THERE are two main views with regard to the place of formation of

Transcription

1 ON PITUITARY SECRETION. BY DOUGLAS COW, Beit Memorial Research Fellow. (From the Pharmacological Laboratory, Cambridge.) THERE are two main views with regard to the place of formation of the active principle (or principles) of the pituitary body. On the one hand it is supposed that it is formed in the posterior lobe of the gland: this view rests chiefly on the fact that extracts of the posterior lobe have far greater physiological activity than extracts of the anterior lobe (Herring(l), Howell(2)). On the other band it is supposed to be formed in the anterior lobe and to pass back into the posterior lobe and so to the cerebrospinal fluid: this view is based chiefly on histological grounds; the cells of the anterior lobe resemble secretory cells, and Guerrini(3) has described changes in the cells indicating secretory processes; again Herring(Z) (4) has traced colloid material from the pars intermedia into the posterior lobe. Experiments on another subject led me to think that extracts of duodenal mucous membrane might stimulate the pituitary body to activity. Consequently I have investigated the action of this extract with the view of determining whether it causes histological changes in the cells of the anterior lobe, and, if so, whether the secretion could be traced histologically on its way to the posterior lobe and then detected physiologically in the cerebrospinal fluid. The experiments were carried out on cats, rabbits and dogs. They consisted in: (a) Perfusing the animal with Ringer's solution containing extract of duodenal mucous membrane for periods varying between a few minutes and three hours (six experiments). (b) Intravenous injections of the extract in hypotonic, isotonic and hypertonic saline solutions at different times before killing the animals (seventeen experiments). 24-2

2 368 D. COW. (c) Intravenous or subcutaneous injections of the extract in unanaesthetized animals (five experiments). The injections in the case of cats were made by Dr W. E. Dixon, whom I have to thank for his kindness. (d) Injection of Indian ink or gelatine-carmine into the fourth ventricle in animals just before they were killed (six experiments). Some of these animals had received an injection of duodenal extract two hours previously. Control experiments were carried out in the case of every variety of experiment under (a), (b) and (c). All the animals were kept under the same conditions for some time before the experiments, and the subsequent treatment of the pituitaries was the same in all respects. The observations were partly histological and partly physiological. The former I may deal with later; but I shall only mention here certain tentative results, which however fall into line with those obtained by physiological experiment. The pituitary bodies were hardened in an aqueous solution of potassium bichromate (2.5 %/o) and mercuric chloride (50/o): and stained by the method of Galeotti(5) in concentrated solution of acid fuchsin in aniline water at 500 C. for five minutes, and then in 1*5 0/0 methyl green in 500/0 alcohol. The pituitaries taken from animals treated with extract of duodenal mucous membrane showed in the anterior lobe a much larger number of cells staining with acid fuchsin and a correspondingly smaller number staining with methyl green. A substance staining with methyl green occurred in clefts in the posterior lobe. It was found that the colour masses injected into the third ventricle found their wav into the cavity of the posterior lobe (in cats) and into clefts similar to those just mentioned. Further the colour mass could be traced into the body of the anterior lobe (Fig. 1). A direct connection between the cleft-like relic of Rathke's pouch and the ventricular cavity was described by Peremeschko(7). Herring (quoted by Cushing and Goetsch(6)) has observed the same connection, though Cushing and Goetsch failed to observe it. It should be noted that the latter observers worked on dogs. In the case of the dogs whose pituitaries I have examined the colour mass could not be traced into the anterior lobe so well as in the cats. It is possible that in the dog the same connecting path exists, but that the slender channels in the posterior lobe connecting with the infundibular cavity are so much longer in the dog than in the cat

