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Lens epithelial cell apoptosis and intracellular Ca2+ increase in the presence of xanthurenic acid.

Malina H, Richter C, Frueh B, Hess OM - BMC Ophthalmol (2002)

Bottom Line: In the control cells gelsolin stained the perinuclear region, whereas in the presence of 10 microM xanthurenic acid gelsolin is translocated to the cytoskeleton, but does not lead to cytoskeleton breakdown.At low (5 to 10 microM) of xanthurenic acid concentration, the elongation of the cytoskeleton was associated with migration of mitochondria and cytochrome c release.We observed an induction of calpain Lp 82 and an increase of free Ca2+ in the cells in a xanthurenic acid concentration-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Swiss Cardiovascular Research Center, Inselspital, CH-3010 Bern, Switzerland. halina.malina@dkf2.unibe.ch

ABSTRACT

Background: Xanthurenic acid is an endogenous product of tryptophan degradation by indoleamine 2,3-dioxygenase (IDO). We have previously reported that IDO is present in mammalian lenses, and xanthurenic acid is accumulated in the lenses with aging. Here, we studied the involvement of xanthurenic acid in the human lens epithelial cell physiology.

Methods: Human lens epithelial cells primary cultures were used. Control cells, and cells in the presence of xanthurenic acid grow in the dark. Western blot analysis and immunofluorescence studies were performed.

Results: In the presence of xanthurenic acid human lens epithelial cells undergo apoptosis-like cell death. In the control cells gelsolin stained the perinuclear region, whereas in the presence of 10 microM xanthurenic acid gelsolin is translocated to the cytoskeleton, but does not lead to cytoskeleton breakdown. In the same condition caspase-3 activation, and DNA fragmentation was observed. At low (5 to 10 microM) of xanthurenic acid concentration, the elongation of the cytoskeleton was associated with migration of mitochondria and cytochrome c release. At higher concentrations xanthurenic acid (20 microM and 40 microM) damaged mitochondria were observed in the perinuclear region, and nuclear DNA cleavage was observed. We observed an induction of calpain Lp 82 and an increase of free Ca2+ in the cells in a xanthurenic acid concentration-dependent manner.

Conclusions: The results show that xanthurenic acid accumulation in human lens epithelial cells disturbs the normal cell physiology and leads to a cascade of pathological events. Xanthurenic acid induces calpain Lp82 and caspases in the cells growing in the dark and can be involved in senile cataract development.

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Increase of free Ca2+ in HuLEC after growth in the presence of xanthurenic acid for 96 hours. HuLEC were stained with Calcium Orange™: (A) control cells, (B) in the presence of 10 μM xanthurenic acid, (C) in the presence of 20 μM xanthurenic acid.
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Figure 7: Increase of free Ca2+ in HuLEC after growth in the presence of xanthurenic acid for 96 hours. HuLEC were stained with Calcium Orange™: (A) control cells, (B) in the presence of 10 μM xanthurenic acid, (C) in the presence of 20 μM xanthurenic acid.

Mentions: Ca2+ increases are associated with cataract development. We investigated intracellular Ca2+ by loading the cells with acetometoxyl ester of Calcium Orange™. This dye becomes fluorescent when hydrolysed in the cell by esterases and conjugated with free Ca2+ Cells were incubated without xanthurenic acid or with xanthurenic acid at concentration of 0.125; 0.25, 0.5; 1; 2; 5, 10, and 20 μM. A presence of xanthurenic acid in the cell culture medium higher then 2 μM provokes an increase of intracellular Ca2+ in comparison with control. In the presence of xanthurenic acid at concentration of 10 μM and 20 μM an intensive staining with Calcium Orange™ was observed indicating an increase of free Ca2+ in the cell in a xanthurenic acid concentration-dependent manner (Fig. 7).


Lens epithelial cell apoptosis and intracellular Ca2+ increase in the presence of xanthurenic acid.

Malina H, Richter C, Frueh B, Hess OM - BMC Ophthalmol (2002)

Increase of free Ca2+ in HuLEC after growth in the presence of xanthurenic acid for 96 hours. HuLEC were stained with Calcium Orange™: (A) control cells, (B) in the presence of 10 μM xanthurenic acid, (C) in the presence of 20 μM xanthurenic acid.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC103667&req=5

Figure 7: Increase of free Ca2+ in HuLEC after growth in the presence of xanthurenic acid for 96 hours. HuLEC were stained with Calcium Orange™: (A) control cells, (B) in the presence of 10 μM xanthurenic acid, (C) in the presence of 20 μM xanthurenic acid.
Mentions: Ca2+ increases are associated with cataract development. We investigated intracellular Ca2+ by loading the cells with acetometoxyl ester of Calcium Orange™. This dye becomes fluorescent when hydrolysed in the cell by esterases and conjugated with free Ca2+ Cells were incubated without xanthurenic acid or with xanthurenic acid at concentration of 0.125; 0.25, 0.5; 1; 2; 5, 10, and 20 μM. A presence of xanthurenic acid in the cell culture medium higher then 2 μM provokes an increase of intracellular Ca2+ in comparison with control. In the presence of xanthurenic acid at concentration of 10 μM and 20 μM an intensive staining with Calcium Orange™ was observed indicating an increase of free Ca2+ in the cell in a xanthurenic acid concentration-dependent manner (Fig. 7).

Bottom Line: In the control cells gelsolin stained the perinuclear region, whereas in the presence of 10 microM xanthurenic acid gelsolin is translocated to the cytoskeleton, but does not lead to cytoskeleton breakdown.At low (5 to 10 microM) of xanthurenic acid concentration, the elongation of the cytoskeleton was associated with migration of mitochondria and cytochrome c release.We observed an induction of calpain Lp 82 and an increase of free Ca2+ in the cells in a xanthurenic acid concentration-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Swiss Cardiovascular Research Center, Inselspital, CH-3010 Bern, Switzerland. halina.malina@dkf2.unibe.ch

ABSTRACT

Background: Xanthurenic acid is an endogenous product of tryptophan degradation by indoleamine 2,3-dioxygenase (IDO). We have previously reported that IDO is present in mammalian lenses, and xanthurenic acid is accumulated in the lenses with aging. Here, we studied the involvement of xanthurenic acid in the human lens epithelial cell physiology.

Methods: Human lens epithelial cells primary cultures were used. Control cells, and cells in the presence of xanthurenic acid grow in the dark. Western blot analysis and immunofluorescence studies were performed.

Results: In the presence of xanthurenic acid human lens epithelial cells undergo apoptosis-like cell death. In the control cells gelsolin stained the perinuclear region, whereas in the presence of 10 microM xanthurenic acid gelsolin is translocated to the cytoskeleton, but does not lead to cytoskeleton breakdown. In the same condition caspase-3 activation, and DNA fragmentation was observed. At low (5 to 10 microM) of xanthurenic acid concentration, the elongation of the cytoskeleton was associated with migration of mitochondria and cytochrome c release. At higher concentrations xanthurenic acid (20 microM and 40 microM) damaged mitochondria were observed in the perinuclear region, and nuclear DNA cleavage was observed. We observed an induction of calpain Lp 82 and an increase of free Ca2+ in the cells in a xanthurenic acid concentration-dependent manner.

Conclusions: The results show that xanthurenic acid accumulation in human lens epithelial cells disturbs the normal cell physiology and leads to a cascade of pathological events. Xanthurenic acid induces calpain Lp82 and caspases in the cells growing in the dark and can be involved in senile cataract development.

Show MeSH
Related in: MedlinePlus