Newcastle upon Tyne. insulin hypoglycaemia, the response to which includes an inhibitory element. (Received 27 July 1953)

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1 168 J. Physiol. (I954) I23, i68-i86 THE EFFECTS OF PYLORIC ANTRECTOMY ON THE SECRETORY RESPONSE OF HEIDENHAIN POUCHES IN DOGS TO CENTRAL VAGAL STIMULATION BY PAMELA A. BURSTALL AND B. SCHOFIELD From the Department of Physiology, Medical School, King's College, Newcastle upon Tyne (Received 27 July 1953) In a previous paper (Burstall & Schofield, 1953) secretory responses of the Heidenhain gastric pouch to psychic stimulation and to insulin hypoglycaemia have been described and their possible mechanism discussed. The excitation could be conveyed from the vagal centres to the pouch by a few vagal fibres reaching it along the blood vessels of the greater curvature, or by gastrin released from the main stomach on vagal stimulation. The present paper describes experiments designed to differentiate between these possibilities by removal of the pyloric antrum, which is the main gastrin-producing area. Most of the work has been done with psychic stimulation by food showing or sham feeding. For the purposes of this paper sham feeding has been treated as a powerful form of psychic stimulation, the term 'psychic' being used in the same sense as in the 'psychic' or 'cephalic' phase of gastric secretion. It is of course recognized that sham feeding involves other than pure psychic stimuli. It was shown previously that psychic stimulation, particularly when potentiated by a previous dose of a stable choline ester, was a more reliable stimulant than insulin hypoglycaemia, the response to which includes an inhibitory element (Burstall & Schofield, 1953). Of the last seven dogs investigated all have responded to psychic stimulation, but only three out of six to insulin hypoglycaemia. METHODS The methods were those described in the previous paper (Burstall & Schofield, 1953), and only modified or new procedures are described here. Their essential feature is that even extremely small amounts of secretion are recovered from the pouch by washing it out continuously over successive 15 min periods with 5 N-HC1 and estimating the acid output by titration of the washings. Preparation and maintenance of animals Pyloric antrectomy. Under thiopentone-ether anaesthesia the pyloric antrum was removed from a point just distal to the pylorus to about 2 cm proximal to the incisura angularis (Fig. 1). This usually involved the removal of up to 2-5 cm of the distal end of the scar left when the original

2 EFFECTS OF ANTRECTOMY ON HEIDENHAIN POUCHES 169 Heidenhain pouch was made. The duodenal stump was closed and the continuity of the alimentary tract restored by an end-to-side anastomosis between the stomach remnant and the descending part of the duodenum. Four of the five antrectomized animals were also oesophagostomized. The surgical procedures have already been described (Burstall & Schofield, 1953). Post-operative care. For the first 3 days the animals were given a fluid diet only. Afterwards they were fed their normal ration but divided for the first week into three meals daily and subsequently into two. The normal regime of one meal per day was resumed after 1 month. After antrectomy alone they remained in good health and gained weight. The combination of antrectomy and oesophagostomy introduced difficulties in maintenance. There was a tendency for stomach contents to be regurgitated after feeding, presumably owing to the reduced capacity of the gastric remnant. Three of the four animals submitted to the combined procedure, dogs B4, J3 and G3 had the slit type of oesophagostomy described by Gregory (195) and could be fed solid food. These all remained in good health, though two of them, B4 and G3, showed some weight loss. The remaining b B D Fig. 1. Diagram of dog stomach. Line AB demarcates the portion used to prepare the Heidenhain pouch, and line CD that removed at pyloric antrectomy. animal, dog S2, had the classic type of oesophagostomy and was maintained on a semi-liquid diet. This animal was more prone to regurgitation, possibly owing to the nature of the diet, and went downhill rapidly after antrectomy. It was destroyed 4 weeks after the operation. Dog B4 was also oesophagostomized before antrectomy. This animal was maintained for 4 months after the antrectomy in good health. To avoid the difficulties of prolonged maintenance of the combined preparation, the last two animals of the series, dogs J3 and G3 were oesophagostomized 4 months after the antrectomy. Both these animals had given excellent responses to the food showing procedure before antrectomy, and the intention was to apply by sham feeding a stringent test of the abolition of the psychic response produced by the operation. After approximately 6 weeks both these oesophagostomies were closed, in order to maintain these animals for an extended period to see if any return of the psychic response or restoration in size of the 'Mechothane'* response occurred. Dog G3 had lost some weight during the oesophagostomy period and this was rapidly restored following closure. Extra care was required after oesophagostomy with antrectomy to protect the skin of the neck from damage by regurgitated food and secretion. Aluminium powder paste was effective for this purpose. Two animals developed appreciable skin reddening and excoriation. One was dog S2 mentioned above, in which excessive regurgitation occurred, the other was J3, in which the skin seemed particularly sensitive to this type of damage. In these animals there was evidence of raised basal acid secretion and enhanced response to 'Mechothane' during this period. This is in * 'Mechothane', Savory and Moore: urethane of,b-methyl choline hydrochloride.

