A novel system to measure labelled CO2 and HCO3- fluxes across epithelia: corneal epithelium as model tissue.
A method that allows for an accurate measurement of 14C-labelled fluxes of HCO3- and/or CO2 across epithelia is described. It is based on the principle that in a closed system the specific activities of HCO3- and CO2 are equal to each other. The hemichambers between which the tissue was mounted were modified Ussing-type chambers that included a capacity for recirculation and mixing of the labelled fluid and gas phases within a closed system. The isolated frog corneal epithelium was used as a model system. In solutions containing 25 mM HCO3- and 5% CO2, the unidirectional fluxes from tear to stroma (t-s) and stroma to tear (s-t) were 3.08 +/- 0.12 and 2.33 +/- 0.11 mu eq h-1 cm-2 (means +/- S.E.S), respectively, with a statistically significant t-s net flux. These fluxes were independent of the presence of Cl- in the bathing solutions. The bilateral addition of methazolamide (10(-4) M) reduced both unidirectional fluxes to about 2.0 mu eq h-1 cm-2. This finding was a strong indication of the presence of carbonic anhydrase (CA) in the corneal epithelium as well as its involvement in the production of the net flux of 14C-label. In nominally CO2-free media, unidirectional t-s and s-t fluxes were 0.19 +/- 0.03 and 0.27 +/- 0.05 respectively. These fluxes were not affected by methazolamide. The s-t net HCO3- flux can result from the activity of a basolaterally located Na(+)-(n)HCO3- cotransporter. The CO2-elicited t-s net flux could also be explained by the presence of this transporter and a recirculation of label across the basolateral membrane.
Candia
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OA
Department of Ophthalmology, Mount Sinai School of Medicine, New York, NY 10029, USA.
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism*; D002255:Carbonic Acid / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism; D004728:Endothelium, Corneal / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004848:Epithelium / Q000201:enzymology / Q000378:metabolism; D066298:In Vitro Techniques; D017136:Ion Transport; D008704:Methazolamide / Q000494:pharmacology; D008954:Models, Biological; D011892:Rana catesbeiana
D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D002245:Carbon Dioxide; D002255:Carbonic Acid; D002256:Carbonic Anhydrases; D008704:Methazolamide