Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies

Tissues from male Wistar rats, fixed with 4% paraformaldehyde and embedded in paraffin, were studied with immunoperoxidase techniques using polyclonal antibodies raised against aldehyde oxidase or xanthine oxidase purified from rat liver. Immunohistochemical studies demonstrated that aldehyde oxidase-bearing cells were strongly stained in renal tubules, esophageal, gastric, intestinal and bronchial epithelium as well as liver cytoplasm. Weak but positive immunoreactivity was observed on the pulmonary alveolar epithelial cells, gastric glands and intestinal goblet cells. In contrast, it was demonstrated that cells with xanthine oxidase were strongly stained in renal tubules, esophageal, gastric, and small and large intestinal and bronchial epithelia etc. Positive immunostaining was also found in adrenal gland, skeletal muscle, spleen and cerebral hippocampus. Immunoreactivity againt aldehyde oxidase was not found in adrenal gland, spleen, mesentery or aorta, while immunoreactivity against xanthine oxidase was not found in mesentery or aorta. Although the significance of this ubiquitous and similar localization of aldehyde and xanthine oxidase seems unclear at present, these results may provide a clue as to the full understanding of the pathophysiological role of these oxidases in tissues.     

Immunohistochemical demonstration of fibre type-specific isozymes of cytochrome c oxidase in human skeletal muscle

Summary The immunohistochemical reaction of monoclonal as well as polyclonal antibodies against cytochrome c oxidase (COX) subunits with serial sections of normal human skeletal muscle was investigated. The stronger reactivity of polyclonal antibodies to COX subunits II–III and VIIbc with type I as compared to type II fibres, correlated well with the higher histochemical reactivity of NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase in type I fibres. In contrast an almost exclusive reaction of a monoclonal antibody against subunit IV with type I fibre and a preponderan reaction of a polyclonal antibody against subunits Vab with type II fibres was obtained. Antibodies against subuntis I, Vb and VIc did not reveal a fibre-type-specific reactivity. The data indicate in human muscle the occurrence of fibre type-specific isozymes of cytochrome c oxidase differing in subunits IV and Va or Vb.     

Immunohistochemical demonstration of fibre type-specific isozymes of cytochrome c oxidase in human skeletal muscle

The immunohistochemical reaction of monoclonal as well as polyclonal antibodies against cytochrome c oxidase (COX) subunits with serial sections of normal human skeletal muscle was investigated. The stronger reactivity of polyclonal antibodies to COX subunits II-III and VIIbc with type I as compared to type II fibres, correlated well with the higher histochemical reactivity of NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase in type I fibres. In contrast an almost exclusive reaction of a monoclonal antibody against subunit IV with type I fibre and a preponderant reaction of a polyclonal antibody against subunits Vab with type II fibres was obtained. Antibodies against subunits I, Vb and VIc did not reveal a fibre-type-specific reactivity. The data indicate in human muscle the occurrence of fibre type-specific isozymes of cytochrome c oxidase differing in subunits IV and Va or Vb.     

Effect of acetyl-l-carnitine on lipid peroxidation and xanthine oxidase activity in rat skeletal muscle

It has been reported that acetyl-l-carnitine (AcCn) can reduce the degenerative processes in the central nervous system of rats, modify the fluidity of membranes and decrease the accumulation of lipofuscins in neurones. In light of these considerations we have assayed the in vitro effect of acetyl-l-carnitine on spontaneous and induced lipoperoxidation in rat skeletal muscle; in addition, the effect of AcCn on XD/XO ratio was evaluated. The presence of AcCn (10–40 mM) in incubation medium significantly reduced MDA and conjugated diene formation in rat skeletal muscle; moreover, a significant decrease in induced MDA levels was observed when microsomal preparation where incubated in the presence of 10–40 mM AcCn. Since a significant reduction of XO activity was detected in the presence of 10–80 mM AcCn, the reduced lipid peroxidation by AcCn seems to be due to an inhibition of XO activity.     

