Babkin, Rubashkin & Savitsch [1909] showed from cytological SECRETION IN THE CAT

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1 80 J. Physiol. (I94I) I00, :6I2.343 THE INFLUENCE OF SECRETIN ON PANCREATIC SECRETION IN THE CAT By E. J. W. BARRINGTON (Rockefeller Foundation Fellow) From the Department of Physiology, McGill University, Montreal, and Department of Zoology, University College, Nottingham (Received 4 February 1941) DESPITE the extensive work which has been carried out on the properties of secretin, there still remains a fundamental uncertainty regarding its mode of action, arising from the existence in mammals of a dual mechanism controlling pancreatic secretion, comprising the parasympathetic secretory fibres running in the vagus, stimulation of which was shown by Pavlov [1897] to evoke a secretion of pancreatic juice, and the hormone secretin, discovered later by Bayliss & Starling [1902]. The latter concluded that the juice obtained by secretin injections was 'normal', with a full equipment of enzymes, and that the nervous mechanism was therefore superfluous, but this view became difficult to reconcile with certain later work which indicated that the output of enzymes was associated especially with vagal stimulation or with the parasympathomimetic action of pilocarpine. Bayliss & Starling [1904], together with de Zilwa [1904], themselves showed that the 'pilocarpine' juice contained a greater quantity of protein solids than did the 'secretin' juice. More recently Still & Barlow (1927) and Zucker, Newburger & Berg [1932] have confirmed this and have shown that the pilocarpine juice also has a higher enzyme content, while Crittenden & Ivy (1937) have shown that the dog's pancreas secretes continuously in the absence both of the secretin mechanism and of secretagogue absorption. Meanwhile Babkin, Rubashkin & Savitsch [1909] showed from cytological observations that an extensive extrusion of granules occurred during vagal stimulation, but that after copious secretion induced by hydrochloric acid acting on the duodenum the cells remained full of fine

2 ACTIOAT OF SECRETIN ON PANCREATIC SECRETION 81 secretory granules. A thorough study of these problems by Mellanby [1925] led him to differentiate sharply between the functions of vagal and secretin stimulation, and to suggest that the metabolism of the enzymes of the pancreas was controlled by the vagi, while the chief, if not the only, effect of secretin was to cause the secretion of the water and inorganic constituents of the juice. According to this view, the granules discharged during secretin secretion are merely washed out by a dilute solution of sodium bicarbonate, the secretin exerting no direct effect upon them. This interpretation, although providing an attractive explanation of the dual mechanism, is in its turn difficult to reconcile with recent work involving the use of secretin as a clinical test for pancreatic function. Agren & Lagerlof (1936) and Diamond, Siegel, Gall & Karlen [1939] found evidence in man for a specific action of secretin upon the enzymes, shown most clearly by a rise in enzyme concentration at a time when the rate of flow of the secretion is diminishing after the initial rise in rate which immediately follows the secretin injection. Voegtlin, Greengard & Ivy [1934] also found that the effect of secretin in the dog and man was not only an increase in the rate of flow and of the output of enzymes, but in some cases an increased concentration of the latter. Finally, Langstroth, McRae & Komarov [1939], in a mathematical approach, have concluded that the mechanisms responsible for the secretion of protein and water in the pancreatic juice of the dog under secretin stimulation are largely independent, except in so far as both depend on secretin for their initiation. They did not investigate the enzyme content of the juice, but if it be assumed that this would largely parallel the protein content, their results are apparently not reconcilable with a passive washing-out of the enzyme material by the fluid. In view of these contradictory interpretations, it has seemed desirable to secure a more precise definition of the mechanism of the action of secretin, and it has been the object of the present investigation to provide this. MATERIAL AND METHODS Acute experiments were carried out on cats, which were anaesthetized with an intraperitoneal injection of nembutal (approximately 05 grain/ kg.), prolonged anaesthesia being maintained, when necessary, by additional intravenous injections. Cannulae were inserted into the right femoral vein, the trachea and the pancreatic duct, while the pylorus and both carotid arteries were tied. In some experiments the bile duct was also cannulated, in order to remove any possible influence of the bile salts 1928], although upon the course of the pancreatic secretion [M4ellanby, PH. C. 6

