by the action of insulin. Loss in adrenocortical lipids is considered an index of

Transcription

1 222 J. Physiol. (I95I) I14, THE ROLE OF HYPOGLYCAEMIA AND OF ADRENALINE IN THE RESPONSE OF THE ADRENAL CORTEX TO INSULIN BY MARTHE VOGT From the Departnent of Pharmacology, University of Edinburgh (Received 29 November 195) In a previous paper (Vogt, 1947), comparison was made of the response of the normal and the denervated adrenal gland of the rat to injections of insulin. By histological means it was possible to follow simultaneously the lipids in the cortex and the chromaffine material in the medulla, both of which are reduced by the action of insulin. Loss in adrenocortical lipids is considered an index of accelerated cortical secretion. This loss was studied in denervated adrenals in order to discover whether, in the normal gland, it was exclusively brought about by the simultaneous release of adrenaline. (The term adrenaline is used in this paper for the total secretion of sympathomimetic amines from the medulla, irrespective of what percentage of noradrenaline it may contain.) The experiments showed that insulin caused loss of cortical lipids in animals with bilaterally denervated adrenals, that is, under conditions where the secretion of adrenaline could not be responsible. Since, however, the histological method did not lend itself to quantitative study, the question remained open whether the share of the adrenaline in the cortical response of the normal rat was large or small. It was also left undecided whether the insulin itself or the hypoglycaemia was responsible for the cortical response of the denervated gland. The object of the present paper is to answer these points. Four types of experiments were performed for the purpose. (1) Comparison of adreno-cortical response to insulin of rats with normal and bilaterally denervated adrenals, using the depletion of ascorbic acid in the adrenal as an index of cortical activity (Sayers, Sayers, Liang & Long, 1945, 1946). (2) Similar comparison of the response of rats with normal and totally demedullated adrenals. (3) Experiments as under (1) and (2), the hypoglycaemic effect of insulin, however, being prevented.

2 ADRENAL CORTEX AND INSULIN 223 (4) Direct estimation of the rate of secretion of cortical hormones in anaesthetized, splanchnotomized dogs made hypo- or hyper-glycaemic. METHODS Rats The experiments were carried out on adult male Wistar rats, most of which weighed about 2 g. when the experiment started. 83 normal rats were used, 38 with denervated, and 68 with demedullated adrenals. The denervation of the adrenals was always carried out bilaterally under ether anaesthesia as described elsewhere (Vogt, 1945), and the animals were used between 22 and 3 days after denervation. For the demedullation, the rats were anaesthetized with ether and the skin of the back incised in the midline. The left adrenal was lifted gently through a small incision in the lumbar musculature by gripping the fat and connective tissue around it in a pair of small forceps. The adrenal was cut in two longitudinally with a pair of scissors, and each half of the gland enucleated, leaving behind only microscopic fragments ofthe cortex. The right adrenal was removed completely. During the first post-operative week, the rats were offered saline to drink and kept at 22 C. After that they were given water and exposed to the usual fluctuations of 'room temperature'. There was a mortality of 5% of animals which failed to regenerate enough adrenal tissue to survive. Between 34 and 6 days were allowed for adrenal regeneration. During the second month after the operation, regeneration still proceeded although slowly. It is, therefore, desirable to use test and control animals after approximately the same interval. Reports in the literature (Greep & Deane, 1949; Gordon, 195) dealing with bilaterauy demedullated rats state that the adrenal ascorbic acid level is normal from the 32nd postoperative day onwards. This does not appear to hold for animals treated as described above and in which regeneration is allowed to proceed in one gland only. The ascorbic acid estimations were made by the method of Roe & Kuether (1943) on trichloroacetic acid extracts of freshly excised adrenal glands. Insulin (Burroughs Wellcome) was injected subcutaneously after an overnight fast. After varying time intervals the rats were killed by a blow on the head, samples of heart blood taken immediately when blood sugar estimations were required, and the adrenal(s) removed. The rats in which hypoglyeaemia was to be avoided were in the early experiments given subcutaneous glucose injections shortly before the insulin. Later this procedure was abandoned and instead the rats, which had not been fasted, were supplied with concentrated glucose solutions from which they could drink ad lib. All doses referred to were per 1 g. body weight, whether this fact is explicitly stated or not. Dog8 The technique for collecting adrenal venous blood and assaying cortical hormones in the plasma has been described earlier (Vogt, 1943). All experiments were performed under chloralose or urethane-chloralose anaesthesia (per kg. body weight 15 ml. of a solution containing 33% urethane, 3-3% chloralose and 9 % NaCl) after induction with ether. The splanchnic nerves were cut on both sides immediately after the laparotomy. The evisceration was carried out in the usual manner, and was followed by the ligation of all acceasible hepatic veins; where the hepatic vein draining a liver lobe was embedded in liver tissue, the whole lobe was tied off. Glucose infusions were made into a femoral vein. When it was intended to restore hypoglyeaemic blood levels to normal, a rapid infusion of isotonic glucose was given which provided the animal with 75 mg. glucose/kg. body weight. This was immediately followed by a slow constant infusion of 5 mg./kg./min., a quantity which maintained approximately normal levels of blood sugar throughout the experiment. The blood sugar estimations were carried out according to the method of Hagedorn & Jensen (1923).