3 PITUITARY SECRETION. 369 that the friction offered by their walls is too great to allow the passage of an injection mass. This explanation appears not improbable when one recalls the fact demonstrated by Herring(4) that, whereas in the cat the posterior lobe is hollow and in direct communication with the third ventricle, that of the dog is solid and the direct communication with the third ventricle is continued only into the infundibular stalk. There is then a channel by which the products of secretion of the anterior lobe can reach the ventricular cavity and the cerebrospinal fluid; and these results suggest that the methyl green staining substance is secreted by the cells of the anterior lobe, passes back through the B Fig. 1. Appearance piesented by a sagittal section through the pituitary body of a cat, which had received an injection of a suspension of Indian ink into the fourth ventricle (by suboccipital puncture) before being killed. Note the black injection-mass within the ventricular cavity of the posterior lobe (A), lying in the fine radially disposed channels within the pars nervosa (B), present also in fine intercolumnar channels in the anterior lobe (C) and also within a definite cleft (D) in the anterior lobe. The black injection is also seen in the pars intermedia envelopment of the infundibular stalk. posterior lobe into the third ventricle and so becomes mixed with the cerebrospinal fluid. Experimental methods. The experiments were carried out on dogs and cats. The animals were anaesthetized with urethane, preceded by A.C.E. mixture in the case of the cats and by ether and morphine in the case of the dogs. A cannula was placed in the fourth ventricle by suboccipital puncture, and samples of cerebrospinal fluid were withdrawn both before and at different times after intravenous injections of extracts of duodenal mucous membrane and of other injections used as controls. A sample of normal cerebrospinal fluid was taken

4 370 D. COW. from each animal and further samples, as a rule one every 30 minutes, up to three hours or more after the injections. The different samples of cerebrospinal fluid were tested as follows: (a) On the isolated uterus of the guinea-pig. (b) On the isolated intestine of the rabbit. (c) On the vascular system of the frog. (d) On the enucleated eyeball of the frog. (e) On the blood-pressure of the intact cat and rabbit. (f) On the urinary flow of the cat and rabbit. (g) On the mammary secretion of the lactating cat. (h) Half of each sample digested with trypsin and subsequently compared with the untreated samples as to their action on the isolated uterus. (i) Evaporated to dryness on a water-bath and the residues taken up in (a) absolute alcohol, (b) ether and (c) water; these again evaporated and their solid residues redissolved in water. Finally the water-soluble, ether-soluble and alcohol-soluble constituents tested on the isolated uterus. Fig. 2. Isolated uterus of guinea.pig suspended in 80 c.c. oxygenated Ringer's solution at 370 C. At the points indicated 10 drops of normal cerebrospinal fluid (L) of a dog, and 10 drops of cerebrospinal fluid collected from the same animal 60 minutes after an intravenous injection of extract of duodenal mucous membrane (0) were added to the bath of Ringer's solution. Upstroke = contraction. Succeeding doses of normal cerebrospinal fluid of 20 and 30 drops produced no contractions. The amount of cerebrospinal fluid obtained from any one animal is not large enough to allow all these tests to be applied to the same

5 PITUITARY SECRETION. 371 samples. The isolated uterus test was applied to all and at least one of the other tests in each case, so that in the aggregate each test was applied at least twice. The results showed that, whilst the normal cerebrospinal fluid as a rule contained no demonstrable active principle (unless large doses, e.g. 3 c.c. of the fluid were used at a time, in which case contractions of the isolated uterus might be evoked), those samples taken about an hour after the injection of extract of duodenal mucous membrane contained an active principle, or principles, which: (a) Produced contraction of the isolated uterus (Fig. 2). (b) Produced contraction of the isolated intestine (Fig. 3). Fig. 3. Isolated intestine of rabbit suspended in 80 c.c. oxygenated Ringer's solution at 370 C. At the points indicated D, A and P were added to the bath, each in 10 drop dose. Upstroke = contraction. D = cerebrospinal fluid of a dog collected minutes aft&r an intravenous injection of extract of duodenal mucous membrane. A = normal cerebrospinal fluid of the same dog. P = one drop of pituitrin (P.D.) in 10 drops Ringer's solution. (c) Constricted the blood-vessels of the frog (Fig. 4). Fig. 4. Drop-record of perfusion through the blood vessels of a frog (cannula inserted in aortic arch-outflow from vena cava). Time-marking = secs. At the points indicated by signal 10 drops of normal cerebrospinal fluid of a dog (lower tracing) and of cerebrospinal fluid of the same dog collected 60 minutes after an intravenous injection of extract of duodenal mucous membrane (upper tracing) were added through a side tube to the perfusion fluid. (d) Dilated the pupil of the enucleated eyeball of the frog. (e) Produced typical, though slight, increase in the blood-pressure of the intact cat (Fig. 5).