3 17 PAMELA A. BURSTALL AND B. SCHOFIELD agreement with the observations of previous workers on the effect of skin irritation on gastric secretion (Babkin, 195). Little reliance can be placed on the results from dog S2 in view of the poor condition of the animal, but in J 3 a comparison was made of the size of the post-antrectomy acid output to 'Mechothane' during the oesophagostomized period with that before and after. The results show a statistically significant enhancement. The mean response during the oesophagostomized period was for five experiments 2-6±-5 ml..1 N-HCI, while for twelve experiments, seven before and five after the oesophagostomized period, it was ml..1-HCl (t=2-68, -2 >P >-1). Additional procedure8 and drugs Irrigation of the pouch with local anaesthetics. It was considered possible that if the response were due to efferent parasympathetic fibres reaching the pouch mucosa it might be possible to block these by prolonged irrigation with solutions containing local anaesthetic drugs. These drugs abolish conduction in both afferent and efferent fibres, though higher concentrations may be required for the latter when they are of larger size. In the case of the vagus nerve, however, the small cardio-inhibitory efferent fibres are more readily blocked than the larger pulmonary afferents (Goodman & Gilman, 1941). Accordingly, procaine hydrochloride, and in two experiments amethocaine hydrochloride were added to the wash fluid. The crystalline salt was dissolved in distilled water and brought with -1 N-NaOH to ph 7- by glass electrode. Sufficient hydrochloric acid and distilled water were then added to give a final concentration of.5-4.% ofthe anaesthetic in.5 N-HCI. This solution was run into the pouch in the normal way and the samples titrated as usual. Control samples of the fluid containing local anaesthetic titrated before use showed similar free acid figures to normal wash fluid. Owing to the presence of buffering power above neutrality they tended to give high figures for total acid and this was avoided by titration in these experiments to ph 7 instead of the usual 8*. Amethocaine hydrochloride at the 2% level interfered more severely with the titration, and in the one experiment carried out with this concentration a different procedure was adopted. Control observations were made using normal wash fluid. The pouch was then washed out with the local anaesthetic fluid for 24 hr but no samples from this period were titrated. Washing with the normal fluid was then resumed and a further group of observations made. Daily collections of gastric juice. All pouch dogs in the laboratory wear rubber bags in which the secretion is continuously collected (Schofield & Watson, 195). Their daily output of juice is routinely determined on all days except those on which experiments are carried out, and these figures have been used to assess the effect of antrectomy on the volume of gastric secretion produced by their normal meals. Mean figures for comparable periods before and after antrectomy have been calculated. No figures from periods during which animals are oesophagostomized have been used for this purpose. Recrystallized insulin. In some experiments a sample of insulin purified by six times recrystallization was used. This was obtained from Boots Ltd. as a sterile solution containing 2 units/ml., the same strength as the Boots standard insulin normally used. TREATMENT OF RESULTS The acid responses encountered in this work are extremely small, and suitable methods for their analysis were fully described in the previous paper (Burstall & Schofield, 1953). Only essential features of the procedure are recapitulated. In each experiment the basal level expressed in ml. -1 N-HCI/15 min was calculated as the mean of the total acid figures for three resting 15 min periods preceding the application of the stimulus. The difference was determined between this basal level and the mean total acid level over the three successive 15 min periods which normally included the maximum acid response to the

4 EFFECTS OF ANTRECTOMY ON HEIDENHAIN POUCHES 171 appropriate stimulus. With the psychic stimulus these were the first, second and third periods after the start of the stimulus. In the insulin response they were the second, third and fourth periods after the injection. This figure is the mean output above basal level for the appropriate three periods. For each series of results an overall mean output based on at least three experiments was calculated and its significance determined by Student's t test (Fisher, 1946). The minimum output acceptable as a positive response to a stimulus in a single experiment was defined as follows. During the first hour following the insulin injection or the start of the psychic stimulus the response must attain a total acid output of at least O15 ml..1 N-HCI above the basal level in each of three successive 15 min periods, or in each of two successive 15 mmn periods it must attain an output of at least -2 ml..1 N-HCI above the basal level. Mean response curves for total acid secretion to psychic stimulation before and after antrectomy were prepared by calculating for all experiments the mean output above basal level for each successive 15 min period after the start of the stimulus. Similar curves were prepared for pepsin secretion, save that pepsin outputs were expressed as a percentage change from the basal output calculated from three or four resting periods. RESULTS Acid secretion Psychic responses of antrectomized animals. It was shown previously (Burstall & Schofield, 1953) that psychic stimulation by food showing or sham feeding was a more reliable stimulus than insulin hypoglycaemia, and the present investigation has been carried out primarily with this method of central excitation. Potentiation of the response by a previous dose of -25 mg 'Mechothane' intravenously has been used as a routine procedure in all postantrectomy experiments save for a few of the earlier ones on dogs T 2 and B 4. Five dogs which had previously responded well to psychic stimulation were antrectomized. In four out of five animals there was a complete absence of response after antrectomy. The positive responses in the fifth animal S2 are considered below. Table 1 shows the results of all the psychic stimulation experiments on these dogs before and after the operation. The most potent psychic stimulus is sham feeding, and save for dog T2, which was not oesophagostomized, all the animals were given post-antrectomy sham meals. Dogs B 4 and S2 were oesophagostomized before antrectomy, and all the post-antrectomy experiments were with sham feeding. Dogs J 3 and G 3 were not oesophagostomized until about 4 months after the antrectomy. Two sham feeding experiments were carried out on the former and three on the latter, all with negative results. The oesophagostomies were then -closed

