Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage

Xanthine oxidase (XO) has been hypothesized to be a potential source of oxygen-derived free radicals during reperfusion of ischemic myocardium based on the fact that allopurinol, a XO-inhibitor, can reduce reperfusion injury. In this communication we report that both allopurinol and oxypurinol, the principle metabolite of allopurinol, prevent the reperfusion injury in isolated pig heart. However, we found that neither pig heart nor pig blood contain any XO activity. Our study showed a direct free radical scavenging action of these XO-inhibitors during ischemia and reperfusion, as judged by the reduction of free radical signals when compared using an Electron Paramagnetic Resonance Spectrometer. Using a Luminometer, we also confirmed that both allopurinol and oxypurinol can scavenge ClO2, HOCl, and significantly inhibit free radical signals generated by activated neutrophils. These XO-inhibitors, however, failed to scavenge O2. and OH. radicals. Our results suggest that these XO-inhibitors salvaged the ischemic-reperfused myocardium by scavenging free radicals, and not by inhibiting XO in the pig heart.     

Effect of calcium overload on the phosphoinositide breakdown in the rat left ventricular papillary muscle

We investigated the effect of Ca²⁺ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca²⁺ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca²⁺-containing medium following either Ca²⁺-containing or Ca²⁺-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [³H]inositol phosphates ([³H]IPs) in the tissues prelabeled with [³H]inositol. Ca²⁺ repletion following Ca²⁺-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [³H]IPs accumulations. Treatment with ouabain following Ca²⁺-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [³H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [³H]IPs were significantly potentiated by prior Ca²⁺-free superfusion instead of Ca²⁺-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [³H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca²⁺-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca²⁺, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.Abbreviations [³H]IPs [³H]Inositol Phosphates - IP Inositol Phosphate - IP₂ Inositol Bisphosphate - IP₃ Inositol Trisphosphate - PI Phosphatidylinositol - PI-4-P Phosphatidylinositol-4-phosphate - PI-4,5-P₂ Phosphatidylinositol 4,5-bisphosphate - PRZ Prazosin - ATR Atropine - INDO Indomethacin - min Minutes     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Predominance of gluconate formation from glucose during germination of Bacillus megaterium QM B1551 spores.

Metabolic pathways of glucose during germination of Bacillus megaterium QM B1551 spores were studied by using specifically labeled glucose and gluconate. The Embden-Meyerhof pathway, the pentose cycle, and the direct oxidation route of glucose to gluconate (the gluconate pathway) were all operative at this stage; among those, gluconate accumulation was most predominant, especially in the early stage. Potassium fluoride, an enolase inhibitor, abolished the catabolism by the Embden-Meyerhof pathway totally without affecting gluconate accumulation. Under these conditions glucose was exclusively oxidized to gluconate. Gluconate thus accumulated could be metabolized further via phosphorylation by gluconate kinase. Remarkable gluconate accumulation was also demonstrated in several other spores requiring alanine as an effective germinant. NADH formed by the direct glucose oxidation may serve as a initial ATP source to phosphorylate glucose in germinating spores.     

Synergistic induction of ornithine decarboxylase by diacylglycerol, A23187, and cholera toxin in guinea pig lymphocytes

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetyl-glycerol (OAG), A23187, and cholera toxin, ornithine decarboxylase activity was induced synergistically, peaking at 6 h. Addition of 12-O-tetradecanoyl-phorbol 13-acetate (TPA), A23187, and dibutyryl cAMP caused the same kind of induction. Cholera toxin potentiated the ability of A23187 to induce ornithine decarboxylase, but not that of OAG. Dibutyryl cAMP augmented the induction caused by A23187 but not by TPA. These results suggest that both the activation of Ca++-sensitive, phospholipid-dependent protein kinase (protein kinase C) and the increase in intracellular levels of Ca++ and cAMP are necessary for this induction. cAMP may potentiate the induction by modulating a Ca++ messenger system other than that for protein kinase C activation.     

Induction of ornithine decarboxylase in guinea-pig lymphocytes. Synergistic effect of diacylglycerol and calcium

Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and trifluoperazine inhibited ornithine decarboxylase induction in lymphocytes activated with phytohemagglutinin or inophore A23187. W-7, a more potent calmodulin antagonist than W-5, suppressed ornithine decarboxylase induction in a higher extent than did W-5. These results suggest that calmodulin may play an important role in ornithine decarboxylase induction in the activated lymphocytes. However, the extent of ornithine decarboxylase induction was greater in cells pretreated with Clostridium phospholipase C and then incubated with ionophore A23187 than in cells incubated with ionophore A23187 without the pretreatment. Moreover, combined treatment of cells with ionophore A23187 and tumor promotor, phorbol 12-myristate 13-acetate, caused synergistic induction of ornithine decarboxylase activity. These results, taken together, suggest that both activations of Ca2+-activated phospholipid-dependent protein kinase by diacylglycerol and of calmodulin-dependent function resulted from an elevation of cytosolic Ca2+ concentration may operate in the induction of ornithine decarboxylase in the activated lymphocytes.     

A simple method for preparing physiologically active mitochondria from plant leaves rich in oils and phenolics

Amberlite XAD-7, a nonionic polyacrylate adsorbent, was found to be a very effective protectant for isolating mitochondria from tissues rich in oils and phenolics. Physiologically active, well-coupled mitochondria were successfully prepared from young green leaf tissues of citrus, apple, pear and tobacco.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - BSA bovine serum albumin - PVP polyvinylpyrrolidone     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.