Potassium Channel and NKCC Cotransporter Involvement in Ocular Refractive Control Mechanisms

Myopia affects well over 30% of adult humans globally. However, the underlying physiological mechanism is little understood. This study tested the hypothesis that ocular growth and refractive compensation to optical defocus can be controlled by manipulation of potassium and chloride ion-driven transretinal fluid movements to the choroid. Chicks were raised with +/−10D or zero power optical defocus rendering the focal plane of the eye in front of, behind, or at the level of the retinal photoreceptors respectively. Intravitreal injections of barium chloride, a non-specific inhibitor of potassium channels in the retina and RPE or bumetanide, a selective inhibitor of the sodium-potassium-chloride cotransporter were made, targeting fluid control mechanisms. Comparison of refractive compensation to 5mM Ba2+ and 10−5 M bumetanide compared with control saline injected eyes shows significant change for both positive and negative lens defocus for Ba2+ but significant change only for negative lens defocus with bumetanide ; ; ; ; ; ). Vitreous chamber depths showed a main effect for drug conditions with less depth change in response to defocus shown for Ba2+ relative to Saline, while bumetanide injected eyes showed a trend to increased depth without a significant interaction with applied defocus. The results indicate that both K channels and the NKCC cotransporter play a role in refractive compensation with NKCC blockade showing far more specificity for negative, compared with positive, lens defocus. Probable sites of action relevant to refractive control include the apical retinal pigment epithelium membrane and the photoreceptor/ON bipolar synapse. The similarities between the biometric effects of NKCC inhibition and biometric reports of the blockade of the retinal ON response, suggest a possible common mechanism. The selective inhibition of refractive compensation to negative lens in chick by loop diuretics such as bumetanide suggests that these drugs may be effective in the therapeutic management of human myopia

cAMP stimulates transepithelial short-circuit current and fluid transport across porcine ciliary epithelium

2016-2017 > Academic research: refereed > Publication in refereed journal201811_a bcmaVersion of RecordPublishe

Characterization of anoctamin in porcine ciliary epithelium

ARVO 2020 : Association for Research in Vision & Ophthalmology Annual Meeting, May 3, 2020 - May 7, 2020, Baltimore, MD, US202011 bcrcMetadata onlyPublishe

Regulation of gap junction coupling in bovine ciliary epithelium

Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 ± 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (Rf) in recipient to donor cell was 0.47 ± 0.09 (n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased Rf by ∼60% to 0.20 ± 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 μM) also reduced LY dye transfer by ∼60%, reducing Rf from 0.41 ± 0.05 (n = 15) to 0.17 ± 0.05 (n = 20) after 10 min. Junctional currents were lowered by ∼50% (n = 6) after 10-min perfusion with 500 μM dibutyryl-cAMP (n = 6); thereafter, heptanol abolished the currents (n = 5). Preincubation with the PKA inhibitor H-89 (2 μM) prevented cAMP-triggered current reduction (n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication