Molecular mechanisms underlying the corneal endothelial pump

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Abstract

The corneal endothelium is responsible for maintaining the hydration of the cornea. This is through a “Pump-Leak” mechanism where the active transport properties of the endothelium represent the “Pump” and the stromal swelling pressure represents the “Leak”. For the “Pump”, Na+, K+ ATPase activity and the presence of HCO3, Cl, and carbonic anhydrase activity are required. Several basolateral (stromal side) anion transporters, apical (facing the aqueous humor) ion channels and water channels have been identified that could support a model for ion secretion as the basis for the endothelial pump, however evidence of sustained anion fluxes, osmotic gradients or the need for water channels is lacking. This has prompted consideration of other models, such as Electro-osmosis, and consideration of metabolite flux as components of the endothelial pump. Although the conditions under which the “Pump” is supported are known, a complete model of the endothelial “Pump” has yet to emerge.

Highlights

► Review of corneal endothelial pump function. ► Evidence supporting an anion transport based pump is limited. ► siRNA and shRNA approaches provide new insights on endothelial function. ► Evidence for alternate endothelial pump models including electro-osmosis & lactate flux.

Introduction

The topic of molecular mechanisms underlying the corneal endothelial pump was extensively reviewed in 2003 (Bonanno, 2003). The purpose here is to summarize the important features of the endothelial pump and provide an update on recent significant findings. Development of new techniques, e.g. siRNA knockdown and in vivo viral transfection of shRNA, has produced more specific testing of pump features. Similarly, the use of knockout models (e.g., AQP1) has prompted significant re-thinking of the nature of solute-fluid coupling. These molecular approaches together with new studies using customary pharmacological approaches for studying corneal endothelial fluid transport have led to re-evaluations of the corneal endothelial pump.

Section snippets

Background

The cornea is the major refractive element of the eye. This requires optical transparency and smooth curved surfaces. The cornea has five layers: the outer epithelium, Bowman’s (basement) membrane, the stroma, Descemet’s (basement) membrane, and the inner surface endothelium. The smooth regular surface, tight packing of cells, relative paucity of organelles (especially mitochondria), and lack of blood vessels in the stratified squamous corneal epithelium (∼50 μm) reduce light scatter thereby

Corneal endothelial pump description

The endothelial pump function is best demonstrated using rabbit corneas mounted in modified Ussing chambers in vitro. If carefully mounted and perfused with appropriate media, the cornea will maintain its thickness for several hours. Another approach is to remove the epithelium, expose the anterior stroma for a short period to Ringer’s solution allowing the stroma to swell, remove the anterior solution and replace it with silicone oil. The corneal thickness will then slowly decrease

The anion (bicarbonate & chloride) secretion model

Fluid secretion that is coupled to ion fluxes is dependent on active transport mechanisms to produce local osmotic gradients that move water across the cellular layer. This is best described for secretory glands and kidney reabsorption processes. More recently, evidence for direct coupling of water to ion fluxes in cotransporters has also been presented (Hamann et al., 2003, Meinild et al., 2000) and could have a significant role in fluid absorption across the intestinal mucosa (Loo et al., 1996

The electro-osmosis model

The conventional view of epithelial cell secretion and absorption of water is that epithelial cell layers create local osmotic differences in the lateral spaces between the cells and/or on the apical surfaces of cells within an unstirred layer. The osmotic gradients generated serve as the driving forces for water movement across the epithelium. However, in many epithelial cells, including the corneal endothelium, there is no evidence that these gradients exist. This has led to the consideration

Aquaporins

The role of aquaporins in fluid transport has been controversial. Knockout models of AQP1, the most ubiquitous aquaporin, showed essentially no phenotype unless the animal was stressed by withholding water (Agre, 1998). Subsequent studies indicated that knockout of aquaporins had effects on secretory tissues if the rate of secretion was relatively high (Ma et al., 2000, Ma et al., 1999). Corneal endothelium has a high density of AQP1 located on both apical and basolateral membranes (Kuang

Fluid transport agonists

The most highly studied fluid transport agonists in corneal endothelium are those that result in increased [cAMP]. Adenosine was the first agonist discovered (Dikstein and Maurice, 1972) and was subsequently shown that it increased [cAMP] in endothelial cells (Riley et al., 1996) through activation of A2b receptors (Tan-Allen et al., 2005). Other agonists that increase [cAMP], e.g. forskolin or the phosphodiester inhibitor rolipram, also cause corneal thinning (Wigham et al., 2000). It is

Facilitated lactate transport

Often overlooked is the fact that in the in vivo cornea there are substantial bulk gradients of ions, e.g. glucose, HCO3 and lactate (Chhabra et al., 2009). [HCO3] is higher in the anterior chamber than in the cornea, especially when the eyelid is open. Thus HCO3 will diffuse into the cornea passively in the opposite direction of the putative bicarbonate secretory mechanism. The cornea gets all of its glucose from the anterior chamber. Glucose in the cornea is consumed thereby maintaining

Conclusion

The corneal endothelial pump is an active transport dependent process that requires both Cl and HCO3, but is highly dependent on HCO3 and facilitated by carbonic anhydrase activity. While a HCO3 secretory model for the pump has been preferred, there are several issues that argue against this interpretation. These include: the lack of apical anion transporters, equivocal measures of net anion fluxes from stroma to anterior chamber (probably due to the extremely leaky nature of the

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