3 2_24 MIARTHE VOGT RE SULTS Effect of adren?al denervation The adrenal ascorbic acid was estimated in normal and in operated animals after a fast, after a dose of 4 i.u. insulin, and after the same amount of insulin preceded by an injection of glucose (15 g. as 1% solution). TABLE 1. Adrenal ascorbic acid (mg./1 g. fresh adrenal) in adult male rats with normal and denervated adrenals (mean and standard error). I Normal Adrenals No. of Ascorbic No. of Treatment rats acid rats Fed, not handled 6 59±13-6 Fasted (16 hr.) 9 428± Fed, saline injected ±22-2 Fasted, 4 i.u. insulin ± Fasted, 4 i.u. insulin and u3 g. glucose Doses per 1 g. body weight. All injected rats were killed 3 hr. after the injection. I )enervated Ascorbic acid 419± ± Normal 4 Adrenal denervated 3 2i Fig. 1. Adrenal ascorbic acid of adult male rats with normal and denervated adrenals. Ordinate: nmg. ascorbic acid/1 g. fresh adrenal. The number of rats used is marked at the foot of each column. Columns 1 and 2: ascorbic acid after a 16 hr. fast; 3 and 4: ascorbic acid 3 hr. after 4 i.u. insulin; and 6: ascorbic acid 3 hr. after 4 i.u. insulin and 5 g. glucose subcutaneously. All doses per 1 g. body weight. Comparison of the responses was made easy by the fact that the initial ascorbic acid content of the glands was not appreciably affected by the operation. Table 1 and Fig. 1, in which the columns represent the concentrations of