3 82 B. J. W. BARRINGTON no significant differences were noted when this procedure was adopted. Unless otherwise stated, both vagi were cut. Secretin solutio4s, adjusted in concentration to the weight of the animal, were injected into the femoral vein in volumes of 1 or 2 c.c. at intervals of 5, 10 or 15 min. according to the nature of the experiment. It is well known that much of the earlier work on the action of secretin suffered from the use of relatively crude preparations containing histamine-like substances and other vaso-depressors which in themselves affect the pancreatic secretion, and it is always open to argument that the enzyme content of the juice in such experiments might result from the action of such substances rather than of secretin itself. Care was therefore taken to ensure, by blood-pressure tests, that the secretin used in the present work had no significant vaso-depressor action, while the following different preparations were used in order to give the results as wide a significance as possible: Secretin A' and A2. Two preparations of the secretin concentrate 'SI' of Ivy, Kloster, Drewyer & Lueth [1930]; A2 (in solutions of 1.0 mg./c.c./3 kg.) was free of vaso-depressors, while Al included not more than an unimportant trace. These were used for the majority of the experiments. Secretin B. The commercial 'Pancreotest' preparation supplied by Messrs Astra, of Sweden. This is a highly purified substance used for clinical tests, and is guaranteed free from antigens, cholecystokinin and vaso-depressors. Secretin C. A highly purified material prepared by Dr S. A. Komarov from the intestine of the dog. After precipitation by sodium chloride and by trichloroacetic acid, subsequent purification followed the suggestions of the Swedish workers [Hammarsten, Agren & Wilander, 1933], and involved successive precipitation by mercuric sulphate, picrolonic acid and picric acid, the final precipitate being converted into the chloride. In some tests this material appeared to be free from vaso-depressors, but others suggested the presence of a small trace which would not be significant for the present work. Secretin D. A crystalline material prepared by Dr Komarov from the 'SI' concentrate according to the procedure of Greengard & Ivy [1938]. This was a very potent preparation, free from vaso-depressors (0.1 mg./c.c./3 kg.) but containing more than one type of crystal. The pancreatic juice was collected, usually in 15 or 30 Mmin. portions, in graduated tubes on ice, and was stored in a refrigerator overnight. All digest mixtures were made up within 24 hr. of the collection of the juice. Standard methods were used for the estimation of the enzymes in the samples, suitably modified so as to provide reasonable sensitivity over the considerable range of concentrations found in the experiments. Standard curves were constructed for each enzyme. using a known series of dilutions of a portion of juice, and from these curves all titration readings were transformed into arbitrary enzyme units in

4 ACTION OF SECRETIN ON PANCREATIC SECRETION 83 which all the results are expressed. The methods are outlined briefly below: Amylase. Following the procedure of Agren & Lagerlof [1936], a buffered starch solution was made up to contain Dibasic sodium phosphate 2-37 g. Monobasic potassium phosphate 2-27 g. Sodium chloride 3-75 g. Soluble starch 5 g. Water to 500 c.c. Aliquot parts of the diluted juice were placed in 25 c.c. Erlenmeyer flasks, 25 c.c. of the starch solution added to each, and the mixtures incubated at 370 C. for 24 hr. The flasks were then transferred to a refrigerator for 1 hr., and the reducing sugar in 1 c.c. of each mixture then immediately estimated by the method of Hagedorn & Jensen. In the tables and figures, the amylase concentration represents enzyme units per 1 c.c. of digest mixture; for convenience of tabulation, the total output of amylase in each portion of juice is expressed as one-tenth of the actual value. Trypsin. A substrate solution was made up to contain 5 % casein and 6 % normal sodium hydroxide. To 25 c.c. of this were added 10 c.c. of veronal-sodium buffer at ph 8-4 [Weil, 1936], 2 c.c. of a 1 in 10 dilution of juice, and a few drops of toluene. The digest mixtures were incubated at 370 C. for 15 hr.; 10 c.c. of each were then added to 10 c.c. of distilled water in Erlenmeyer flasks, and the amino-acid content estimated by the formol-titration method, using 02 N sodium hydroxide. The trypsin concentration is expressed as units per 10 c.c. of digest mixture. Lipase. A substrate solution was made up t9 contain 10 % triacetin and 0-08 % sodium glycocholate [Anrep, Lush & Palmer, 1925]. To 2 c.c. were added in a test tube 1 c.c. of Clark & Lubs' phosphate buffer at ph 8, 1 c.c. of a l in 10 dilution of juice, 4 drops of phenol red and 1 drop of toluene. The digest mixtures were incubated at 370 C. for 24 hr., and then titrated back to ph 8 with 0 05 N sodium hydroxide. The lipase concentration is expressed as units per total volume of digest mixture. Reliance has been placed chiefly on the estimation of amylase, as the selected method gave much greater sensitivity than could be obtained for the other enzymes. Where the latter were also estimated, the general shapes of the curves for the several enzymes were in close agreement, in accordance with the generally accepted principle of the parallel secretion of the pancreatic enzymes. That this parallel secretion occurs during secretin stimulation is shown in Fig. 1; such slight variations as were 6-2

