Abstract
Purpose: :
It is thought that fluid transport across the corneal endothelium is driven by transcellular HCO3– transport. However, fluid is transported residually in nominally HCO3––free solutions. Our aim was to identify the mechanisms of this residual transport.
Methods: :
To determine fluid transport, corneal thickness was measured with a computer–controlled specular microscope at 5–minute intervals. Fluid transport was calculated from the rates of corneal thickness changes.
Results: :
Experiments were designed to disable stepwise all cellular elements that could contribute to fluid transport, including HOC3–, Cl–, and Na+ pathways, and carbonic anhydrases. In nominally HCO3– free solution, fluid transport remains at 48 % of control. The addition of a cocktail of Cl– channel inhibitors (50 µM NPPB and 100 µM niflumic acid) leads to further inhibition but fluid transport still proceeds at 30 % of the control rate. Importantly, addition of the carbonic anhydrase inhibitor ethoxyzolamide (0.2 mM), which inhibits both the intracellular carbonic anhydrase II and the membrane–associated apical carbonic anhydrase IV, does not affect fluid transport significantly (25% of the control rate). Therefore, the residual fluid transport observed cannot be due to hydration of intracellular CO2 to HCO3– by carbonic anhydrase and subsequent HCO3– transport as postulated earlier (Kuang et al.,1990; Bonanno, 1994). In contrast, however, residual fluid transport can be inhibited completely by the further addition of an inhibitor (100 µM benzamil) of the epithelial Na+ channel ENaC.
Conclusions: :
In nominally HCO3– free solutions containing Cl– channel and carbonic anhydrase inhibitors fluid transport continues in the absence of transcellular anion transport and consequently in the absence of solute transport. Under these conditions fluid transport cannot be driven by local osmotic gradients. The results are, however, consistent with fluid transport taking place by an electro–osmotic mechanism (Sanchez JM et al., J Membr Biol 2002) in which the transcellular electrical current includes as components apical anion efflux and Na+ influx.
Keywords: cornea: endothelium • ion transporters • ion channels