6 372 D. COW. (f) Produced a distinct increase in diuresis in the intact cat (Fig. 5). (g) Produced a marked increase in mammary secretion in the lactating cat. Fig. 5. Cat-urethane-carotid B-P (figs. in mm. Hg). Drop-record of urine-flow. At the point indicated 4 c.c. of cerebrospinal fluid (withdrawn from a dog minutes after an intravenous injection of boiled extract of duodenal mucous membrane) were injected intravenously. (h) Was inactivated by digestion with trypsin (Fig. 6). (i) Was soluble in water, and insoluble in absolute alcohol and in ether (Fig. 7). The following protocols give details of some of these experiments: Exp. 1. Samples of cerebrospinal fluid collected from a dog as follows: A-normal c.s. fluid. B-c.s fluid 5-15 minutes after extract of duod. mucous m. (intraven.). C,, 50-60,,.... D,, ,, A horn of the uterus of a guinea-pig was suspended in a bath of oxygenated Ringer's solution at 370 C. in the usual way 1. A, B, C and D each produced maximal contractions of the uterus in five drop doses. These samples were therefore diluted, one drop of c.s. fluid in 1 c.c. of Ringer's solution, when it was found that five drops of A produced no effect whilst two drops of D produced maximal contractions. A piece of isolated intestine of a rabbit was similarly suspended in oxygenated Ringer's solution. In this case it was found that whilst 10 drops of A produced no effect 10 drops of D produced typical contractions and increased movements. 5 c.c. of C and D mixed were injected intravenously into a rabbit, whose carotid blood-pressure was recorded: a slight but appreciable increate of blood-pressure was recorded, which was not produced by injecting an equal volume of Ringer's solution, or of Sample A. Exp. 2. Samples of c.s. fluid collected from a dog as follows: F-normal c.s. fluid. G-c.s. fluid minutes after intravenous injection of boiled extract of duod. mucous membrane. Ten drop doses of both F and G produced no effect on an isolated horn of uterus: 20 drops of G produced contractions of the uterus whilst repeated 20 drop doses of F had no effect. 1 The bath of Ringer's solution contained in all cases 80 c.c. of fluid.

7 PITUITARY SECRETION. 373 The blood vessels of a frog were perfused with normal saline solution under constant pressure, the fluid entering by the aorta and leaving by a wound in the vena cava: a drop record was taken of the rate of flow. It was found that the addition of 1 c.c. of G to the perfusion fluid produced a 20 0/0 diminution in the rate of flow, whereas a similar dose of F was without effect. Fig. 6. Fig. 7. Fig. 6. Isolated uterus of guinea-pig suspended in.80 c.c. oxygenated Ringer's solution at 3700C. At the point indicated J, K, L, M and M' were added to the bath of Ringer's solution in doses of 10 drops each. Upstroke = contraction. J -normal cerebrospinal fluid of a dog. K =cerebrospinal fluid of a dog 5-20 minutes after an intravenous injection of boiled extract of duodenal mucous membrane. L =cerebrospinal fluid of a dog minutes after an intravenous injection of boiled extract of duodenal mucous membrane. M = cerebrospinal fluid of a dog minutes after an intravenous injection of boiled extract of duodenal mucous membrane. M' = M previously digested with trypsin. The separate tracings are superimposed. Fig. 7. Isolated uterus of guinea-pig suspended in 80 c.c. oxygenated Ringer's solution at 370 C. At the points indicated the various samples were added to the bath of Ringer's solution, 3E and 3A in 2 c.c. doses, the remainder in 20 drop doses. Upstroke = contraction. 1 W, IA and IE = the water-, absolute alcohol- and ether-soluble constituents of normal cerebroispinal fluid of a dog. 3W, 3A, and 3E = the water-, absolute alcohol- and ether-soluble constituents of the cerebrospinal fluid collectedl from the same, dog minutes after an intravenous injection of boiled extract of duodenal mucous membrane.

