Ascorbate-mediated enhancement of reactive oxygen species generation from polymorphonuclear leukocytes: modulatory effect of nitric oxide

Recent studies from our laboratory have demonstrated that ascorbate potentiated enzymatic synthesis of nitric oxide (NO) from polymorphonuclear leukocytes (PMNs). NO is known to modulate various function of PMNs such as chemotaxis, adherence, aggregation, and generation of reactive oxygen species (ROS). The role of ascorbate in the PMN phagocytosis, ROS generation, and apoptosis was thus evaluated in the present study. Ascorbate and its oxidized and cell-permeable analog, dehydroascorbate (DHA), did not affect the phagocytosis but enhanced ROS generation and apoptosis following treatment with Escherichia coli or arachidonic acid. A detailed investigation on the DHA-mediated response indicated that inhibitors of DHA uptake, reduced nicotinamide adenine dinucleotide phosphate oxidase, NO synthase, or ROS scavengers attenuated ROS generation. In DHA-treated cells, enhanced generation of peroxynitrite was also observed; thus, ascorbate-mediated ROS and reactive nitrogen species generation might mediate cytotoxicity toward the ingested microbes and subsequently, augmented PMN apoptosis. Results of the present study have helped in delineating the role of ascorbate in the modulation of NO-mediated ROS generation from PMNs

L-Citrulline mediated relaxation in the control and lipopolysaccharide-treated rat aortic rings

The present study was undertaken to investigate relaxant effect of -citrulline in phenylephrine precontracted endothelium intact thoracic aortic rings obtained from control or lipopolysaccharide (1 mg/kg)-treated rats. L-Citrulline produced 40&#177; 3% (n=36) and 60&#177; 5% (n=24) relaxations in control and lipopolysaccharide-treated rings, respectively. Nitric oxide (NO) release and cyclic guanosine-3',5'-monophosphate levels from the rings were also increased following treatment with L-citrulline. Inhibition of guanylate cyclase, L-citrulline recycling to L-arginine or denudation of the endothelium, significantly reduced L-citrulline-induced relaxations both in control and lipopolysaccharide-treated rings. Treatment of rings with protein synthesis inhibitors prevented relaxations to L-citrulline. Inhibitor of Ca<SUP>2+</SUP>-activated K<SUP>+</SUP> channels, tetrabutylammonium or precontraction of the rings with KCl (80 mM), significantly attenuated L-citrulline mediated relaxations in control and lipopolysaccharide-treated rings. Thus, L-citrulline seems to exert significant relaxation by supplementing the release of NO due to its recycling to L-arginine, which gets further augmented after lipopolysaccharide treatment

Role of ascorbate in the regulation of nitric oxide generation by polymorphonuclear leukocytes

We have recently demonstrated that NO-mediated polymorphonuclear (PMN)-dependent inhibition of rat platelet aggregation is significantly enhanced in the presence of ascorbate. Consequently, the present study was undertaken to elucidate the underlying mechanisms involved in ascorbate-mediated potentiation of NO synthesis in PMNs. We observed that ascorbate or its oxidized product, dehydroascorbate (DHA), enhanced NOS activity, as measured by nitrite content, diaminofluorescein fluorescence or conversion of L-[3H]arginine to L-[3H] citrulline in rat, monkey, and human PMNs. The increase in NO generation following ascorbate treatment was due to the intracellular ascorbate as iodoacetamide-mediated inhibition of DHA to ascorbate conversion attenuated the DHA-mediated increase in NO synthesis. The augmentation of NOS activity in the PMN homogenate by tetrahydrobiopterin was significantly enhanced by ascorbate, while ascorbate alone did not influence the NOS activity. Ascorbate-mediated enhancement of NOS activity in the cultured PMNs was significantly reduced in the presence of biopterin synthesis inhibitors. Ascorbate, thus, seems to regulate the NOS activity in the PMNs through tetrahydrobiopterin

Inhibition of platelet aggregation by rat globin

This study was undertaken to identify and characterize the anti-aggregatory protein factor present in rat polymorphonuclear leukocytes (PMNs) supernatant. Since the purified protein exhibited sequence homology to beta globin, globin was also isolated from rat blood by acid-acetone precipitation and was purified on Superdex-75 column in FPLC. Elution of rat globin on the gel filtration column yielded two peaks of approximately 60 and 30 kDa as observed in the PMNs supernatant. Purity of globin and eluted fractions was further evaluated by SDS-PAGE. Platelet aggregation induced by agonists viz. adenosine-5'-diphosphate (ADP; 2-5 μM), arachidonic acid (AA; 10 μM), A23187 (2.50 μg/ml) was inhibited by globin and the purified fractions. ADP-induced rise in intracellular calcium levels and expression of CD62 on the platelets were reduced by both globin and active fraction of PMNs supernatant. Results obtained suggest that globin or globin-related protein present in the PMNs supernatant inhibits platelet aggregation response