Kinetics of myo…Inositol Transport in Corneal Endothelial Cells: Diverse Effects of Sugars and Implications in Corneal Deutergence

Abstract

Kinetics of myo-inositol (MI) uptake into primary cultures of bovine corneal endothelial cells (CEC) were studied. Confluent corneal endothelial cells accumulated ³H-MI in a time dependent and saturable process. At a narrow range of external concentrations of ³H-MI (4–50 µM), the Na ⁺-dependent MI uptake followed saturation kinetics. The apparent K_m value was 20 µM with a maximum velocity (V_max) of 16 pmol/20 min/µg DNA. At low external ³H-MI concentrations the uptake was dependent on Na ions, but at higher levels the Na ⁺-independent fraction of MI uptake significantly increased. The uptake was sensitive to removal of Ca ions and to the presence of inhibitors such as n-ethyl maleimide, phlorizin, ouabain, and amiloride (an inhibitor of Na ⁺/H⁺ exchanger). The sensitivity of MI uptake toward inhibitors and ionic changes in the bathing media was reduced as external concentrations of ³H-MI increased. Citrate at 0.5 mM increased the uptake, suggesting involvement of mitochondrial oxidative metabolism in the MI uptake. Percent release of radioactivity by 2 min, after an initial 40-min incubation with 20 µM ³H-MI, was 6.6% ± 0.8 or 35% ± 4 when release media contained BSS alone or BSS containing 5 mM nonradioactive MI, respectively. Efflux of radioactivity from the cells also was enhanced when release media contained 40 mM glucose. Glucose and galactose as well as nonmetabolizable glucose analogues, such as 3O-methyl glucose or α- methyl glucose, at high concentrations (40 mM), acutely (in the incubation media) or chronically (in the growth media) inhibited MI uptake into CEC, and the extent of inhibition was inversely proportional to the external levels of ³H-MI. However, glucose at lower levels (≤10 mM) slightly increased MI uptake. These studies indicated that the uptake of MI into corneal endothelial cells was an Na⁺-dependent active process at a narrow range of external radioactive MI concentrations. Higher levels of MI were taken up by the cells via a passive diffusion mechanism, independent of carrier protein(s). Glucose influenced the uptake of MI in a complex manner. The increased MI efflux by glucose or by MI was perhaps due to the limited capacity of CEC for accumulation or compartmentalization of this or other solutes/osmolytes, a phenomenon that may be related to the role of CEC in maintenance of corneal deutergence. High glucose-induced inhibition of Na ⁺-dependent MI uptake may be in part due to glucose regulation of Na⁺ fluxes and cell volume. Hyperglycemia of diabetes may induce changes in transport properties of CEC, resulting in corneal complications such as polymegatism.

Key Words:

• Corneal endothelial cells
• myo-inositol
• culture
• diabetes
• glucose
• galactose
• deutersence
• uptake
• polymegatism
• Na ⁺-dependent
• simple diffusion
• Sorbinil