The Effects of Posterior Pituitary Extracts on Water Balance in Bufo Carens and Xenopus Laevis, together with some General Considerations of Anuran Water Economy

In a previous communication (Ewer, 1951) it was noted that in both Bufo regularis and B. bufo pitressin has a greater water-balance effect than has pitocin, whereas in various species of Rana the reverse is the case. It was suggested that high pitressin sensitivity might be a characteristic of the genus Bufo, and that this might be correlated with the more terrestrial habit of toads, as compared with frogs. Before accepting this generalization as correct further data on other species of Anura are necessary; and a comparison of a terrestrial with an aquatic species also seemed desirable. B. carens occurs, although in small numbers only, in the neighbourhood, and is in general habit similar to B. regularis-, the totally aquatic Xenopus laevis is also obtainable locally. The effects of pitressin and pitocin injection on water balance in these two species have therefore been investigated.

Other points in the physiology of the animals which were considered to be of interest in relation to their adaptations to terrestrial or aquatic life were also studied. These include the normal rate of water uptake, and the maximal rate of water uptake after desiccation of uninjected animals. It has further been found (Ewer, 1952) that in Bufo regularis, when the animals are kept in a damp atmosphere but without access to free water, that pituitrin injection causes resorbtion of fluid from the bladder and accumulation of fluid in the lymph sacs. The relative efficacy of pitressin and pitocin in causing these effects has now been investigated.

The species used were Bufo regularis Reuss, B. carens Smith and Xenopus laevis (Daudin). The pituitary extracts used were Parke Davis pitressin, pitocin and pituitrin. All experiments were carried out at 26°C.

The method used for studying the effects of pitressin and pitocin injections on water uptake and urine output was the same as that described previously (Ewer, 1950, 1951). The rates of water uptake and urine production after injection of the pituitary extract are expressed as percentages of the normal rate, measured over a 3 hr. period immediately preceding injection. These values are referred to as the relative water uptake and relative urine production. The relative water uptake and urine production are calculated for the first hour after injection, and for the total period from the time of injection until the animal’s weight has reached a maximum. In the case of Xenopus there is no weight gain after injection of the extracts, and the values have therefore been calculated for i hr. and for 2 hr. following injection.

The method of investigating the effects of pitressin and pitocin on lymph accumulation and fluid resorbtion from the bladder was as previously described (Ewer, 1952). The animal was first kept in water for 1 hr. to ensure a state of balance. The bladder was then emptied, the cloaca ligatured and the animal returned to water for a further 2 hr. The increase in weight at the end of this period has been found to be approximately equal to the weight of urine in the bladder. The animal was now injected with the appropriate extract, and was placed in a moist chamber for 2 hr. At the end of this period it was killed by decapitation, and the fluid in the femoral lymph sacs and in the body cavity and also the urine in the bladder were collected and weighed. The weight of lymph is expressed as a percentage of the animal’s original weight, while the quantity of urine resorbed from the bladder is expressed as a percentage of the bladder content at the time of injection, and also as weight resorbed per hour per 100 g. body weight.

With Bufo carens experiments have been carried out at two dosages, 1 and 0·1 i.u./100 g. body weight. Only a small number of specimens could be obtained, and therefore only three sets of experiments at each dosage were carried out. The results are given in Tables 1 and 2. Since there are only three readings the standard errors of the means cannot be used in assessing the significance of the differences found between the effects of pitressin and pitocin. The values for the two doses may, however, be considered together, and the variance analysed. When this is done pitressin is found to have a significantly greater effect than pitocin upon relative water uptake (F 1/8 = 5·99), relative urine production (F 1/8 = 6·43) and maximum weight increase (E 1/8= 15·72). These results closely resemble those previously obtained with B. regularis (Ewer, 1950).