Activated neutrophils increase microvascular permeability in skeletal muscle: role of xanthine oxidase

To determine the role of xanthine oxidase in the microvascular dysfunction produced by activated granulocytes, we examined the effect of xanthine oxidase depletion or inhibition on the increase in microvascular permeability produced by infusion of the neutrophil activator phorbol myristate acetate (PMA). Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient for total plasma proteins (sigma) in rat hindquarters subjected to PMA infusion in xanthine oxidase-replete and -depleted animals, in animals pretreated with the xanthine oxidase inhibitor oxypurinol, and in animals depleted of circulating neutrophils by pretreatment with antineutrophil serum (ANS). Xanthine oxidase depletion was accomplished by administration of a tungsten-supplemented (0.7 g/kg diet) molybdenum-deficient diet. In animals fed the tungsten diet, muscle total xanthine dehydrogenase plus xanthine oxidase activity was decreased to less than 10% of control values. Estimates of sigma averaged 0.84 +/- 0.04 in control hindquarters, whereas PMA infusion was associated with a marked increase in microvascular permeability (decrease in sigma to 0.68 +/- 0.03). PMA infusion also caused an increase in the amount of the radical-producing oxidase form of xanthine oxidase (from 3.9 +/- 0.05 to 5.6 +/- 0.4 mU/g wet wt). ANS pretreatment attenuated this permeability increase (sigma = 0.77 +/- 0.04) and diminished the rise in xanthine oxidase activity (4.9 +/- 0.5 mU/g wet wt). Xanthine oxidase depletion with the tungsten diet or pretreatment with oxypurinol had no effect on this neutrophil-mediated microvascular injury (sigma = 0.69 +/- 0.06 and 0.67 +/- 0.03, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

The localization of VAMP5 in skeletal and cardiac muscle

Vesicle-associated membrane protein 5 (VAMP5) is a member of the SNARE protein family, which is generally thought to regulate the docking and fusion of vesicles with their target membranes. This study investigated the expression and localization of the VAMP5 protein. Immunoblotting analyses detected the VAMP5 protein in skeletal muscle, heart, spleen, lung, liver, and kidney tissue, but not in brain or small intestine tissue. Through the immunofluorescence microscopy of skeletal muscle, we found that the expression level of VAMP5 varies among fibers. Most of the fibers with high expression levels of VAMP5 were categorized as type IIa fibers on the basis of their myosin heavy chain subtypes. In addition, the expression patterns of VAMP5 and glucose transporter 4 (GLUT4) were similar. In cardiac muscle, we determined that VAMP5 was localized to the vicinity of intercalated discs. These results suggest that VAMP5 plays local roles in membrane trafficking in skeletal and cardiac muscle.     

Immunohistochemical localization of smooth muscle myosin in normal human tissues

Immunohistochemical localization of smooth muscle myosin, an immunologically distinct contractile protein, was achieved using rabbit anti-human uterine smooth muscle myosin antibodies. In immunodiffusion studies and in cryostat sections, these antibodies were highly specific and reacted with smooth muscle myosin but not with platelet, skeletal muscle, or cardiac muscle myosin. To evaluate comprehensively the structural profile of smooth muscle elements in normal human tissues, an indirect immunoperoxidase technique (peroxidase-antiperoxidase) was applied to a wide variety of specimens. Parallel studies comparing cryostat sections with fixed (10% formalin, B5, Bouin's, or Zenker's solution) paraffin-embedded tissues revealed optimal immunoreactivity, sensitivity, and specificity of staining for smooth muscle myosin using frozen tissues. Strong immunoreactivity was present in muscular tissues such as blood vessels and the muscularis of gastrointestinal and genitourinary tracts. Distinct delineation of smooth muscle elements, including individual smooth muscle cells, and their specific patterns of alignment and organization, were observed, e.g., cells comprising the muscularis mucosae and extending into the lamina propria of the gastrointestinal tract, and myoepithelial cells of skin, exocrine glands, and breast. This method provides excellent morphologic preservation and readily permits unambiguous identification of individual cells containing smooth muscle myosin.     

Flux control of cytochrome c oxidase in human skeletal muscle

In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 +/- 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 +/- 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 +/- 0.03 to 0.36 +/- 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme.     

