The binding of [3H]ouabain to the serosal side was studied in a chambered preparation of frog skin, free of connective tissue, while the short circuit (Isc) was concurrently monitored. Both ouabain binding and Isc inhibition proceeded as hyperbolic functions of time. A plot of the number of ouabain molecules bound vs. the corresponding values of Isc inhibition (%) yielded a straight line, yet showed that 1/3 of the binding occurred before any inhibition of Isc. Upon separation of the skins into 2 groups based upon initial Isc(Isci) - high, >20 μA/cm2 and low, <10 μA/cm2, the authors observed 2 distinct populations. The high Isci skins bound very little ouabain before inhibition of Isc whereas low Isci skins bound 1/2 of the total number of sites before exhibiting any inhibition of Isc. These observations strongly suggest that the Na,K-ATPase is directly involved in the generation of Isc, and at low Isc, inhibition of some pumps by ouabain causes a 'recruitment' of other pumps to increase their turnover rate and maintain Isc relatively unaffected. In addition, the binding of ouabain also displayed various characteristics that were consistent with known properties of the Na,K-ATPase: increased intracellular K/Na concentrations, whether achieved through the addition of amiloride or removal of Na from the outside medium, led to a significant decrease in ouabain binding rate relative to paired controls; and ouabain binding, either with normal or decreased intracellular Na, was significantly reduced in the presence of elevated K in the serosal bathing medium. Finally, the number of ouabain molecules bound to the frog skins was not correlated with their initial Isc values, indicating that the spontaneous skin-to-skin variation in Isc was not related to the number of functional pump sites but, rather, to their turnover rate.
|Original language||English (US)|
|Number of pages||21|
|Journal||Journal of General Physiology|
|State||Published - 1978|
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