In the case of Xenopus laevis experiments were carried out at dosages of 2·5 and 0·25 i.u./100 g. body weight. The results are given in Tables 3 and 4. The injections were not followed by any consistent anti-diuretic effect, and indeed the urine flow usually increased after injection, keeping pace with the increased rate of water uptake, so that there was no increase in weight. Pitressin and pitocin both cause an increase in rate of water uptake, although the responses are considerably smaller than those shown by the two species of Bufo. The difference found between the effects of pitressin and pitocin are not significant at either dosage. In responding approximately equally to the two extracts Xenopus differs from any of the species of either Bufo or Rana which have been investigated to date.

Some preliminary experiments suggested that the lymph accumulation response might be seasonably variable. Experiments were therefore carried out on three pairs of animals at the end of each month from April to October. The results are given in Table 5. The individual variation is very great, but pitressin gives consistently higher values, both for the weight of lymph accumulated and the resorb-tion of urine. There are indications that the responses are least marked in winter (June-August) and increase once spring has begun, and the animals emerge from their winter hiding and are active again. The maximum is reached at the end of May. There are, however, not sufficient data to lay any great stress on the seasonal changes. The values for all months may be summed, the means calculated and the significance of the differences between the effects of pitressin and pitocin assessed by the t test. When this is done the difference in total fluid accumulated is found to be just significant (P=0·2), while the difference in urine resorbtion is highly significant (P∠0·001). A better comparison may be made by making an analysis taking into account the variation from month to month. When this is done a value of F 1/2O = 8·16^* is found for total lymph accumulation. This is significant at the 1 % level. The significance of the variation between months may also be tested, and gives a value of F 4/20 = 7·50, which is also significant at the 1 % level. Thus in Bufo regularis pitressin is more effective than pitocin in causing lymph accumulation and urine resorbtion. This is in harmony with its greater water-balance effect in this species.

A few qualitative experiments were performed on B. carens. This species also shows the lymph accumulation response after pituitrin injection. Out of seven animals injected with pituitrin at a dosage of 3 i.u./100 g. body weight four showed large distension of the lymph sacs, two gave a definite but smaller response, and in one the response was doubtful.

The rate of water uptake, expressed as percentage body weight per hour, varies with the size of the animal. Small specimens, with a relatively greater surface area, have naturally the highest rates of water uptake. The rates of uptake per unit surface area would be expected to be more constant, but since conversion factors have not been worked out for the species under consideration the values are given as per 100 g. body weight per hour, and are grouped into various weight classes. The results are given in Table 6 and Fig. 1. The rate of water uptake of B. regularis is significantly greater than that of Xenopus laevis, while the single value for Bufo carens does not differ significantly from the value for B. regularis of the same weight.

It is difficult to compare these values with those of other workers, since the experimental temperatures have not been the same. This difference between more terrestrial and more aquatic species has, however, been noted previously. Rey (1937) gives values of 22, 9 and 5 mg./cm.²/hr. for B. vulgaris Laur. = B. bufo (L.), Rana temporaria L. and R. esculenta L. respectively, while Jorgensen (1950) finds the rate of Bufo bufo to be about twice that of Rana temporaria.

In § (2) it was noted that the response of Xenopus to posterior pituitary extracts was slight compared with those of the two species of Bufo. It was thought that the two genera might also show a difference in their ability to take up water after they had been subjected to desiccation. Experiments to test this point were therefore carried out on B. regularis and Xenopus. To ensure full hydration the Bufo were kept in water for an hour before an experiment was started; Xenopus is, of course, normally kept in water. The animal was weighed, its bladder having first been emptied by means of a cannula. It was then left in a wire-covered dish exposed to the draught of an electric fan until it had lost between 10 and 20 % of its original weight. It was then replaced in water and weighed again after half an hour, after i hr. and after 2 hr. or until it had regained its original weight. The rate of water uptake is expressed as the weight increase per 100 g. body weight per hour and can be compared with the normal rate of uptake of an animal of the same weight found from Fig. 1. The results are given in Table 7. The two genera behave very differently. Bufo regularis after desiccation is capable of taking up water at a remarkable rate, the highest value found being 36 % per hour, representing more than seven times the normal rate. Xenopus, on the other hand, shows very little ability to increase its rate of water uptake; in only three out of the seven experiments was there any increase, the maximum being 127 % of the normal rate. The decrease shown in the other experiments is probably due to the drying of the sticky mucous secretion on the surface of the animal, which may hinder water uptake. These data strongly suggest that in Bufo regularis the high rate of water uptake after desiccation is not a passive phenomenon, brought about solely by the increase in the osmotic pressure of the body fluids, but very probably involves the secretion of water-balance principle from the posterior lobe of the pituitary. It would be of considerable interest to repeat these experiments on hypophysectomized animals.

