The Permeability of the Skin of the Aquatic Anuran Xenopus Laevis (Pipidae)

Among the Amphibia, the Pipidae are of special physiological and phyletic interest as they are considered to be the most aquatic of the anurans (Darlington, 1957) and they may represent the remnants of an ancestral anuran stock (Griffiths, 1963). Xenopus laevis, the African clawed-toad, is a species of one of two extant genera of the Pipidae, and, except during periods of aestivation, it lives an entirely aquatic existence.

The skin of amphibians is unusual among the vertebrates as it is quite permeable and makes an important contribution to their osmoregulation. Its properties are, however, known to differ in a way which may be related to their normal manner of life (see Bentley, 1971). Although the electrical properties and osmotic permeability of the skin of Xenopus have been previously investigated (Maetz, 1963; Bentley & Main, 1972), there appear to be no precise measurements of its permeability to solutes and water. We have found that the skin of this species has a relatively low permeability to Na, Cl and water, as compared to other amphibians, and while it is responsive to neurohypophysial hormones, aldosterone is without effect.

African clawed-toads (Xenopus laevis) were obtained from a biological supplier (Nasco, Fort Atkinson, Wisconsin) and kept in a tank of tap water at 19–20 °C. They were not fed and were used within 1 week of their arrival.

In vitro preparations of the dorsal and ventral surface skin were made following double pithing of the toads. The isolated skin was then mounted as a diaphragm, either tied on to the end of a lucite cylinder (surface area 10 cm²), for the determination of diffusional water flow (with tritiated H₂O) and urea permeability (with [¹⁴C-]urea), or placed in an Ussing-type chamber (surface area 3 cm²). The latter preparation was used for the measurement of the electrical potential difference (PD) and short-circuit current (SCC), as well as unidirectional fluxes of ²²Na and ³⁶CL To minimize ‘edge damage’ effects double parafilm rings were placed between the edge of the tissues and the chamber.

The PD and SCC across Xenopus skin was measured using Ringer-agar bridges connected through calomel and Ag-AgCl cells, to a potentiometric recorder and an automatic voltage clamp. The SCC was continually recorded on a strip chart recorder.

Unidirectional fluxes (j_in, outside to inside and j_out, inside to outside) of Na and Cl across the dorsal and ventral skin of the toads were determined under short-circuited conditions as described previously (Yorio & Bentley, 1977) using ²²Na and “Cl as tracers. Thirty minutes were allowed for isotope equilibration and samples were taken from the ‘cold’ side at 30 min intervals for four successive periods, or when vasotocin (AVT) was present, eight periods (four control plus four following the addition of AVT). Owing to the duration of the experiments different preparations of the skin from different toads (from the same batch and kept under identical conditions) were used to measure J_ln and J_out. An estimate of the net flux can be obtained from the difference of J_ln and J_out and in the instance of Na this was found to be similar to the measured SCC in the 12 preparations (Table 1).

For the determination of unidirectional fluxes of tritiated-water (³H_aO) and [¹⁴C-] urea, the dorsal and ventral surface skins of Xenopus laevis were mounted as diaphragms see above) and the measurements made as described previously (see Yorio & Bentley, 1977). The isotopes (1 ρCi/ml) were added to the epidermal side and samples were taken from the trans side every 0·5 min for ³H_aO and every minute for [¹⁴C-] urea for five successive periods. The fluxes were steady over the five periods indicating that equilibration with the tissue occurred very rapidly. Each value represents the mean of these five periods. The same skin preparation was used when studying the effects of the neurohypophysial hormone, vasotocin, on both ³H_aO and [¹⁴C-]urea fluxes.

The isotopes were counted, where appropriate, in a Nuclear Chicago scintillation spectrometer or a Beckman Autogamma counter. The fluxes were determined from the specific activity of the ‘hot’ solution. The isotopes (²²Na, ³⁶Cl, ³H₂O and [¹⁴C-] urea) were obtained from New England Nuclear, Boston, Mass.

The Ringer’s solution had the following composition (HIM): NaCl, 111; KC1, 3·35; CaCl_a, 2·7; NaHCO₃, 4·0 and glucose, 5·0. This solution was aerated and the pH was about 8·0.

The following hormones were used: 8-arginine vasotocin (standardized by its ability to increase the rat’s blood pressure, Schwartz/Mann, Orangeburg, N.Y.) and D-aldosterone (CIBA Pharmaceutical Co., Summit, New Jersey).

