The Water-Balance Principle of Crustacean Eye-Stalk Extracts

The pars nervosa of the pituitary gland is present in all classes of craniates and its active principles, viz. the antidiuretic, oxytocic and water-balance hormones, have been identified and estimated in mammals, birds, reptiles, amphibians, elasmobranchs and teleosts (Heller, 1941a, 1942, 1945). In Amphioxus the adenohypophysis is represented but apparently not the neurohypophysis (de Beer, 1926). A structure similar to the latter seems, however, to occur in ascidians (de Beer, 1926; Butcher, 1930). Homology of this structure, the subneural gland, with the vertebrate neurohypophysis has frequently been postulated on developmental grounds, first apparently by Julin (1881), and this conclusion is supported by the work of Butcher (1930) and of Bacq & Florkin (1935) who found that extracts of the subneural gland raised the blood pressure of the cat and stimulated the uterus of the guinea-pig. More recently Gray & Ford (1940) reported that extracts of crustacean eye-stalks produce an increase of water uptake in frogs similar to that observed after the injection of neurohypophysial extracts. There appears thus the possibility that posterior pituitary-like hormones occur in a class of invertebrates lacking in any structure homologous to the neurohypophysis. However, the similarity between the ‘water-balance effect’ of extracts of crustacean eye-stalks and that of extracts of vertebrate pituitaries is scarcely sufficient to characterize the eye-stalk factor as a posterior pituitary-like principle. It was therefore decided to investigate whether crustacean eye-stalk extracts showed any of the other actions of vertebrate neurohypophysial preparations.

Eye-stalks of the brackish water-crab, Carcinus moenas, and of the fresh-water crayfish, Potamobius fluviatilis, were used.

The method of extraction resembled closely that for mammalian posterior pituitary glands described in the British Pharmacopoeia (1932). Immediately after removal, the eye-stalks were dropped into acetone where they remained until the next day. The acetone was changed twice during the subsequent 24 hr. The material was then removed and dried in an evacuated desiccator over phosphorus pentoxide. The dry eye-stalks were stored at 5 ° C. either in sealed ampules or in a desiccator over phosphorus pentoxide. For the preparation of extracts a number of dry eye-stalks were powdered in an agate mortar, the powder weighed and washed into a hard glass boiling tube with 0 ·5 c.c. per eye-stalk of a solution containing 0 ·6% NaCl and 0 ·25% acetic acid. The extracts were boiled for 3 min., were then filtered and the residue washed twice with 0 ·5 c.c. of 0 ·6% NaCl solution. All extracts were assayed on the day of preparation. Control extracts from claw or leg tissue were prepared in an identical manner.

English frogs (Rana temporaria) of an average weight of 20 g. were used. Several hours before the experiment started each frog was placed in a covered glass jar containing about 1 in. of tap water. Changes of weight of frogs kept under these conditions are small and inconsistent (Heller, 1941 b). As a rule cross-tests were used to compare the effect of any preparations on the weight increase of a series of frogs. Usually five of a series of ten frogs were injected with one preparation, the other five with the other extract. After an interval of 48 hr. the grouping was reversed. Injections were made into a ventral lymph sac. The volume of each injection was kept uniform in any one experiment. Weighings were made 1 hr. before the injection, immediately before the injection, immediately after the injection and subsequently at hourly or half-hourly intervals. Before each weighing the frogs were carefully dried and the bladder thoroughly emptied.

Virgin guinea-pig uteri were used. The solution in which they were suspended was that recommended in the British Pharmacopoeia (1932), with the exception that magnesium chloride was omitted.

Adult male rats were used. The animals were starved for 18 hr. before the experiment started but water was allowed ad lib. They received 4% of their body weight of water by stomach tube, immediately followed by subcutaneous injection of the extracts which had been neutralized with NaOH and diluted to 2 c.c. The rats were then placed singly in glass metabolism cages fitted with urine/faeces separators. The urine was collected under paraffin. Chloride in the urine was determined by the method of Volhard (1878).

Intravenous injections into unanaesthetized rabbits were employed (Heller, 1942).

Spinal cats were used.

Dudley’s (1920) test was applied. It consists in mixing a volume of extract with an equal volume of 2 N-sodium hydroxide. The mixture is allowed to stand for 2 hr. at room temperature, and is then carefully neutralized with 2 N-hydrochloric acid and diluted for use.

Pituitrin and Pitocin (Parke, Davis and Co.) were used as the standard (mammahan) neurohypophysial preparations.

Fisher’s ‘t’ test was applied to estimations of the significance of differences of means. ‘Small sample’ methods were used for the calculation of ‘t’ (Mainland, 1938). Allowance for the number of samples in any one series of experiments was made by determining the probability (P) from the tables of Fisher & Yates (1943). A probability of 5% was taken as the division between significance and non-significance, so that when P < 0 ·05, the difference between the values compared was regarded as significant.