4 ADRENAL CORTEX AND INSULIN 225 ascorbic acid, illustrate the striking similarity between the responses of the two groups of animals. Both showed large depletion when insulin alone was given and a smaller fall when glucose, in a dose which brings the blood sugar of the normal rats back to 67 % of the normal, was injected as well. None the les, this does not mean that the responses were, in fact, comparable, because the conditions were not. The operated rats reacted to the insulin with severe hypoglyeaemic coma, whereas the controls manifested no more than the slightest loss of muscular tone. The adrenaline which the normal rats were able to release prevented a dangerous fall in blood sugar, and for them, therefore, the insulin was not as serious a stress as for the operated rats. Thus it was hardly warranted to conclude that the adrenaline normally plays no part in the response of the cortex, and a more detailed analysis, particularly regarding the speed of the reaction in animals with and without adrenal nerves, was desirable. This was carried out on demedullated rather than on denervated adrenals, in order to counter the possible objection that a few nerve fibres might have escaped interruption in the procedure of denervation. Effect of adrenal demedullation Regeneration of glandular tissue. During the first 5 weeks following removal of one adrenal and demedullation of the other, most animals regenerate adrenal tissue amounting to a little less than one normal gland, and thus have only about half the normal quantity of cortical tissue available. In the next few weeks the adrenal continues to grow slowly, but does not always reach the size of two normal glands, even if allowance is made for the absence of medullary tissue. The ascorbic acid content of glands which have been allowed 5 weeks for regeneration is about 16 %/ less than that of normal resting glands and tends to rise as the interval allowed increases. With such a shifting baseline ascorbic acid depletion after stress is best expressed as percentage of the figure for a group of 'resting' glands of precisely the same history. That calculation also avoids comparison of figures in which the ascorbic acid is expresed per gram of total adrenal. (normal rats) with figures in which it is expressed per gram of cortical tissue (demedullated rats). Response to insulin. When calculating the percentage depletion due to insulin, it is necessary to decide what constitutes a 'resting' gland. Ideally, the effect of insulin on normal or demedullated (fasting) rats should be compared with controls from both groups subjected to fasting and saline injection. Certain difficulties arise, however, because demedullated rats suffer appreciable hypoglyeaemia as a result of fasting. As will be shown later, this means that, at the time of injecting the insulin, the level of ascorbic acid is lower than in normal rats. Owing to the fact that, however serious a stress, the fall in ascorbic acid does not go below a certain concentration, unequally depleted glands do not give the same response to equal stress stimuli. Thus, in order to start from PH. cxiv. 15

5 226 MARTHE VOGT 226MAHEVG comparable states of the adrenals, a 'resting' gland was considered to be that of a saline-injected, fed rat. Hence, the depletion caused by various treatments was expressed as a percentage of the adrenal ascorbic acid concentration of groups of fed rats injected with saline. mg. ' NNormal 8 Demedullated Fig. 2. Adrenal ascorbic acid of adult male rats with normal and demedullated adrenals. Ordinate: adrenal ascorbic acid as percentage of the figure for controls which were fed and injected subcutaneously with saline. The number of rats used is entered at the foot of each column. The figures above the columns represent the mean blood sugar of each group in mg./1 ml. Columns 1 and 2: controls, fed, injected with NaCl; 3 and 4: rats after a 16 hr. fast; 5 and 6: rats fasted, 3 hr. after -2 i.u. insulin; 7 and 8: rats fasted, 1 hr. after -2 i.u. insulin; 9 and 1: rats fasted, 3 hr. after -1 i.u. insulin; 11 and 12: rats fed, 3 hr. after -1 i.u. insulin. The first observation, then (Fig. 2 and Table 2), is the high sensitivity of the adrenal ascorbic acid of demedullated rats to fasting. The blood sugar is only about 14 % lower than that of the normal fasting rat, yet this mild hypoglyeaemia is sufficient, without release of adrenaline, to cause a considerable cortical response. The same observation may be found in a recent paper by Gordon (195) who estimated adrenal ascorbic acid in demedullated rats. On this response to fasting the action of insulin is superimposed. Two doses of insulin were given (.2 and 41 i.u.) and some animals killed after a long (3 hr.) and some after a short (1 hr.) interval. In all insulin treated groups of demedullated rats, the ascorbic acid depletion was of the same order and not much greater than the depletion caused by fast alone. None the less, these depletions may represent maximal responses, as the concentrations recorded in Table 2 suggest. Since the medulla contains very little ascorbic acid, the figure of 235 mg. per 1 g. cortex, for instance (after X1 i.u. insulin),