5 172 PAMELA A. BURSTALL AND B. SCHOFIELD to facilitate prolonged maintenance of the animals. It is intended to investigate these two for a possible return of the psychic response. At the time of writing, 9 months after antrectomy, there is no sign of this. Since, with the exception of dog S 2, there was complete abolition of the psychic response after antrectomy, the results of the two types of stimulation have not been separated in the table. TABLE 1. General classification of results Psychic stimulation Insulin stimulation* A A,1 AA Before antrectomy After antrectomy Before antrectomy After antrectomy Dog Total Positive Total Positive Total Positive Total Positive T B G Insulin negative J Insulin negative S2t Insulin negative * Standard insuiin experiments only; experiments after atropine not included. t The antrectomy on this animal was incomplete. Dog S2 was destroyed shortly after antrectomy, since its condition deteriorated after the operation. At post-mortem it was found that the removal of the antrum had been incomplete. Figures from this dog have therefore been excluded from the analyses of post-antrectomy results. The condition of dog S2 has already been described in the section dealing with maintenance of animals. It was destroyed in the fourth week after antrectomy. No experiments are normally performed until at least 1 month after a major operation, but in this case as the condition of the animal seemed to be deteriorating three preliminary tests were carried out. These showed an enhanced response to 'Mechothane' and persistence of the sham-feeding response. Post-mortem examination showed that a portion of antral mucosa had been left attached to the duodenal stump. This had been invaginated at closure and now appeared as a congested polyp-like mass projecting into the duodenum. In this animal difficulty had been encountered in mobilizing the pylorus at the original operation. Woodward, Bigelow & Dragstedt (195) have shown that even small amounts of unresected antral mucosa may have an appreciable effect on pouch secretion. There was reddening of the skin on the neck and upper chest in this animal as a result of the regurgitation of food and secretion which would enhance secretory responses (see Methods section). In view of the incomplete antrectomy and the conditions described above, no use has been made of the post-antrectomy results on this dog. In Table 2 the post-antrectomy responses to psychic stimulation on the four animals completely antrectomized have been assessed according to the methods previously described and compared statistically with those occurring before the operation. In each case there was a highly significant. A pair of potentiated food-showing experiments before and after antrectomy are shown in Fig. 2. A mean post-antrectomy response curve has also been prepared based on thirty-seven experiments on the four dogs with complete antrectomy. In the upper half of Fig. 3 it is compared with the corresponding

6 EFFECTS OF ANTRECTOMY ON HEIDENHAIN POUCHES 173 curve for forty-five experiments on six pre-antrectomy dogs (which include the four) published previously (Burstall & Schofield, 1953). 'Mechothane' responses after antrectomy. The acid secreted in response to the TABLE 2. Acid responses to psychic stimulation before and after pyloric antrectomy Before antrectomy Mean acid output No. above basal level No. of over 3 periods (ml. of Dog expts. -1 N-HCI/15 min) expts. T2 8-26±-7 4 After antrectomy Mean acid output above basal level over 3 periods (ml. 1 N-HCI/15 min) 5±4 B ± >P G3 8-32± ± *1>P J3 1-4±-4 9-1± >P Dog T2.-' Statistical comparison t P Conclusion >P>-2 Significant Highly significant Highly significant Highly significant 4 E LA ' 3 'Ir o* 1 E (a) * _ ~ ~~ : 1ti 'Mechothane' -25 mg i.v. * ~ -s. 2 Hours 3 Shown food 4 3 c-.e Lr 2 " c -, 1.'. - c LA z I r ae 2 A v (b) r@--e ~ ~ ~......; r,f:_ii_iz IIZII. 1 'Mechothane' -25 mg i.v. 2 / /A 3 Hours Shown food 2 -.E_ Ln 1 -_. Z t, Fig. 2. Two experiments on the same dog to show the response to psychic stimulation (a) before, and (b) after, pyloric antrectomy. Potentiation was used in each case and an appreciable in acid and pepsin outputs to 'Mechothane' can be seen after antrectomy., total acid; --- free acid;... pepsin. The horizontal fine interrupted line indicates the base-line of the pepsin scale. The same conventions and symbols are used in all the other diagrams illustrating results of single experiments.. v X-