5 84 E. J. W. BARRINGTON noted in the present work"need indicate nothing more than differences in sensitivity of the methods, or the absence of optimum activating conditions in the digest mixtures, although there is some evidence that under pathological conditions this parallelism may break down [Diamond et al. 1939] , ,000 28, A , ,000 5 _0-3,000 12, \ 2, t I-ij3 - I 7 O 0 10t i I alf-hour portions Fig. 1. Cat 23, weight 3-1 kg. Parallel fluctuations in output of amylase, trypsin and lipase during secretin administration. 1 c.c. of secretin A2 injected each 10 min., except for portion 6, for which the rate was quadrupled. Juice collected in 30 min. portions. Ordinates: Enzyme output in arbitrary units. In order to check the condition of the pancreas at the end of some of the experiments, small pieces of the organ were fixed overnight in Helly's fluid, embedded in wax and sectioned in the usual way, and stained in iron haematoxylin. EXPERIMENTAL The volume and enzyme content of the juice obtained by repeated administration of secretin (A1) over a period of 6 hr. is shown in Table 1. This experiment, begun after an hour's restitution which followed upon several secretin injections, clearly demonstrates that after 6 hr. of secretion under secretin stimulation there is still a readily measurable quantity

6 ACTION OF SECRETIN ON PANCREATIC SECRETION 85 TABLE 1. Cat 11, weight 3-7 kg.; 2 c.c. secretin A' (0.8 mg./c.c.) each 15 min.: juice collected in 30 min. portions (1-12) Volume (c.c.) Amylase concentration Amylase output Volume (c.c.) Amylase concentration Amylase output of enzymes in the juice. The progressive fall in enzyme output is characteristic of such experiments, and agrees with the protein-nitrogen readings for the pancreatic juice of the dog obtained by Komarov, Langstroth & McRae [1939] under comparable experimental conditions. Similar results were obtained in other experiments, including ones in which secretin C was administered for 4- hr. and D for 5j hr. At any stage during such an experiment faradic stimulation of the vagus will cause an increase in both the concentration and total output of enzymes. This is shown in Table 2, where portion 9 was the result of TABLE 2. Cat 21, weight 3 kg.; 2 c.c. secretin A2 (1 mg./c.c.) each 15 min.; juice collected in 30 min. portions (1-9). After taking portion 8, each vagus stimulated for 1 min. (coil 10), repeated after 15 min Volume (c.c.) Amylase concentration Amylase output Lipase concentration S Lipase output combined vagal and secretin stimulation and shows a markedly increased activity over the previous portions which were the result of secretin stimulation alone. This parasympathetic effect, already referred to, has been taken as evidence that the fall in enzyme output during secretin stimulation is not due to a true exhaustion of the gland. Anrep et at. [1925] believed that the changes in the concentration of the enzymes in similar acute experiments on the dog depended upon the general condition of the animal and of the blood supply to the gland, low enzyme concentration being associated with low blood pressures, but they used a crude secretin containing vaso-depressors, and their conclusion is not applicable to the present work. Mellanby [1925], using a much purer preparation, regarded the effect as supporting his view that the enzyme output was determined by the parasympathetic stimulation and not by the secretin. However, another explanation would be that the output

