On the distribution of Na+-pump sites in the frog skin

Exposure of the outside of the isolated frog skin to a Ringer's solution, made hypertonic by the addition of mannitol, causes a rapid and sustained increase in transepithelial permeability through a structural distortion-a focal blistering-of the "tight" junctions of the outermost living cell layer. [(3)H]ouabain, used as an autoradiographic marker for the Na+-pump (Na+-K+-adenosine triphosphatase), is usually unable to penetrate the frog skin from the outside solution, but when added to a hypertonic mannitol- Ringer's solution in the outside bath it readily penetrates the epithelium, presumably through the opened shunt pathway. Radioautographic analysis of [(3)H]ouabain binding sites revealed that most of ouabain enters from the outside solution binds to the sites on the cell membranes of the stratum spinosum, as was the case when it was applied from the inside bath in an earlier study. The outer living cell layer, the first to be exposed to ouabain, does not appear to be the major site for the Na+-pump, and therefore, is not likely to be responsible for most of the active pumping of Na+. This result demonstrates that previous failure to show a high density of Na+-pump sites on the cells of the outermost layer, when [(3)H]ouabain was applied from the inside solution, was not due to the inability of the marker to reach these cells at a sufficient concentration to reveal all pump sites. These results provide further support for a model of Na+-transport across the frog skin which distributes the active pump step on the inward facing membranes of all living cells.