Uptake experiments were performed after the RCECs reached
confluence in 8 to 9 days. Uptake of
3H-CsA by
cultured RCEC was measured as reported previously by Tsuji et
al.
21 32 Briefly, the cultured cell layer was washed three
times with 1 ml incubation buffer (Hanks’ balanced salt solution
([HBSS pH 7.4]; osmolarity 315 mOsm/kg; at 37°C) whose composition
was (in millimolar): 1.3 CaCl
2, 5.0 KCl, 0.3
KH
2PO
4, 0.8
MgCl
2, 138 NaCl, 0.3
Na
2HPO
4, 5.6
d-glucose, and 10 HEPES. Cultured RCECs were preincubated
at 37°C for 30 minutes in this incubation solution. Immediately after
the end of the preincubation period, the solution was removed by
suction and replaced with 250 μl of the same medium containing
3H-CsA.
14C-Mannitol was
used as the extracellular space marker. To terminate uptake, the RCECs
were washed with 1 ml ice-cold incubation medium at a designated time.
RCECs in each well were solubilized by incubating them overnight in 500μ
l 1 N NaOH at 37°C, and samples were taken for quantifying
3H-CsA and
14C-mannitol
content. The same procedure was followed to evaluate efflux. Efflux was
determined from the remaining amount of
3H-CsA.
Protein content was determined by modified Lowry et al. method using
bovine serum albumin as the standard.
33 Transport studies
were performed after RCECs reached confluence in 8 to 9 days. It was
measured as previously described.
31 The cells were grown
on inserts and then were washed three times with transport buffer
(HBSS; pH 7.4) at 37°C and placed in a 12-well cluster plate, which
was maintained at 37°C. They were then preincubated at 37°C for 30
minutes in the transport buffer. Immediately after the end of the
preincubation period, the solution on the donor side was removed by
suction, and was replaced with fresh incubation medium containing
3H-CsA (0.5 ml tear [T] side, 1.5 ml stromal[
S] side). At appropriate time intervals over 4 hours, samples were
withdrawn from the receiver side, and replaced with an equal volume of
HBSS.
14C-Mannitol was used as the paracellular
permeable marker. Concerning the adsorption of CsA to cell culture
plates, dish, and pipet tips, 0.1% polysorbate 80 and 0.1%
dimethyl sulfoxide were added in HBSS to inhibit absorption of CSA. The
maximum recovery of
3H-CsA was approximately
95%. The radioactivity was measured by using a liquid scintillation
counter (Tri-Carb 2100TR; Packard, Meriden, CT). The transepithelial
electrical resistance (in ohms per square centimeter) on Transwell COL
was measured with an electrical resistance meter (Millicell ERS;
Millipore, Bedford MA). The transepithelial electrical resistance was
152.4 ± 4.0 Ω/cm
2 (
n =2 4,
mean ± SEM).