Nitric Oxide Synthesis Is Increased in Cybrid Cells with m.3243A>G Mutation

Abstract

Nitric oxide (NO) is a free radical and a signaling molecule in several pathways, produced by nitric oxide synthase (NOS) from the conversion of l-arginine to citrulline. Supplementation of l-arginine has been used to treat MELAS (mitochondrial encephalopathy with lactic acidosis and stroke like syndrome), a mitochondrial disease caused by the m.3243A>G mutation. Low levels of serum arginine and endothelium dysfunction have been reported in MELAS and this treatment may increase NO in endothelial cells and promote vasodilation, decreasing cerebral ischemia and strokes. Although clinical benefits have been reported, little is known about NO synthesis in MELAS. In this study we found that osteosarcoma derived cybrid cells with high levels of m.3243A>G had increased nitrite, an NO metabolite, and increased intracellular NO, demonstrated by an NO fluorescent probe (DAF-FM). Muscle vessels from patients with the same mutation had increased staining in NADPH diaphorase, suggestive of increased NOS. These results indicate increased production of NO in cells harboring the m.3243A>G, however no nitrated protein was detected by Western blotting. Further studies are necessary to clarify the exact mechanisms of l-arginine effect to determine the appropriate clinical use of this drug therapy.

Pulmonary artery endothelial cell dysfunction and decreased populations of highly proliferative endothelial cells in experimental congenital diaphragmatic hernia

“…The DAF-FM compound was washed off the cells, and phenol red-and serum-free media was placed on the cells. Following incubation, fluorescent intensity was measured and compared between control (n ϭ 3 animals) and CDH (n ϭ 3 animals) cells as previously described (7,13,28). VEGF protein (25 ng/ml) was used as a positive control to stimulate NO production.…”

NO control of mitochondrial function in normal and transformed cells

“…This down-regulation was only found in the presence of COX deficiency, as other patients with Complex II or I deficiency did not have alterations in NOS activity (99). However, these mechanisms may be more complex because a study with cultured osteosarcoma derived cybrid cells with a mitochondrial deficiency due to the m.3243A>G mutation, showed an increase of intracellular NO and nitrate/nitrite in cultured media when compared the control cells (107). On the other hand, another study with SH-SY5Y (neuroblastoma derived) cybrid cells, carrying the same mutation (m.3243A>G) showed a significant reduction of nitrite/nitrate concentration, which suggested that these cells had a reduced NOS activity (108).…”

L-Arginine Affects Aerobic Capacity and Muscle Metabolism in MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-Like Episodes) Syndrome

“…It has been previously suggested that L-arginine supplementation in MELAS may work by enhancing nitric oxide mediated vasodilation of blood vessels with intrinsically impaired capacity for vasodilation [ 4 ]. A recent study found elevated baseline levels of intracellular nitric oxide in m.3243A>G cybrid cells, bringing into dispute the latter role of L-arginine supplementation and raising the possibility of an alternative metabolic role [ 30 ]. Our finding of improved aerobic metabolism supports a benefit from arginine supplementation beyond nitric oxide mediated vasodilation.…”