7 174174PAMELA A. BURSTALL AND B. SCHOFIELD standard dose of -25 mg 'Mechothane' was markedly reduced in the four animals completely antrectomized. In Table 3 the mean total acid output in response to this dose is compared for each dog before and after antrectomy. The s range from -48 to -83%. Histamine responses after antrectomy. To exclude the possibility of a general decline in the responsiveness of the pouch after antrectomy, a series of experiments was carried out with histamine stimulation before and after antrectomy -c- Pre-antrectomy Post-antrectomy S 45 expts. 37 expts. E D dogs 4 dogs u ~~~~~ Resting_,I +8% +6% X 32 expts. 3 expt. a. 2% 1Hour Hour,ZHu EZHo Psychic Psychic stimulation stimulation Fig. 3. Diagrams showing mean responses of acid and pepsin to psychic stimulation before and after pyloric antrectomy. The acid results are in the upper and the pepsin results in the lower half. Each heavy horizontal line represents the mean output over the period covered. Acid is expressed in absolute units, pepsin as percentage change from the resting level. The total number of experiments on which each curve is based is given at its end. Not all experiments continued for the same time and where the number of experiments in a given period is less than the total the actual number is indicated by an adjacent numeral. One S.E. on either side of every mean is indicated by cross-hatching. on dogs J 3 and G 3. The mean total acid output in response to a dose of. 1 mg histamine acid phosphate injected subcutaneously in 1 ml. 9 % (w/v) sodium chloride solution is shown in the lower part of Table 3. There is clearly no in the histamine response. On the contrary, that in dog J 3 is significantly enhanced. Daily output ofgastricjuice after antrectomy. The daily output of gastric juice was recorded on all dogs except on days when experiments were performed. Save when they were oesophagostomized the dogs were on a uniform diet before and after antrectomy. The mean volume for thirty daily collections, consecutive save for occasional interruptions due to experiments was deter-

8 EFFECTS OF ANTRECTOMY ON HEIDENHAIN POUCHES 175 mined before and after antrectomy in three dogs, T 2, J 3 and G 3. The second series was commenced 4 weeks after the operation. The other animals were oesophagostomized for the whole period after antrectomy. The results in Table 4 show a significant decrease in each animal of about 5 %, and there is surprisingly good agreement between the figures for the three animals. There has been no sign of recovery of the response in dogs J3 and G3 which have been followed for 9 months after antrectomy. TABLE 3. Effects of pyloric antrectomy on acid secretion in response to 'Mechothane' and histamine No. Mean acid No. Mean acid Statistical comparison of output (ml. of output (ml.,- Stimulus Dog expts. -1N-HC1) expts. -1 N-HCl) t p 'Mechothane' T F± >P -25 mg i.v. B4 6 3*3± 5 9 1F7±* *2>P> 1 Histamine acid phos. 1 mg s.c. Before antrectomy After antrectomy G ±*8 12-7±t >P J J ±* >P G3 4 2*8±1* ± > P > -6 J3 5 2*5± ± >P TABLE 4. Daily output of secretion (thirty results in each case) Volume (ml.) (Statistical comparison Before After, _A_l Dog antrectomy antrectomy t P Conclusion G3 159± >P Highly significant J ± >P Highly significant T >P Highly significant Conclusion Highly significant Highly significant Highly significant Highly significant Increase not significant Increase highly significant % in vol. -48 Responses to insulin hypoglyeaemia. The first dog to be antrectomized (T 2) was insulin positive, and though after antrectomy the psychic response was totally abolished, a large but modified positive response to hypoglycaemia produced by standard insulin persisted. The acid secretion rose slowly, reached a peak in the fourth 15 min period after insulin and then declined very slowly over about 3 hr. The animal had previously given an extended response in the procaine experiment in Fig. 6a. This unexpected result suggested that the psychic and insulin responses might be produced by different mechanisms. It was thought possible that there might be a direct-acting stimulant in the insulin preparation, and it was decided to investigate the action of atropine on the response to hypoglycaemia. After doses of 2-6 mg of atropine sulphate C%/ change in mean output

9 176 PAMELA A. BURSTALL AND B. SCHOFIELD a reduced secretion of acid persisted which rose and fell slowly as did all postantrectomy insulin responses in this dog. Responses after antrectomy with and without previous atropine are illustrated in Fig. 4b and c. For comparison, the largest response before antrectomy on this dog is shown in Fig. 4a. It was intended next to test this animal with a highly purified sample of insulin, and a six times recrystallized preparation was obtained. Before the dog could be tested, however, it died suddenly from acute peritonitis arising from the torsion of an intestinal loop by adhesions. It had previously been in excellent health. Subsequently four dogs, B4, G3, J3 and R4, were tested with standard insulin. Only B 4 and R 4 were positive to this stimulation, and these were also tested with the recrystallized preparation. Both secreted some acid with the latter stimulus, but in only one of them, B4, did a satisfactory response reaching the positive level occur (Fig. 4d). A response to standard insulin occurring in this animal about the same time is shown in Fig. 4e. Even in this animal the response to the recrystallized material did not remain positive but declined with repeated testing until it completely disappeared. Over the same period the responses to standard insulin and to psychic stimulation continued unimpaired. In Fig. 5 are shown the last two experiments performed on dog B4 before it was antrectomized, 3 months after those shown in Fig. 4d and e. There is no response to the recrystallized insulin, though a large response to sham feeding immediately preceded it (5a) and a large and somewhat drawnout response to standard insulin was recorded 4 days later (5b). The blood sugar curves of these two experiments are of very similar form. Dog R 4 never gave a positive response with the recrystallized material. Neither would have been classed as insulin positive on the results with the recrystallized material. The collected results on these two animals are shown in Table 5. They show that the mean response to recrystallized insulin was in both cases significantly less than that to the standard preparation, though similar blood sugar depressions occurred in each case and the two series of experiments on each dog were carried out concurrently. When dog B 4 was given standard insulin after atropine there was the suggestion of a long-drawn-out elevation of the acid level (Fig. 4g). It was not seen with the recrystallized preparation after atropine even in an experiment (Fig. 4f) carried out 2 days after the significant response to this material shown in Fig. 4 d. After antrectomy dog B 4 never gave a positive response to standard insulin (see Table 1). Five experiments were carried out. Three were completely negative, as Fig. 4h, but the last two also showed a very small long-drawn-out secretion which failed to reach the positive level, Fig. 4i. Both this and the pre-antrectomy response after atropine, though they are well below the levels required for significance, show some resemblance to the long-drawn-out post-antrectomy response of dog T2. Dog R4 was not