In most of the early studies of the effects of posterior pituitary extracts on anuran water balance no distinction was made between renal and dermal effects, and the net weight increase alone was measured. This weight increase after injection of posterior pituitary extracts is known as the Brunn reaction. The magnitude of the Brunn reaction is obviously a function of both the renal and dermal responses, since unless there is an antidiuretic response an increase in water uptake would not result in a weight increase, as the excess water would merely be excreted by the kidney. Steggerda (1937) has pointed out that there appears to be a correlation between the magnitude of the Brunn effect and the habits of the animal, the more terrestrial species showing the larger responses. With this view the present findings are in full accord; the totally aquatic Xenopus shows no weight increase, while the two species of Bufo have large Brunn reactions. Table 8 summarizes the available data on the Brunn reaction, and these in general support Steggerda’s conclusion. Jorgensen (1950), in discussing this question, is doubtful whether the correlation between Brunn reaction and degree of terrestrial adaptation is valid, since the findings of certain workers seem to contradict it. These cases, however, require further consideration. Novelli (1933) finds a weight increase of 18 % in B. arenarum Hensel, and 24-28 % in Lepto-dactylus occelatus (L.). These values cannot be accepted, since Novelli made his first weighings 10 hr. after injection, and there is no indication in his work of his having previously determined that the weight is at a maximum at this period. Other workers have generally found that species of Bufo show their maximal weight gain 4 or 5 hr. after injection, and Novelli’s value of 18 % is therefore probably not the maximum weight attained. Bělehrádek & Huxley’s (1927) experiments with Amblystoma tigrinum are too scanty to be of any value. Jorgensen further quotes Howes’s (1940) value of 14 % weight increase in newly metamorphosed Bufo bufo, but does not mention that in fully adult specimens Howes found a value of 40 %.

The values of 22 % for the two species of Bufo found in the present work are low for this genus. This is probably due to the fact that the experimental temperature was high, namely 26° C. Boyd & Brown (1938) have shown that the magnitude of the Brunn reaction varies with temperature, being less at higher temperatures. Unfortunately, few workers have stated the temperatures at which they worked; and the very different values found for Rana temporaria by Brunn (1921) and Rey (1935) may possibly be due to their having worked at different temperatures.

The effects of posterior pituitary extracts on anuran water balance may be considered from two aspects : their bearing on the identity of the active principles of the anuran posterior pituitary, and the biological significance of the responses.

