ATP activates an apical-to-basolateral nonselective cation current across the skin of larval bullfrogs (Rana catesbeiana) with similarities to currents carried by some P2X receptors. A functional P2X receptor was cloned from tadpole skin RNA that encodes a 409-amino acid protein with highest protein homology to cP2X(8). RT-PCR showed that this transcript was found in skin, heart, eye, brain, and skeletal muscle of tadpoles but not in skin, brain, or heart of adults. After transcribed RNA from this clone was injected into Xenopus oocytes, application of ATP activated a transient current similar to other P2X receptors and the ATP-activated transient in short-circuit current (I(sc)) across intact skin. The agonists 2-methylthio-ATP and adenosine-5'-O-(thiotriphoshate) also activated transient currents. alpha,beta-Methylene-ATP and ADP were poor agonists of this receptor. Suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS) were potent antagonists, and PPADS showed an irreversible blockade of this receptor to agonist activation. Under external Na(+)-free, Ca(2+)/Mg(2+)-free conditions (N-methyl-D-glucamine replacement, 0.5 mM EGTA), ATP activated a steadily increasing inward current. Fluorescence microscopy showed that propidium was entering the cells, suggesting that a relatively large pore size was formed under zero divalent conditions. This clone has some characteristics consistent with previously described ATP-activated I(sc) in the tadpole skin. Because the clone is not found in adult skin, it may have some exclusive role in the tadpole such as sensory reception by the skin or triggering apoptosis at metamorphosis.

For many years the adult frog skin has been used as a model system to study transepithelial sodium transport. In other sodium transporting tissues the three homologous subunits of the sodium channel have been cloned. The aim of this study was to clone and characterize the amiloride inhibitable sodium channel (ENaC) in adult bullfrog (Rana catesbeiana) skin. Three transcripts corresponding to the alpha, beta, and gamma subunits of ENaC were cloned and sequenced. Co-expression of all three in Xenopus oocytes yielded a functional frog sodium channel (fENaC). Amiloride sensitivity and current voltage relationships suggested that its characteristics were similar to other ENaCs. Subunits from the Xenopus sodium channel (xENaC) and fENaC were combined in all possible triplets. Although functional amiloride inhibitable sodium channels were formed in every case, the amiloride sensitivities were not identical. Subunit combination studies suggested that the alpha subunit made a major contribution to amiloride sensitivity but interactions of beta and gamma were also seen. When the amiloride sensitivities of intact skin from adult R. catesbeiana and Xenopus laevis were compared, Rana also had a consistently higher affinity. Comparison of fENaC and xENaC sequences may provide insight into which amino acids beyond those already identified are critical for amiloride binding.

Bullfrog tadpoles respond to apical application of 100 microM amiloride, acetylcholine (ACh) or ATP with a sharp transient inward (apical to basolateral) cation current. In adult skin, amiloride blockable transepithelial Na+ transport is upregulated by the hormone aldosterone. Tadpoles were treated in vivo with aldosterone and changes in short circuit current (Isc) in response to apical application of ATP were determined. Bullfrog tadpoles were exposed to aldosterone (10(-6) M) for periods ranging from 3 h to 60 h. Skins from 60-h aldosterone-treated animals showed a two- to three-fold increase in apical ATP-activated short circuit current when compared to animals treated with vehicle alone. Sodium replacement with a large, nonpermeable cation resulted in no measurable increase in Isc after exposure to ligand, consistent with ATP activation of an inward cation current and not chloride efflux. Activation/desensitization time courses and treatment with blockers revealed no measurable differences between aldosterone-treated and non-treated skins. Activation by amiloride and ACh gave essentially identical results. Studies with RT/PCR showed significant increases over controls of levels of mRNA associated with P2X channels. Given these data, our working hypothesis is that all three ligands activate the same process that exhibits both purinergic and cholinergic characteristics. These data are consistent with aldosterone upregulation of ATP gated channels expressed in the apical membrane of larval frog skin.

The epithelium of the larval bullfrog Rana Catesbeiana contains an apically located non‐selective cation channel. This channel has been shown to be activated by a variety of compounds including acetylcholine (ACh), amiloride and ATP. The purpose of this present study was to further characterize the tadpole channel in terms of interactions between these three ligands at their sites of activation. Tadpole skins were mounted in Ussing chambers with NaCl Ringers bathing the basolateral side. Perfusion of ligands in Ca++ free Ringers was done alone and in various combinations to test for interactions such as potentiation and suppression of ligand effects. ATP was found to potentiate the effects of amiloride and ACh at low concentrations, yet at high concentrations the responses were not additive as would be expected if two different populations of channels were being activated. ACh and amiloride perfusion resulted in a suppression of the ACh response. The addition of two ligands had little effect upon the desensitization time constant. Each of the ligands largly cross‐desensitized the other ligands. These results are consistent with a scenario in which all three ligands activate the same response.