10 EFFECTS OF ANTRECTOMY ON HEIDENHAIN POUCHES 177 antrectomized. No acid secretion whatever was recorded when the recrystallized insulin was given in the antrectomized animals. The fact that hypoglycaemia is a considerably weaker stimulant when induced by highly purified insulin than when produced by the standard preparation suggests that with the latter material there is another stimulant 2 Dog T2 Pre-antrectomy 1 (a) S.I. (large response) Ir ~~~~~~~Post-antrectomy S.l. c E v- I z EL- 1 - (c) m, I I1 Shown food t Post-antrectomy S.I. after Atr. O Doi:B4B4 ~ Atr. t t 1 3 i 1 h (d) Pre-antrectomy IR.l. O Ll l Shown food t 1_ (e) J.1 O _ 1 _ (f) I (g) tr Pre-antrectomy S Pre-antrectomy R.I. after Atr. Atr. t Pre-antrectomy 3 75 mg i.v. S.I. after Atr. O 1 (h) -r Atr. It 3-75 mg i.v. Post-antrectomy S.l. O _ Sham meal t 1F- k'j Post-antrectomy S.I. 1 - Dog J 3 Sham meal Post-antrectomy S.l. 1 (k) I T Insulin 1-5 U/kg. i.v. A I 1 Post-antrectomy R.l Hours Fig. 4. Collection of individual results to show responses, before and after pyloric antrectomy, with standard and recrystallized insulin. Total acid figures only are shown. (a-c), dog T2; (cl-i), dog B4; (j) and (k), dog J3. S.I., standard insulin; R.I., recrystallized insulin; Atr., atropine. PH. CXXIII. 12

11 178 PAMELA A. BURSTALL AND B. SCHOFIELD mechanism. To take the simplest view, this may be an impurity having a direct stimulant action on the pouch. This explanation could also account for the occurrence of small responses to the standard preparation alone after Dog B4 *. 8~~~~~~~~~~~8 E E a). E - -.-_ - s--- z 2t Hours Sham meal R.I. 1-5 U/kg i.v. 8 (b).8 E _ bo Z~~ -* 5 ~~~~~~~~~~5 ~ u 2. o z.1 ~~~~~~~~~~~~~~~~~ * * Hours S.I. 1-5 U/kg i.v. Fig. 5. Two experiments on dog B4 carried out 3 months after those in 4d and e. In (a) there is no response to recrystallized insulin (R.I.), though a large response to sham feeding immediately preceded it. In (b), 4 days later, there is a large and somewhat drawn-out response to standard insulin (S.I.)., blood sugar values. The horizontal heavy interrupted line indicates the critical blood sugar level of 5 mg%. The blood sugar curves of these experiments are of very similar form. TABLE 5. Standard insulin Comparison of responses to standard and recrystallized insulin Recrystallized insulin Mean acid out- Mean acid output above put above basal level basal level Statistical comparison of Mean blood Results over 3 periods Mean blood Results over 3 periods mean acid outputs sugar min. (ml. -1 sugar mn., (ml..1 A _ Dog (mg %) Total Pos. N-HCI/15 min) (mg %) Total Pos. N-HCl/15 min.) t P Conclusion B ± ± ± ±t >P Highly significant difference R4 33-8±1: ±-9 39-±4t2 3 8± >P>-2 Significant difference antrectomy and after atropine. The large responses after antrectomy found only in one dog, T 2, are difficult to explain but might be accounted for as follows. (1) This dog might have been particularly sensitive to the direct stimulant. Though its responses to insulin were very variable, it was much the most sensitive animal to this stimulus of the whole

