Effect of amiloride, ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia
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Effect of amiloride, ouabain, and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia.
The active Na+ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na+−K+ pump at the basolateral membrane. In isolated epithelia with a very small Na+ efflux, the appearing Na+-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na+ transport. It was found that blocking the passive diffusion of Na+ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na+ transport, whereas the appearing flux (the rate of the Na+−K+ pump) decreased with a halftime of 1.9 min. Addition of the Na+−K+ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na+ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K+ channel inhibitor Ba++, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na−K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.
It is argued that in the short period, where the Ba++-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (ΔSCC) is equal to the net K+ flux via the Na+−K+ pump, and the coupling ratio (β) of the Na+−K+ pump can be calculated from the following equation β=SCCt=0/ΔSCC where SCCt=0 is the steady-state SCC before the addition of Ba++.
Key wordsfrog skin sodium flux ouabain amiloride barium
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