DPOFA, a Cl⁻/HCO₃⁻ exchanger antagonist, stimulates fluid absorption across basolateral surface of the retinal pigment epithelium

Abstract

Background: Retinal detachment is a disorder of the eye in which sensory retina separates from the retinal pigment epithelium (RPE) due to accumulation of fluid in subretinal space. Pharmacological stimulation of fluid reabsorption from subretinal space to choroid across the RPE has been suggested as a treatment strategy for retinal detachment. DPOFA, (R)-(+)-(5,6-dichloro 2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy]acetic acid, is an abandoned drug capable of inhibiting Cl⁻/HCO₃⁻ exchanger activity. We hypothesized that DPOFA may increase fluid absorption across basolateral surface of the RPE.

Methods: Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA for six different transporters that may act as Cl⁻/HCO₃⁻ exchangers was conducted in bovine and human RPE to confirm that RPE from two species expresses the same repertoire of Cl⁻/HCO₃⁻ exchanger isoforms. The degree of amino acid homology between orthologous human and bovine RPE-specific isoforms was calculated after performing protein alignments. Transport of fluid across bovine RPE-choroid explants mounted in the Ussing chamber was used to assess the ability of DPOFA to modulate fluid absorption across the RPE.

Results: Using RT-PCR we showed that three isoforms (SLC4A2, SLC4A3, and SLC26A6) are strongly expressed in human and bovine RPE preparations. Amino acid comparisons conducted for RPE-specific isoforms support the use of bovine RPE-choroid explants as an adequate experimental system for assessing fluid absorption activity for DPOFA. Our data is consistent with the fact that DPOFA stimulates fluid absorption across the RPE in bovine RPE-choroid explants.

Conclusions: DPOFA seems to stimulate transport of water across the RPE in bovine RPE-choroid explants. Additional experiments are required to establish dose-dependent effect of DPOFA on fluid absorption in the bovine RPE-choroid experimental system.

Figure 1

Chemical structure of DPOFA. The compound is also known in the literature as B-3(+) [20] and L-644,711 [18].

Figure 2

Expression analysis of Cl^-/HCO3^- exchanger isoforms in human and bovine RPE. RT-PCR amplification was conducted on human (A) and bovine (C) RPE cDNA preparations. Panels B and D show the results of control PCR amplification with human (B) and bovine (D) isoform-specific primers performed with water in place of cDNA. Lanes 1-6 indicate PCR reactions performed with human (panels A, B) and bovine (panels C, D) oligonucleotides specific for SLC4A1, SLC4A2, SLC4A3, SLC26A3, SLC26A4, SLC26A6, respectively. Lane 7 represents amplification of the control GAPDH fragment performed with human (panels A, B) or bovine (panels C, D) oligonucleotide primers. Lane 8 contains size markers (100 b.p ladder) with arrow indicating a position of the 500 b.p. band.

Figure 3

Comparison of changes in water pumping rates in bovine RPE-choroid explants induced by DPOFA and vehicle treatment. DPOFA at 1-20 μM concentrations was added to apical and basal baths of the chamber. The number of experiments and change in pumping rate values are shown in Table 1. Mean values are plotted with error bars depicting Standard Error of Means (SEM). The change in pumping rate from apical to basal side of the RPE-choroid explant was calculated at 10, 15, 20, and 30 minutes after drug and vehicle addition. While no statistically significant difference between compound doses could be discerned, we were able to detect the statistically significant (p = 0.013) increase in fluid absorption after drug treatment versus vehicle control during the first 20 minutes post drug/vehicle addition when the data for all DPOFA concentrations within the 1-20 μM were pooled for the analysis.