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Thor Eysteinsson, Sunna Skarphedinsdottir, Sighvatur Saevar Arnason; Adrenergic but not purinergic receptors affect ion transport across the mouse retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6348.
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To develop procedures to examine the effects of adrenergic and purinergic receptors on ion transport across the mouse retinal pigment epithelium (RPE), as measured by the RPE short-circuit current (ISC) and transepithelial resistance (TER).
Sheets of RPE from normal mice with the retina attached were mounted in Ussing chambers with a small aperture of 0.031 cm2 (EasyMount, Physiological Instruments Inc.). An aerated Krebs solution kept at 38°C was placed on each side of the tissue. The ISC and the TER were recorded with electrodes placed on each side of the tissue and connected to a voltage clamp apparatus. All drugs were applied for 30 min on both apical and basolateral sides.
The mean initial ISC (n=7) was -17,6 ± 4,5 μA/cm2, with the apical side negative, and TER was 82,8 ± 7,6 Ohm*cm2. The mouse RPE preparations remained stable for 2-3 hours. A low [Cl-] in the Krebs reduced the ISC. Reducing [K+] on the apical side decreased the ISC, an effect that was inhibited by 1 mM BaCl2. The Na-K-Cl cotransporter blocker bumetanide (100 µM) reduced the ISC from -16.1 ± 5.2 to -14.5 ± 5.4 μA/cm2 (P = 0.005). The Na-K-ATPase blocker ouabain (1 mM) induced a biphasic response, first an increase in the ISC to -17.4 ± 2.6 μA/cm2 in 6 minutes, and then a decrease in 30 minutes to to -7,74 ± 4,45 μA/cm2, which was 36% of ISC levels before applying ouabain (P <0,001). The purine agonist ATP (100 µM) had no significant effects on the ISC or TER, while epinephrine (100 µM) raised the ISC by 5%: -16,1 ± 4,1 to -17,0 ± 4,2 μA/cm2 (P = 0,006).
The ISC, and the TER can be recorded reliably from the murine RPE with our procedures. The ion transport across the murine RPE as measured by ISC is likely to be primarily Cl- transport, with some K+ contribution and the Na-K-ATPase as an important driving force. The results indicate that adrenergic but not purinergic receptors affect murine RPE ion transport.
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