Dysregulation of mitochondrial calcium signaling and superoxide flashes cause mitochondrial genomic DNA damage in Huntington disease.

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder characterized by the progressive loss of striatal medium spiny neurons. Indications of oxidative stress are apparent in brain tissues from both HD patients and HD mouse models; however, the origin of this oxidant stress remains a mystery. Here, we used a yeast artificial chromosome transgenic mouse model of HD (YAC128) to investigate the potential connections between dysregulation of cytosolic Ca(2+) signaling and mitochondrial oxidative damage in HD cells. We found that YAC128 mouse embryonic fibroblasts exhibit a strikingly higher level of mitochondrial matrix Ca(2+) loading and elevated superoxide generation compared with WT cells, indicating that both mitochondrial Ca(2+) signaling and superoxide generation are dysregulated in HD cells. The excessive mitochondrial oxidant stress is critically dependent on mitochondrial Ca(2+) loading in HD cells, because blocking mitochondrial Ca(2+) uptake abolished elevated superoxide generation. Similar results were obtained using neurons from HD model mice and fibroblast cells from HD patients. More importantly, mitochondrial Ca(2+) loading in HD cells caused a 2-fold higher level of mitochondrial genomic DNA (mtDNA) damage due to the excessive oxidant generation. This study provides strong evidence to support a new causal link between dysregulated mitochondrial Ca(2+) signaling, elevated mitochondrial oxidant stress, and mtDNA damage in HD. Our results also indicate that reducing mitochondrial Ca(2+) uptake could be a therapeutic strategy for HD.

[Effects of 50 Hz sinusoidal magnetic field on Ca2+ release channel ryanodine receptor of sarcoplasmic reticulum vesicles].

OBJECTIVE: To investigate the effects of sinusoidal magnetic field on isolated sarcoplasmic reticulum (SR) calcium release channel (RyR1) function. METHODS: With the Ca2+ dynamic spectrum and isotope labeled methods, the Ca2+ release and [(3)H]-Ryanodine binding, the initial rates of NADH oxidation and the production of superoxide of SR exposed to 50 Hz sinusoidal magnetic field (MF) were investigated respectively. RESULTS: 0.4 mT, 50 Hz sinusoidal MF exposure for 30 min increased SR Ca2+ release initial rate about 35% from (10.82 +/- 0.89) pmol.mg(-1) pro.s(-1) to (14.69 +/- 1.21) pmol.mg(-1) pro.s(-1); and the [(3)H]-Ryanodine binding by about 15% from (2.13 +/- 0.05) pmol/mg pro to (2.45 +/- 0.07) pmol/mg pro, which regulated by 1 mmol/L NADH with 1 mmol/L NAD+. Meanwhile MF upregulated the rate of NADH oxidation by about 22% from (0.88 +/- 0.11) x 10(-4) FI/s to (1.07 +/- 0.13) x 10(-4) FI/s and upregulated the production of superoxide by about 32% from (0.99 +/- 0.09) x 10(-5) FI/s to (1.31 +/- 0.06) x 10(-5) FI/s. CONCLUSION: 0.4 mT sinusoidal MF increases the activity of RyR1 within the low redox potential environment, and promotes NADH oxidase activity and superoxide production.

Swim training sensitizes myocardial response to insulin: role of Akt-dependent eNOS activation.

OBJECTIVES: Physical activity has been well known to benefit heart function. The improved autonomic nervous activity is considered to be mainly responsible for this beneficial effect. However, the precise mechanism behind the intrinsic myocardial responsiveness to exercise is still unclear. This study was designed to examine the effect of swim training on myocardial response to insulin with a special focus on the endogenous endothelial nitric oxide synthase (eNOS)-nitric oxide (NO) cascade. METHODS: Adult male Sprague-Dawley (SD) rats were subjected to a 10-week free-loading swim training (3 h/day, 5 days/week). Contractile response to insulin at the levels of cardiomyocytes and isolated perfused heart, myocardial glucose uptake and post-insulin receptor signaling cascades were evaluated. RESULTS: Swim training enhanced cardiac contractile response to insulin in cardiomyocytes and isolated perfused heart, respectively. The improved cardiac response was accompanied by facilitated insulin-stimulated glucose uptake, GLUT4 translocation and upregulation of Akt and eNOS expression (p<0.01). Treatment with insulin resulted in a 3.6- and 2.2-fold increase of eNOS phosphorylation (p<0.01), as well as a 3.0- and 1.9-fold increase of Akt phosphorylation in exercise and sedentary groups, respectively (p<0.01). In addition, exercise significantly facilitated insulin-induced myocardial NO production (p<0.01 vs. sedentary). Moreover, pretreatment with either LY294002, a phosphatidylinositol-3 kinase (PI-3K) inhibitor or L-NAME, a NOS inhibitor, abolished the exercise-induced sensitization of myocardial contractile response to insulin, insulin-induced NO production and phosphorylation of Akt and eNOS. CONCLUSION: These results demonstrate that swim training is capable of sensitizing myocardial contractile response to insulin via upregulation of Akt- and eNOS signaling cascades.

Calcium signaling in physiology and pathophysiology.

Calcium ions are the most ubiquitous and pluripotent cellular signaling molecules that control a wide variety of cellular processes. The calcium signaling system is represented by a relatively limited number of highly conserved transporters and channels, which execute Ca2+ movements across biological membranes and by many thousands of Ca2+-sensitive effectors. Molecular cascades, responsible for the generation of calcium signals, are tightly controlled by Ca2+ ions themselves and by genetic factors, which tune the expression of different Ca2+-handling molecules according to adaptational requirements. Ca2+ ions determine normal physiological reactions and the development of many pathological processes.