12 EFFECTS OF ANTRECTOMY ON HEIDENHAIN POUCHES 179 series, though not the most sensitive to psychic stimulation. Before antrectomy it had given on two occasions a response larger than those obtained afterwards. (2) Variations might occur in the amount of direct acting stimulant in the insulin preparations, and experiments were carried out on other dogs to investigate this with material from several different batches. A few suggestive results were obtained, but nothing convincing, since in any case there is a wide variation in the insulin responses with the sample in the same dog. (3) The sensitivity of the pouch to the direct stimulant might have been raised after antrectomy. This is pure speculation since no histamine series was carried out on dog T2, but in the two animals which were given histamine before and after antrectomy there was some enhancement of the response, in one case to the highly significant increase of + 8%. It is interesting that this dog J3, which was completely negative to both forms ofinsulin before antrectomy, showed after antrectomy on both occasions on which it was tested a tiny long-drawn-out elevation of acid secretion with standard insulin (Fig. 4j). No sign of this occurred with the recrystallized preparation (Fig. 4k). Effect of local anaesthetics on psychic and insulin responses. The effect of irrigation of the pouch for long periods with wash fluids containing local anaesthetics has been investigated. A preliminary control experiment with dog T 2 showed that the acid response to -25 mg 'Mechothane' intravenously after 21 hr irrigation with 1 % procaine hydrochloride was only reduced by 3 % compared with a similar response just before the irrigation commenced. In the first subsequent experiment a response to insulin failed to appear after 2 hr irrigation with this solution, but in further experiments both psychic and insulin responses were observed in spite of the procaine concentration being raised up to 4 %. One of these experiments is illustrated (Fig. 6a). In view of the poor mucosal penetrant properties of procaine, amethocaine was tried. In an experiment with I % amethocaine hydrochloride, the 'Mechothane' response after 1 hr irrigation was enhanced and a subsequent foodshowing response came through strongly (Fig. 6b). When the amethocaine level was raised to 2 %, the food showing response was abolished, but the 'Mechothane' response was almost abolished too. No further experiments with this procedure were carried out. Pepsin secretion Resting pepsin secretion after antrectomy. Basal pepsin outputs were calculated from three or, where possible, four resting periods at the start of experiments. Mean basal outputs for each dog have been calculated before and after antrectomy. There is a wide scatter in the results of each dog and considerable variation in level between dogs. After antrectomy the basal output was significantly reduced in one dog, T 2, but not in the other three. One animal, G3, showed an increase, but not to a significant level. The results are shown in Table 6. Psychic response after antrectomy. In common with the acid, the pepsin output in response to sham feeding was abolished. The results are presented as a mean response curve for thirty experiments on the four completely antrectomized dogs. The curve is compared with the corresponding one from 12-2

13 18 PAMELA A. BURSTALL AND B. SCHOFIELD thirty-two experiments on five dogs pre-antrectomy, in which the four are included (Fig. 3, lower half)..e _ 2 I xl z 1 4 EO D (a) Dog T2 I % procaine Dz,2 T 3 Shown food Insulin 1F5 U/kg iv. W. =7 Hours 4 8 bo so E 8- co JJ~ S 'Mechothane' Hours 'Mechothane' Shown food 25mg i.v. 25mg i.v. Fig. 6. Two experiments in which the pouch was irrigated with solutions of local anaesthetics. (a) Irrigation throughout with wash fluid containing 1% procain HC1. Persistence of psychic and insulin responses. (b) Irrigation with wash fluiid containing.5% amethocaine HC1 began after 2 hr. Subsequently acid response to 'Mechothane' enhanced and response to psychic stimulation persisted. Blood sugar values indicated as in Fig. 5. TABLE 6. Effect of pyloric antrectomy on the basal secretion of pepsin Before antrectomy After antrectomy Mean basal Mean basal pepsin output pepsin output Statistical comparison k No. of (Hunt No. of (Hunt Dog expts. units/hr) expts. units/hr) t P Conclusion T ± >P Highly significant B ± ± >P>-5 Reduction not significant G ± ± >P> 5 Increase not significant J ± ± >P>-3 Reduction not significant Pepsin responses to 'Mechothane' after antrectomy. The effect of antrectomy on the pepsin response to 'Mechothane' varied among the four dogs. The mean results are shown in Table 7. In two dogs there was a significant

14 EFFECTS OF ANTRECTOMY ON HEIDENHAIN POUCHES 181 and in two there was not. The corresponding figures for in the acid response are shown alongside them. The two dogs which showed no pepsin, B4 and G3, both showed considerable acid s, that in G3 being the largest of all. TABLE 7. Effects of pyloric antrectomy on pepsin secretion in response to 'Mechothane' (-25 mg i.v.) Before antrectomy After antrectomy ~ ~ % change in Mean pepsin Mean pepsin Statistical comparison mean output No. of output No. of output, Dog expts. (Hunt units) expts. (Hunt units) t P Conclusion Pepsin Acid T ± ± OO1>P Highly significant B ± ± >P>*7 No change G3 5 75± ± *6 >P>5 No change J ± ± >P Highly significant Effect of insulin hypoglycaemia on pepsin secretion after antrectomy. The same depressant effect on pepsin secretion was produced after antrectomy as previously reported in unantrectomized animals. The effects of standard and recrystallized insulin were similar. DISCUSSION For reasons which have been previously discussed (Burstall & Schofield, 1953) it was at first thought that a small direct parasympathetic innervation of the pouch was the most likely explanation of the responses to central vagal stimulation which had been observed. The small series of experiments with local anaesthetics was the first carried out to investigate this possibility. It was realized that the pouch-washing method in use was ideally suited for prolonged irrigation of the mucosa with local anaesthetic solutions in order to block efferent secretory fibres reaching it. If the response to central vagal stimulation had been abolished by this procedure but the response to a direct stimulus such as 'Mechothane' or histamine been unaffected by a similar or longer period of irrigation, this would have been good evidence of the response being dependent on such fibres. The failure in practice of the local anaesthetic to abolish the secretion was merely consistent with the alternative explanation of the response, that it was mediated by the gastrin mechanism. In view of the variability of the insulin response, no significance is attached to its abolition on one occasion. It was then decided to carry out an investigation of the effect of antrectomy on the response. Its persistence in these circumstances would be very strong evidence for the direct innervation hypothesis, since the principal source of gastrin would have been removed. Its abolition, also, would be good evidence for the gastrin hypothesis. It was intended to rely primarily on the psychic stimulus, and carry out only corroborative experiments with insulin.