6 * " " *; E Ca ce - " r. "C$ Ca-S VI I i C.5.;- 1 U+; Ca5 :!V t Cm - ONr C-o = = o to ~4 C- 5o Co I- 1 Oq oo Co oc1 N- m p.. O~C d > I 4 CD N t X 'N - oo ~ rc I.. - o -" C c4 b E o -Ho -H-H -H c O cs s ON c Co Ca Qm cq eq m x I' *~ ~ ~~~~~~*qn x t- N CS -4." 1._ c G N e NM N McN rc Ca " C 4-C o e "S._ " ;--4 z " C O o ~ ~. t N. d NN1 CO11 X Co o~ncob < C s oxwed n ~~~~~t mt, -C Ca Ca ) 5- -O >U 1 r) mea._- b " ~o" ~.. CO " 5-3 e E. * :.*;.5 = Os.._._._._ CQ -z, u 15-2

7 228 MARTHE VOGT would correspond to a figure of 235 mg. per 125 to 15 g. total gland. This is about as low as ascorbic acid ever falls in normal rats in this type of experiment. The lowest figure observed in the demedullated glands was a depletion to 61 % of the initial value. The greater percentage depletion found in normal glands ought not, however, to be interpreted as a greater response to stress of the normal gland but as a consequence ofthe lower initial value in the regenerated demedullated gland. In the normal controls, killed after 3 hr., the effect of different doses was reflected in the response, both ascorbic acid and blood sugar showing recovery after injection of the smaller dose (Fig. 2). No such recovery was seen in the demedullated rats; their clinical signs, too, were severe whether the dose was large or small. There was no indication that in demedullated rats the cortical response developed more slowly than in normals. This, however, may have been because the main fall in ascorbic acid took place overnight during the hours of fasting, and the insulin contributed but little to the total depletion. The experiments also give us some information on the role played by the cortex in counteracting hypoglycaemia under conditions where no adrenaline is available for this purpose. Since an overnight fast does not produce hypoglycaemia in rats after adrenal denervation, but does so after adrenal demedullation, the fall in blood sugar cannot merely be attributed to the deprivation of adrenaline, but to the combined effect of lack of adrenaline and reduction in size of the adrenal cortex which, in most of these animals, amounted to nearly one-half. The demedullated rats also tolerated insulin less well and developed coma with half the dose producing coma in the rats having undergone denervation only. In a few demedullated rats regeneration was poor and produced glands weighing only 2-3 mg. (about one-tenth of the normal size). In these rats, even the smallest dose of insulin (.1 i.u.) was not tolerated, and the animals died in hypoglycaemic coma. Combined administration qf insulin and glucose The experiments represented in the last column of Fig. 2 were carried out in order to see whether, in the demedullated rat, hypoglycaemia was the sole factor responsible for the effect of insulin on cortical secretion. In the normal animal, Gershberg & Long (1948), by giving glucose, succeeded in preventing ascorbic acid depletion 1 hr. after 1 i.u. insulin/1 g. In the experiments of the first section of this paper, partial inhibition of ascorbic acid depletion was obtained in normal and in denervated adrenals by injecting subcutaneously a quantity of glucose sufficient to diminish, but not to abolish, the hypoglycaemia resulting from 4 i.u. insulin. As the volume of glucose solution required to prevent any degree of hypoglycaemia would have been unmanageably large for injection, attempts were made to determine the dose of glucose necessary to prevent the hypoglycaemic effect of smaller doses of insulin. The results

