Nystatin‐induced nitric oxide production in mouse macrophage‐like cell line RAW264.7

Abstract

Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the iκB/NF-κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-κB pathway without affecting the condition of lipid rafts.Key words lipopolysaccharide, nitric oxide, nystatin, RAW264.7 cells.NO acts as a releasing factor that mediates vasodilation, a neuronal messenger molecule, and a major regulatory molecule and principal cytotoxic mediator of the immune system (1-5). A large amount of NO is synthesized by an inducible isoform of NO synthase (iNOS) that, once expressed, produces NO for long periods of time (3, 6). iNOS-mediated NO production is enhanced by and LPS in vitro (6, 7). NO has emerged as an important cytotoxic and cytostatic effector in host defense against a number of pathogens, including viruses, bacteria, fungi, and parasites.Nystatin is clinically used as anti-fungal drug. On the other hand, nystatin is also known as a lipid depleter and List of Abbreviations:Akt, protein kinase B; COX-2, cyclooxygenase-2; CTB, cholera toxin B; ERK1/2, extracellular signal-regulated kinase 1/2; GPI, glycosylphosphatidylinositol; IFN, interferon; IκB, inhibitor of nuclear factor κB; iNOS, inducible NO synthase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MyD88, myeloid differentiation response gene 88; NF-κB, nuclear factor κB; NO, nitric oxid; PKB, protein kinase B; PI3K/Akt, phosphatidylionsitol 3-kinase/Akt; TLR, Toll-like receptor; TNF, tumor necrosis factor. often utilized in lipid rafts studies (8,9). Lipid rafts are glycolipid-enriched membrane domains that are rich in cholesterol and sphingolipids. Certain proteins associated with cellular signaling processes have been shown to associate with lipid rafts (10). Proteins that have shown association to the lipid rafts include GPI-anchored proteins, doubly-acylated tyrosine kinases of the Src family, and transmembrane proteins. Lipid rafts have been implicated in a number of physiological and pathological processes. Unexpectedly, we found that non-cytotoxic doses of nystatin caused NO p...

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