7 86 E. J. W. BARRINGTON depends upon both the intensity of the secretin or other stimulus, and also upon the amount of zymogen present in the gland cells. If this were so, under constant secretin stimulation the enzyme output would fall as a result of the progressive reduction of the zymogen content of the gland, a line of argument which has received interesting mathematical treatment at the hands of Langstroth et al. [1939], although, as they themselves clearly recognize, certain of their assumptions may be over-simplified. This last hypothesis suggests two possible consequences. First, variations in the intensity of the secretin administration should produce variations in output, and that this does in fact occur is shown in Table 3. TABLE 3. Cat 7, weight 3 kg.; 2 c.c. secretin Al (0.6 mg./c.c.) each 5 min. (portions 1-4), then 1 c.c. each 10 min. (portions 5-8), then 2 c.c. each 5 min. (portions 9-12); juice collected in 30 min. portions. After taking portion 8, faradic stimulation (secondary coil at 10 cm.) applied for 1 min. to each vagus and repeated after 15 min. interval. Total period of stimulation, 4 mi Volume (c.c.) Amylase concentration Amylase output Volume (c.c.) Amylase concentration Amylase output HIere, in the first phase of the experiment (portions 1-4), there was the usual fall in output: in the second phase (portions 5-8) the rate of secretin injection was reduced to one-quarter of the previous rate, and this was accompanied by a fall in concentration and output and a more gradual but equally clear fall in volume; in the third phase (portions 9-12) the secretin injections were restored to their original rate, and there was an immediate increase in output and volume. A second possible consequence is that after prolonged administration of secretin, with corresponding loss of zymogen from the gland, the falling output might increasingly approximate to the rate of synthesis of fresh material, and would finally become constant, output and synthesis balancing each other. In practice, the animals are found to vary so much in their sensitivity to secretin that there is no certainty of attaining this stage without the use of an extravagant amount of secretin, but in certain experiments the output has actually been found to maintain relative constancy over a number of samples. Table 2 shows such a condition extending over 3j hr. (portions 3-8), during which the enzyme output fluctuated around a constant level rather than showing a steady fall as

8 ACTION OF SECRETIN ON PANCREATIC SECRETION 87 in Table 1. It is probably significant that this experiment was begun with the pancreas already considerably exhausted after a series of secretin injections not recorded in the table. Clearly, whatever may be the correct explanation of this constancy, continued fall in output is not an inevitable accompaniment of secretin administration, and the possibility of secretin directly stimulating the discharge of enzymes cannot, there. fore, be excluded. Mellanby [1925] showed that section of the vagi during repeated administration of secretin caused a considerably diminished output of enzymes, and held this as further support for the view that the output was determined by vagal impulses. An experiment of this type is shown in Table 4; the vagi were cut after the collection of portion 5, and the TABLE 4. Cat 10, weight 3 kg.; 2 c.c. secretin Al (0*6 mg./c.c.) each 15 mim.; juice collected in 30 min. portions (1-10). Vagi cut after collecting portion Volume (c.c.) * Amylase concentration Amylase output Trypsin concentration Trypsin output Volume (c.c.) * Amylase concentration * Amylase output Trypsin concentration Trypsin output results confirm the occurrence of some reduction in output. The significance of this is not, however, quite clear, since the rate of flow also diminished and later increased, possibly as a result of the mechanical irritation stimulating the vagal endings in the smooth muscle fibres of the pancreatic ductules [Anrep, 1916]. In any case, the essential fact is that the discharge of enzymes is still continuing, at only a slowly diminishing rate, 31 hr. after the section of the vagi, and this, in conjunction with the numerous experiments in which the nerves were cut from the beginning, indicates that secretin can effect discharge of enzymes in the absence of vagal impulses, even although the presence of the latter may augment that discharge. Finally, in order to eliminate the possibility that the enzyme content of the juice during prolonged secretini stimulation with the vagi cut might be due to stimulation of the vagal endings by parasympathomimetic substances in the secretin preparation rather than to the action of the secretin itself, some experiments were carried out upon atropinized