15 182 PAMELA A. BURSTALL AND B. SCHOFIELD Further difficulties, however, were encountered with the insulin procedure. It had been previously pointed out (Burstall & Schofield, 1953) that need for an injected stimulant was a serious disadvantage, since, at the very low secretion rates observed in this work there is no adequate control of the possibility of a direct peripheral action of the hormone or some contained impurity. Evidence suggestive of such an impurity has now been obtained. Highly purified insulin has been shown in two animals to be a less effective stimulant to the Heidenhain pouch than the standard preparation in doses producing similar blood sugar depression. Both these animals, though clearly positive to standard insulin, would have been classed as negative on their results with the highly purified material. It appears therefore that the insulin response of the Heidenhain pouch may depend on three separate factors, two stimulant, one vagal and one possibly direct, and the inhibitory influence of hypoglyeaemia. This complex origin may account for its great variability. In view of the very small total size of the response it is almost impossible to determine which factor may be of predominant importance, but the above results suggest that a direct factor may play an important part in the ordinary insulin response. On the other hand, in the concentrations present it can hardly be a very powerful stimulant even allowing for the inhibitory influence, for out of eleven Heidenhain pouches tested only five have given consistent positive responses to standard insulin. It is admitted that the delayed, long-drawn-out acid secretion produced by standard insulin in some animals after antrectomy and after atropine is not of the form expected to result from the uncomplicated action of a single intravenous dose of a direct stimulant. No explanation of this can be given and the minute size of these responses, save in the isolated case of dog T 2, makes it unlikely that much more information will be obtained by the present methods. Porter, Longmire & French (1953) have recently postulated a posterior hypothalamus-pituitary-adrenocortical system pathway by which a delayed acid secretion reaching a peak in 24-3 hr and lasting up to 5 hr is produced by the insulin hypoglyeaemia stimulus. The present delayed responses reached their peaks in 1-1 hr and declined in 2-4 hr, but this and their very small size might possibly be accounted for by species differences and by the use of different preparations. Porter et al. (1953) appear to have recorded the delayed responses from vagotomized whole stomachs in monkeys. The presence of a synergistic stimulant impurity in the standard insulin could account for the fact that these delayed responses in the present work were only observed with this preparation. If the above mechanism can be confirmed a further complication is added to the already complex mechanism suggested above for the insulin response of the Heidenhain pouch. In the circumstances it seems that there is little advantage in the use of insulin hypoglycaemia as a differentiating stimulus in investigations of the present type.

16 EFFECTS OF ANTRECTOMY ON HEIDENHAIN POUCHES 183 In contrast to the rather unsatisfactory state of the insulin experiments the results obtained from the psychic stimulation experiments have been unequivocal and strongly support the gastrin hypothesis. The five dogs which were antrectomized all gave clear-cut and consistent responses to psychic stimulation before the operation. Afterwards these were completely abolished in four of the animals. In the fifth, in which they persisted, the antrectomy was found to have been incomplete, but it must be remembered that the condition of this animal at the time was poor, and its responses may have been enhanced owing to the presence of skin damage round the oesophagostomy. Three of the four were tested after antrectomy by sham feeding, which is the most effective stimulant to the vagal nuclei so far employed, without the slightest trace of a response being produced. Two of them have been followed now for over 9 months without any sign of its return. A general depression in the excitability of the pouches following antrectomy has been excluded by investigating in two of them the response to histamine before and after the operation. In neither case was there any and in one case the response was significantly enhanced. Langlois & Grossman (195) and Woodward & Dragstedt (1953) investigated the effect of antrectomy on the response of the Heidenhain pouch to histamine and also found no. The view that the pyloric antrum is concerned in the acid response to vagal stimulation, initiated by Straaten (1933), was strongly supported by the work of Uvnas (1942). His interpretation, however, was not confirmed by Babkin & Schachter (1944). Since then a good deal of supporting evidence has accumulated which was discussed in the previous paper (Burstall & Schofield, 1953). If the release of gastrin from the antrum forms an important physiological mediator of the vagal secretory influence, however, it is surprising that the response of the Heidenhain pouch to vagal stimulation is so very small. This may be accounted for by the hypothesis that gastrin released by vagal stimulation normally acts synergistically with the direct vagal innervation to the parietal cell and in its absence produces only a minimal effect. Uvnais (1942) first put forward the idea of synergistic action between gastrin and the direct vagal innervation, and even Babkin & Schachter (1944) were prepared to concede that the vagus might release gastrin as well as acetylcholine during the complex process of acid secretion. Both Linde (195) and Lim & Mozer (1951) suggested that the amount of gastrin released from the antrum during vagal stimulation may be subthreshold for the parietal cells of the Heidenhain pouch when their function is tested by normal methods. This might be because the quantity concerned is extremely small, or because of the absence of a synergistic effect from the direct innervation, or because of an unbalanced influence exerted by the intact sympathetic innervation of the pouch. It must be emphasized that although these results with psychic stimulation strongly support the gastrin release hypothesis they do not exclude the possi-

