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 (IS) by the cells. We determined the kinetic constant (k) of the change in IS on exposure to a 10% hypoosmotic challenge to calculate the osmotic permeability (Pf) of the plasma membrane. At 37 degrees C, we found values of k = 0.68 +/- 0.07 s-1 and Pf = 93.3 +/- 33 microns/S (n = 13). The sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS; 1 mM) reduced Pf by 75%; 5 mM dithiothreitol reversed such inhibition. The activation energy (Ea) of Pf in the range 10-37 degrees C was 4.7 +/- 0.7 kcal/mol (n = 5). The high Pf, values, the inhibition by pCMBS, and the low Ea 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 Pf values from the rate of volume increase on exposure to hypoosmotic medium. In control oocytes injected with 50 nl of water, Pf was 13.4 +/- 0.3 microns/S (n = 63). In oocytes injected with poly(A)+ RNA (50 ng/oocyte in 50 nl water), Pf was 40.9 +/- 1.6 microns/S (n = 72).(ABSTRACT TRUNCATED AT 250 WORDS)
Echevarría
M
M
Department of Physiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Kuang
K
K
Iserovich
P
P
Li
J
J
Preston
G M
GM
Agre
P
P
Fischbarg
J
J
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004727:Endothelium / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008433:Mathematics; D008565:Membrane Proteins / Q000096:biosynthesis*; D051379:Mice; D008954:Models, Biological; D016376:Oligonucleotides, Antisense; D009865:Oocytes / Q000502:physiology; D011061:Poly A / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014867:Water / Q000378:metabolism*; D014982:Xenopus laevis
C496021:Aqp1 protein, mouse; D020346:Aquaporins; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D014867:Water; D051398:Aquaporin 1; D011061:Poly A; D002731:4-Chloromercuribenzenesulfonate; D012313:RNA; D004229:Dithiothreitol