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 t he plasma membranes of these cells have water channels. We cultured BC EC on glass plates and monitored the intensity of the light scattered (I(s)) by the cells. We determined the kinetic constant (k) of the cha nge in I(s) on exposure to a 10% hyposmotic challenge to calculate the osmotic permeability (P(f)) of the plasma membrane. At 37-degrees-C, we found values of k = 0.68 +/- 0.07 s-1 and P(f) = 93.3 +/- 33 mum/s (n = 13). The sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMB S; 1 mM) reduced P(f) by 75%; 5 mM dithiothreitol reversed such inhibi tion. The activation energy (E(a)) of P(f) in the range 10-37-degrees- C was 4.7 +/- 0.7 kcal/mol (n = 5). The high P(f), values, the inhibit ion by pCMBS, and the low E(a) strongly suggest the presence of water channels. Therefore, we tested whether the injection of poly(A)+ RNA p repared from CBCEC into Xenopus laevis oocytes results in the expressi on of water channels. Four days after injection, we measured oocyte P( f) values from the rate of volume increase on exposure to hyposmotic m edium. In control oocytes injected with 50 nl of water, P(f) was 13.4 +/- 0.3 mum/s (n = 63). In oocytes injected with poly(A)+ RNA (50 ng/o ocyte in 50 nl water), P(f) was 40.9 +/- 1.6 mum/s (n = 72). This mRNA -dependent increase in P(f) was inhibited by 62% with 1 mM pCMBS and b y 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 ident ify such water channels, we coinjected CBCEC-poly(A)+ RNA with an exce ss 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 i n P(f). Our results strongly suggest that CHIP28 water channels accoun t for most of the osmotic permeability of the plasma membranes of CBCE C.