The skin of Xenopus laevis is known to display, in vivo, a transcutaneous PD, (inside positive) and a short-circuit current (SCC) which is usually assumed to result from an active transport of Na, though Cl could also be involved (Maetz, 1963 ; Bentley & Main, 1972). We measured the unidirectional transcutaneous fluxes of Na and Cl (Table 1). The Cl fluxes were similar in both directions but the influx of Na exceeded the efflux. The net estimated Na flux was equivalent to the measured SCC. There thus appears to be an active uptake of Na, but not Cl, across the skin of Xenopus. The effluxes of Na and Cl were quite low compared to those in other amphibians.

Neurohypophysial and adrenocortical hormones can increase the rate of Na transfort across the skin of many amphibians. Vasotocin, as observed previously (Maetz, 1963; Bentley & Main, 1972), increases the SCC and this was found to reflect an increased influx of Na. The rise in Na influx was 0·48 ±0-08 μequiv cm⁻² h⁻¹ (5), which equals 12 ρA/cm⁻² and is nearly equivalent to the observed increase of 10 ρA in the SCC. In contrast to other anurans (Table 2) aldosterone had no effect on the SCC across the skin of Xenopus.

These experiments were performed on skin from the ventral surface of the animals but dorsal skin behaved similarly, suggesting that the permeability of the integument is relatively uniform in this species.

Non-ionic solutes such as water and urea can pass across amphibian skin. The unidirectional fluxes of these molecules was measured in ventral skin from Xenopus (Table 3). Permeability to water was relatively low and although this process was observed to increase in the presence of vasotocin it was of relatively limited magnitude and barely attained levels seen in the absence of the hormone in other species. The unidirectional flux of urea was similar to that observed in other species and was not changed in the presence of vasotocin. Skin from the dorsal surface exhibited similar properties.

Osmoregulation by vertebrates in fresh water involves several types of processes which are concerned with: solute losses across the integument, active accumulation of ions from the external media and the osmotic uptake and subsequent excretion of water by the kidneys. The strategy adopted by Xenopus laevis appears to primarily involve a restricted permeability of its integument to water and ions rather than any enhanced ability to actively take up Na or Cl. The cutaneous permeability to another type of solute, urea, was however not remarkable.

It has previously been observed that the skin of Xenopus exhibits a transcutaneous PD with a relatively low SCC and high electrical resistance (Maetz, 1963 ; Bentley & Main, 1972; Bentley, 1975). These properties reflect the ionic permeability observed in the present experiments. Passive permeability to Na and Cl was low. There was no evidence of active Cl transport, such as has been observed in some anurans, but there was a net active uptake of Na which was equivalent to the observed SCC. The active Na uptake was, however, not high in Xenopus, despite its aquatic manner of life where a vigorous process of active Na uptake could conceivably be useful. Aldosterone can regulate transcutaneous Na accumulation across anuran skin (Crabbé, 1964) but this hormone was ineffective in Xenopus, though Na transport could be strongly stimulated by vasotocin. In other aquatic anurans active cutaneous uptake of Na has also been observed to be either absent, as in the mudpuppy Necturus maculosus (Bentley & Yorio, 1977) or low, as in the mud eel, Siren lacertina and the congo eel, Amphiuma means (Bentley, 1975). The limited efflux, or ‘leakage’, of Na and Cl in Xenopus may make a more important contribution to its osmoregulation in hypo-osmotic media than active ion uptake.

The permeability of the skin of Xenopus to water was low compared to more terrestrial species such as Bufo marinus, Rana pipiens and Agalychnis dacnicolor (Table 3). It was, however, similar to that observed in the urodele Necturus maculosus which is also aquatic. Neurohypophysial hormones, such as vasotocin, have been observed to elicit large increases in cutaneous permeability to water in many amphibians, especially more terrestrial species (see Bentley, 1971). This response was also seen in Xenopus but the permeability of the skin remained low even in the presence of this hormone. Vasotocin is readily released and appears in high concentrations in the blood of Xenopus (Bentley, 1969). If a strong response to this hormone existed it could result in a massive increase in the accumulation of water. A limited rate of water uptake across the skin reduces the need for its renal excretion and as some salts inevitably remain in the urine this property will also indirectly limit losses of ions via the kidneys. In Xenopus a low cutaneous permeability to water, as well as ions, may thus be of primary importance in its ability to osmoregulate in its natural freshwater environment.

Supported by National Science Foundation Grant No. BMS75-07684.