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. 2010 Nov 4:16:2279-85.

The effect of topical amiloride eye drops on tear quantity in rabbits

Shuya Hara  1 , Akihiro Hazama, Masao Miyake, Takashi Kojima, Yasumasa Sasaki, Jun Shimazaki, Murat Dogru, Kazuo Tsubota
Affiliations

The effect of topical amiloride eye drops on tear quantity in rabbits

Shuya Hara et al. Mol Vis. .

Abstract

Purpose: To investigate the presence of epithelial sodium channels (ENaC) in rabbit and human conjunctival epithelium and to test the effects of topical amiloride, a potassium-sparing diuretic that blocks the ENaC, on tear quantity in rabbits.

Methods: Both healthy normal human and rabbit conjunctival tissues underwent immunohistochemistry staining for ENaC-α and γ subunits as well as for reverse transcription-polymerase chain reaction (RT-PCR) for detection of ENaC-α and ENaC-γ subunit mRNA expression. Rabbits were instilled topical amiloride eye drops and tear function tests were performed before and after instillations.

Results: Immunohistochemical staining for ENaC-α subunit in all rabbit eyes showed positive staining in apical and basal conjunctival epithelial cells. Human conjunctival epithelia revealed positive staining with ENaC-α antibody especially in the apical and basal layers. Immunohistochemistry staining with ENaC- γ antibody also revealed positive staining of the conjunctival epithelial cells especially in the basal layers. The ENaC-α mRNA was detected in samples from healthy white rabbit conjunctival epithelia, and ENaC-α and ENaC- γ mRNAs were detected in samples from healthy human conjunctival epithelia. The mean tear quantity showed a significant increase at 15 and 30 min compared to the pre-instillation value in eyes assigned to amiloride eye drops. The mean tear quantity at 15 and 30 min was significantly higher in the amiloride group compared to the control eyes.

Conclusions: Topical amiloride application appears to increase the quantity of preocular tears owing to inhibition of conjunctival epithelial sodium channels.

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Figures

Figure 1
Figure 1
Expression of ENaC subunits in rabbit conjunctiva in immunohistochemical stainnings. ENaC-α (A and I), ENaC-β (B and J), and ENaC-γ (C and K) were positive at apical layer of conjunctiva. As a positive control, ENaC-α (E and M), ENaC-β (F and N), and ENaC-γ (G and O) were positive at inner medullas of kidney. Isotype control specimen showed no positive staining in conjunctiva (D and L) and kidney (H and P). Scale bars in A-H represent 20 μm and scale bars in I-P represent 40 μm.
Figure 2
Figure 2
Expression of ENaC-α and housekeeping gene (ACTB) in rabbit conjunctiva.
Figure 3
Figure 3
Immunohistochemistry stainings of ENaC- α and ENaC-γ subunits in human conjunctiva.
Figure 4
Figure 4
Expression of CIC-2, ENaC-α, ENaC-γ, and housekeeping gene (ACTB) in human conjunctiva.
Figure 5
Figure 5
The change of Schirmer test value in topical amiloride eye drop experiment.
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