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Influence of GSH synthesis inhibition on temporal distribution of NAD+/NADH during vascular endothelial cells proliferation.

Busu C, Atanasiu V, Caldito G, Aw TY - J Med Life (2014 Oct-Dec)

Bottom Line: The redox couples reduced glutathione (GSH)/oxidized glutathione (GSSG), NADH/NAD+, and NADPH/NADP+ play major functions in the intracellular redox balance.Inhibition of GSH synthesis by buthionine sulfoximine (BSO) and sustained low cellular GSH significantly increased nuclear NADH levels (p<0.01), which correlated with lower nuclear GSH and prolonged cell cycle S-phase.These results provide important insights into GSH control of vascular endothelial growth and restitution, key processes in the restoration of the endothelium adjacent to the post-injury lesion site.

View Article: PubMed Central - PubMed

Affiliation: "Carol Davila" University of Medicine and Pharmacy, Medical School, Biochemistry Department, Bucharest, Romania ; Department of Molecular & Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA.

ABSTRACT
Pathological conditions states such as stroke, diabetes mellitus, hypertension, dyslipidemia are associated with increased levels of free radicals that alter normal function of the vascular endothelium and perturb vascular homeostasis. The redox couples reduced glutathione (GSH)/oxidized glutathione (GSSG), NADH/NAD+, and NADPH/NADP+ play major functions in the intracellular redox balance. Any decrease in tissue or systemic GSH levels under the aforementioned pathologies would enhance oxidative damage to the vascular endothelium. Beside their role as coenzyme that participate in cellular metabolism, pyridine nucleotides serve also as substrate for enzymes involved in DNA repair and longevity. There is scant data on NAD+/NADH kinetics and distribution during human cells proliferation. Here, we determined the influence of cellular GSH status on the early dynamics of nuclear-to-cytosol (N-to-C) NAD+ and nuclear NADH kinetics (6 h interval) over 72 h of endothelial cell proliferation. The IHEC cell line was used as a surrogate for human brain micro vascular endothelial cells. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) and sustained low cellular GSH significantly increased nuclear NADH levels (p<0.01), which correlated with lower nuclear GSH and prolonged cell cycle S-phase. When BSO was removed the pattern of nuclear NAD+ resembled that of control group, but nuclear NADH concentrations remained elevated, as in GSH deficient cells (p<0.01). The coincidence of high nuclear NADH and lower nuclear NAD+ with S-phase prolongation are suggestive of CtBP and NAD+-dependent DNA repair enzyme activation under conditions of decreased cellular GSH. These results provide important insights into GSH control of vascular endothelial growth and restitution, key processes in the restoration of the endothelium adjacent to the post-injury lesion site.

No MeSH data available.


Related in: MedlinePlus

Time course of cytosolic (A) and nuclear (B, C) GSH and NAD+, NADH from 0 to 72 h in cytosolic and nuclear fractions from IHEC cells treated with BSO to maintain low GSH (Treated) or treated with BSO that was removed to allow GSH recovery (Reversal). Line drawing represents GSH data reproduced from ref. 5 and included here for direct comparison with changes in cytosolic & nuclear NAD+ and NADH. Filled square represents NAD+; filled circle represents NADH. Results are mean ± SEM for 6 (GSH) or 5 (NAD+, NADH) different determinations. Vertical error bars represent mean ± SEM of GSH, NAD+, NADH
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Figure 2: Time course of cytosolic (A) and nuclear (B, C) GSH and NAD+, NADH from 0 to 72 h in cytosolic and nuclear fractions from IHEC cells treated with BSO to maintain low GSH (Treated) or treated with BSO that was removed to allow GSH recovery (Reversal). Line drawing represents GSH data reproduced from ref. 5 and included here for direct comparison with changes in cytosolic & nuclear NAD+ and NADH. Filled square represents NAD+; filled circle represents NADH. Results are mean ± SEM for 6 (GSH) or 5 (NAD+, NADH) different determinations. Vertical error bars represent mean ± SEM of GSH, NAD+, NADH

Mentions: The control group maintained a relatively constant concentration of nuclear NAD+ between 36-66h (Fig. 2B), a time frame that corresponded with two rounds of cell cycle as showed by S phase peaks (Fig. 3A). In the reversal group constant levels of nuclear NAD+ were maintained between 42-72h (Fig. 2B), which correlated with a lengthened S phase of the cell cycle (Fig. 3B)


Influence of GSH synthesis inhibition on temporal distribution of NAD+/NADH during vascular endothelial cells proliferation.

