ABSTRACT
In Bufo regularis the injection of either pitressin or pitocin is followed by an increase in the rate of water absorption through the skin, together with a marked decrease in urine flow. The response to pitressin is greater than that to pitocin.
The effect of pitocin in increasing water uptake can be attributed to the activity of the 5-10% of the pressor fraction which it contains.
A mixture of pitressin and pitocin has the same effect on water balance as a corresponding dose of pituitrin. Pitressin alone has a greater anti-diuretic effect than it has when pitocin is added.
Taking into account the antagonistic action of pitocin to pitressin it is possible to account for the anti-diuretic action of pitocin in terms of its pressor fraction content.
It is concluded that the pressor fraction is the main factor responsible for both the dermal and the renal components of the water-balance effect produced in B. regularis by injection of mammalian posterior pituitary extracts. This conclusion is discussed in relation to the findings of other workers.
I. INTRODUCTION
In Anura injection of pituitrin is followed by an increase in weight if the animals are kept in water. In a previous communication (Ewer, 1950), it was shown that in Bufo regularis this is the result both of an increased rate of water uptake through the skin and a decreased rate of urine elimination through the kidneys. The possibility existed that the renal and dermal components of this water-balance effect might be due to different fractions of the pituitary extract, and the desirability of investigating separately the effects of the pressor and the oxytocic fractions was pointed out. The experiments described below were carried out in order to investigate this point.
II. MATERIAL AND METHODS
The species used was B. regularis Reuss, and the pituitary preparations were Parke Davis pitressin and pitocin. All experiments were carried out at 26° C.
The method used was the same as that described previously (Ewer, 1950). Water uptake and urine production were measured at hourly intervals for 4 hr. before injection. The appropriate injection was then made into the crural lymph sac, and measurements were continued at hourly intervals until the animal’s weight had reached a maximum. As before, the values for the first hour, when recovery from the anaesthetic may not be complete, were discarded. The mean values for the 3 hr. preceding injection give the normal rate of water uptake and urine production for the animal. The values for the post-injection period are expressed as percentages of the normal values for the same animal, and are referred to as the relative water uptake and relative urine production. In the present experiments the value for the relative water uptake in the hour following injection has been calculated, as well as the mean value for the whole period from the time of injection until the animal’s weight reaches a maximum.
III. RESULTS
(1) General
Experiments were carried out at a series of dosages ranging from 0·1 to 25 i.u./100 g. body weight for pitressin and from 1 to 15 i.u./100 g. for pitocin. Fig. 1 shows the results of two typical experiments, one with pitressin, the other with Pitocin injection. The general picture is similar to that previously found with pituitrin injection. Injection is followed by a sharp rise in the rate of water uptake accompanied by a rapid fall in urine production; the two processes result in an increase in the weight of the animal. Water uptake is maximal in the hour following injection and thereafter returns gradually to normal in the course of a few hours. Urine production remains negligible, or may even cease completely, for some time, but its recovery to normal (or to a value greater than normal) is very sudden. The period indicated in the graphs as the ‘duration of anuresis’ can therefore easily be determined. The point at which the effect on water uptake ceases is less definite, and the duration of this effect cannot always be determined with certainty. In calculating the mean values given in the tables any experiments in which the duration of the increased uptake is doubtful have been neglected.
The summed results for all dosages studied are given in Tables 1 and 2. It can be seen from these tables that both water uptake and urine production are affected by both extracts. It can also be seen that at the lowest dosages used, where a definite effect on water uptake is just manifested, there is already a very strongly marked anti-diuretic effect. The threshold for the renal effect is therefore presumably lower than for the dermal effect.
(2) Effects on water uptake
Fig. 2 shows the relative water uptake for the first hour after injection, and for the total post-injection period after pitressin and after pitocin injection. Pitressin has the greater effect. The maximum rate of uptake after pitressin injection is 231·2 ±15·64 at a dosage of 5-10 i.u./100 g. body weight, as compared with 189·5 ±11·61 at a dosage of 1-5 i.u./100 g. of pitocin. This difference is of doubtful significance (P > 0·05), but since all the values for relative water uptake in the first hour after pitressin injection are greater than those following pitocin injection the greater effect of pitressin is clear. In addition, pitressin is effective at lower concentrations than is pitocin, giving a definite effect at 0·1 i.u./100 g., while approximately 0·5 i.u./100 g. of pitocin is required to produce a comparable effect. With both extracts the effect becomes less when very high dosages are used : this is more marked in the case of pitocin, so that pitressin is effective over a greater range of dosages than is pitocin.
