J Korean Ophthalmol Soc.  2008 Jun;49(6):979-986. 10.3341/jkos.2008.49.6.979.

Ischemic Preconditioning and the Role of Protein Kinase C in Cultured Retinal Ganglion Cell Line

• Na KD
• Kang SY
• Seong GJ
• Hong S
• Chun MJ
• Kim CY

• KMID: 2110880
• DOI: http://doi.org/10.3341/jkos.2008.49.6.979

Abstract

PURPOSE: To investigate the cellular protective effects of hypoxic preconditioning against oxidative stress in a staurosporine-differentiated RGC-5 cell line and the relevance of protein kinase C subtype expression.
METHODS
The minimum staurosporine concentration and exposure time necessary to morphologically fully differentiate RGC-5 cells were determined. Cytotoxic injury was provided by oxidative stress with 800 micrometer hydrogen peroxide (H2O2) for 15 hours to morphologically fully-differentiated cells. The cytoprotective effect of hypoxic preconditioning was found by exposing the cell line to 0.3% oxygen for different periods of time. Quantifiable changes in the expression of mRNAs and proteins of the isoenzymes alpha, beta, gamma, delta, epsilon, zeta of protein kinase C were determined before and after 1, 2, 15, and 24 hours of hypoxic preconditioning.
RESULTS
Axonal growth in RGC-5 cells after the induction of differentiation with staurosporine caused these cells to resemble neurons. The minimal concentration and exposure time to staurosporine that evoked full differentiation of RGC-5 cells was exposure to 2 micrometer staurosporine for 1 hour. An LDH assay demonstrated that hypoxic preconditioning had neuroprotective effects against hydrogen peroxide-induced oxidative stress. Protein and mRNA levels of PKC isoforms alpha and epsilon increased after preconditioning.
CONCLUSIONS
Hypoxic preconditioning of staurosporine-differentiated RGC-5 cells had a cytoprotective effect against oxidative stress. The associated increase of mRNA and proteins of PKC isoenzymes alpha and epsilon suggest some functional relevance of these isoenzymes to the cytoprotective effects conferred by hypoxic preconditioning.

Keyword

Ischemic preconditioning; Oxidative stress; Protein kinase C; RGC-5

MeSH Terms

Axons
Cell Line
Hydrogen
Hydrogen Peroxide
Ischemic Preconditioning
Isoenzymes
Neurons
Neuroprotective Agents
Oxidative Stress
Oxygen
Protein Isoforms
Protein Kinase C
Protein Kinases
Proteins
Retinal Ganglion Cells
Retinaldehyde
RNA, Messenger
Staurosporine
Hydrogen
Hydrogen Peroxide
Isoenzymes
Neuroprotective Agents
Oxygen
Protein Isoforms
Protein Kinase C
Protein Kinases
Proteins
RNA, Messenger
Retinaldehyde
Staurosporine

Figure

• 

  Figure 1. The assessment of optimal level and duration in staurosporine treatment for differentiation of RGC-5: The shape of RGC-5 cell changes from fibroblast-like shape to neuron-like shape showing axon and multiple synapse with treatment of staurosporine. Staurosporine 1.0 µg for 2 hours seemed to be the minimum duration and the level for the appropriate morphological differentiation. Staurosporine 0 µg: 1 hr (A), 2 hr (B), 6 hr (C), 24 hr (D); Staurosporine 0.5 µg: 1 hr (E), 2 hr (F), 6 hr (G), 24 hr (H); Staurosporine 1.0 µg: 1 hr (I), 2 hr (J), 6 hr (K), 24 hr (L); Staurosporine 2.0 µg: 1 hr (M), 2 hr (N), 6 hr (O), 24 hr (P).

• 

  Figure 2. Cytotoxicity of ischemic preconditioning (0.3% oxygen state) against oxidative stress (800 µM H2 O2 for 15 hr) in differentiated RGC-5 as demonstrated with LDH assay. With prolonged duration of ischemic preconditioning, RGC-5 survival increased up to 8 hrs of ischemic preconditioning after which cytotoxicity increased. Cytotoxicity after oxidative stress was statistically significantly decreased at 2 to 24 hours of ischemic preconditioning when compared to control (* p<0.05 by Mann-Whitney U test).

• 

  Figure 3. Changes of PKC iso-enzyme mRNA before and after ischemic preconditioning (B-PC, before preconditioning; PC0hr, just after the preconditioning; PC1hr, 1 hour after preconditioning; PC2hr, 2 hours after preconditioning; PC15hr, 15 hours after preconditioning; PC24hr, 24 hours after preconditioning); We can see the elevation of level of PKC α, β, γ, δ, ε mRNA 1 or 2 hours after PC (preconditioning, 0.3% oxygen 8 hrs). The elevations are decreasing with time after PC.

• 

  Figure 4. Changes of PKC iso-enzyme protein before and after ischemic preconditioning (B, before preconditioning; 0, just after the preconditioning; 1, 1 hour after preconditioning; 2, 2 hours after preconditioning; 15, 15 hours after preconditioning; 24, 24 hours after preconditioning). PKC ε increased until 2 hours after the preconditioning, and PKC α increased from 15 hours after the preconditioning. These changes are relatively correlated with increasing tendency of mRNA.

Reference

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