Busu C, Atanasiu V, Caldito G, Aw TY - J Med Life (2014 Oct-Dec)

Time course of cytosolic (A) and nuclear (B, C) GSH and NAD+, NADH from 0 to 72 h in cytosolic and nuclear fractions from IHEC cells treated with BSO to maintain low GSH (Treated) or treated with BSO that was removed to allow GSH recovery (Reversal). Line drawing represents GSH data reproduced from ref. 5 and included here for direct comparison with changes in cytosolic & nuclear NAD+ and NADH. Filled square represents NAD+; filled circle represents NADH. Results are mean ± SEM for 6 (GSH) or 5 (NAD+, NADH) different determinations. Vertical error bars represent mean ± SEM of GSH, NAD+, NADH
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4316149&req=5

Figure 2: Time course of cytosolic (A) and nuclear (B, C) GSH and NAD+, NADH from 0 to 72 h in cytosolic and nuclear fractions from IHEC cells treated with BSO to maintain low GSH (Treated) or treated with BSO that was removed to allow GSH recovery (Reversal). Line drawing represents GSH data reproduced from ref. 5 and included here for direct comparison with changes in cytosolic & nuclear NAD+ and NADH. Filled square represents NAD+; filled circle represents NADH. Results are mean ± SEM for 6 (GSH) or 5 (NAD+, NADH) different determinations. Vertical error bars represent mean ± SEM of GSH, NAD+, NADH
Mentions: The control group maintained a relatively constant concentration of nuclear NAD+ between 36-66h (Fig. 2B), a time frame that corresponded with two rounds of cell cycle as showed by S phase peaks (Fig. 3A). In the reversal group constant levels of nuclear NAD+ were maintained between 42-72h (Fig. 2B), which correlated with a lengthened S phase of the cell cycle (Fig. 3B)

Bottom Line: The redox couples reduced glutathione (GSH)/oxidized glutathione (GSSG), NADH/NAD+, and NADPH/NADP+ play major functions in the intracellular redox balance.Inhibition of GSH synthesis by buthionine sulfoximine (BSO) and sustained low cellular GSH significantly increased nuclear NADH levels (p<0.01), which correlated with lower nuclear GSH and prolonged cell cycle S-phase.These results provide important insights into GSH control of vascular endothelial growth and restitution, key processes in the restoration of the endothelium adjacent to the post-injury lesion site.

View Article: PubMed Central - PubMed

Affiliation: "Carol Davila" University of Medicine and Pharmacy, Medical School, Biochemistry Department, Bucharest, Romania ; Department of Molecular & Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA.

ABSTRACT
Pathological conditions states such as stroke, diabetes mellitus, hypertension, dyslipidemia are associated with increased levels of free radicals that alter normal function of the vascular endothelium and perturb vascular homeostasis. The redox couples reduced glutathione (GSH)/oxidized glutathione (GSSG), NADH/NAD+, and NADPH/NADP+ play major functions in the intracellular redox balance. Any decrease in tissue or systemic GSH levels under the aforementioned pathologies would enhance oxidative damage to the vascular endothelium. Beside their role as coenzyme that participate in cellular metabolism, pyridine nucleotides serve also as substrate for enzymes involved in DNA repair and longevity. There is scant data on NAD+/NADH kinetics and distribution during human cells proliferation. Here, we determined the influence of cellular GSH status on the early dynamics of nuclear-to-cytosol (N-to-C) NAD+ and nuclear NADH kinetics (6 h interval) over 72 h of endothelial cell proliferation. The IHEC cell line was used as a surrogate for human brain micro vascular endothelial cells. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) and sustained low cellular GSH significantly increased nuclear NADH levels (p<0.01), which correlated with lower nuclear GSH and prolonged cell cycle S-phase. When BSO was removed the pattern of nuclear NAD+ resembled that of control group, but nuclear NADH concentrations remained elevated, as in GSH deficient cells (p<0.01). The coincidence of high nuclear NADH and lower nuclear NAD+ with S-phase prolongation are suggestive of CtBP and NAD+-dependent DNA repair enzyme activation under conditions of decreased cellular GSH. These results provide important insights into GSH control of vascular endothelial growth and restitution, key processes in the restoration of the endothelium adjacent to the post-injury lesion site.

No MeSH data available.


Related in: MedlinePlus