To clarify the mechanism of oxidative stress in skeletal muscle atrophied by immobilization, we measured the activities of antioxidant enzymes and xanthine oxidase (XOD) and carried out the cytochemical study of hydrogen peroxide in a typical slow red muscle, the soleus. Male Wistar rats (15 wk old), of which ankle joints of one hindlimb were immobilized in the fully extended position, were killed after 4, 8, or 12 days. The activities of Mn-containing superoxide dismutase (Mn-SOD), Cu-Zn-containing superoxide dismutase (Cu-Zn-SOD), Se-dependent glutathione peroxidase (Se-GSHPx), glutathione S-transferase, catalase, and glutathione reductase were measured spectrophotometrically. The XOD activity and the concentrations of hypoxanthine, xanthine, and urate were measured using a high-performance liquid chromatography. The cytochemical study of hydrogen peroxide in short-term organ culture was performed using an electron microscope. Increased Cu-Zn-SOD and decreased Mn-SOD in atrophy might reflect increased generation of superoxide anions in the cytoplasm rather than in the mitochondria. The source of superoxide anions in the cytoplasm might be the increased superoxide-producing XOD. Enhanced generation of superoxide anions and increased Cu-Zn-SOD activity in atrophy suggested the enhanced generation of hydrogen peroxide in the cytoplasm. Due to the unchanged activity of Se-GSHPx and the unchanged or slightly increased activity of catalase in atrophy, the ability to degrade hydrogen peroxide might not increase so much. Hence, hydrogen peroxide is expected to be increased in atrophy. The cytochemical study supported this expectation.(ABSTRACT TRUNCATED AT 250 WORDS)

The movements of trace elements and the level of oxidative stress in the soleus, a typical slow red muscle which, atrophied by immobilization, were investigated in designated intervals. Male Wistar rats (14 wk old) whose one ankle joints were immobilized in the extended position were killed after 4, 8, and 12 days. Fe, Zn, Mn, and Cu concentrations and the levels of thiobarbituric acid-reactive substance (TBARS) and glutathione were measured. The rate of atrophy increased rapidly until the 8th day and slowly after that. In whole muscle, Fe concentration kept increasing, and Zn and Mn increased temporarily. Their subcellular distributions also changed; especially, the Fe level of the microsomal fraction kept increasing and reached threefold at 12 days. Increased TBARS and glutathione disulfide and decreased total glutathione indicated the increased oxidative stress in atrophy, which might result from an increased Fe level, especially that of the microsomal fraction. Vitamin E injection lessened the rate of atrophy, which showed that oxidative stress accelerated muscle atrophy. This might be mediated by increased intracellular Ca. Also metallothionein was induced in muscle atrophy.

Electron probe X-ray microanalysis using freshly frozen hydrated and dried thin sections of dog submandibular gland was performed to determine the distribution of elements and water in the acinar cells of resting and stimulating states. The results obtained are as follows : (a) The secretory granules contained high concentrations of Ca and S while high concentrations of K and P were present in the cytoplasm and/or nucleus of acinar mucous cells of the gland in the resting state. (b) With pilocarpine stimulation, the concentration of Ca increased in the cytoplasm and decreased in the secretory granules, while there was an increase in the concentration of Na and Cl in both the cytoplasm and secretory granules of the cells. (c) The local dry-mass fractions of acinar cells, estimated by comparing the continuum radiation of X-ray spectrum from the frozen hydrated sections with that from the frozen dehydrated sections, were approximately 20 and 33 % in the cytoplasm and secretory granules of resting acinar cells, respectively, and each value was not significantly altered under conditions of stimulation having a tendency to decrease slightly. Therefore, the passive Na and Cl influx and the cytoplasmic Ca flowed in from extracellular spaces and released from secretory granules, an intracellular calcium store, by secretory stimulation probably triggers the passive or active Na and Cl extrusion and consequently the osmotic water flux from the basal part of acinar cells to the secretory granules and the lumen, as well as the serial exocytosis of the granules in the luminal side of the acinar cells.Key Words: electron probe X-ray microanalysis, dog salivary gland, pilocarpine stimulation, electrolyte transport, intracellular calcium store.Saliva is primarily secreted in the acini of the salivary glands and is followed by a secondary process of reabsorption in the duct system (YOUNG and MARTIN,

Changes in electrolytes of pig pancreatic acinar cells following application of gastrin-cholecystokinin (CCK) were investigated using the technique of X-ray microanalysis of hydrated and dehydrated sections of freshly frozen pancreas. After stimulation by CCK (10(-9) M), Na and Cl increased significantly in the cytoplasm [Na, from 10 mmol/kg wet wt. (48 mmol/kg dry wt.) to 19 mmol/kg (95 mmol/kg); Cl, from 22 mmol/kg (105 mmol/kg) to 49 mmol/kg (245 mmol/kg)] as well as in the luminal interspace [Na, from 53 mmol/kg (189 mmol/kg) to 65 mmol/kg (283 mmol/kg); Cl, from 65 mmol/kg (232 mmol/kg) to 102 mmol/kg (443 mmol/kg)]. In the secretory granules Cl increased significantly from 30 mmol/kg (86 mmol/kg) to 67 mmol/kg (203 mmol/kg). K decreased significantly from 120 mmol/kg (571 mmol/kg) to 81 mmol/kg (405 mmol/kg) in the cytoplasm, while both increased from 38 mmol/kg (109 mmol/kg) to 58 mmol/kg (176 mmol/kg) in the granules and from 46 mmol/kg (164 mmol/kg) to 48 mmol/kg (209 mmol/kg) in the luminal interspace. Ca increased significantly in the cytoplasm as well as in the luminal interspace, and decreased significantly in the secretory granules. CCK evoked Ca release from secretory granules in the secretory pole of acinar cells. The values were measured from dehydrated sections, and agreed well with those from hydrated sections. The effect of furosemide, an inhibitor of the Na+-K+-2Cl- co-transporter, on the ion transport of acinar cell was studied. When furosemide (10(-5) M) was added to the external solution, the cytoplasmic Cl and Ca concentrations decreased significantly, while there was a little decrease in Na and K concentrations under the secretory condition. These results indicate that Na+-K+-2Cl- co-transport, and Na+, Cl- and K+ exits into the lumen are involved in the mechanism of ion secretion in pig pancreatic acinar cells.