8 ADRENAL CORTEX AND INSULIN 229 with normal rats are shown in Table 3. 5 g. glucose proved sometimes (Exp. 3), but not always (Exp. 4) sufficient to prevent a fall in blood sugar 3 hr. after the injection of -2 i.u. insulin. Adrenal ascorbic acid levels were higher than without glucose, particularly in Exp. 3, but far from normal. Since it seemed impracticable to inject larger volumes of fluid, the amount of glucose administered was raised by using 15% instead of 1% solutions. This effectively prevented any hypoglyeaemia (Exp. 5), but the ascorbic acid was lower than ever, and it was quite obvious that these procedures produced emotional and other disturbances in the rat which caused a greater degree of 'stress' than the hypoglycaemia. Not much was gained by reducing the amount of insulin and the concentration of glucose to one-half (see Exps. 6 and 7). Injections were therefore abandoned, and the procedure adopted of not fasting the rats and also supplying them, as soon as the insulin had been injected, with concentrated glucose solution to drink. In this way it was possible to obtain (Exp. 8), in spite of injecting insulin, ascorbic acid values similar to those of control rats given an injection of saline (Exp. 1). Thus, in the normal rat, after these precautions, the only remaining effect of the insulin injection was that due to the emotional stimulus of a subcutaneous injection. When precisely the same experiment was carried out on demedullated rats (last column of Fig. 2, see also Table 2), a small, but significant, additional ascorbic acid depletion was observed. The mean blood sugar of these rats was lower and more variable than that of normal rats subjected to the same treatment. It is th-us likely that the slight depression in the ascorbic acid level was a result of fluctuations in the blood sugar occurring, in spite of the intake of food and glucose, when insulin is given to rats with demedullated adrenals. Hypoglycaemia in the eviscerated dog Since the early work of Poll (1925), all the information available on the action of insulin and hypoglycaemia on the adrenal cortex has been obtained on rodents. In view of that fact, it appeared desirable to try and confirm in the dog the main result of the present work, namely that the effect of insulin on the adrenal cortex is produced by hypoglyeaemia, and that the hypoglyeaemia causes an accelerated production of cortical hormone whether or no adrenaline is released simultaneously. In the dog, direct estimation of the rate of cortical secretion can be carried out by hormone assay in adrenal venous blood; this distinct advantage over the indirect method of ascorbic acid determinations, however, is outweighed by the fact that anaesthesia and operative procedures produce an abnormal baseline for the effects to be investigated. It was shown elsewhere (Vogt, 1951), that the rate of cortical secretion of the isolated, perfused adrenal of the dog is not affected by either hypo- or hyper-

9 23 MARTHE VOGT glyeaemia. Thus any alteration in cortical activity by varying blood sugar levels is effected through stimulation or inhibition of the pituitary. The first problem was to find a way of producing hypoglycaemia in the dog in an acute experiment without the use of insulin. This was done by cutting the splanchnic nerves as soon as the animal was anaesthetized and carrying out an evisceration which included ligation of all hepatic veins. If the hepatic veins were left patent, no hypoglyeaemia ensued even if an interval of 3 hr. was allowed between evisceration and collection of the first blood sample. Obviously sugar was diffusing from the anoxic liver into the general circulation, a phenomenon observed in cats 25 years ago by Best, Dale, Hoet & Marks (1926). TABLE 4. Effect of glucose infusions on rate of cortical hormone secretion in the hypoglycaemic (eviscerated) dog. Blood glucose (mg./1 ml.) Before During Rate of cortical Exp. no. infusion infusion hormone secretion* Comments Increased Blood pressure low. No cortical hormone in adrenal plasma before infusion Unchanged Unchanged Unchanged Unchanged Unchanged t A first sample of adrenal plasma was tested 5-13 min., and a second sample min. after restoration of the blood glucose to normal, and these samples were compared with plasma taken during the hypoglycaemia. Table 4 records the results of these experiments. The restoration to normal, or slightly above normal, of low or very low blood sugars had no effect on the rate of cortical secretion, and certainly did not, as one might have expected, diminish it. In Exp. 1 of the table there was, in fact, a rise in hormone output when the blood sugar was raised. In this animal the blood pressure was very low throughout the experiment and the simplest explanation of this isolated result is to assume that the combined effects of low blood pressure and low blood sugar had so depressed the activity of either the pituitary or the adrenal that a supply of glucose restored the failing glandular activity. That explanation is supported by the observation that this was the only experiment in which the first sample of adrenal venous blood did not contain a detectable quantity of cortical hormone. In a few experiments, glucose infusions were made at a time when the blood sugar had not yet fallen to a hypoglycaemic level, and adrenal secretion tested at different intervals after the blood glucose had been raised to a concentration ranging between 18 and 262 mg./1 ml. Occasionally an acceleration of cortical secretion resulted from the hyperglycaemia but the changes were neither sustained nor consistent.

