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Sighvatur Saevar Arnason, Sunna B Skarphedinsdottir, Thor Eysteinsson; The role of purinergic P2X receptors and Ca++-dependent chloride channels in ion transport of mouse retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1882.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelium (RPE) is important for normal function of the retina, one of which is to transport water and Cl- from the subretinal space to the choroid, across the RPE cells. It has been suggested that apical P2X purinergic receptors (P2XR) transduce second messenger signals, such as Ca++, into stimulation of Cl- transport across the RPE. The purpose of this study was to assess the function of P2X receptors and Ca++-dependent chloride channels (CaCC) in the mouse RPE.
Healthy mice (C57BL/6J) where euthanized and the RPE together with the retina, choroid and sclera was mounted in special miniature epithelial Ussing chambers with an aperture of 0.031 cm2 (EasyMount, Physiological Instruments) with normal Krebs on both sides kept at 38°C and aired with 5%CO2-95%O2. The tissue was voltage clamped to zero (WPI) to measure the short-circuit current (Isc) as an indicator of net ion transport. Every 4th minute a 1 mV pulse was passed to estimate the transepithelial resistance (TER). The number of mice was 4-6 in each experiment series. The results are presented as mean +/- SEM. Statistical significance was tested by a paired t-test. The Ca++ dependent chloride channel blocker CaCCA01 (Tocris) was added to both sides of the RPE at two different doses, 0.58 mM and 1.15 mM, each tested over a 30 minute period. The P2XR agonist β,γ-Met ATP (Sigma) followed by the P2XR antagonist PPADS (Tocris) where added to the apical side of the RPE and tested over a 30 minute period each.
CaCCA01 applied apically to the mouse RPE caused a significant decrease and reversal in the Isc, from -8.2+/- 2.4 to 4.7 +/- 0.9 µAmp/cm2 at the higher dose, 1.15 mM (p < 0.05), and an increase in the TER (53 +/- 8 to 76 +/- 10 Ohm*cm2 (p < 0.05; n=4). Neither β,γ-Met ATP (1 mM) nor PPADS (0.5 mM) induced any change in the initial Isc of -10.5 +/- 2.1 µAmp/cm2, but both induced a small but significant change in the TER, from 47 +/- 6 to 55 +/- 9 and 58 +/- 9 Ohm*cm2 , respectively (p < 0.05; n=6).
The results indicate that the CaCC on the apical side are open in the short-circuit state and drive a part of the ion transport in the mouse RPE. Our experiments also suggest that CaCC are situated on the apical side. On the other hand, P2XR on the apical side do not mediate ion transport across the RPE.
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