Korean J Ophthalmol.  2019 Oct;33(5):414-421. 10.3341/kjo.2019.0057.

Effect and Mechanism of Phosphodiesterase Inhibitors on Trabecular Outflow

• Kim JW
• Lee JB
• Lee SH

• KMID: 2459522
• DOI: http://doi.org/10.3341/kjo.2019.0057

Abstract

PURPOSE
Phosphodiesterase (PDE) inhibitors increase matrix metalloproteinase (MMP) production by inhibiting re-uptake of adenosine and may potentiate nitric oxide (NO) activity. This study was performed to investigate the effects and mechanisms of PDE inhibitors on trabecular outflow in cultured human trabecular meshwork cells (HTMCs).
METHODS
Primary HTMC cultures were exposed to 0, 20, and 50 µM dipyridamole (DPD) or theophylline (TPN). Permeability through the HTMC monolayer was assessed using carboxyfluorescein. The production of NO was assessed using the Griess assay and MMP-2 levels were measured via Western blotting.
RESULTS
DPD significantly increased permeability accompanied with increased nitrite concentration and MMP-2 levels (all p < 0.05). TPN increased nitrite but did not affect permeability or MMP-2 levels significantly (p > 0.05). When treated with DPD and TPN together, both permeability and nitrite production were increased; however, MMP-2 levels showed no difference compared to DPD exposure alone (p > 0.05).
CONCLUSIONS
DPD increased trabecular permeability accompanied with increased nitrite production and MMP-2 levels. PDE inhibitors may increase trabecular outflow by increasing MMP-2 levels and by potentiating NO activity through cyclic GMP in HTMC.

Keyword

Dipyridamole; Matrix metalloproteinases; Nitric oxide; Theophylline; Trabecular meshwork

MeSH Terms

Adenosine
Blotting, Western
Cyclic GMP
Dipyridamole
Humans
Matrix Metalloproteinases
Nitric Oxide
Permeability
Phosphodiesterase Inhibitors*
Theophylline
Trabecular Meshwork
Adenosine
Cyclic GMP
Dipyridamole
Matrix Metalloproteinases
Nitric Oxide
Phosphodiesterase Inhibitors
Theophylline

Figure

• 

  Fig. 1 Effect of phosphodiesterase inhibitors on permeability through the trabecular cell monolayer. Exposure to 20 or 50 µM dipyridamole (D) or theophylline (T) significantly increased the concentration of carboxyfluorescein in the outer well (permeability) compared to the non-exposed control (-). Co-exposure to 20 µM D and T, or 50 µM D and T further increased permeability. Carboxyfluorescein intensity of the outer chamber was normalized to the mean value obtained using a non-exposed control (permeability 100%, *p < 0.05).

• 

  Fig. 2 Effect of phosphodiesterase inhibitors on the production of nitric oxide. Exposure to 20 or 50 µM dipyridamole (D) or theophylline (T) increased the concentration of nitrite significantly compared to the non-exposed control (-). Co-exposure to 20 µM D and T or 50 µM D and T further increased the concentration of nitrite (*p < 0.05).

• 

  Fig. 3 Effect of dipyridamole (D) on the expression of (A) endothelial nitric oxide synthase mRNA and (B) matrix metalloproteinase 2 mRNA. Exposure to 20 or 50 µM dipyridamole did not significantly affect the expression of endothelial nitric oxide synthase mRNA or matrix metalloproteinase 2 mRNA compared to non-exposed control (-) (p > 0.05).

• 

  Fig. 4 Effect of phosphodiesterase inhibitors on the activity of matrix metalloproteinase (MMP)-2 levels measured by Western blotting. Exposure to 50 µM dipyridamole (D) significantly increased MMP-2 activity compared to non-exposed controls (-). Co-exposure to 20 µM D and theophylline (T), or 50 µM D and T further increased MMP-2 production (*p < 0.05).

• 

  Fig. 5 Effect of theophylline (T) on the matrix metalloproteinase 2 levels measured by Western blotting. Exposure to either 20 or 50 µM T did not significantly affect the matrix metalloproteinase 2 levels compared to non-exposed controls (-).

• 

  Fig. 6 Effect of endothelial nitric oxide synthase inhibitor on the production of (A) nitric oxide and (B) permeability of trabecular meshwork cell monolayer. Exposure to 20 µM dipyridamole (D) with or without 10 µM Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, L) increased the production of (A) nitrite and (B) permeability compared to non-exposed controls (-) (*p < 0.05). Co-exposure to 20 µM D and 10 µM L-NAME compared to exposure to 20 µM D alone, the production of nitrite and permeability showed no statistically significant differences (p = 0.864, 0.815).

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