Pitocin and pitressin are not complete separations of the oxytocic and pressor fractions of pituitrin: each contains some 5-10% of the other. It therefore becomes necessary to consider whether the effect of pitocin could in reality be due to its pressor fraction content. If this is so, and there are no other complicating factors, then the curves for the effects of pitressin and pitocin on water uptake should be superposable by alteration of the dosage scale. There is, however, the complication of diminishing effectiveness with increasing dosage. Since this is more marked with pitocin than with pitressin the former extract exerts a large effect over a very small range of dosages, while the latter is highly effective over a wide range, and the curves are therefore of quite different shapes. Whether the reduced response represents a true reversal of action at high dosages, or is the result of some damage to the animal, has not been investigated. Since its mechanism is not understood it is desirable in comparing the effects of pitressin and pitocin to consider only the lower dosages, where the complication of diminishing effectiveness with increasing concentration does not arise. If pitocin owes its activity to the presence of 5-10% of the pressor fraction then a dosage of 0·5-1 i.u./100 g. of pitocin should have approximately the same effect as pitressin at a dosage of 0·1 i.u./100 g. ; and at
5-10 i.u./100 g. its effect should be similar to that of pitressin at 0·5-1 i.u./100 g. The figures are as follows :
Pitocin: 0·5-1 i.u.; 1st hour 119·5 ± 8·97; total period 102·66+ 7.15
Pitressin: 0·1 i.u.; 1st hour 130·7 ± 12·40; total period 119·3 ± 14·90
Pitocin: 5-10 i.u.; 1st hour 173 0 ±15·96; total period 142.7 ± 16·46
Pitressin: 0·5-1 i.u.; 1st hour 188·5 ± 8·19; total period 152·5 ± 9·54
In no case is the difference between the effects of the two extracts significant. It therefore appears that as far as its effect on water uptake is concerned the activity of pitocin may be attributed to its content of the pressor fraction.
(3) Effects on urine production
Fig. 3 shows the relative urine production and the duration of anuresis after pitressin and after pitocin injection. The effect of pitressin is much greater than that of pitocin, both in intensity and in duration. These differences are significant. For both extracts for the range from 0·5 to 15 i.u./100 g. the responses seem to be almost independent of the dosage. Analysing variance for relative urine production over this range we find that, as expected, dosage has little effect (F3/3 = 4·3), whereas the difference between the effects of pitressin and pitocin is highly significant (F1/3 = 46·0). Similarly, in the case of duration of anuresis the effect is again almost constant over the range of dosages considered (F 3/3 =1·1), while the difference between the effects of pitressin and pitocin gives a value of (F 1/3 = 9·1), which is probably significant.
Fig. 4 shows the maximum weight increase of the animals after injection of the two extracts. Here again over the range studied the effect is almost uniform for all doses (F 3/3 = 3·8), while pitressin and pitocin are significantly different in their effects (FI/3 = 53·4).
At first sight it would appear impossible for the anti-diuretic effect of pitocin to be due to the pressor fraction which it contains. As before, the effects of Pitocin at 0·5-1 and at 5-10 i.u./100 g. must be compared with those of pitressin at 0·1 and 0·5-1 i.u./100 g. respectively. In addition, a comparison may be made of the effects of pitocin at 10-15 and pitressin at 1-5 i.u./100 g. The figures for relative urine production are:
Pitocin: 0·5-1 i.u.; 51·0± 10·30
Pitressin: 0·1 i.u.; 17·2± 3·80
Pitocin: 5-10 i.u.; 28.4± 6 07
Pitressin: 0·5-1 i.u.; 25·8± 5·92
Pitocin: 10-15 i.u.; 42·5 ± 8·42
Pitressin: 1-5 i.u.; 18·3 ± 2.51
The difference between pitocin and pitressin is highly significant in the first case (P< 0·003); in the second case it is not significant, and in the third case the difference is significant (P < 0·001). For the duration of anuresis the figures are :
Pitocin: 0·5-1 i.u.; 1.1 ±0·17
Pitressin: o.1 i.u.; 2.16±0·17
Pitocin: 5-10 i.u.; 1.9 ±0.26
Pitressin: 0·5-1 i.u.; 1-83 ±0·40
Pitocin: 10-15 i.u.; 1·3 ±0·40
Pitressin: 1-5 i.u.; 2·66±0·42
In the first case the difference is highly significant (P<0·001), in the second case it is not significant, while in the last case the difference is probably significant (P< 0·02). The second comparison is probably the least reliable, since here the pitocin experiments include one unusually low value, and the pitressin experiments one particularly high value. Since the difference between the effects of pitocin and pitressin, both on duration of anuresis and on relative urine production, are highly significant at the lowest and significant at the highest pairs of dosages compared, it would appear to be impossible to attribute the effects of pitocin solely to its pressor fraction content. Moreover, the effect of pitocin is less, not greater, than is to be expected on the basis of its pressor fraction content alone. This suggests the possibility that pitocin may antagonize the action of the pressor fraction. If this is so, then the anti-diuretic effect of pitocin will be less, and the urine flow therefore greater, than would be expected on the basis of its 5-10% pressor fraction content. Experiments to test this point were therefore carried out.