Cross-talk between calcium and reactive oxygen species signaling.

Calcium [Ca2+] and reactive oxygen species (ROS) constitute the most important intracellular signaling molecules participating in the regulation and integration of diverse cellular functions. Here we briefly review cross-talk between the two prominent signaling systems that finely tune the homeostasis and integrate functionality of Ca2+ and ROS in different types of cells. Ca2+ modulates ROS homeostasis by regulating ROS generation and annihilation mechanisms in both the mitochondria and the cytosol. Reciprocal redox regulation of Ca2+ homeostasis occurs in different physiological and pathological processes, by modulating components of the Ca2+ signaling toolkit and altering characteristics of local and global Ca2+ signals. Functionally, interactions between Ca2+ and ROS signaling systems can be both stimulatory and inhibitory, depending on the type of target proteins, the ROS species, the dose, duration of exposure, and the cell contexts. Such extensive and complex cross-talk might enhance signaling coordination and integration, whereas abnormalities in either system might propagate into the other system and undermine the stability of both systems.

Ca2+ sparks and Ca2+ glows in superior cervical ganglion neurons.

AIM: Ca2+ release from the endoplasmic reticulum (ER) is an integral component of neuronal Ca2+ signaling. The present study is to investigate properties of local Ca2+ release events in superior cervical ganglion (SCG) neurons. METHODS: Primary cultured SCG neurons were prepared from neonatal rats (P3-P7). Low concentration of caffeine was used to induce Ca2+ release from the ER Ca2+ store, and intracellular Ca2+ was recorded by high-resolution line scan confocal imaging and the Ca2+ indicator Fluo-4. RESULTS: Two populations of local Ca2+ release events with distinct temporal characteristics were evoked by 1.5 mmol/L caffeine near the surface membrane in the soma and the neurites of SCG neurons. Brief events similar to classic Ca2+ sparks lasted a few hundreds of milliseconds, whereas long-lasting events displayed duration up to tens of seconds. Typical somatic and neurite sparks were of 0.3- and 0.52-fold increase in local Fluo-4 fluorescence, respectively. Typical Ca2+ glows were brighter (deltaF/F0 approximately 0.6), but were highly confined in space. The half maximum of full duration of neurite sparks was much longer than those in the soma (685 vs 381 ms). CONCLUSION: Co-existence of Ca2+ sparks and Ca2+ glows in SCG neurons indicates distinctive local regulation of Ca2+ release kinetics. The local Ca2+ signals of variable, site-specific temporal length may bear important implications in encoding a 'memory' of the trigger signal.

The timing of endocytosis after activation of a G-protein-coupled receptor in a sensory neuron.

Endocytosis is a fundamental cellular event in membrane retrieval after exocytosis and in the regulation of receptor-mediated signal transduction. In contrast to the well-studied depolarization-induced membrane recycling, little is known about the kinetics of ligand-induced endocytosis of G-protein-coupled receptors in neurons. Here we investigated the kinetics of ligand-receptor binding-induced endocytosis in rat sensory neurons using a membrane capacitance assay. The time constant of ADP-induced endocytosis of P2Y-receptors was determined as 1.7 s. The ADP-induced endocytosis was blocked by antagonists against P2Y, phosphorylation, and clathrin. However, block of dynamin was without effect. The ADP-induced endocytosis was confirmed independently by a single vesicle image technique using a styryl FM2-10. Finally, the receptors were internalized in response to ADP, as determined by GFP-labeled P2Y. We conclude that ligand-receptor binding leads to rapid endocytosis in the cytoplasm of rat dorsal root ganglion neurons.

[Microscopic mechanism of excitation-contraction coupling in cardiac myocytes].

Cardiac excitation-contraction coupling (ECC) is, in essence, a communication process between sarcolemmal voltage-dependent L-type calcium channels (LCCs) and ryanodine receptors (RyRs) of sarcoplasmic reticulum (SR) by the mechanism of calcium-induced calcium release (CICR). Recent advances displayed more information about the microscopic signaling between LCCs and RyRs. In calcium release couplons, the calcium influx through the opening of LCCs by membrane depolarization forms calcium sparklets locally which then act on the adjacent SR RyRs. Stochastic activation of RyRs discharges calcium sparks from different calcium couplons, which summate into global calcium transients. Therefore, ignition of calcium sparks by calcium sparklets constitutes the elementary events in ECC. This review focuses on the intermolecular signaling between LCCs and RyRs, to describe the microscopic view of CICR and ECC.

Blockade of paeoniflorin on sodium current in mouse hippocampal CA1 neurons.

AIM: To study the blockade of paeoniflorin (Pae) on I(Na) in the acutely isolated hippocampus neurons of mice. METHODS: The whole-cell patch clamp technique was used. RESULTS: Pae inhibited I(Na) in frequency-dependent and concentration-dependent manners, with an IC50 of 271 micromol/L. Pae 0.3 mmol/L shifted the activation potential of the maximal I(Na) from -40 mV to -30 mV, shifted the steady-state activation and inactivation curves toward more positive and negative potentials by 10.8 mV, and 18.2 mV, respectively, and postponed the recovery of I(Na) inactivation state from (4.2+/-0.7) ms to (9.8+/-1.2) ms. CONCLUSION: Pae inhibited I(Na) in mouse hippocampus neurons.