8 374 D. COW. Exp. 3. Samples of c.s. fluid collected from a cat as follows: L-normal c.s. fluid. M-c.s. fluid 15 minutes after ext. of duod. mucous membrane (intraven.). N,, 30,, ,. P,, 90,. Q, 120,,,J.. In 20 drop dose L produced no effect on the isolated uterus of a guinea-pig, whilst a 10 drop dose of 0 produced maximal contractions. Five drops of 0 injected into saline solution perfusing through a frog's blood vessels produced a vely decided constriction, whilst 20 drops of L were without effect. Exp. 4. Samples of c.s. fluid collected from a dog as follows: Z-normal c.s. fluid. Y- c.s. fluid 30 mins. after ext. of duod. mucous membrane (intraven.). X,, 60,, W,, 120,..... Five drops of X produced contractions of the isolated uterus, whilst 10 drop doses of Z produced no effect. Six enucleated eyeballs of frogs were prepared and placed each in a separate capsule containing: 1. Frog's Ringer's solution. 2. Rabbit's Ringer's solution. 3. Five per cent. pituitrin (P.D.) in frog's Ringer's solution. 4. Five per cent. Z in frog's Ringer's solution. 5. Five per cent. Y,,.. 6. Five per cedt. X These were left in the dark for 60 minutes, after which time 1, 2 and 4 showed con. stricted pupils whilst the pupils of 3, 5 and 6 were widely dilated. Exp. 5. Samples of c.s. fluid collected from a dog as follows: J-normal c.s. fluid. K-c.s. fluid 0-20 minutes after boiled extract of duod. mucous m. (intraven.). L,, 2-60.,,,,, M,, ,... N,, ,... Each of these samples was divided into two equal portions to one of which trypsin uas added (1 drop trypsin to 1 c.c. of c.s. fluid) and all were incubated at 370 C. for 18 hours. Those samples to which trypsin was added were labelled J'. K', etc. Tested on the isolated uterus M produced maximal contractions in five drop dose whilst M' produced no effect in 10 drop dose. None of the other samples whether treated with trypsin or not produced any effect in 10 drop doses. Exp. 6. Samples of c.s. fluid collected from a dog as follows: 1-normal c.s. fluid. 2-c.s. fluid 0-30 mins. after boiled extract of duod. mucous m. (intraven.). 3,, ,,,,,, Each of these samples was divided into three equal parts which were evaporated to dryness on a water-bath: one part of each was treated with water, one with absolute alcohol and one with ether, and labelled 1W, IA and IE; 2W, 2A and 2E; 3W, 3A and 3E respectively. These were again evaporated to dryness and each redissolved in Ringer's solution to the amount of the original volume of c.s. fluid.

9 PITUITARY SECRETION. 375 Tested on the isolated uterus 3W produced maximal contractions in 10 drop dose, whilst none of the others produced any effect in 20 drop doses. In 2 c.c. doses both 3A and 3E failed to produce any effect. These samples were also injected intravenously into a lactating cat, whose bloodpressure, urine flow and mammary secretion were recorded. 4 c.c. of 3W produced a slight but absolutely distinct increase in blood-pressure, a marked increase in urine flow, and evoked a considerable flow of milk. None of the other samples produced any effect. Cushing and Goetsch(6) claimed to have demonstrated the presence of the active principles of the posterior lobe of the pituitary in the cerebrospinal fluid. Carlson and Martin(s) however raised several objectionis to the results of Cushing and Gooetsch, more particularly in that they (C. and G.) used pathological cerebrospinal fluid, in that they concentrated the fluid verv strongly (from 50 c.c. to 2X5 c.c.) and in that they injected the cerebrospinal fluid of one animal into an animal of different species. Whilst admitting that their failure to demonstrate the presence of pituitary active principles in the cerebrospinal fluid does not necessarily negative its presence therein, Carlson and Martin express their opinion that the concentration of pituitary active principles should be high in the blood and low in the cerebrospinal fluid. Whilst the normal cerebrospinal fluid of those animals which I have employed contains in minute quantities only any active principle similar to that evolved by the pituitary (2 c.c. to 3 c.c. of normal cerebrospinal fluid in 80 c.c. of Ringer's solution are as a rule necessary to produce any effect on the most sensitive test organ- -the uterus of the guinea-pig), given a suitable hormone which will provoke the pituitary body to activity, the cerebrospinal fluid will under certain conditions yield an active principle which fulfils the usual tests for pituitary active principle and does not fulfil the tests for other known substances with actions similar to that of the active principle of the pituitary body. In carrying out these tests I have throughout made use of the obvious controls, e.g. when injecting cerebrospinal fluid into an animal I have also injected similar amounts of the serum of the animal from which the cerebrospinal fluid was obtained. Similarly when testing my fluids on the isolated uterus and on other test organs I have used as controls normal cerebrospinal fluid and other substances. Again I have given injections of duodenal extract to animals in which the pituitary had previously been destroyed (by cautery). In these cases no change could be demonstrated in the pressor activity of the cerebrospinal fluid collected before and after the injection.

