Competition Between Potassium and Rubidium Ions for Penetration of the Midgut of Hyalophora Cecropia Larvae

It is obviously an advantage to use Rb-86 when working with the midgut, so we have retested the competition between K and Rb fluxes. A 100% agreement between the fluxes could not be found, the values varied and on the average a 50 − 60% Rb flux was found for a 100% K flux. The variations between different guts were, however, much larger so the mean value cannot be used for a specific experiment. It made no difference if we considered the active K flux from blood-side to lumen or the flux from lumen to blood-side.

Fifth-instar larvae of Hyalophora cecropia (L) of 10 − 15 g were used for the experiments. They were grown on artificial diet (Riddiford, 1968) and on leaves of willow (Salix babylonica). The larvae were chilled on ice for at least 1 h before the gut was removed and placed in the apparatus described by Harvey, Haskell & Zerahn (1967) and Harvey & Zerahn (1972). Generally the midguts were short-circuited, but a few experiments were performed on unshorted guts.

Potassium flux was measured by labelling with ⁴²K and the rubidium flux by labelling with ⁸⁶Rb. The necessary amount of K⁴² and Rb⁸⁶ were mixed and simultaneously added to the solution. In this way K and Rb were influenced identically, for example, by inaccurate short-circuiting.

The K⁴² was measured with a Nal crystal in a scintillation well counter and pulse height analyser (Selectronic) adjusted to minimize the effects of ⁸⁶Rb gamma rays. After decay of the ⁴²K the ⁸⁶Rb was counted on a Packard beta spectrometer (Tricarb) with an accuracy of 2 % or better. Radio tracers were added to the solution of one side of the gut at the start and samples taken from the other side at times given in the tables.

The concentration of K was identical on both sides as was the concentration of Rb. The ratio of the concentration of the two ions was varied from experiment to experiment to test the influence of the concentration on the K/Rb flux ratio. The bathing solution used with ⁴²K had the following composition: K+Rb = 32 HIM; sucrose, 166 mM; Cl, 30 HIM; HCO₃, 2 mM.

Table 1 shows the measured Rb/K flux ratios. To correct for the concentration differences between K and Rb in the bathing solutions the Rb flux is multiplied by the K/Rb concentration ratio. The ratio is thus given for a 1 mM solution of both ions or for the rate constants. From Table 1 it can be seen that the mean values of the Rb/K ratio do not vary significantly with time. Rb carries only 56% of that expected for a perfect tracer for K. Because of individual variation, between 19 and 98%, an appropriate mean value cannot be obtained.

The inability of Rb to substitute for K might be true for the active K transport only. Accordingly flux experiments from lumen to blood-side were made. The results shown in Table 2 show that Rb does not behave differently when it traces for K from the blood-or from the lumen side.

Larvae are reared on leaves or on artificial diet. It is conceivable that differences in rearing might change the Rb/K ratio for transport. Table 3 summarizes results for animals reared on willow and show that under these conditions Rb is also not a reliable tracer for K. With the midgut at its normal potential the flux of K from blood-side to lumen can be diminished by a factor of 2 − 5. Nevertheless the Rb/K ratio is still far from 1 even with a potential over 100 mV across the midgut.

The above results indicate that Rb is not a perfect tracer for K. Only about half of the K flux is measured when Rb is used as a tracer for K, regardless of the Rb concentrations. However, the wide biological variations between the guts implies that while Rb partially reflects the K flux, a constant factor between Rb flux and K flux cannot be found. The conclusion is that Rb cannot be used quantitatively as a tracer for K. This is true regardless of whether the active K flux is measured from blood-side to lumen or from lumen to blood-side.

The diffusion rates in water for potassium and rubidium are very close, so if the ions were passing the gut by diffusion they should compete almost equally, We know that the transport of K is active and accordingly the Rb/K ratio is often far from 1. But for the K flux from lumen to blood-side we do not know how the K crosses the membrane. It is interesting to note that under short-circuit conditions the Rb/K ratio is about the same as for the active K flux. We must conclude that in general K does not cross the gut by diffusion, but more probably uses the same route as the active K flux, possibly as a leak through the K pump.

I thank Dr Koefoed-Johnsen and Prof. H. H. Ussing for helpful criticism in preparing the manuscript and Dr Leon Pape for correcting the language. For technical assistance with the experiments my best thanks to Mrs Susanne Jensen.