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. 2000 Feb;84(2):199–204. doi: 10.1136/bjo.84.2.199

Fluid transport by cultured corneal epithelial cell layers

H Yang 1, P Reinach 1, J Koniarek 1, Z Wang 1, P Iserovich 1, J Fischbarg 1
PMCID: PMC1723367  PMID: 10655198

Abstract

BACKGROUND/AIMS—Fluid transport across the in vitro corneal epithelium is short lived, hence difficult to detect and characterise. Since stable rates of fluid transport across several cultured epithelial cell layers have been demonstrated, the behaviour of confluent SV40 transformed rabbit corneal epithelial cells (tRCEC) grown on permeable supports was examined.
METHODS—Fluid transport was determined with a nanoinjector volume clamp; the specific electrical resistance of the layers was 184 (SEM 9) Ω cm2. tRCEC layers transported fluid (from basal to apical) against a pressure head of 3 cm H2O for 2-3 hours.
RESULTS—In the first hour, the rate of fluid transport was 5.2 (0.5) µl/h/cm−2 (n=23), which is comparable with that found in other epithelia. Fluid transport was completely inhibited in 15-30 minutes by either 100 µM ouabain (n=6), 50 µM bumetanide (n=6), or 1 µM endothelin-1 (ET-1; n=6). Preincubation with 10 µM BQ123 (an ETA receptor antagonist) obviated inhibition by ET-1 (n=6). ET-1 also caused a 22% decrease in specific resistance.
CONCLUSIONS—Fluid transport appears to depend on transepithelial Cl transport since (1) their directions are the same (stroma→tear), and (2) both bumetanide and ouabain inhibit it with similar time course. tRCEC appear useful to investigate aspects of the physiology and pharmacology of fluid transport across this layer, including receptor mediated control of this process.



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Figure 1  .

Figure 1  

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Schematic cross sectional diagram of the experimental setup used to measure fluid transport across epithelial cell layers (tRCEC). The right half of the chamber has been omitted so as to provide labels for the various elements within the chamber compartments.

Figure 2  .

Figure 2  

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(A) An individual recording of fluid transport. Upward deflections correspond to fluid moving in the basolateral to apical direction, from the bottom to the upper chamber. Each deflection represents the amount of fluid transported in 12 seconds. (B) Average fluid flow recorded from 23 experiments.

Figure 3  .

Figure 3  

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Relation between the rate of fluid transport and electrical resistance of the 23 cultured layers referred to in Figure 2B. Each point denotes the maximal rate for a given experiment, averaged for a period of 30 minutes; labels beside symbols denote the experiment number. A regression line fitted to the data is shown, along with its variables.

Figure 4  .

Figure 4  

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Recording of fluid transport as in Figure 2. Top: a representative experiment (n=6), showing the inhibitory effect of 100 µM ouabain added to the apical side of a cell layer. Bottom: similarly, a representative experiment (n=6) showing the inhibitory effect of 50 µM bumetanide ouabain added to the apical side.

Figure 5  .

Figure 5  

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(A) An individual recording shows the inhibitory effect of ET-1 on fluid transport. ET-1 (final concentration: 1 µM) was added to the apical side of the cells. (B) Effects of ET-1 on average rates of fluid flow (n= 6). This panel also shows the average specific electrical resistance of the layers before and after addition of 1 µM ET-1.

Figure 6  .

Figure 6  

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(A) BQ123 added to the apical side (final concentration: 10 µM) forestalls the inhibitory effect of 1 µM ET-1. (B) Effects of BQ123 (n=6).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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