The oxytocic and pressor fractions of mammalian posterior pituitary extract are chemically similar; Waring & Landgrebe (1950) state that ‘it seems likely that the specific activities of the posterior lobe are due to closely related polypeptides of molecular weight about 2000’. Since most previous work has been performed on species of Rana there has developed a tendency to assume that the amphibian water-balance principle is a single entity, and that it is most closely related to the oxytocic fraction of mammalian posterior pituitary extract. Heller (1950) defines amphibian water-balance activity in terms of the effects on Rana, but points out the desirability of investigations on more species of Anura. The results of the present investigation show that the Anura vary in their sensitivities to the oxytocic and pressor fractions of mammalian extracts; while Rana is most sensitive to the oxytocic fraction, it has now been shown that the genus Bufo is most sensitive to the pressor fraction, while Xenopus responds equally to both. In addition Jorgensen (1950) has shown that extracts of Rana temporaria and Bufo bufo pituitaries differ in their anti-diuretic potencies whichever species is used as the test animal. These data suggest that within the Anura the water-balance principle is not identical in different genera but that in all cases the chemical resemblance to pitocin and pitressin is close. In some genera the animal’s own water-balance principle may be assumed most closely to resemble the pressor fraction, while in others the resemblance is closer to the oxytocic fraction of mammahan extract. The greater response will be expected to be shown to the mammalian extract which most closely resembles the animal’s own water-balance principle. Which fraction evokes the greater response in any particular genus cannot therefore be regarded as having any significance other than indicating its relationship to the secretions of the posterior pituitary in the genus concerned. Heller & Smith (1948) obtained an extract from crabs’ eye stalks which caused an increase in weight in Rana temporaria, but had neither oxytocic nor antidiuretic effects in mammals. In this case we must assume that the eye-stalk extract sufficiently resembled the natural water-balance principle of Rana to have an effect, but was not sufficiently similar to the mammalian hormones to be effective in this class.

The two species of Bufo which have been investigated are highly adapted to terrestrial fife, and can exist for long periods without access to ponds or streams. A comparison of the water economy of these species with the less terrestrial Rana and the aquatic Xenopus may therefore be expected to throw some light on the physiological adaptations of Anura to life on land. The normal rate of water uptake in Bufo is extremely high, and becomes even higher after desiccation, whereas that of Xenopus is low and shows little or no increase after desiccation. The terrestrial toad can therefore readily absorb free water when this becomes available and can take advantage of temporary supplies provided by rain and dew. Such a high normal rate of uptake would, however, be unsuited to an aquatic form; it would merely throw an increased amount of work on the kidneys, since the water would have to be pumped out as fast as it entered.

In its responses to pituitary extracts Bufo shows a number of characteristics which appear to be correlated with its terrestrial habit. The increase in water uptake following injection is very considerable, and there is a well-marked antidiuretic effect. As a result there is a large weight increase, the animal being able not only to take up water rapidly, but to hold an increased amount of fluid for some time. This extra fluid, as has previously been shown, is held mainly in the lymph sacs (Ewer, 1950). This accumulation of fluid in the lymph sacs will take place after injection of posterior pituitary extracts even if the animal has no access to external supplies of water. In these circumstances if the bladder be full, the fluid in it can be resorbed, thus providing a further method of conserving water.

Heller (1950) points out that the water-conserving role of the secretions of the posterior pituitary constitutes an example of the way in which, within the different classes of the vertebrates, a hormone may perform a similar function by acting on morphologically non-homologous effectors. In mammals water conservation is achieved by increased resorbtion in the kidney tubule. In the Amphibia the antidiuretic effect appears to be brought about by constriction of the afferent glomerular arterioles (Heller, 1950), while increase in water absorbtion occurs not in the kidney, but in the skin. In addition, it has now been shown that in Bufo the water-conserving mechanisms include the resorbtion of fluid from the bladder and the regulation of the volume of the lymph. Whether this latter is achieved by effects on the smooth muscle of the lymph hearts, or by changes in capillary permeability or blood pressure, is not yet clear. It may, however, be pointed out that the converse of Heller’s statement also holds: to wit, that the same secretions in different classes of vertebrates may, by acting on morphologically similar structures, fulfil diverse functional roles. In the Anura the effects of posterior pituitary secretions on smooth muscle (of renal blood vessels and possibly lymph hearts) assist in water conservation, while in mammals the smooth muscle responses are concerned with the regulation of blood pressure and the physiology of the pregnant uterus.

This work was carried out during the tenure of a Research Grant from the South African Council for Scientific and Industrial Research.