Open AccessMechanisms of Ion Transport Across the Mouse Retinal Pigment Epithelium Measured In Vitro
Author(s) -
Sunna Björg Skarphedinsdottir,
Þór Eysteinsson,
Sighvatur S. Árnason
Publication year - 2020
Publication title -
investigative ophthalmology and visual science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.935
H-Index - 218
eISSN - 1552-5783
pISSN - 0146-0404
DOI - 10.1167/iovs.61.6.31
Subject(s) - bumetanide , ouabain , retinal pigment epithelium , transepithelial potential difference , ion transporter , chemistry , retinal , biophysics , ussing chamber , endocrinology , medicine , in vitro , ion , biology , sodium , biochemistry , organic chemistry
Purpose To examine ion transport across the mouse retinal pigment epithelium (RPE), measured by the short-circuit current (I SC ) 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-cm 2 aperture) and Krebs solution. The I SC 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 I SC was −12.0 ± 3.9 µA/cm 2 (basolateral negative), and mean TER was 67.1 ± 8.0 ohm·cm 2 . RPE preparations remained stable for 3 hours, with I SC decreasing by 0.078 ± 0,033 µA/cm 2 /hr. Adenosine triphosphate (100 µM) increased I SC by 2.22 ± 0.41 µA/cm 2 (P = 0.003). Epinephrine (100 µM) increased I SC by 1.14 ± 0.19 µA/cm 2 ( P = 0.011). Bumetanide (100 µM) reduced I SC by 1.72 ± 0.73 µA/cm 2 ( P = 0.027). Ouabain (1 mM) induced a biphasic response: an I SC increase from −7.9 ± 2.4 to −15.49 ± 2.12 µA/cm 2 and then a decrease to −3.7 ± 2.2 µA/cm 2 . Ouabain increased TER by 15.3 ± 4.8 ohm·cm 2 . These compounds were added sequentially. Apical [K + ] o at zero mM transiently increased I SC by 3.36 ± 1.06 µA/cm 2 . Ba ++ decreased I SC from −10.4 ± 3.1 to −6.6 ± 1.8 µA/cm 2 ( P = 0.01). Ba ++ reversed the K + -free response, with I sc decreasing further from −5.65 ± 1.24 to −3.37 ± 0.79 µA/cm 2 ( P = 0.029). Conclusions The I SC 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.