Regulation of MI Transport in Retinal Pigment Epithelium by Sugars, Amiloride, and pH Gradients: Potential Impairment of Pump-Leak Balance in Diabetic Maculopathy

Abstract

Impairment of transport and metabolism of retinal pigment epithelium (RPE) has been recognized to play a role in the development of diabetic macular edema. To understand the mechanism(s) of action of high glucose levels in alteration of RPE metabolism, primary cultures of RPE cells were used as an in vitro model of diabetic retinopathy/maculopathy. RPE cells were grown with 5 mM (control) or 40 mM glucose (a monosaccharide that enters the cells), or 40 mM sucrose (a disaccharide that does not enter the cells), and the extent of Na⁺-dependent active transport of an osmolyte ([³H]-myo-inositol, MI, 10µM) into cells was determined. While 40 mM glucose down-regulated ³H-MI transport, 40 mM sucrose stimulated it, compared to 5 mM glucose feeding. Addition of 1 mM amiloride, an inhibitor of Na⁺/H⁺ exchanger, in the incubation media, significantly inhibited MI transport. Cells treated with high sucrose or high glucose were more sensitive toward amiloride inhibition, compared to controls. Inhibition of either pump or leak pathway alone was not sufficient to completely inhibit MI transport, but simultaneous inhibition of both pathways, by amiloride and ouabain (1 mM each), strongly inhibited osmolyte accumulation. The strongest inhibition of uptake occurred when 150 mM NaCl in the incubation media was replaced by 150 mM choline-Cl, and the percent inhibition of uptake, with choline-Cl, was highest with sucrose-fed cells, compared to normal or high glucose-fed cells. Imposition of a pH gradient [pH_i (6.1) <pH₀ (8.0)] across the cell membrane, a condition that stimulates Na⁺/H⁺ exchange activity, also reduced MI accumulation. Cellular water content, measured by the extent of [³H]-3-O-methyl glucose uptake, in the presence of balanced salt solution (BSS), BSS containing half the ionic strength (hypotonic solution), or BSS containing 20 mM K⁺, for induction of cell swelling, varied when cells were fed with various sugars. Cells fed with high glucose were less sensitive toward media tonicity compared to normal. These results suggested that in cultured RPE cells, changes in Na⁺/H⁺ exchanger activity (intracellularly or extracellularly), through its inhibition by amiloride, its activation via intracellular acidification, or perhaps by chronic feeding with high sucrose or high glucose, affected the Na⁺-dependent active accumulation of MI. A metabolic factor involved in the development of diabetic macular edema is perhaps associated with glucose-induced alterations in Na⁺ fluxes (e.g., changes in Na⁺/H⁺ exchanger activity), which can secondarily influence osmolyte accumulation, impairment of pump-leak balance, and/or intracellular pH. Glucose-induced altered osmolyte/solute or ion transport systems in RPE might be intimately involved in the development of diabetic macular edema.

Key Words:

• Retinal pigment epithelium
• culture
• osmolyte
• myo-inositol
• glucose
• amiloride
• transport
• sucrose
• diabetic maculopathy
• retinopathy
• Na⁺/H⁺ exchanger
• pump-leak balance
• macular edema