Oxytocic activity. It is stated that 1 c.c. of oxytocin contains 12 units. single, multivalent, active principle, or whether a number of active

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1 SOME PROPERTIES OF THE SEPARATED ACTIVE PRINCIPLES OF THE PITUITARY (POSTERIOR LOBE). BY J. H. GADDUM (National Institute for Medical Research). EXTRACTS of the posterior lobe of the pituitary gland have been found to produce a great diversity of physiological effects and, in the course of time, a large number of experiments have been performed with the object of determining whether all these effects could be attributed to a single, multivalent, active principle, or whether a number of active substances were present. The former view was upheld by Abel(l) and his collaborators, while the latter has received much support from work done in this country. In a recent paper, Kamm, Aldrich, Grote, Rowe and Bugbee(2) have brought forward conclusive evidence of the separation of the substance which produces a rise in the blood-pressure of a dog from that which produces a contraction of the uterus of a guinea-pig. These workers give an account of previous work in this direction, together with the details of the method by which two separate purified preparations, "Vasopressin" and " Oxytocin," are being manufactured. I am indebted to Messrs Parke, Davis and Co. for the opportunity of investigating further the physiological properties of these substances. Pressor activity. It is stated that vasopressin contains 25 international standard units per c.c., and oxytocin less than 1 unit per c.c. These results, obtained on a dog under chloralose, have been confirmed by comparing the preparations with the British standard on the blood pressure of a spinal cat. The pressor effect of the solution prepared from 041 unit (i.e mg.) of the standard was indistinguishable from that of 1/250 c.c. of the vasopressin solution. The rise in blood-pressure produced by large doses of oxytocin was more prolonged than that due to small doses of vasopressin, and it seems possible that it is an effect of the oxytocic principle itself, and is not due to imperfect separation of two principles. Oxytocic activity. It is stated that 1 c.c. of oxytocin contains 12 units of oxytocic activity. My estimate was 13 units per c.c. These results do not differ by a significant amount. Vasopressin contains less than 1 unit per c.c.

2 .-~~~~~~~~~~~~ PITUITARY (POSTERIOR LOBE). 435 Diuretic action. The observation of Kamm and his co-workers that this effect is produced by much smaller doses of vasopressin than of oxytocin has been confirmed. The diuretic effect of large doses of oxytocin, on a cat under ether, was, like the pressor effect, more prolonged than that of small doses of vasopressin. There is as yet no evidence as to whether or not the substance which produces, under certain conditions, an antidiuretic effect can be separated from the other principles, and there is little justification for speaking, as certain authors do, of a "diuretic-antidiuretic effect." The two effects are quite distinct, and should be studied separately. Depressor activity, Vasopressin showed no depressor effect on a cat, even when given in repeated doses. 1 c.c. was mixed with an equal volume of twice normal NaOH. After some hours the solution was neutralised and injected intravenously into a cat. There was no sign of pressor or depressor effect, though mg. of histamine produced a definite depression. 1 c.c. oxytocin (containing 12 units) showed no depressor effect in the same experiment, when similarly treated with soda, whereas mg. of histamine still produced a definite fall of pressure. On the other hand, when 12 units of the untreated oxytocin solution were injected into a cat, in which the blood vessels were still in high tone as the result of several large doses of vasopressin, a definite fall of blood-pressure occurred (cf. Fig. 1). The effect was equal to that produced in this cat 12 uiiiits ins ing. OxVtoCifl histainine h istamnine Fig. 1. Blood-pressure of spinal cat which had previously received several injections of vasopressin. Depressor effect of the intravenous injection of a large dose of oxytocin compared with that of histamine. by mg. of histamine, but it was evidently not due to histamine itself, since, if there had been so much histamine present, a definite fall of blood-pressure would have been produced after treatment with alkali. Similar falls of blood-pressure have been found to occur after a second

3 436 J. H. GADDUM. injection of Abel's purified pituitary tartrate(l). For some unknown reason the reaction is not given by all cats. The effect is possibly analogous to the depressor action of pituitary preparations on the fowl. This was first described by Paton and Watson(3). It has been reinvestigated by Hogben(4), who has shown that the substance producing it is present in the pars neuralis and to a less extent in the pars intermedia, but not in other tissues. He also showed that it is destroyed by treatment with NaOH, and is accordingly not histamine. It was found that a fall of arterial blood-pressure was produced in a fowl under ether by large doses both of vasopressin and of oxytocin (cf. Fig. 2). After several injections the fowl became much less sensitive, Mig. 2. Oxytocin and vasopressin both produce a fall of blood-pressure when injected intravenously in the fowl, and the preparation soon becomes much less sensitive. Oxytocin then produces the effect in much smaller doses than vasopressin. and a direct comparison of the effects of the two preparations showed that oxytocin was active in much smaller doses than vasopressin. In