Immunohistochemical localization and vascular effects of vasoactive intestinal polypeptide in skeletal muscle of the cat

Summary Scattered vasoactive intestinal polypeptide (VIP) — immunoreactive nerves were found in the striated muscle of the hind limb of the cat, where they usually were associated with small blood vessels. VIP-immunoreactive nerves were also demonstrated in the sciatic nerve; after nerve ligation an abundance of intensely immunoreactive VIP fibres were seen proximal to the ligation. Intraarterial infusion of VIP into the isolated hind limb of the cat had dramatic effects on different sections of the vascular bed. Thus, VIP dilated the resistance vessels leading to a marked increment in muscle blood flow. VIP also relaxed the capacitance vessels causing regional pooling of blood; it increased the capillary surface area available for fluid exchange. Infusions of VIP at a dose of 8 g/min significantly inhibited the vasoconstriction induced by electrical stimulation of the regional sympathetic nerves. It is suggested that local nervous release of VIP may act as a modulator of vascular tone in skeletal muscle.     

Inhibition of xanthine oxidase reduces hyperglycemia-induced oxidative stress and improves mitochondrial alterations in skeletal muscle of diabetic mice

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of diabetes and more recently in mitochondrial alterations in skeletal muscle of diabetic mice. However, so far the exact sources of ROS in skeletal muscle have remained elusive. Aiming at better understanding the causes of mitochondrial alterations in diabetic muscle, we designed this study to characterize the sites of ROS production in skeletal muscle of streptozotocin (STZ)-induced diabetic mice. Hyperglycemic STZ mice showed increased markers of systemic and muscular oxidative stress, as evidenced by increased circulating H(2)O(2) and muscle carbonylated protein levels. Interestingly, insulin treatment reduced hyperglycemia and improved systemic and muscular oxidative stress in STZ mice. We demonstrated that increased oxidative stress in muscle of STZ mice is associated with an increase of xanthine oxidase (XO) expression and activity and is mediated by an induction of H(2)O(2) production by both mitochondria and XO. Finally, treatment of STZ mice, as well as high-fat and high-sucrose diet-fed mice, with oxypurinol reduced markers of systemic and muscular oxidative stress and prevented structural and functional mitochondrial alterations, confirming the in vivo relevance of XO in ROS production in diabetic mice. These data indicate that mitochondria and XO are the major sources of hyperglycemia-induced ROS production in skeletal muscle and that the inhibition of XO reduces oxidative stress and improves mitochondrial alterations in diabetic muscle.     

Xanthine oxidoreductase and xanthine oxidase in human cornea

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.     

Human cardiac and skeletal muscle spectrins: differential expression and localization.

We describe multiple human cardiac and skeletal muscle spectrin isoforms. Cardiac muscle expresses five erythroid alpha,beta spectrin-reactive isoforms with estimated MR's of 280, 274, 270, 255, and 246 kD, respectively. At least one nonerythroid alpha-spectrin of MR 284 kD is expressed in heart. While skeletal muscle shares the 280, 270, and 246 kD erythroid spectrins, it expresses an immunologically distinct 284 kD nonerythroid alpha-spectrin isoform. The 255 kD erythroid beta-spectrin isoform is specific for cardiac tissue. By immunocytochemistry, both erythroid beta- and nonerythroid alpha-spectrins are localized to costameres, the plasma membrane, and the neuromuscular junctional region.     

The mass-spectrometric identification of hypoxanthine and xanthine (`oxypurines'') in skeletal muscle from two patients with congenital xanthine oxidase deficiency (xanthinuria)

1. The presence of hypoxanthine and xanthine in the skeletal muscle of two patients with congenital xanthine oxidase deficiency (xanthinuria) was demonstrated by high-resolution mass spectrometry. 2. Evidence was obtained for the presence of a trace of hypoxanthine only in normal muscle. 3. Dry pulverized tissue was introduced directly into the mass spectrometer and preliminary chemical processing of the tissue was therefore unnecessary. 4. The criteria for the mass-spectrometric identification of hypoxanthine and xanthine in the tissue and the significance of the observations are discussed.     

Neuregulin receptor ErbB2 localization at T-tubule in cardiac and skeletal muscle.