10 376 D. Cow. The fact that hyperactivity of the pituitary may be evoked equally by boiled or by unboiled extract of duodenal mucous membrane, both of course being filtered before injection, would suggest that the hormone is not nucleo-protein: and this is confirmed by the fact that various other animal extracts, more particularly orchitic extract, do not affect the pituitary in this way. The fact that the cerebrospinal fluid as withdrawn from the suboccipital cannula does not contain an increase in pressor substance until after the lapse of more than 30 minutes from the time of the injection of the hormone is, I think, explainable by the distance to be traversed by the cerebrospinal fluid on its journey from the lneighbourhood of the infundibulum to the suboccipital region, and to the fact that after the first gush, which takes place as soon as the puncture is made, the flow of cerebrospinal fluid is-slow (1 drop in about 120 seconds is the average rate). The somewhat considerable variation in strength of active principle in the different cerebrospinal fluids which I have examined is probably accounted for by a combination of various factors. Possibly some of the pituitaries were in a state of exhaustion or semi-exhaustion before the experiments were begun. The degree of activity of the hormones employed to stimulate the pituitaries probably varied in different instances. In some cases any particular sample of cerebrospinal fluid may have consisted of fluid collected at a time when the pituitary was in full activity: in other cases the most active sample may have been diluted, before it reached the cannula, with cerebrospinal fluid secreted either before or after the period of greatest pituitary activity. Again it is possible that in certain cases a degree of pituitary activity may be masked by a concurrent degree of activity of the secretion of the cerebrospinal fluid itself; in this way a considerable dilution might take place. There is no evidence that the hyperactivities of the two secretions go hand in hand. It does not follow of course, because the active principle of the pituitary is secreted into the cerebrospinal fluid, that it is not also secreted into the blood stream, though I believe that all attempts to demonstrate an excess of active principle in the internal jugular vein blood over carotid blood have failed. It would seem more likely that the active principle, possibly secreted rapidly by the gland, is passed into the cerebrospinal fluid, so that its gradual diffusion from this fluid into the blood stream mav allow of a gradual and prolonged rather than a sudden and short-lived action on the organs which it stimulates.

11 PITUITARY SECRETIO1V 377 Work on this subject is being continued in this laboratory in an investigation of the action of other pituitary hormones. CONCLUSIONS. 1. Certain definite channels of communication exist (in the cat at ainy rate) between the ventricular cavity in the infundibular stalk and spaces in the anterior lobe of the pituitary. 2. Under certain conditions an active principle (or principles) is demonstrable in the cerebrospinal fluid, which principle fulfils every test for pituitary active principle that has been applied. 3. The products of activity of the pituitary body are secreted into the cerebrospinal fluid. REFERENCES. (1) Herring. Quart. Journ. of Exper. Physiol. vii. p (2) Howell. Journ. of Exper. Med. m. p (3) Guerrini. Lo Sperimentale (4) Herring. Quart. Journ. of Exper. Physiol. I. p (5) Galeotti. Arch. f. mikr. Anat. XLVIII. p (6) Cushing and Goetsch. Amer. Journ. of Phvsiol. xxvii. p (7) Peremeschko. Arch. f. path. Anat. xxxvm. p (8) Carlson and Martin. Amer. Journ. of Physiol. xxix. p The references quoted do not pretend to embrace all previous work on the subject: this is very voluminous and such references are to be found in any up-to-date work on the subject, such as that of Swale-Vincent or Biedl.