Binding of [3H]ouabain to split frog skin: the role of the Na,K-ATPase in the generation of short circuit current.

Abstract

A s s T R A C X The binding of [SH]ouabain to the serosal side was studied in a chambered preparation of frog skin, free of connective tissue, while the short circuit (I~) was concurrently monitored. Both ouabain binding and 1~ inhibition proceeded as hyperbolic functions of time. A plot of the number of ouabain molecules bound vs. the corresponding values of Iae inhibition (percent) yielded a straight line, yet showed that one-third of the binding occurred before any inhibition of Ise. Upon separation of the skins into two groups based upon initial Ise(l~l)-high, >20 /~A/cm 2 and low, <10 /~A/cm 2, we observed two distinct populations. The high I~t skins bound very little ouabain before inhibition of lsc whereas low Isel skins bound one-half of the total number of sites before exhibiting any inhibition ofl~. These observations strongly suggest that (a) the Na,K-ATPase is directly involved in the generation of Ise, and (b) at low Ise, inhibition of some pumps by ouabain causes a "recruitment" of other pumps to increase their turnover rate and maintain Ise relatively unaffected. In addition, the binding of ouabain also displayed various characteristics that were consistent with known properties of the Na,K-ATPase: (a) 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 (b) 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 Ise values, indicating that the spontaneous skin-to-skin variation in lae was not related to the number of functional pump sites but, rather, to their turnover rate.

Na, K-ATPase and the development of Na+ transport in rat distal colon

“…If we suppose that the pump density in the young epithelium is 69 x 1012 sites/cm 2 and in that of adults 784 • 1012 sites/cm 2, the maximum turnover rate would be 500 Na+/site 9 min in adult and 6400 Na § site 9 min in young rats. These values are within the range reported for other epithelia such as rabbit colon [31] and tadpole and adult frog skin [4,30], however, much smaller than the turnover rate of purified Na,K-ATPase, 30,000 Na+/site ' min. Hence cellular factors appear to control the turnover of the pump.…”

“…Also the comparison of I)c and activity of Na,K-ATPase converted to the same unit (p~mol Na+/hr 9 cm2), which shows that the maximal pumping rate based on enzymatic measurements is four times higher in 10-day-old and more than 10 times higher in adult rats, indicates the possible regulation of turnover by the cell. Colonocytes may have an intracellular pool of Na,K-ATPase which is variable during development as was demonstrated in some other cells [43], or there may be inactive pumps at the surface of the cells [4]. It is possible that the higher pump turnover rate reflects the action of corticosteroids because this turnover is higher in the distal colon of adult rabbits with secondary hyperaldosteronism than in animals with unstimulated adrenal glands [31].…”

Effect of amiloride, ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

“…Furthermore, the cells lose K-during ouabain poisoning, which would increase the K + concentration in the extracellular space. An increase in the extracellular K + concentration reduces the binding of ouabain to the Na + -K + pump (Cala, Cogswell & Mandel, 1978). Since these secondary changes would influence the rate of ouabain binding to the Na +-K § pump and the rate of the pump, complex changes in the active appearing flux during ouabain poisoning has to be expected.…”

Rheogenic sodium transport in a tight epithelium, the amphibian skin.

“…is abolished by ouabain, a specific inhibitor of the Na-K-activated ATPase. The effect is manifest as early as 3-5 min after addition of 104 M-ouabain, indicating a high velocity of binding to, and blockage of the ATPase (see also Cala, Cogswell & Mandel, 1978). It seems highly unlikely that changes of intra-extracellular ion composition during such short periods account for the effect of ouabain.…”