The Effect of Salt Adaptation and Amiloride on the In Vivo Acid-Base Status of the Euryhaline Toad Bufo Viridis

The euryhaline toad Bufo viridis can be adapted to extremely high salinity (Stoicovici & Pora, 1951; Tercafs & Schoffeniels, 1962; Gordon, 1962; Katz, 1973). Under these conditions there is a great increase in the osmolarity of the blood; both the concentration of NaCl and of urea increase, and there are marked changes in transport properties of the skin (Katz, 1975). The control of acid-base status in amphibia is different from that in higher vertebrates-notably mammals; amphibian kidneys are much less effective (Yoshimura et al. 1961), and the urinary bladder may play a role in the acid-base regulation in some species or subspecies, but not in others (Ziegler, Ludens & Fanestill, 1974). The skin, which is endowed with an Na⁺/H⁺ exchange mechanism operating under open-circuit conditions (Garcia-Romeu, 1971 ; Ehrenfeld & Garcia-Romeu, 1977), may also participate in amphibian acid-base regulation. This study is concerned with the in vivo acid-base status of the blood in the toad Bufo viridis during adaptation to high salinity, and compares this with the effect of amiloride. A preliminary report has been published (Katz, 1979).

* Present address : Department of Biology, the Technicon, Haifa.

Toads (Bufo viridis) were collected in Jerusalem (Israel) and were kept outdoors with free access to tap water. They were fed twice a week with maggots. During the experiments the toads were kept in the laboratory (19 – 25 °C) and were not fed. Salt adaptation was carried out gradually (Katz, 1973). Toads were immersed to a depth of 2-3 cm, first in NaCl solution of 230 mOsm for 5–7 days and then directly into a 500 mOsm solution. For the collection of blood a permanent cannula was implanted into the dorsal artery as detailed elsewhere (Katz, 1980). Blood was collected anaerobically from unrestrained animals and the pH, and were determined immediately, using a Radiometer BMS 3 MK 2 blood microsystem and Radiometer electrodes, attached to PHM 73 pH/blood gas monitor. All electrodes were recalibrated after each measurement and readings were corrected accordingly. Osmolarity was determined on a Knauer (Berlin) semimicro osmometer, and chloride on a Radiometer CMT 10 chloride titrator. Na⁺/H+ exchange was determined in vitro on abdominal skins under open-circuit conditions with low (2 mm) sodium on the outside, according to the method of Ehrenfeld & Garcia-Romeu (1977). The internal solution contained (in mm): NaCl, 83; NaHCO₃, 24; Na₂HPO₄, 4; KC1, 2·5; KH₂PO₄, 1·3; CaCl₂, 4; MgSO₄, 4; glucose, 11, gassed with 95%O₂/s%CO₂ mixture, pH 7·4. In the external solution there was only 2 mm NaCl buffered with imidiazole (2 mm) to pH 7·4, and the solution was gassed with air. The unidirectional influx of sodium was determined with ²²Na added to the mucosal side, and hydrogen net fluxes were obtained from the changes in H⁺ concentration on each side during the flux period. H⁺ was titrated with 2 mm NaOH in 2 ml samples with a Radiometer ATSI/TTA61 autopipetting titration system, after 30 min bubbling air to eliminate respiratory CO₂. Potential difference was measured with calomel electrodes through agar bridges. Amiloride was from Merck, Sharp & Dohme. Student’s t test was used for statistical analysis.

Fig. 1 shows the time course of changes in the pH, HCO₃⁻, and osmolarity of the blood in 4 toads during adaptation to high salinity. There is a significant reduction in the pH and HCO₃⁻, while the osmolarity nearly doubled, after 6 days in 500 mOsm NaCl (the toads can live under these conditions for over a month; Katz, 1973). Transferring the toads back to tap water resulted in a rapid return of these parameters to their previous control values. Table 1 summarizes the pH, blood gases and the osmolarity of the blood in toads which were adapted to high salinities. It should be pointed out that the control group had a free access to tap water, while the others were immersed 2–3 cm in the solution. This has been found to have no effect on the acid-base status of the blood (Katz, 1980). There is a considerable acidification of the blood and a simultaneous reduction in the concentration of HCO₃⁻ at the high salt conditions (500 mOsm), characteristic of metabolic acidosis. did not change, and neither was there a change in the ⁠. The osmolarity of the plasma, on the other hand, increased from 370 to 565 mOsm/kg H₂O, and the concentration of the chloride doubled. A calculation of the ionic contribution to the osmolarity in Table 1 reveals that there is always some 20 – 70 mm of other osmolyte, which is contributed by the urea in the blood (Katz, 1973). Two days after transference back to tap water, all variables returned to their previous control values.

