Korean J Ophthalmol.  2012 Oct;26(5):378-382. 10.3341/kjo.2012.26.5.378.

Effect of Amiloride to Retinal Toxicity Induced by Tissue Plasminogen Activator

• Kim US
• Oh HS
• Kwon OW
• Chung I
• Lee SH
• Lee JH

• KMID: 1397489
• DOI: http://doi.org/10.3341/kjo.2012.26.5.378

Abstract

PURPOSE
The effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated.
METHODS
Primary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 microm) in addition to commercial tPA or L-arginine treatment.
RESULTS
After exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 +/- 5.1 pmole/mL and 63.1 +/- 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 +/- 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 microm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 microm amiloride and 78.4% with 100 microm amiloride (p = 0.015).
CONCLUSIONS
L-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 microm protects against L-arginine-induced retinal cell death.

Keyword

Amiloride; Arginine; Retinal toxicity; Tissue plasminogen activator

MeSH Terms

Amiloride/*pharmacology
Analysis of Variance
Animals
Arginine/toxicity
Cell Death/drug effects
Cells, Cultured
Cyclic GMP/pharmacology
Enzyme-Linked Immunosorbent Assay
Mice
Retina/cytology/*drug effects
Tissue Plasminogen Activator/*toxicity

Figure

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  Fig. 1 (A) Fluorescence micrograph (Hoechst-PI stain) of cultured retinal cells treated with L-arginine (L-arg). L-arg-induced cell death showed nuclear fragmentation, the typical apoptotic nuclear pattern. Cells with blue fragmented nuclei are early apoptotic cells, and cells with pink fragmented nuclei are late apoptotic cells (arrows). (B) The graph shows the level of cell death in primary cultured retinal cells at 24 hours after adding tissue plasminogen activator (tPA) alone, tPA plus L-arg, and L-arg alone. Cell death was assessed by lactate dehydrogenase (LDH) assay. Significant cell death occurred at 24 hours after treatment with tPA plus L-arg and L-arg alone. The data represent the means ± SD of the three independent experiments and were statistically analyzed by a one-way ANOVA and the Student-Newman-Keul's test using SigmaStat software. *p < 0.01 compared with control.

• 

  Fig. 2 (A) Cyclic guanosine monophosphate (cyclic-GMP) formation after exposure to tissue plasminogen activator (tPA) plus L-arginine (L-arg) and L-arg alone. The graph shows the cyclic-GMP concentration (pmole/mL) in primary cultured retinal cell media at 0, 12, or 24 hours after adding 10 µg/400 µL tPA (containing L-arg) and 5.0 mM L-arg. Cyclic-GMP concentration was assessed by ELISA assay. The concentration of cyclic-GMP was significantly elevated at 12 hours after treatment with 5.0 mM L-arg and at 24 hours after adding 10 µg/400 µL tPA plus L-arg and 5.0 mM L-arg. *p < 0.05 and **p < 0.01 compared with same time control groups (closed circle; control group, open circle; tPA-arg and reverse triangle; L-arg). (B) Measurement of cell death in cultures treated with various concentrations of cyclic-GMP. The graph shows the level of cell death in primary cultured retina cells at 24 hours after adding cyclic-GMP in various concentrations. Cell death was assessed by lactate dehydrogenase (LDH) assay. Concentrations of 0, 0.01, 0.05, 0.1, 0.5, or 1.0 mM cyclic-GMP were added, respectively. Added cyclic-GMP did not induce cell death. All data are presented as mean ± SE and statistically analyzed by a one-way ANOVA and the Student-Newman-Keul's test using SigmaStat software. conc = concentration.

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  Fig. 3 Measurement of cell viability of cultured retinal cells pretreated with various concentrations of the cyclic nucleotide-gated channel blocker, amiloride, followed by tissue plasminogen activator (tPA) plus L-arginine (L-arg, 10 µg/400 µL) or L-arg alone (5 mM). The data represent the mean ± SD of independent experiments and were statistically analyzed by one-way ANOVA and the Student-Newman-Keul's test using SigmaStat software. *p < 0.05 compared with control (first column, no treatment; second column, 1 µm amiloride; third column, 10 µm amiloride; and fourth column, 100 µm amiloride). LHD = lactate dehydrogenase.

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