10 ADRENAL CORTEX AND INSULIN 231 DISCUSSION Adrenal denervation in rats abolishes neither the ascorbic acid depletion nor the loss of sudanophilic material caused by insulin in the adrenal cortex. Since both these responses of the adrenal are assumed to be due to a release of adrenocorticotrophic hormone (A.C.T.H.) from the anterior pituitary, it follows that insulin can release A.C.T.H. by means other than stimulating secretion of adrenaline. A number of experiments were designed in order to decide whether hypoglycaemia was the cause of this release of A.C.T.H. In the denervated adrenal, ascorbic acid depletion by insulin was less when the hypoglycaemia had been partly checked by injecting glucose. Attempts were then made completely to avoid insulin hypoglycaemia in rats with demedullated adrenals. Like rats with denervated adrenals, these animals react to insulin by exhibiting ascorbic acid depletion, as was recently also shown by Gordon (195). By providing them with food and glucose at the time of injecting the insulin, this depletion can be very much reduced, though not entirely abolished. It is probable that, in these animals, which have no adrenaline to accelerate glycogenolysis, a fleeting hypoglycaemia occurs, in spite of feeding, when insulin is injected. This hypoglycaemia may cause the small residual ascorbic acid depletion. The protection afforded by glucose fully confirms the observation by Gershberg & Long (1948) of the effect of glucose on the adrenal cortex of normal rats given insulin. The results on the denervated or demedullated adrenals are, however, not compatible with the conclusion reached by these two workers, namely that it is only the liberation of adrenaline and not the fall in blood glucose which determines the release of A.C.T.H. by insulin. This point has also been contested by Gordon (195) in his work on demedullated rats subjected to stress. The low insulin tolerance of rats in which regeneration of the demedullated adrenals was incomplete, and the comparison of the susceptibility to insulin after denervation and after demedullation show a direct dependence of the insulin tolerance on the amount of adrenal cortex present. In fact, blood sugar determinations made after a small dose of insulin might be used to measure the amount of regenerated tissue present at different intervals after demedullation. The ascorbic acid content of such poorly regenerated glands is always low, and much lower than any values encountered even in 'stressed' larger glands. These facts suggest great caution in the interpretation of results obtained on transplanted adrenals. Langley (195), for example, found that the transplantation of adrenals prevented the accumulation of liver glycogen which occurs in normal rats exposed to low 2 tension and which is produced by an increased secretion of cortical hormone. The conclusion drawn was that the lack of cortical response was the result of having, by the transplantation,

11 232 MARTHE VOGT denervated the adrenals. Their results may also be interpreted as a lack of response due to a reduced total quantity of cortical tissue, which would already be working to full capacity before the stress was applied. The experiments on the eviscerated dog did not show any dependence of the rate of cortical secretion on the blood sugar level. The restoration of a normal blood sugar after a prolonged period of hypoglycaemia did not slow down cortical activity, as might have been expected from the accelerating effect of hypoglycaemia in the conscious rat. The rate of release of A.C.T.H. and, therefore, of cortical secretion, under conditions of operative stress, is known to be far above normal, and it is quite possible that the further stress of hypoglycaemia does not alter that speed at all, or not sufficiently to cause changes which the crude method of assay at present available will detect. This explanation of the negative findings is as likely to be correct as one which attributes the lack of pituitary response in the dog either to peculiarities of the canine species or to the absence of the liver. The present state of our knowledge does not permit a correlation between the functions of the liver and the pituitary response to hypoglycaemia, since the way in which the activity of the pituitary is stimulated is not at all understood. If utilization of cortical hormones by tissues is involved (Sayers & Sayers, 1947), it is possible that the removal of a large metabolic organ has indeed a decisive influence. In normal animals, the following picture emerges of the role played by the adrenal gland in response to injected insulin. The contribution made by the cortex in counteracting insulin hypoglycaemia can be shown in the absence of the medulla. It is probably effected through conversion of protein into carbohydrate rather than mobilization of glycogen (Engel, 1949; Bondy, 195). The contribution of the medulla is complex. By its action on glycogen breakdown, the adrenaline is indispensable for the prevention of prolonged severe hypoglycaemia. It is a contributing but not an essential factor for the release of A.C.T.H. from the pituitary. SUMMARY 1. Rats in which both adrenals have been denervated show the same fall in adrenal ascorbic acid level after an injection of insulin as do normal rats, though in the former the hypoglycaemia is much more pronounced. 2. Unilaterally adrenalectomized rats in which the remaining adrenal has been demedullated, have, even 34-6 days after the operation, less cortical tissue than normal rats. These animals develop mild hypoglyeaemia during a 16 hr. fast and their adrenals show a greater percentage depletion of ascorbic acid, at that time, than is found in normal fasted rats. Further and probably maximal depletion ensues when these demedullated rats are given insulin. This shows that release of A.C.T.H. follows an injection of insulin in the complete absence of adrenal medullary tissue.

