Effects of vasopressin and aldosterone on amiloride binding in toad bladder epithelial cells

Methods have been devised to measure the binding of [$^{14}$C]amiloride to isolated cells from bladders of toads, Bufo marinus. This agent blocks transepithelial sodium transport across bladders by preventing sodium entry to the transporting mechanism. A saturable binding component has been found with an affinity of 5.6 $\times $ 10$^{7}$ M$^{-1}$ in the presence of 1.1 mM Na$^{+}$, which corresponds to the affinity of amiloride when used as a transport inhibitor at the same sodium concentration. In freshly isolated cells the capacity of the binding sites is 3.6 $\times $ 10$^{5}$ sites/cell, but this value falls to about one third in aged suspensions. When cells are treated with vasopressin (100 mU/ml) somewhat less specific binding is measured at an amiloride concentration giving 50% occupancy. The results are consistent with the view that vasopressin moves the binding curve to the right along the concentration axis, reducing the affinity of amiloride by a factor of approximately two, while leaving the total capacity unaffected. The affinity of amiloride when used as an inhibitor of transport is also found to be reduced by a factor of two by vasopressin, and complete inhibition of transport can still be achieved. d-Aldosterone in vitro increases the number of amiloride binding sites in isolated cells by approximately 115%, and results from transport studies indicate that there is no significant change in the affinity of amiloride after d-aldosterone treatment. Inhibitors of transcription and translation (actinomycin D and cycloheximide) prevent the increase in amiloride binding caused by d-aldosterone. In vivo the effects of d-aldosterone are more complex, but it is shown that the steroid increases the transport capacity of the tissue, when this is expressed as the number of amiloride binding sites per unit mass of tissue. The results are discussed in terms of the ways in which the two hormones may alter the entry of sodium into the epithelial cells, and so in turn affect transepithelial sodium transport.