J Periodontal Implant Sci.  2011 Feb;41(1):17-22. 10.5051/jpis.2011.41.1.17.

Synergic induction of human periodontal ligament fibroblast cell death by nitric oxide and N-methyl-D-aspartic acid receptor antagonist

• Seo T
• Cha S
• Woo KM
• Park YS
• Cho YM
• Lee JS
• Kim TI

• KMID: 2212131
• DOI: http://doi.org/10.5051/jpis.2011.41.1.17

Abstract

PURPOSE
Nitric oxide (NO) has been known as an important regulator of osteoblasts and periodontal ligament cell activity. This study was performed to investigate the relationship between NO-mediated cell death of human periodontal ligament fibroblasts (PDLFs) and N-methyl-D-aspartic acid (NMDA) receptor antagonist (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK801).
METHODS
Human PDLFs were treated with various concentrations (0 to 4 mM) of sodium nitroprusside (SNP) with or without 200 microM MK801 in culture media for 16 hours and the cell medium was then removed and replaced by fresh medium containing MTS reagent for cell proliferation assay. Western blot analysis was performed to investigate the effects of SNP on the expression of Bax, cytochrome c, and caspase-3 proteins. The differences for each value among the sample groups were compared using analysis of variance with 95% confidence intervals.
RESULTS
In the case of SNP treatment, as a NO donor, cell viability was significantly decreased in a concentration-dependent manner. In addition, a synergistic effect was shown when both SNP and NMDA receptor antagonist was added to the medium. SNP treated PDLFs exhibited a round shape in culture conditions and were dramatically reduced in cell number. SNP treatment also increased levels of apoptotic marker protein, such as Bax and cytochrome c, and reduced caspase-3 in PDLFs. Mitogen-activated protein kinase signaling was activated by treatment of SNP and NMDA receptor antagonist.
CONCLUSIONS
These results suggest that excessive production of NO may induce apoptosis and that NMDA receptor may modulate NO-induced apoptosis in PDLFs.

Keyword

Cell proliferation; Mitogen-activated protein kinase; N-methyl-D-aspartate receptor; Periodontal ligament

MeSH Terms

Apoptosis
Blotting, Western
Caspase 3
Cell Count
Cell Death
Cell Proliferation
Cell Survival
Culture Media
Cytochromes c
Dizocilpine Maleate
Fibroblasts
Humans
Hydrogen
Maleates
N-Methylaspartate
Nitric Oxide
Nitroprusside
Osteoblasts
Periodontal Ligament
Protein Kinases
Proteins
Tissue Donors
Caspase 3
Culture Media
Cytochromes c
Dizocilpine Maleate
Hydrogen
Maleates
N-Methylaspartate
Nitric Oxide
Nitroprusside
Protein Kinases
Proteins

Figure

• 

  Figure 1 Effect of sodium nitroprusside (SNP) on the viability of periodontal ligament fibroblasts (PDLFs). PDLFs showed morphological changes following treatment with SNP and SNP+200 µM (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK801) for 16 hours (A). PDLFs were treated with SNP at indicated concentrations for 16 hours and the cell viability was determined by MTS assay (B). Magnification ×100. a)Indicates significantly different from non-treated cells (P<0.05). OD: optical density.

• 

  Figure 2 Sodium nitroprusside (SNP) altered the expression of apoptosis marker proteins. Periodontal ligament fibroblasts were treated with SNP and (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d] cyclohepten-5, 10-imine hydrogen maleate (MK801) for 16 hours. The cell lysates were performed to Western blot analysis using anti-Bax, anti-cytochrome c, anti-caspase-3, and anti-β-actin antibodies. β-actin was used as internal control.

• 

  Figure 3 Sodium nitroprusside (SNP) induced phosphorylation of mitogen-activated protein kinases in periodontal ligament fibroblasts (PDLFs). PDLFs were treated with SNP and (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK801) for 16 hours. The cell lysates were performed to Western blot analysis using anti-c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SARK), anti-extracellular-signal-regulated kinase (ERK), anti-mitogen-activated protein kinases (p38), anti-phosphorylated JNK/SARK, anti-phosphorylated ERK or anti-phosphorylated p38 antibodies.

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