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mRNA from frog corneal epithelium increases water permeability in Xenopus oocytes
1Department of Ophthalmology, Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Purpose:
To investigate the existence of a water channel in the frog corneal epithelium by studying the osmotic water permeability (Pf) of Xenopus oocytes expressing the mRNA message from frog corneal epithelium.
Methods:
Total RNA was obtained from corneal epithelium by a single-step phase separation method, and poly A+ RNA was isolated using oligo-dT columns. This mRNA was injected into the oocytes. After a 48-hour incubation, oocyte volume changes elicited by a hypoosmotic solution were measured with a computerized video system.
Results:
Oocytes injected with 50 nl mRNA (1 microgram/microliter) showed a significant increase in Pf compared to water-injected controls (8.4 +/- 1.5 to 17.5 +/- 1.9 cm.sec-1 x 10(-4), P < 0.005). mRNA-injected oocytes exposed to a higher external [Cl-] showed a heightened permeability. Furthermore, Pf of oocytes exposed to a solution containing the recognized water-channel blocker HgCl2 was significantly lower than the Pf of mRNA-injected oocytes not exposed to HgCl2.
Conclusions:
Evidence was found for a water channel in the frog corneal epithelium because oocytes injected with the epithelial mRNA manifested increased water permeability. The increase in water permeability was larger in the presence of external Cl- and was inhibited by HgCl2. This finding correlates with measurements of Pf in the intact epithelium in which apical Cl- induced an increase in transepithelial water permeability prevented by HgCl2.