Cultured bovine corneal endothelial cells express CHIP28 water channels

Cultured bovine corneal endothelial cells (CBCEC) transport fluid from the basal to the apical surface. In this study, we examined whether the plasma membranes of these cells have water channels. We cultured BCEC on glass plates and monitored the intensity of the light scattered (I(s)) by the cells. We determined the kinetic constant (k) of the change in I(s) on exposure to a 10% hyposmotic challenge to calculate the osmotic permeability (P(f)) of the plasma membrane. At 37°C, we found values of k = 0.68 ± 0.07 s^-1 and P(f) = 93.3 ± 33 μm/s (n = 13). The sulfhydryl reagent p- chloromercuribenzenesulfonate (pCMBS; 1 mM) reduced P(f) by 75%; 5 mM dithiothreitol reversed such inhibition. The activation energy (E(a)) of P(f) in the range 10-37°C was 4.7 ± 0.7 kcal/mol (n = 5). The high P(f), values, the inhibition by pCMBS, and the low E(a) strongly suggest the presence of water channels. Therefore, we tested whether the injection of poly(A)⁺ RNA prepared from CBCEC into Xenopus laevis oocytes results in the expression of water channels. Four days after injection, we measured oocyte P(f) values from the rate of volume increase on exposure to hyposmotic medium. In control oocytes injected with 50 nl of water, P(f) was 13.4 ± 0.3 μm/s (n = 63). In oocytes injected with poly(A)⁺ RNA (50 ng/oocyte in 50 nl water), P(f) was 40.9 ± 1.6 μm/s (n = 72). This mRNA-dependent increase in P(f) was inhibited by 62% with 1 mM pCMBS and by 78% with 0.3 mM HgCl₂; the posterior addition of 5 mM dithiothreitol (DTT) or restored P(f) to near-control values. In an attempt to identify such water channels, we coinjected CBCEC- poly(A)⁺ RNA with an excess of an antisense oligonucleotide for mouse CHIP28. This resulted in nearly full abolition of the expressed P(f). Coinjection with a sense oligonucleotide for CHIP28 did not affect the RNA- dependent increase in P(f). Our results strongly suggest that CHIP28 water channels account for most of the osmotic permeability of the plasma membranes of CBCEC.