9 88 E. J. W. BARRINGTON animals. The atropinization was checked by faradic stimulation either of the submaxillary gland through the chorda tympani, the duct of the gland being cannulated,'or of the pancreas through the cut vagi, a portion of juice being collected in the latter case and tested for the absence of any increase in enzyme output. The result of one of these experiments is seen in Fig. 3, which shows that enzymes continue to be secreted for 5j hr. even during paralysis of the parasympathetic nerve endings.. It may be concluded from the experiments recorded above that pancreatic juice secreted under the stimulus of secretin contains enzymes which are discharged independently of any vagal stimulus, but this conclusion still sheds no light upon the fundamental question as to whether that discharge is a result of direct action of secretin upon the cell inclusions or merely of the washing-out of the granules by the fluid passing through the cell. It is therefore necessary next to inquire whether any relationship can be traced between the rate of flow and the output of enzymes, for if the latter are merely washed out by the bicarbonate solution then an increased rate of flow might be expected to result in an increased output of enzymes per unit time. Indeed, it has already been seen (Table 3) that a diminished rate of flow may be associated with a fall in output. The most convenient way of varying the rate of flow of the fluid is by varying the rate of injection of secretin, as shown in Table 3. During the second phase of this experiment, when the secretin stimulation was reduced to one-quarter of the previous rate, the volume, concentration and output were all lowered, but it is noticeable that while the output fell abruptly, the volume (i.e. rate of flow) fell much more gradually and there is thus little indication of any direct relationship between them. With a resumption of the original rate of injection (portions 9-12) there was a sharp increase in volume and total output, but again with no clear relationship between them, for the latter fell slightly while the former continued to increase (portions 10-12). Other experiments show a further point, not strikingly shown in Table 3, and that is that an increase in the rate of injection may produce an increase in concentration of the enzymes as well as of volume and total output. This is well seen in Figs. 2 and 3, where the rate of secretin injection was quadrupled for portions 11 and 6 respectively. Such an effect of secretin on concentration is strongly suggestive of a direct action upon the cell inclusions rather than of an indirect effect of washing-out, and Diamond et al. (1939), who found secretin to affect the concentration of enzymes in human pancreatic juice, have argued in the same way.

10 ACTION OF SECRETIN ON PANCREATIC SECRETION 89 Such observations, however, cannot be conclusive, and it has seemed more profitable to devise some means of dissociating the rate of flow from the secretin stimulus by varying the former while maintaining the latter constant. For this purpose, advantage has been taken of the secretagogue action of sodium nitrite, the effect of which in augmenting pancreatic secretion appears to be a result of vasodilatation [Barlow, 1927]. 150 Amylase concentration 6 13_ 5 110_ 4 90_ 3 70 Ai 2 Vlme 1 3 i Half-hour portiono Fig. 2. Cat 18, weight 3 kg. Effect on volume of secretion and on amylase concentration of injecting secretin A1 and sodium nitrite. Juice collected in 30 min. portions. Here and in Fig. 3 the total amylase output in each portion ofjuice is: volume x concentration x 400. For further 9xplanation see text. One experiment involving the use of this salt is shown in Fig. 2. Portions 1-7 (inclusive) were taken during a constant rate of injection of secretin, and show the usual decline in enzyme concentration and output. During the period represented by portions 8-10, sodium nitrite (3 mg./15 min.) was added to the secretin. The result was an increased rate of flow (volume) but a further diminution of total enzyme output and therefore a fall in concentration. Portion 9 is particularly striking, for here, by accident, slightly less secretin than normal was injected, and this was marked by a slight fall in volume and output. Finally, portion 11 'Was the result of a maintenance of the nitrite injections at the same rate, but a quadrupling of the secretin injections; here there was a sharp rise in volume, concentration and output. In this experiment, then, which has been confirmed, the rate of flow was increased in two different ways, first

