Active urea transport in toad skin is coupled to H⁺ gradients

Active transport of urea exists inwardly through the skin of the toad Bufo viridis and is enhanced by adapting the toads to hypertonic saline solutions. In this paper, we report our studies on the energy coupling of this active transport system. We have shown previously that this system is independent of sodium transport. We thus studied the possibility of coupling to proton transport by studying the following: 1) the effect of acidifying the epithelium by means of CO₂ and protonophores, 2) the effect of alkalinizing the epithelium with the anion-exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and by removal of chloride from the serosal medium, 3) the effect of inhibiting the proton-translocating ATPase of the skin, 4) dependence on mucosal pH. We found that 1) acidification of the epithelium by means of 5% CO₂ and the protonophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nigericin inhibited urea influx; 2) alkalinization of the epithelium by means of the disulfonic stilbene DIDS and by removal of serosal chloride increased urea influx; 3) inhibition of the proton-translocating ATPase of the skin by means of 1,3-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide (NEM), and sodium orthovanadate inhibited urea transport; 4) urea influx was unaffected by alkalinizing the external medium to 8.5 or by acidifying to 6.5; it was significantly suppressed by a mucosal pH of 5.5 These results suggest that active influx of urea in toad skin may be coupled to the proton gradient across the epithelium and are compatible with a cotransport system with protons. The electrochemical proton gradient that drives this system may be generated by the proton-translocating ATPase of the skin. We cannot, however, exclude the possibility of an alternative system, such as a urea-ATPase, which is pH sensitive.