12 ADRENAL CORTEX AND INSULIN By injecting glucose immediately before giving insulin, it is possible, in rats with normal and denervated adrenals, to reduce the ascorbic acid depletion due to insulin alone. As however the concentration and volume of the glucose solution injected into normal rats are increased, the protection afforded is diminished as a result of the disturbance caused to the animals. The effect of an injection of insulin on adrenal ascorbic acid level can however be completely prevented by omitting the fast and offering the rats a glucose solution to drink. 4. The same procedure will greatly reduce, though not completely abolish, the ascorbic acid depletion in the demedullated rat. The difference in the response from that of the normal rat is best explained by assuming that, in the operated rat, feeding does not entirely prevent small fluctuations in the blood sugar when insulin has been injected. 5. The conclusion regarding the release of A.C.T.H. by insulin is that, in the rat without adrenal medulla, it is due to the hypoglyeaemia alone, and, in the normal rat, to the combined effect of adrenaline and (a much milder) hypoglyeaemia. 6. Under the conditions of operative stress and anaesthesia required for collecting adrenal venous blood in the eviscerated dog, a release of A.C.T.H. as a result of hypoglyeaemia cannot be demonstrated. Grateful acknowledgement is made of a grant from the Medical Research Council which defrayed part of the expenses of this work. REFERENCES Best, C. H., Dale, H. H., Hoet, J. P. & Marks, H. P. (1926). Proc. Roy. Soc. B, 1, 55. Bondy, P. K. (195). Endocrinology, 45, 65. Engel, F. L. (1949). Endocrinology, 45, 17. Gershberg, H. & Long, C. N. H. (1948). J. cin. Endocrinol. 8, 587. Gordon, M. L. (195). Endocrinology, 47, 13. Greep, R.. & Deane, H. W. (1949). Endocrinology, 45, 42. Hagedorn, H. C. & Jensen, B. N. (1923). Biochem. Z. 135, 46. Langley, L. L. (195). J. clin. Endrocrinol. 1, 82. Poll, H. (1925). Anat. Anz. 6, Erg. H., 229. Roe, J. H. & Kuether, C. A. (1943). J. biol. C(hem. 147, 399. Sayers, G. & Sayers, M. A. (1947). Endocrinology, 4, 265. Sayers G., Sayers, M. A., Liang, T.-Y. & Long, C. N. H. (1945). Endocrinology, 37, 96. Sayers, G., Sayers, M. A., Liang, T.-Y. & Long, C. N. H. (1946). Endocrinology, 38, 1. Vogt, M. (1943). J. Physiol. 12, 341. Vogt, M. (1945). J. Phy8iol. 14, 6. Vogt, M. (1947). J. Phy8iol. 16, 394. Vogt, M. (1951). J. Phy8iol. 113, 129.