Previous studies have indicated that ErbB receptors for neuregulins play an important role in cardiac development and muscle spindle formation during embryogenesis; however, little is known about their functions in adulthood. Recent reports indicate that breast cancer therapy with humanized monoclonal ErbB2 antibody induces cardiomyopathy, suggesting that ErbB2 serves as a crucial signaling receptor, even in the adult heart. Here, we examine ErbB2 expression and localization in both cardiac and skeletal muscle of adult mice via immunoblot and immunohistochemistry. ErbB2 was detected as a band approximately 185 kD molecular mass in each cardiac and skeletal muscle extraction. Confocal images of double labeling showed that ErbB2 was colocalized with caveolin-3 in cardiac muscle and with dihydropyridine receptor in skeletal muscle, suggesting that ErbB2 was localized at the T-tubule. In addition, immunoelectron micrographs clearly demonstrated that ErbB2 was located at the T-tubule in both types of muscle. Taken together, the results of the present study suggest that neuregulin-ErbB2 signaling plays a role in the physiological function of cardiac and skeletal muscle, even in adulthood.     

Immunohistochemical localization of D-aspartate oxidase in porcine peripheral tissues

d-Aspartate (d-Asp) is an endogenous substance in mammals. Degradation of d-Asp is carried out only by d-aspartate oxidase (DDO). We measured DDO activity in porcine tissues, and produced an anti-porcine DDO antibody to examine the cellular localization of DDO. All the tissues examined showed DDO activities, whereas the substrate d-Asp was not detected in kidney cortex, liver, heart, and gastric mucosa. In the kidney, intensive immunohistochemical staining for DDO was found in the epithelial cells of the proximal tubules. In the liver, the epithelial cells of interlobular bile ducts, liver sinusoid-lining cells with cytoplasmic processes, and the smooth muscle cells of arterioles were strongly stained for DDO. In the heart, cardiomyocytes and the smooth muscle cells of arterioles showed DDO-immunoreactivity. In the gastric mucosa, only the chief cells were DDO-positive. These newly identified DDO-positive cells seem to actively degrade d-Asp to prevent an excess of d-Asp from exerting harmful effects on the respective functions of porcine tissues.     

Comparative localization of aldehyde oxidase and xanthine oxidoreductase activity in rat tissues

The distribution of aldehyde oxidase activity was evaluated in unfixed cryostat sections from tissues of male Wistar rats using a tissue protectant, polyvinyl alcohol, with Tetranitro BT as a final electron acceptor. The distribution of aldehyde oxidase activity was compared with that of xanthine oxidoreductase. The enzyme histochemical method demonstrated aldehyde oxidase activity in the epithelium of the tongue, renal tubules and bronchioles, as well as in the cytoplasm of liver cells. Such activity was not detected in oesophagus, stomach, spleen, adrenal glands, small or large intestine or skeletal and heart muscle fibres. In contrast, xanthine oxidoreductase activity was demonstrated in the tongue, renal tubules, bronchioles, oesophageal, gastric, small and large intestinal epithelial cells, adrenal glands, spleen and liver cytoplasm but not in skeletal and heart muscle fibres. The significance of the ubiquitous distribution of aldehyde oxidase activity, especially in surface epithelial cells from various tissues, except for the gastrointestinal tract, is unclear. However, aldehyde oxidase may possess some physiological activity other than in the metabolism of N-heterocyclics or of certain drugs. © 1998 Chapman & Hall     

A monoclonal antibody against cytochrome c oxidase distinguishes cardiac and skeletal muscle mitochondria

The mitochondrial enzyme cytochrome c oxidase (COX) in eukaryotes consists of at least seven subunits, three of which (I-III) are encoded by mitochondrial DNA (mitDNA) and the others (IV-VII) by the nuclear genome. There is increasing evidence that COX in mammals exists in multiple tissue-specific forms, presumably specified by nuclearly encoded subunits. We performed immunologic studies in human cardiac and skeletal muscle, using a monoclonal antibody raised against subunit IV of COX purified from human cardiac muscle. In immunotitration studies, the antibody bound with high affinity to mitochondria from cardiac muscle, but reacted only weakly with mitochondria from skeletal muscle. Similarly, immunocytochemical studies showed prominent mitochondrial staining in frozen sections of heart, but no staining in sections of mature skeletal muscle. Although this antibody did not stain mitochondria in mature skeletal muscle, it clearly stained mitochondria in myoblasts and immature myotubes of human muscle cultures, suggesting that mitochondria in immature muscle cells are different from those in mature muscle, and similar to heart mitochondria. Immunotitration data using either native or denatured COX protein from heart or skeletal muscle showed similar immunoreactivity. These studies indicate that the epitope for recognition by this antibody is exposed in mitochondria from heart and immature muscle cells, but masked in mitochondria from mature skeletal muscle.