11 90 E. J. W. BARRINGTON by the injection of nitrite, the secretin stimulus remaining constant, and secondly by the increase of secretin, the nitrite remaining constant. The fact that only the second increase caused a rise in concentration and output of enzymes strongly implies that these are determined by the direct action of secretin and not by the rate of flow of fluid through the cell. One objection to the above experiment suggests itself, and that is that the nitrite might exert some undefined inhibitory effect on the discharge of zymogen, and thus prevent the rise in output which would otherwise result from the increased rate of flow of fluid in portions To meet 5 80 varayla8e concentration i Hialf-hour portions Fig. 3. Cat 19, weight 3-1 kg. Effect on volume of secretion and on amylase concentration of injecting secretin A2 and sodium nitrite into an atropinized animal. Juice collected in 30 min. portions. 2 mg. atropine injected after collecting portion 1, 1 mg. after portion 5, 1 mg. after portion 11. For further explanation see text. this, the experiment shown in Fig. 3 was devised, the animal being also atropinized to remove any possible vagal influence. Portions 1-5 (inclusive) were collected under constant secretin stimulation (1 c.c. each 10 min.). For portion 6 the secretin stimulus was quadrupled, and this resulted in a sharp rise in volume, concentration and output. Portions 7-9 were collected under the original secretin stimulus, and show a return to the original values. For portions 10 and 11 sodium nitrite (3 mg. each 10 min.) was added to the secretin, and this resulted in a rise in volume but not in concentration or output. For portion 12 the nitrite was maintained and the secretin quadrupled, and there was the expected rise in

12 ACTION OF SECRETIN ON PANCREATIC SECRETION 91 volume, concentration and output. Calculation shows that the total output in portion 6 is 4-4 times that in portion 5, while that in portion 12 is 3*9 times that in portion 11; these increases are of the same order, and there is thus no indication of any inhibitory effect induced by the nitrite. As may be seen from Fig. 3, the increasq in concentration in portion 12 as compared with 11 is considerably greater than that in 6 compared with 5. Finally, it may be noted that the increased volume seen in portion 3, and due apparently to some effect of the atropine [Mellanby, 1925], is not accompanied by any increase in concentration or output. It may be concluded from these and similar experiments that an increase in the rate of secretion of pancreatic juice is not necessarily accompanied by any increase in the output of enzymes, but that the latter is determined, independently of the rate of flow, by the direct action of secretin upon the cell inclusions. DIscussIoN There is little to add by way of discussion to the above account. It has been shown that secretin, as much as the vagus nerves, has a stimulatory effect upon the inclusions of the pancreatic cells and therefore plays a direct part in the control of enzyme output. This is a conclusion of some interest for the comparative physiologist, for it has been suggested [Babkin, 1931] that in the skate the pancreatic secretion may not be under nervous control. In lampreys the zymogen cells corresponding to those of the pancreas of higher vertebrates appear still to be located in the gut epithelium [Barrington, 1936], and it is ttus conceivable that the secretin mechanism may have evolved first as a local hormone [Collip, 1938], the nervous mechanism being added later. This, however, is quite speculative, and much further work would be needed to confirm any such suggestion. It seems impossible to decide what the relative importance of the two stimuli may be in the normal feeding of the mammal, although there is no reason to suppose that it is mirrored by their apparent relative importance under certain experimental conditions, for while faradic vagal stimulation and pilocarpine injections are known to cause extensive discharge of zymogen granules, they may well represent a more vigorous stimulation than would be physiologically normal, and indeed it is known that after strong pilocarpine stimulation the pancreatic cells of the mouse require at least 3-4 hr. restitution before becoming capable of fresh extrusion [Hirsch, 1932]. The major difficulty in estimating the importv-' ance of secretin under physiological conditions lies in a lack of knowledge