4 PITUIT?4RY (POST$RIOR LOBE). 437 confirmation of Hogben's results it was found that this effect of oxytocin was certainly not due to histamine, which must be given in relatively large doses to produce a depressor action in the fowl. The effect also disappeared after treatment with alkali. These two depressor actions, on the cat and on the fowl, are the only two actions, apart from that on the uterus, which have been found to be produced by smaller doses of oxytocin than of vasopressin, and it is possible that they are both due to the oxytocic principle itself. This would accord well with the fact that such effects are produced by Abel's purified preparations, in which the oxytocic titre appears to be extraordinarily high. The depressor action on the fowl of large doses of vasopressin may also be due to the oxytocic principle, which is known to be present as an impurity. Action on the bowel. Since pituitary preparations are used in the treatment of paralysis of the bowel, it was of importance to know which of these preparations was likely to be the more efficacious in this respect. It was found that, though both preparations produced shortening of an isolated loop of the rabbit's intestine, and an increase in the peristalsis recorded by a modification of Trendelenburg's method5), vasopressin produced these effects in smaller doses than oxytocin. The effect is rather irregular and is apt to disappear after large doses, and sometimes spontaneously. The colon appeared to be more sensitive than the ileum, and the ileum than the jejunum. In the colon there was usually Treated with alkali Uitreated Treiatel with alkali L'Untrate(id Each dose rep)reseiits oine uint of vasopressiin Fig. 3. Length of a strip of rabbit's ileum in 100 c.c. Tyrode's solution. Vasopressin produces an increase in the contractions and loses this property after treatment with cold NaOH. PH. LXV. 28

5 438 J. H. GADDUM. an inhibition before the stimulation. The effect on peristalsis was more regular than that on length of a segment, and was produced by smaller doses. The substance producing these effects was certainly not histamine, because quantities of histamine, hundreds of times larger than could possibly have been present, had no such effect: further, the active substance was destroyed by treatment with cold alkali. It was not the oxytocic principle, because, although the effects were produced by comparatively small doses of the international standard, a quantity of oxytocin containing twice as much of the oxytocic principle had no effect. On the other hand, this action may be due to the pressor principle. It was found that equipressor doses of the standard and of vasopressin produced similar effects on the bowel. It was not found possible to make a precise estimate of their relative potencies. I unit 2 units un1uit 3tandard oxytocin ndard 0.2 uni-to 0.25 ulit 0.2 unt Mtanisopresistand Fig. 4. Length of strips of rabbit's jejunum in 100 c.c. of Tyrode's solution. Stimulant action not due to oxytocic principle, but may be due to pressor principle. The intravenous injection of 2 units of oxytocin in a rabbit, having a celluloid window in the abdominal wall, previously inserted by Dr Dale by aseptic operation under aneasthesia, had no appreciable effect on the intestinal movements, but an injection of 2 units of vasopressin produced a marked increase of peristalsis. Previous work on this question is summarised in a paper by Mac - donald(6), who found that pituitary preparations had little or no specific effect on the isolated cat's intestine. This observation has been confirmed.

6 PITUITARY (POSTERIOR LOBE). 439 Dilator action on frog's melanophores. This effect was described by Hog ben and Winton (7). Vasopressin had a distinct action when 1 unit was injected subcutaneously into a frog (R. temporaria). One unit of the standard had a similar effect, but oxytocin had little or no action in this dose. Both substances were compared quantitatively with the Units per litre Units per litre 7-5 vasopressin 10 vasopressin 12*5 vasopressin 200- oxytocin saline 500 oxytocin.fig. 5. Portions of skin of Rana esculenta after 3 hours in dilutions of pituitary. Five units of the standard have more effect than 7-5 units of vasopressin, and slightly less than 10. Two hundred units of oxytocin have less effect than 5 units of standard, but oxytocin has some effect, compared with saline. 28-2

7 440 J. H. GADDUM. standard by immersing pieces of frog's skin for several hours in different dilutions of the pituitary preparations in saline. The comparison of the symmetrical pieces shown in Fig. 5 showed that, in concentrations measured by pressor activity, the standard had more than 1F5 times, and slightly less than twice as much action on the melanophores as vasopressin. It also showed that oxytocin had some effect on the melanophores, but that, in concentrations measured by oxytocic action, it had less than 1/40 of the action of the standard. Identical results have been obtained in other tests, and it is thus apparent that the activity of the different preparations on the melanophores does not bear a constant proportion either to their pressor or to their oxytocic activity. This confirms the results of other workers, which have shown that the action on the melanophores is due to a separate substance. A. J. Clark(8) found that this substance does not pass so readily as the others through collodion membranes. Several workers (7, 9), have found that it occurs in relatively higher concentration in the pars intermedia than in the pars neuralis. It appears to have been almost completely excluded from oxytocin, but in vasopressin it has been concentrated to about one-half the same extent as the pressor principle itself. SUMMARY. "Oxytocin" and "Vasopressin" have been subjected to a more detailed physiological study than that accorded them by their makers, and various conclusions of physiological importance have been drawn. "Oxytocin" has, in addition to its action on the uterus, a depressor action on the fowl and, in certain circumstances, on the cat. "Vasopressin" has, in addition to its effects on the blood-pressure and on the kidney, a specific stimulant action on the bowel of a rabbit and a dilator action on the melanophores of the frog. The latter effect is apparently due to a different principle, so that vasopressin is not yet a physiologically pure preparation. REFERENCES. 1. Abel. Bull. Johns Hopkins Hospital, 35. p Kamm, Aldrich, Grote, Rowe and Bugbee. Journ. Amer. Chem. Soc. 50. p Paton and Watson. This Journ. 44. p ; 4. Hogben. Quart. Journ. exp. Physiol. 15. p Garry. Arch. f. exp. Path. u. Pharmak p Macdonald. Quart. Jourm. exp. Physiol. 15. p Hogben and Winton. Biochem. J. 16. p Clark. This Journ. 58. p. xviii van Dyke. Arch. f. exp. Path. u. Pharmak p