Sueli M. Senna

Beneficial effect of glutamine on exercise-induced apoptosis of rat neutrophils.

INTRODUCTION/PURPOSE The effect of a single bout of intensive exercise on apoptosis of rat neutrophils and the possible prevention by glutamine administration was examined. The experiments were performed in sexually immature and sexually mature male rats as to examine the possible involvement of sexual maturation in the effect of exercise. METHODS Exercise was carried out on a treadmill for 1 h before rats were killed by decapitation. Aqueous solution of glutamine was given by gavage (1 g.kg-1 body weight), 1 h before exercise. Neutrophils were obtained by intraperitoneal lavage with phosphate-buffered saline (PBS), 4 h after injection of oyster glycogen solution. The cells were then analyzed for apoptosis by flow cytometry and fluorescence microscopy. Pro- and antiapoptotic gene expression was evaluated by reverse transcriptase chain reaction (RT-PCR). RESULTS Neutrophils obtained from immature and mature exercised rats showed an increase in DNA fragmentation, chromatin condensation, and phosphatidylserine externalization. This suggests that all neutrophils suffered apoptosis. To study the possible mechanism involved, the production of reactive oxygen metabolites, expression of genes involved in apoptosis and mitochondrial transmembrane potential were examined. Acute exercise raised reactive oxygen metabolites production by neutrophils. Exercise did not change the expression of antiapoptotic (bcl-xL) and apoptotic (bax and bcl-xS) genes in neutrophils from immature rats but caused a significant increase of bax and bcl-xS expression and provoked a significant decrease of bcl-xL expression in cells from mature rats. Exercise also induced a marked loss of mitochondrial depolarization in neutrophils. Oral glutamine supplementation partially prevented the exercise-induced apoptosis in neutrophils from sexually immature and mature rats. CONCLUSION The protective effect of glutamine on neutrophil apoptosis induced by acute exercise possibly occurs by preservation of mitochondrial function.

Glutathione metabolism and glutathione S‐conjugate export ATPase (MRP1/GS‐X pump) activity in cancer

Mg2+‐dependent vanadate‐sensitive glutathione S‐conjugate ATPase (GS‐X pump) activity is a common feature of some ATP‐binding cassette (ABC) transporters, such as the multidrug resistance‐associated protein (MRP1) gene product, that exports biologically active electrophiles after their conjugation with intracellular glutathione (GSH) from normal and cancer cells. Antitumor electrophiles (e.g. naturally occurring cyclopentenone prostaglandins and anticancer chemicals) can be intracellularly conjugated with GSH via a glutathione S‐transferase catalyzed reaction and be eliminated through GS‐X pumps thus threatening cancer chemotherapeutics. Since different sensitivities to antitumor electrophiles are shown by different cell types, the ability of several human cancer cell lines to produce and export S‐(2,4‐dinitrophenyl)‐glutathione (DNP‐SG) conjugate through the GS‐X pump, using whole cells and inside‐out membrane vesicle preparations, is investigated. Different cancer cell lines exhibited characteristically different GS‐X pump activity. In particular, HEp‐2 larynx carcinoma cells possess an elevated DNP‐SG export rate through the GS‐X pump compared with HeLa, K562, U937 or HL‐60 cells, which exhibit the lowest activity. The differences in DNP‐SG export rates are not due to decreased glutathione S‐transferase activity or impaired de novo synthesis of GSH. The findings suggest that the GS‐X pump may be involved in the modulation of the biological activity of both naturally occurring electrophiles and anticancer drugs. The differential expression of GS‐X pumps may lead to an improved understanding of multidrug resistance and may be exploited in the development of new therapeutic strategies for the treatment of cancer patients.

Effects of prostaglandins and nitric oxide on rat macrophage lipid metabolism in culture: implications for arterial wall-leukocyte interplay in atherosclerosis.

Macrophages/foam cells have a pivotal role in atherogenesis although little is known about the way lipid imbalance, a hallmark of atherosclerosis, leads to lipid accumulation in these cells. Modified low‐density lipoproteins are associated with macrophage lipid dysfunction in atherosclerosis, but a possible role for altered lipogenesis leading to lipid accumulation remains to be elucidated. Since endothelium‐derived nitric oxide (NO) and prostaglandins (PGs) are physiological autacoids whose production may be impaired in atherosclerosis, the effects of these mediators on de novo lipid synthesis in 24‐h cultured rat peritoneal macrophages is investigated. In resident (unstimulated) cells, 1 μM PGE2 and the stable analog of PGI2 carbaprostacyclin (cPGI2, 1 μM) deviated the overall [1‐14C]acetate from incorporation into cholesterol, free fatty acids and triacylglycerols favoring the formation of phospholipids. In inflammatory (thioglycollate‐elicited) macrophages, these eicosanoids likewise reduced tac‐incorporations into all the lipid fractions tested. Also, cPGI2 and PGE2 reduced [4‐14C]cholesterol uptake from inflammatory cells but did not interfere in 14C‐cholesterol export. The PGE2‐derivative PGA2 (10‐20 μM) reduced 14C‐incorporations into all the lipids in resident cells while it enhanced phospholipid synthesis by up to 129% at the expense of reduced incorporations into the other test lipids. The NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP, 1‐10 μM), when added to macrophages in the presence of superoxide dismutase (SOD, to avoid the reaction of superoxide with NO), significantly reduced lipogenesis especially in inflammatory cells. These findings suggest that endothelium‐derived NO and PGs may be associated with macrophage lipid accumulation by modulating lipogenesis and cholesterol uptake within these ceils.