Abstract
Abstract: :
Purpose:Electro–osmosis through the paracellular junctions could play an important role in corneal endothelial fluid transport. In support of this, we have found a net flux of fluorescein in the same direction as that of fluid transport. Here we study the expression of several claudins and assess the endothelial permeability for different sizes of fluorescein isothiocyanate (FITC) dextrans across RCE monolayers. Methods: Nested PCR: claudin cDNA was reverse transcribed from total RNA of CRCE using rTth DNA polymerase RNA PCR kit in the presence of MnCl2, and the 3’ claudin–1 "downstream" primer for claudin–1 and the degenerated 3’ claudin primer AGSCRYCADSGGGTYRTAGAAGTC for claudins–2 to –8. rTth DNA polymerase was also used for PCR amplification enhanced by chelating Mn2+ and adding MgCl2. The PCR was performed using 5’ claudin–1 "upstream" primer for claudin–1 and the 5’ degenerated primer RAGGGSCTSTGGATGDMSTGYGTG for claudin–2 to –8. The second PCR amplification was done using the first PCR as a template, with claudin–specific primers. 3). Transepithelial electrical resistance (TER) and paracellular permeability (Papp): CRCE cells were grown on grown to confluence on 24 mm permeable membrane inserts and the TER was measured with an Endohm chamber and an EVOM meter. The permeability of the CREC monolayer was assessed by measuring the rate at which FITC–dextrans were transported across the layer using a PTI fluorometer. Results: Mice corneal endothelium expresses claudins 1,2,3,8, while RCE expresses claudins 1,2,3,4,8. The TER value of RCE reached 28.5 ± 0.6 ohm.cm2 at 6–7 days after seeding. The paracellular permeability values (Papp) of RCE (cm/s) for (a) sodium–fluorescein and FITC–dextrans of (b) 4, (c) 20 and (c) 40 KDa were (a) 7.0 ± 0.5 x 10–4, (b) 4.1 ± 0.6 x 10–6, (c) 1.7 ± 0.1 x 10–6, and (d) 8.9 ± 0.5 x 10–7. 10 mM EGTA reduced TER by 80% at 1 hr, with partial reversal after 24 hr. 20 uM Cytochalasin D (cytD) reduced TER by 50% at 1 to 2 hr, irreversibly. RCE treated with EGTA and CytD displayed higher Papp to FITC–dextrans of 20 Kda; EGTA had a much higher effect. Conclusion: The expression of claudin 2 fits with the leakiness of corneal endothelial junctions. The roles of the additional claudins expressed (1, 3, 4 and 8) remain to de determined. Disruption of the intercellular junctions is accompanied by reversible TER decrease and paracellular permeability increase.
Keywords: cornea: basic science • cornea: endothelium • ion transporters