Mechanisms of Ion Transport across the Mouse Retinal Pigment Epithelium Measured in Vitro

Abstract

PURPOSE. To examine ion transport across the mouse retinal pigment epithelium (RPE), measured by the short-circuit current (ISC) and transepithelial resistance (TER). METHODS. Sheets of RPE from mice (C57BL6/J) with retina, choroid, and sclera attached were mounted in Ussing chambers (0.031-cm2 aperture) and Krebs solution. The ISC and TER were recorded with voltage clamps. Receptors implicated in ion transport were blocked or stimulated by ligands applied to both sides. RESULTS. The mean initial ISC was −12.0 ± 3.9 μA/cm2 (basolateral negative), and mean TER was 67.1 ± 8.0 ohm·cm2. RPE preparations remained stable for 3 hours, with ISC decreasing by 0.078 ± 0,033 μA/cm2/hr. Adenosine triphosphate (100 μM) increased ISC by 2.22 ± 0.41 μA/cm2 (P = 0.003). Epinephrine (100 μM) increased ISC by 1.14 ± 0.19 μA/cm2 (P = 0.011). Bumetanide (100 μM) reduced ISC by 1.72 ± 0.73 μA/cm2 (P = 0.027). Ouabain (1 mM) induced a biphasic response: an ISC increase from −7.9 ± 2.4 to −15.49 ± 2.12 μA/cm2 and then a decrease to −3.7 ± 2.2 μA/cm2. Ouabain increased TER by 15.3 ± 4.8 ohm·cm2. These compounds were added sequentially. Apical [K+]o at zero mM transiently increased ISC by 3.36 ± 1.06 μA/cm2. Ba++ decreased ISC from −10.4 ± 3.1 to −6.6 ± 1.8 μA/cm2 (P = 0.01). Ba++ reversed the K+-free response, with Isc decreasing further from −5.65 ± 1.24 to −3.37 ± 0.79 μA/cm2 (P = 0.029). CONCLUSIONS. The ISC and TER can be recorded from the mouse RPE for 3 hours. Adrenergic and purinergic receptors affect murine RPE ion transport. Sodium–potassium adenosine triphosphatase plays a role in net ion transport across mouse RPE, and Na-K-2Cl cotransporter activity partly accounts for transepithelial ion transport. Mimicking light-induced changes, low subretinal [K+]o increases ion transport transiently, dependent on K+ channels.