Fig. 1 shows that extracts of eye-stalks of Carcinus moenas had a small but highly significant effect on the water uptake of normal frogs (t for 60 min. after injection =5 ·26, P < 0 ·001 ; t for 90 min. after injection = 3 ·89, P < 0 ·001). Injections of the extract of ten eye-stalks of Potamobius fluviatilis per frog in a series of ten animals had a similar effect (results given as means with their standard errors, control experiments in brackets); percentage change in body weight 60 min. after injection = +3 ·9±1 ·50 ( + 0 ·4±0 ·21); 90 min. after injection =+3 ·1 ±0 ·50 ( – 0 ·1 ±0 ·39); 120 min. after injection =+2 ·0± 1 ·06 ( – 0 ·6 ±0 ·35) and 180 min. after injection = +1 ·2 ± 1 ·06 ( – 0 ·9 ±0 ·32); t for 60 min. after injection = 3 ·40, P < 0 ·02, t for 90 min. after injection=5 ·08, P < 0 ·001. It is thus evident that a ‘water-balance’ principle occurs in the eye-stalks of both the brackish water Carcinus and the fresh-water Potamobius.

Fig. 2 shows that the intravenous injection of an extract of ten eye-stalks of Carcinus had hardly any inhibitory effect on the water diuresis of an unanaesthetized rabbit, a result which was also obtained in five other animals. The small inhibitory effect noted in some of these experiments (see Fig. 2) was of the same magnitude as that produced by control extracts. It was, in any case, considerably smaller than the inhibition produced by 1 milli-unit of pituitrin. Two experiments with Potamobius eye-stalk extracts gave equally negative results. Experiments aimed at demonstrating a possible blood-pressure raising action of eye-stalk extracts in spinal cats were inconclusive since the eye-stalk extracts were found to contain traces of histamine-like impurities. However, it was sufficiently clear from the diuresis experiments that the crustacean eye-stalk extracts contained no factor comparable with the vertebrate’s vasopressor-antidiuretic principle.

The amphibian water-balance effect of mammalian posterior pituitary extracts has been shown to be exerted by the oxytocic and not, as one would expect, by the vasoprenor-antidiuretic fraction (Heller, 1945). It was therefore of particular interest to test the eye-stalk extracts of oxytocic activity. Fig. 3 shows that the effect of the extract of one eye-stalk of Carcinus on the weight increase of frogs was approximately equal to that of 20 milli-units of pitocin. However, it will be clear from Fig. 4 that little or none of the oxytocic action of Carcinus eye-stalk extracts can be ascribed to a posterior pituitary-like principle. The extracts exerted an oxytocic activity equivalent to less than 2 milli-units pitocin per eye-stalk, but even this small effect was unspecific (control extracts of Carcinus leg tissue had much the same effect) and was not abolished by treatment with cold alkali (Dudley’s test).

Moreover, extracts of eye-stalks of Carcinus or Potamobius failed to show the chloruretic action of the oxytocic fraction of mammalian posterior pituitary glands on rats (Kuschinsky & Bundschuh, 1939; Dicker & Heller, 1946). Six rats, for example, which had each been injected subcutaneously with an extract of ten Carcinus eye-stalks excreted a mean of 0 ·43 ±0 ·130 mg. CI/100 g. in 2 hr., six animals injected with control extracts excreted 0 ·75 ± 0 ·201 mg. Cl/100 g, in the same time.

The injection of extracts of eye-stalks of the brackish water-crab, Carcinus moenas, and of the fresh-water crayfish, Potamobius fluviatilis, was found to increase the body weight of frogs immersed in water. This effect resembles that produced by neurohypophysial extracts of vertebrates, which increase the uptake of water in amphibians (Heller, 1945). It seems likely that the increase in body weight of frogs injected with crustacean eye-stalk extracts was produced in the same manner : urine flow in such animals—though not accurately measured—was not noticeably inhibited and no specific vaso-active substances were found in the eye-stalk extracts.

The eye-stalk extracts had neither an antidiuretic action in rabbits nor an oxytocic action on the isolated guinea-pig uterus; they failed to exert a chloruretic effect in rats. There is thus no evidence that the crustacean and the vertebrate water-balance factors are homologous principles. However, the possibility that the water-balance factor of crustacean eye-stalk extracts is related to that in vertebrate pituitary extracts cannot be dismissed so easily. While the water-balance activity of mammalian neurohypophysial extracts follows closely their oxytocic potency (Heller, 1930; Oldham, 1936; Boyd & Brown, 1938) it has been shown (Heller, 1941) that this parallelism does not necessarily hold for non-mammalian pituitaries. For example, frog pituitary extracts which per gland exerted a water-balance effect equivalent to that of 800 milli-units of the standard (mammalian) posterior pituitary extract, contained less than 40 milli-units of oxytocic activity. These results suggest that the vertebrate water-balance factor may have to be differentiated not only from the antidiuretic but also from the oxytocic pituitary hormone. It is therefore conceivable that a substance occurs in crustacean eye-stalk extracts which is related to the water-balance principle of vertebrate pituitaries but unaccompanied by any of the other neurohypophysial activities.

Recent investigations (Heller, 1945; Jorgensen, Levi & Ussing, 1946) suggest an extrarenal regulatory action of the neurohypophysis on the water and mineral metabolism of lower vertebrates. The results of the present investigation would seem to raise the possibility that an eye-stalk hormone plays a similar role in crustaceans.

The authors are indebted to the Colston Research Committee which defrayed the costs of this investigation. They are much obliged to Dr J. A. Kitching of the Department of Zoology for reading the manuscript.