17 184 PAMELA A. BURSTALL AND B. SCHOFIELD bility that the Heidenhain pouch does receive directly a few vagal fibres. As was previously pointed out (Burstall & Schofield, 1953), there is anatomical evidence for this possibility and the two hypotheses are not mutually exclusive. The few observations on the effect of antrectomy on the insulin response are equivocal, but for the reasons given already it is felt that little significance should be attached to these results. Whether, along with the antral mechanism, vagal fibres play a part in the acid responses which have been observed is a matter for speculation, but it seems clear that the psychic responses do not take place in the absence of the hormonal influence. No conclusion at this stage can be drawn from the abolition after antrectomy of the pepsin response to psychic stimulation, since its mechanism is uncertain (Burstall & Schofield, 1953). The in the volume of secretion in response to the animal's daily meal observed after antrectomy in the present work agrees with the recent observations of Woodward et al. (195) who reported a of 84% in a true Heidenhain pouch and 81 % in insulin negative Pavlov pouches. It is at variance with those of Grindlay (1941) who found no change in volume or acidity in the meal secretion of Heidenhain pouches after pylorectomy. The s in the present series of approximately 5 % in the three dogs were very consistent. The discrepancy between these figures and those of Woodward et al. (195) is not surprising since the meal secretion remaining after antrectomy is likely to depend on a number of factors such as the diet and the size and placing of the gastro-duodenal ostium which may influence the rate of passage of the food through the stomach remnant and upper small intestine. There has been no sign of recovery of the meal response in the two animals followed for 9 months. Recently, Woodward & Dragstedt (1953) have reported no recovery in a prolonged study extending in the case of one animal over 5 years. The s in the acid response to 'Mechothane' observed in the present study are in agreement with the observations of Langlois & Grossman (195), and support their conclusion that the greater part of the acid response to this drug is mediated by the gastrin mechanism. The effect of antrectomy on the pepsin response to 'Mechothane' varied among the four completely antrectomized dogs. Since some in pepsin output might be expected to occur as a direct consequence of the in acid secretion, it seems reasonable to place most stress on the results in the two animals in which there was no fall in pepsin output. Both these animals showed significant s in acid secretion, one having the largest of the series, -83 %. In the two animals in which significant s of pepsin output did occur, these were less than the corresponding figures for acid. These results therefore do not support the possibility that the pepsin response to 'Mechothane' is also largely mediated by a hormone released from the pyloric antrum.

18 EFFECTS OF ANTRECTOMY ON HEIDENHAIN POUCHES 185 SUMMARY 1. Experiments have been carried out to determine whether the response of the Heidenhain pouch to central vagal stimulation is mediated by direct vagal innervation of the pouch or by release of gastrin from the pyloric antrum. A potentiated psychic stimulus, previously shown to be more reliable than insulin hypoglycaemia, has been principally used. 2. In preliminary observations the response was not abolished by irrigation of the pouch with local anaesthetic solutions. This is consistent with the gastrin hypothesis. 3. In four out of five dogs the response to psychic stimulation was completely abolished by pyloric antrectomy. The response to histamine was not reduced, which excludes a general decline in the excitability of the pouch. These results strongly support the gastrin hypothesis, but the possibility of a contributory effect from direct vagal innervation has not been excluded. 4. In two Heidenhain pouch dogs hypoglyeaemia was a considerably weaker stimulant when induced by highly purified insulin than when produced by the standard preparation. It is suggested that the presence in the latter of a stimulant impurity might account for this. 5. Two insulin positive dogs were antrectomized. In one the response was abolished and in the other a modified response persisted which was reduced but not abolished by atropine. Factors concerned in the insulin response are discussed, and in view of its complex origin it is suggested that little stress should be laid on these results. 6. In the four completely antrectomized dogs the acid response to 'Mechothane' was reduced by %, which is in agreement with the view that this response is largely mediated by the gastrin mechanism. The lack of a in pepsin output in two of the animals does not support the possibility that the pepsin response to this drug is similarly brought about by a hormone from the pyloric antrum. 7. The daily volume of juice secreted by the pouches was approximately halved after antrectomy. This has persisted in two animals for over 9 months. The resting output of pepsin was significantly reduced in one out of four dogs. We express our thanks to the Medical Research Council for a grant covering expenses and to King's College Research Fund for certain apparatus. The work was carried out during the tenure of a Junior Luccock Research Fellowship by P.A.B. We are very grateful to Dr G. I. Hobday of the Research Department, Boots Pure Drug Co. Ltd., Nottingham, for a supply of six times recrystallized insulin. Our thanks are also due to Miss Shirley Smith and Miss Patricia George for their most valuable technical assistance.

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