Effects of denervation on the contents of cholesterol and membrane systems involved in muscle contraction in rabbit fast-twitch sarcotubular system.

Denervated fast-twitch rabbit muscles were progressively losing their fresh weight and the yield of sarcotubular protein was increasing. The activity of Ca(2+)-ATPase was affected but very slightly, the basal Mg(2+)-ATPase and the Mg(2+)-ATPase/Ca(2+)-ATPase ratio however increased together with a simultaneous depression of the membrane-bound acetylcholinesterase activity. We did not observe any differences in density properties of sarcotubular fractions between control and denervated muscle. However, a relative enrichment in SM and H fraction could be seen after denervation with small changes in the content of the Ca(2+)-pump protein, increased levels of calsequestrin and cholesterol, mostly in the heavy and the SM fraction. After denervation the binding sites for 3H-PN-200-110 did not show any changes in receptor affinity, but the number of putative Ca(2+)-channels increased twice along with a depression of 3H-ouabain binding sites. We suggest that the denervation of fast-twitch muscle leads to the hypertrophy of the junctional sarcoplasmic reticulum and the T-system. Changes in the cholesterol content, in the number of putative Ca(2+)-channels and in Na+, K(+)-ATPase can affect the muscle contraction.     

Oxidative modification of rat cardiac mitochondrial membranes and myofibrils by hydroxyl radicals

The effect of hydroxyl radicals generated by the FeSO4/H2O2 system on structural properties of proteins and membranes was studied in rat cardiac mitochondria and myofibrils. Exposure of mitochondria to 0.1 mmol/l FeSO4/EDTA plus 1 mmol/l H202 at 37 degrees C for 30 or 60 min caused conjugated diene formation, but it was not accompanied by accumulation of fluorescent lipid-protein conjugates. On the other hand, fluorescence measurements revealed radical-induced and time-dependent loss of tryptophans and production of bityrosines. Under the same conditions, the gradual decrease in tryptophan flurescence and increase in bityrosine formation was also observed in radical-treated myofibrils. These results suggest that *OH radicals can alter the mitochondrial and myofibrillar function via oxidation of amino acid residues and might be implicated in the pathogenesis of myocardial injury.     

Effect of hyperhomocysteinemia on rat cardiac sarcoplasmic reticulum

Molecular and Cellular Biochemistry - Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia...     

Study of the oxidative stress in a rat model of chronic brain hypoperfusion

A multiple analysis of the cerebral oxidative stress was performed on a physiological model of dementia accomplished by three-vessel occlusion in aged rats. The forward rate constant of creatine kinase, k_for, was studied by saturation transfer ³¹P magnetic resonance spectroscopy in adult and aged rat brain during chronic hypoperfusion. In addition, free radicals in aging rat brain homogenates before and/or after occlusion were investigated by spin-trapping electron paramagnetic resonance spectroscopy (EPR). Finally, biochemical measurements of oxidative phosphorylation parameters in the above physiological model were performed. The significant reduction of k_for in rat brain compared to controls 2 and 10 weeks after occlusion indicates a disorder in brain energy metabolism. This result is consistent with the decrease of the coefficient of oxidative phosphorylation (ADP:O), and the oxidative phosphorylation rate measured in vitro on brain mitochondria. The EPR study showed a significant increase of the ascorbyl free radical concentration in this animal model. Application of -phenyl-N-tert-butylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin traps revealed formation of highly reactive hydroxyl radical (OH) trapped in DMSO as the CH₃ adduct. It was concluded that the ascorbate as a major antioxidant in brain seems to be useful in monitoring chronic cerebral hypoperfusion.     

Oxidative alternations in rat heart homogenate and mitochondria during ageing

Our understanding of the role played by reactive oxygen and nitrogen species in disease pathology and ageing is still insufficient. Reactive oxygen species and reactive nitrogen species can initiate protein and lipid oxidative damage that may be the most important contribution to ageing and age-related heart diseases. In the present study, we investigated the effect of ageing on oxidative damage of protein amino acid residues and lipids in heart homogenate and mitochondria of 4- and 26-month-old Wistar rats. Levels of dityrosine and levels of lysine conjugates increased in heart homogenate during ageing, although levels of conjugated dienes did not change. We observed significantly oxidative modification of tryptophan in heart mitochondria and increased levels of dityrosine with advancing age. However, levels of lysine conjugates, conjugated dienes as well as relative level of cytochrome c oxidase were unchanged in heart mitochondria during ageing. The results of this study suggest a different mechanism of oxidative modification in heart compartments during ageing and moreover, mitochondria and other cellular compartments are targets for oxidative modifications.     

Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.     

Distribution of secretory component in hepatocytes and its mode of transfer into bile

Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound ¹²⁵I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [¹⁴C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile.     

Carnosine: An Endogenous Neuroprotector in the Ischemic Brain

1. The biological effects of carnosine, a natural hydrophilic neuropeptide, on the reactive oxygen species (ROS) pathological generation are reviewed.2. We describe direct antioxidant action observed in the in vitro experiments.3. Carnosine was found to effect metabolism indirectly. These effects are reflected in ROS turnover regulation and lipid peroxidation (LPO) processes.4. During brain ischemia carnosine acts as a neuroprotector, contributing to better cerebral blood flow restoration, electroencephalography (EEG) normalization, decreased lactate accumulation, and enyzmatic protection against ROS.5. The data presented demonstrate that carnosine is a specific regulator of essential metabolic pathways in neurons supporting brain homeostasis under unfavorable conditions.