(4) Pitocin-pitressin antagonism
The first point investigated was whether a mixture of pitocin and pitressin gave the same effect as an equivalent dose of pituitrin. Experiments were done at a dosage of 2·5 i.u./100 g. The results obtained in a series of seven experiments using pituitrin and seven experiments using a mixture of pitocin and pitressin are given in Table 3. There is*no significant difference either in rate of water uptake for the first hour only and for the total period, or in relative urine production and duration of anuresis. A mixture of pitocin and pitressin therefore has the same effect as an equal dose of pituitrin.
The values found using the mixture of 2·5 i.u. pitocin+ 2·5 i.u. pitressin were then compared with those previously found using pitressin alone at a dosage of 1-5 i.u./100 g. The figures for pitressin alone are included in Table 3. The relative water uptake, both for the first hour and for the total period, is greater after the injection of pitocin + pitressin than it is after the injection of pitressin alone. The differences in response are significant, P being <0·01 for the first hour and < 0·001 for the total period. This is in agreement with the results discussed in § 2, in which the effect of pitocin on water uptake was found to correspond with what is to be expected on the basis of its pressor fraction content.
In the case of the anti-diuretic responses, on the other hand, it can be seen that pitressin alone has-a greater effect than it has when given together with pitocin. The differences are significant both for relative urine production and for duration of anuresis, the values of P being <0·01 and < 0·001 respectively. Pitocin therefore significantly antagonizes the anti-diuretic effect of pitressin. Since this is so pitocin will have less anti-diuretic activity than would be expected solely on the basis of its pitressin content. This has been found to be the case. In the absence of pure extracts of the pressor and oxytocic fractions complete certainty cannot be reached ; but it nevertheless seems probable that the effect of pitocin on urine formation, as well as its effect on water uptake, is due not to the activity of the oxytocic principle, but rather to the pressor fraction which it contains.
Another point of interest emerges if we compare the lengths of time for which the increased water uptake and the decreased rate of urine flow last after the injection of the two extracts. The values at the lowest dosages, which are near the threshold for an effect on water balance, are neglected in each case. The figures on which the following calculation is based are those for the individual experiments whose means are given in Tables 1 and 2. After pitressin injection anuresis lasts slightly longer than increased water uptake. The mean value for (duration of anuresis—duration of increased water uptake) is + 0.4 hr. The difference in duration of the two effects is, however, not significant (P>0·05). After pitocin injection, on the other hand, anuresis does not last as long as increased water uptake: the mean value for (duration of anuresis—duration of increased uptake) is—0·6 hr., and the difference is significant (P<0·02). These results are in accordance with expectation. As previously noted, the kidneys are more sensitive than the skin and react at lower concentrations. The renal effect might therefore be expected slightly to outlast the dermal effect, as may be the case after pitressin injection. With pitocin injection there is the added complication that the oxytocic fraction antagonizes the renal but not the dermal effects of the active pressor fraction. In these circumstances it is not surprising to find that the dermal effect persists longer than the renal.
IV. DISCUSSION
With the exception of Novelli (1933), previous workers have found that pitocin has a stronger water-balance effect in Anura than pitressin (Heller, 1930; Steggerda & Essex, 1934; Oldham, 1936; Boyd&Brown, 1938; Sawyer, Travis & Levinsky, 1950). The present results, and particularly the conclusion that the activity of pitocin can all be attributed to its pressor fraction content, are thus in direct conflict with the findings of almost all earlier workers. It is therefore of considerable interest to find that, while the present work was in progress, Jørgensen (1950) was independently investigating the same question. Jørgensen used a different method, different species of Anura (Bufo bufo and Rana temporaria), and his pituitary extracts were Pitupartin AB and Insipidin AB. Nevertheless, his results for Bufo bufo are extremely similar to those described above. In this species he finds insipidin (the pressor extract) much more active than pitupartin (the oxytocic extract) ; and concludes that the effect of pitupartin on water uptake can be explained as being due to its content of the pressor fraction. As regards the renal effect Jørgensen could not reach a definite conclusion, but he too finds evidence that pitupartin antagonizes the anti-diuretic action of insipidin. In Rana temporaria Jørgensen found the two extracts to be approximately equally active.