13 92 E. J. W. BARRINGTON as to its effect upon the cell inclusions. It has been said that after prolonged secretin administration the cells of the pancreas are still in a 'resting' condition [Mellanby, 1925], but Netik [1937] has presented some evidence for a decided reduction of zymogen granules in the dog after such treatment. An examination of sections of material from some of the cats used in the present work shows that although an abundance of granules is still present, there is sufficient variation in the appearance of individual cells to accord with the assumption of a discharge of zymogen from them. Some variation has been noted in the zymogen content of the pancreatic cells of the mouse, apparently resulting from the spontaneous secretory activity of the 'resting' gland [Hirsch, 1932]. It is not desired to press this point here, or to emphasize the cytological picture, since there is an obvious need for a statistical study of the effects of secretin upon the cell in a more convenient animal than the cat, and such work is now in progress. At present nothing can be suggested as to the history of the cells during their activity. It is essential first to know, for example, whether the effect of secretin is to cause a few cells to discharge completely their granules, or whether a large number of cells discharge a small proportion of their contents, and other questions readily suggest themselves. Until they have been answered, it must be emphasized that the suggestion put forward above, as to the setting up of a balance between synthesis and output, is essentially speculative, and is intended merely to aid discussion of the experimental results. SUMMARY In atropinized cats, or in cats with both vagi cut, enzymes continue to be discharged in the pancreatic juice after at least 6 hr. continuous secretion under secretin stimulation. A continued fall in enzyme output does not necessarily occur under these conditions. If the rate of flow of the pancreatic juice is increased either by administration of sodium nitrite or by increasing the rate of secretin injection, only the latter results in an increased concentration and output of enzymes. It is concluded that secretin directly stimulates the discharge of zymogen, and that the enzyme content of 'secretin' juice does not result merely from the passive washing-out of the zymogen by the fluid passing through the pancreatic cells. The work was carried out at Montreal during the tenure of a Rockefeller Foundation Fellowship, and I am much indebted to Prof. B. P. Babkin for the hospitality of his laboratory and for his guidance and criticism. I am indebted also to Dr S. A. Komarov

14 ACTION OF SECRETIN ON PANCREATIC SECRETION 93 for providing me with a sample of secretin, and for kindly preparing some crystalline material from the 'SI' secretin concentrate, and to Mr M. J. Schiffrin for assistance in the preparation of that concentrate. REFERENCES Agren, G. & Lagerlof, H. [1936]. Acta med. scand. 90, 1. Anrep, G. V. [1916]. J. Physiol. 50, 421. Anrep, G. V., Lush, J. L. & Palmer, M. G. [1925]. J. Phy8iol. 59, 434. Babkin, B. P. [1931]. Contrib. Canad. Biol. Fi8h. N.S. 7, 1. Babkin, B. P., Rubaschkin, W. J. & Savitsch, W. W. [1909]. Arch. mikr. Anat. 74, 68. Barlow, 0. W. [1927]. Amer. J. Phy8iol. 81, 189. Barrington, E. J. W. [1936]. Proc. Roy. Soc. B, 121, 221. Bayliss, W. M. & Starling, E. H. [1902]. J. Physiol. 28, 325. Bayliss, W. M. & Starling, E. H. [1904]. J. Physiol. 30, 61. Collip, J. B. [1938]. Amer. J. Dig. Dis8. Nutr. 5, 587. Crittenden, P. J. & Ivy, A. C. [1937]. Amer. J. Physiol. 119, 724. Diamond, J. S., Siegel, S. A., Gall, M. B. & Karlen, S. [1939]. Amer. J. Dig. Dis. Nutr. 6, 366. Greengard, H. & Ivy, A. C. [1938]. Amer. J. Physiol. 124, 427. Hammarsten, E., Hammarsten, H., Agren, G. & Wilander, 0. [1933]. Biochem. Z. 264,275. Hirsch, G. C. [1932]. Z. Zellformch. 15, 36. Ivy, A. C., Kloster, G., 15rewyer, G. E. & Lueth, H. C. [1930]. Amer. J. Physiol. 95, 35. Komarov, S. A., Langstroth, G. 0. & McRae, D. R. [1939]. Canad. J. Res. 17, 113. Langstroth, G. 0., McRae, D. R. & Komarov, S. A. [1939]. Canad. J. Bes. 17, 137. Mellanby, J. [1925]. J. Physiol. 60, 85. Mellanby, J. [1928]. J. Phy8iol. 64, 331. Netik, J. [1937]. BulU. Histol. Appl. Physiol. et Path. 14, 149. Pavlov, I. P. [1897]. The Work of the Digestive Glands (Russian). St Petersburg. (2nd English edition, 1910, p. 60.) Still, E. U. & Barlow, 0. W. [1927]. Amer. J. Physiol. 81, 341. Voegtlin, W. L., Greengard, H. & Ivy, A. C. [1934]. Amer. J. Physiol. 110, 198. Weil, L. [1936]. Bi7ochem. J. 30, 5. Zilwa, L. A. E. de [1904]. J. Physiol. 31, 230. Zucker, T. F., Newburger, P. G. & Berg, B. N. [1932]. Proc. Soc. exp. Biol., N.Y., 30, 166.