose that the down-regulation of rpS6 signal transduction may account for a substantial part of the observed increase in cellular tolerance to brain ischemia that occurs during hibernation torpor and after ischemic preconditioning, via a suppression of protein synthesis and/or energy consumption.

Protein SUMOylation is massively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells.

Hibernation torpor provides an excellent natural model of tolerance to profound reductions in blood flow to the brain and other organs. Here, we report that during torpor of 13-lined ground squirrels, massive SUMOylation occurs in the brain, liver, and kidney. The level of small ubiquitin-related modifier (SUMO) conjugation coincides with the expression level of Ubc9, the SUMO specific E2-conjugating enzyme. Hypothermia alone also increased SUMO conjugation, but not as markedly as hibernation torpor. Increased SUMO conjugation (induced by Ubc9 overexpression, ischemic preconditioning (PC)+/-hypothermia) was necessary and sufficient for tolerance of SHSY5Y neuroblastoma cells to oxygen/glucose deprivation (OGD) ('in vitro ischemia'); decreased SUMO conjugation (induced by a dominant-negative Ubc9) severely reduced tolerance to OGD in these cells. These data indicate that post-translational modification of proteins by SUMOylation is a prominent feature of hibernation torpor and is critical for cytoprotection by ischemic PC+/-hypothermia in SHSY5Y cells subjected to OGD.

Sources of activator calcium ions in the contraction of smooth muscles in Aplysia kurodai.

The physiological and pharmacological properties of contraction and the ultrastructure of buccal mass retractor muscle (I4) and gill-pinnule closure muscle (GPCM) in Aplysia kurodai were studied to learn more about the sources of activator Ca2+ in molluscan smooth muscle. Acetylcholine (ACh) and high K+-induced contractions were reduced by lowering the external Ca2+ concentration, and eliminated by the removal of extracellular Ca2+. Nifedipine appreciably reduced ACh- and high K+-induced contractions, while amiloride decreased only ACh-induced contractions and had no significant effect on high K+-induced contractions. When nifedipine and amiloride were applied together, either type of contraction was still appreciable. Serotonin (5-HT) could potentiate subsequent ACh- and high K+-induced contractions in I4; potentiated tension was significantly reduced by nifedipine and amiloride, whereas 5-HT inhibited ACh-and high K+-induced contractions in GPCM. The potentiating effects of 5-HT may be mediated by the activation of the Ca2+-channel to increase the influx from extracellular Ca2+. Caffeine caused contractions in Ca2+-free solution in both muscles. Electron microscopy revealed sarcolemmal vesicles underneath the plasma membrane in both muscle fibers. Electron microscopical cytochemistry demonstrated that pyroantimonate precipitates were localized in the sarcolemmal vesicles and in the inner surface of plasma membranes in the resting fibers. Present results indicate that the contractions of I4 and GPCM fibers are caused not only by Ca2+-influx but also by Ca2+ release from the intracellular storage sites, such as the sarcolemmal vesicles and the inner surface of plasma membranes.

The influences of L(+)-lactate and pH on contractile performance in rabbit glycerinated skeletal muscle.

To know whether L(+)-lactate directly induces the decrease in muscle contractile performance, several parameters of cross-bridge function were measured at various concentrations of lactate and pH in glycerinated rabbit psoas and soleus muscles at three different temperatures (5, 20, 28 degrees C). At all pHs studied (pH 7.0, 6.5, 6.0, and 5.5), isometric tension, unloaded velocity of shortening, and stiffness of a fiber during active and resting state in the presence of 50 mM lactate were not virtually different from those in the absence of lactate, but pH had remarkable effects on these parameters. The active stiffness decreased only slightly, and the small resting stiffness appeared at low pH; they were not affected by the presence of lactate. The present results indicate that the lactate anions may not have marked influence on the interaction between actin and myosin, and the concomitant change in pH with the production of lactate may remarkably affect it, as far as they were examined under the existing conditions of the experimental solutions.

Identification and characterization of a novel mitochondrial tricarboxylate carrier.

Here we report the identification and functional characterization of a novel mitochondrial tricarboxylate carrier protein, designated BBG-TCC, in rat brain. The cDNA encodes the predicted protein of 342-amino acid residues with five putative membrane-spanning domains. The protein has apparent similarity with a mitochondrial tricarboxylate carrier TCC, but is distinct from the other mitochondria anion transporters. BBG-TCC shows a citrate transport activity. It is specifically expressed in the brain and localizes in the mitochondria of Bergmann glial cells. In contrast, the expression of TCC is rather ubiquitous and strong in neuronal cells in the brain. This new family of proteins may contribute to biosynthesis and bioenergetics in the brain.

Expression of mitochondrial tricarboxylate carrier TCC mRNA and protein in the rat brain.

The tricarboxylate carrier protein catalyzes an electroneutral exchange across the mitochondrial inner membrane of tricarboxylate, dicarboxylate or phosphoenolpyruvate. We examined expression and localization of mitochondrial tricarboxylate carrier TCC mRNA and protein in the rat brain. TCC mRNA was ubiquitously expressed in all rat tissues examined and was abundant in brain, liver and kidney. TCC protein as well as mRNA was widely expressed in brain, and the protein expression was strong in neuronal cells in the hippocampus, the olfactory bulb, the corpus mamillare and the cerebellum. Our results suggest that this tricarboxylate carrier protein may contribute to biosynthesis and bioenergetics in neuronal cells in brain.

Peg3/Pw1 is involved in p53-mediated cell death pathway in brain ischemia/hypoxia.

Emerging evidence has shown that tumor suppressor p53 expression is enhanced in response to brain ischemia/hypoxia and that p53 plays a critical role in the cell death pathway in such an acute neurological insult. However the mechanism remains unclear. Recently it was reported that Peg3/Pw1, originally identified as a paternally expressed gene, plays a pivotal role in the p53-mediated cell death pathway in mouse fibroblast cell lines. In this study, we found that Peg3/Pw1 expression is enhanced in peri-ischemic neurons in rat stroke model by in situ hybridization analysis, where p53 expression was also induced by immunohistochemical analysis. Moreover, we found that p53 was co-localized with Peg3/Pw1 in brain ischemia/hypoxia by double staining analysis. In human neuroblastoma-derived SK-N-SH cells, Peg3/Pw1 mRNA expression is enhanced remarkably at 24 h post-hypoxia, when p53 protein expression was also enhanced at high levels. Subcellular localization of Peg3/Pw1 was observed in the nucleus. Adenovirus-mediated high dose p53 overexpression induced Peg3/Pw1 mRNA expression. Overexpression of Peg3/Pw1 reduced cell viability under hypoxic conditions, whereas that of the C-terminal-deleted mutant and anti-sense Peg3/Pw1 inhibited hypoxia-induced cell death. These results suggest that Peg3/Pw1 is involved in the p53-mediated cell death pathway as a downstream effector of p53 in brain ischemia/hypoxia.