In view of this high measure of agreement it seems legitimate to conclude that, at least in the species Bufo bufo and B. regularis, one substance is predominantly responsible for both the dermal and renal components of the water-balance effect; and that in mammalian pituitary extracts this substance is the pressor fraction. In this connexion it is to be noted that Heller & Smith (1948) obtained from crabs’ eye-stalks an extract without anti-diuretic action in rabbits and devoid of oxytocic activity which caused an increase in body weight in Rana temporaria. Heller & Smith conclude that the increase in weight is due to an increase in rate of water uptake, since the extract caused no very marked effect on urine flow in the frog. It should be pointed out that the activity of the frog’s kidneys must have been affected, as otherwise an increased water uptake would have resulted in an increased urine output, and the animal would not have gained in weight.
If the results of those workers who have found the oxytocic more active than the pressor extract are examined it emerges that they have all used species of Rana. Heller (1930) used R. esculenta, Steggerda & Essex (1934), Oldham (1936), Boyd & Brown (1938) and Sawyer et al. (1950) all worked with R. pipiens. Novelli (1933), on the other hand, who found pitressin the more effective, was working with Bufo arenarum. The present work concerns B. regularis, and Jørgensen, who found insipidin more active than pitupartin in B. bufo, found Rana temporaria about equally sensitive to the two extracts. These facts strongly suggest that the genera Bufo and Rana differ in their responses to pituitary extracts. Bufo is highly sensitive to pitressin, whereas Rana is much less so. Whether Bufo regularis reacts at all to the oxytocic fraction is not clear. No such effect can be demonstrated using pitocin, for the sensitivity to pitressin is so high that the responses to the contaminating 5-10% of the pressor fraction completely mask any possible responses to the oxytocic fraction. From Jorgensen’s results the same would appear to hold for B. bufo.
In Rana the response to pressor extracts is slight, and is less than the response to oxytocic extracts. This genus might therefore be sensitive only to the oxytocic fraction, its small response to pitressin being due to the oxytocic fraction content of the extract. This possibility has been discussed and rejected by Heller (1945), and is moreover not compatible with Jorgensen’s finding that R. temporaria responds approximately equally to the two extracts. If it were true, pitupartin should have been five to ten times as active as insipidin. Further investigation of this question is desirable.
The biological significance of this physiological difference between the two genera is probably to be found in the greater degree of terrestrial adaptation shown by toads. Jorgensen has shown that Bufo bufo is more sensitive to anuran pituitary extracts than is Rana temporaria. He concludes that the secretion of ‘water-balance principle’ by the posterior pituitary is of biological significance in the Anura, and that there is a correlation between the importance of such pituitary control of water balance and the habitats of the various species, a high development of such control being characteristic of those species living in the driest habitats.
In this connexion the work of Howes (1940) is of interest. Howes studied the water-balance reaction in developmental stages of Bufo bufo bufo and found that the response to pituitary extracts was not shown by the larvae, but developed gradually as metamorphosis took place. His experiments were carried out on various stages up till 30 hr. after tail resorption had been completed. Although Howes worked mainly with unfractionated pituitary extract he also used pitressin and pitocin in ‘confirmatory experiments’. No details are given, but he remarks that ‘the pitocin was more active’ than the pitressin. Since Jørgensen (1950) has found that in fully grown specimens of this species the pressor extract is the more active it would appear that the high sensitivity to pitressin characteristic of the genus is developed only at a later stage than those dealt with in Howes’s experiments. It is tempting to see in this an indication that the high sensitivity of Bufo to the pressor fraction is the most recently acquired specialization of the hormonal mechanism of water-balance regulation.
The high sensitivity of the various species of Bufo to pitressin is therefore probably a reflexion of the importance of pituitary regulation of water balance in this genus. The results of the present investigation on B. regularis are in harmony with this view. B. regularis is a species which can exist for long periods without access to ponds or streams. In such circumstances the ability rapidly to increase its rate of water uptake is of advantage to the animal, since it will be able to take up water readily when rain or dew provide a temporary supply.
Since Bufo shows a higher sensitivity than Rana both to mammahan and to anuran pituitary extracts the former genus is preferable to the latter as a test animal in any experiments designed to find out whether any preparation does or does not possess the power of eliciting the water-balance effect in Anura.
V. SUMMARY
This work was carried out during the tenure of a Research Grant from the South African Council for Scientific and Industrial Research.