It is now established that sodium transport is markedly diminished under conditions óf adaptation to high salinity (Katz, 1975) and that this may result in a diminution of H⁺ secretion (Garcia-Romeu, 1971). Table 2 summarizes in vitro measurements of the Na⁺/H⁺ exchange across the isolated skin of the toad using the method of Ehrenfeld & Garcia-Romeu (1977). There was a linear and quite significant (r = 0 · 83) correlation between the net fluxes of these two cations, in skins from toads in deionized water:

f_netH⁺ = (– 29 ± 26) – (0 · 99 + 0 · 20) f_netN_a⁺ (mean ± s.E.M. from 14 separate skins). This gives a i : i exchange ratio between the two cations under these conditions. Amiloride, which blocks sodium transport across the skin (Eigler, Kelter & Renner, 1967), had a marked effect on the two fluxes in skins from toads adapted in deionized water (Table 2A). The potential difference across the skin and the secretion of protons disappeared, while the net flux of sodium reversed. The reversal in the net flux is the result of increased efflux, with 120 mm sodium on the inside and only 2 mm on the outside, which is favoured when the entry pathway for sodium is blocked by amiloride (Biber & Mullen, 1977).

The net fluxes across the skin of animals adapted for 10 days in 600 mOsm NaCl are completely abolished (Table 2, B). The effect of amiloride on these skins was seen mainly on the efflux of sodium, which increased by an order of magnitude. It shows that despite the great reduction in the amiloride-sensitive sodium transport across the skin (i.e. entry sites; Katz, 1975), the recycling mechanism (Hviid-Larsen, personal communication, 1980) is operating rather effectively. These experiments (Table 2, A, B) were carried out under similar experimental conditions (the osmolarity and the composition of the bathing solutions) and may therefore be compared. This comparison demonstrates a similarity in the net fluxes of sodium and hydrogen ions between the skins from deionized water-adapted toads treated with amiloride, and the skins from the high-salt-adapted toads, under control conditions. Since the effect of amiloride on the net fluxes of sodium and hydrogen in vitro was similar to the effect of salt adaptation, it was decided to determine the effect of this drug on the acid-base status of the blood in vivo. Table 3 summarizes the results of experiments in which toads were immersed for 2 –4 days in tap water containing 5 ×10 ⁻⁴M amiloride. It can be seen that there is a marked reduction in the pH and the concentration of HCO₃⁻, while the of the blood did not change. These effects are characteristic of metabolic acidosis. The osmolarity of the blood even decreased a little. Comparison of Tables 1 and 3 shows that the acid-base status of the blood in vivo is affected similarly by the two experimental conditions (e.g. salt adaptation and amiloride).

The present study gives an account of the acid-base status and the blood gases all the toad in vivo under conditions of adaptation to high salinity. A great deal of the CO₂ elimination in amphibia is carried out through the skin (Krogh, 1904,Rahn & Howell, 1976) and is characteristic of the bimodal gas exchange of this group (Lenfant, Johansen & Hanson, 1970). The skin of amphibia is therefore of prime interest, being endowed with both respiratory exchange mechanisms, and with solutes and water-exchange mechanisms (Krogh, 1904, 1934; Garcia-Romeu, 1971; Ziegler et al. 1974).

Salt adaptation of the euryhaline toad B. viridis (Katz, 1973) is accompanied by marked physiological changes. The results reported in this study show that the blood gases (i.e. and ⁠) in the toad are not affected either by adaptation in high salt solution for over 6 days (they can live under these conditions for over a month; Katz, 1973), or in tap water containing amiloride (Tables 1 and 3). These conditions do not, therefore, affect the ability of the respiratory and circulatory systems to sustain their function in maintaining normal levels of the blood gases. In fact Jackson & Braun (1979) have shown recently that the regulation of blood in the bullfrog is carried out mostly, if not solely, by the pulmonary system.

In fish, Renzis & Maetz (1973), and Cameron (1978), have postulated an analogous role to the gills in regulating the acid-base status of the blood.

In conclusion, the present study has demonstrated the effect of salt adaptation the in vivo acid-base-status of the blood in the toad. The metabolic acidosis was raated to an inhibition of hydrogen secretion through the skin, as a result of the inhibition of sodium transport under these conditions. A quantitative assessment of the relative contribution of the various organs (i.e. the kidneys, the urinary bladder and the skin), to the regulation of acid-base status under adaptation to high salinities will throw more light on the regulatory mechanisms involved.

Yael Shimoni gave invaluable assistance in the cannulation of the toads. I would like to thank Dr F. Garcia-Romeu, from Nice, for fruitful discussions and critical reading of the manuscript.