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Studies on the Secretion and Motility of Brunner’s Gland Pouches
GASTROENTEROLOGY
Vol. 51, No.4 Printed in U.S.A .
Copyright © 1966 by The Williams & Wilkins Co.
STUDIES ON THE SECRETION AND MOTILITY OF BRUNNER'S GLAND POUCHES A. R. CooKE, M.R .A.C.P.,
AND
M. I. GRossMAN, M .D., PH .D.
Resea1'ch, Veterans Administ1'ation Center and Department of Physiology, University of Califomia Cente1' fo 1' the H ealth Sciences, Los Angeles, California
In 1935 Florey and Harding1 showed that the secretory activity of Brunner's glands was controlled by a humoral mechanism, but the hormone has not yet been identified. Florey and Harding 2 believed the agent to be secretin but subsequently Blickenstaff et aJ.3 presented evidence to show that there were possibly two humoral factors-one stimulating secretion directly and the other increasing secretion indirectly by stimulating motility. The purpose of this study was to investigate further the nature of the hormonal agent or agents regulating Brunner's glands using various mucosal extracts, including preparations of gastrin, secretin, and pancreozymin.
Methods Three mongrel dogs were used . Under sodium pentobarbital anesthesia the abdominal cavity was opened and a double-walled mucosal septum was made at the pylorus. The duodenum was divided just proximal to the entry of the bile duct and the distal end of the first part of the duodenum was brought through a separate small incision in the abdominal wall as the pouch fistula. The duodenal stump was closed and intestinal continuity was reestablished by a side-to-side gastroduodenostomy. For another series of experiments, 1 animal was Received April 11, 1966. Accepted June 7, report of this work has been published in Gastroenterology 48 : 864, 1965. Address requests for reprints to: Dr. M. I. Grossman, Veterans Administration Center, Los Angeles, California 90073. This work was supported in part by National Science Foundation Grant GB 667. The authors wish to thank Professor R. A. Gregory, Liverpool, for his gift of gastrin I, and Ray Lichter and John Washington for technical assistance. 1966. A preliminary
506
prepared with the same kind of pouch of Brunner's glands plus a pouch of the fourth part of the duodenum. Experiments were not started until 4 to 6 weeks after surgery. Before each experiment, the animals were fasted for at least 18 hr. During tests the dogs stood in a sling harness. The juice was collected by a plastic funnel which led to a graduated centrifuge tube . The funnel surrounded but did not touch the e;<;posed mucosa of the opening of the pouch. Basal secretion was collected for four 15-min periods and then the test agents were given either by a single intravenous injection or as a constant intravenous infusion. The test substances were dissolved in 0.15 M sodium chloride and a constant infusion of 30 ml per hr was maintained by means of a peristaltic pump (Harvard Apparatus Company, Dover, Mass.). Combined motility and secretory studies were done in a number of instances. A waterfilled balloon (10 mm by 5 mm) was inserted into the pouch and was connected to a strain gauge (Statham, P23BB) by means of a waterfilled polythene tube. The contractions were recorded on a recording potentiometer (Sargent and Company, Chicago, Ill.) set at a paper speed of Y2 inch per min. The balloon was kept in position by attaching the polythene tube to the collecting funnel. A motility index was determined by multiplying the height by the numbed of contractions during three separate 5min periods before and after the test agent was given. The average of t he before and after periods was calculated and expressed as t he mean motility index (MI). Percentage change of motility was expressed as follows: o/o change of motility [(Mia - Mib) j Mib] X 100; Mia mean motility index after test drug; Mib mean motility index before test drug. Percentage change of secretion was determined using the formula : o/o change of secretion = [ (Va - Vb) j Vb] X 100;
=
= =
507
BRUNNER'S GLAND POUCHES
October 1966
= =
Va mean volume during 15-min period after giving the test agent; Vb mean volume during 15-min period before giving the test agent. The volume of juice collected every 15 min was recorded to the nearest 0.1 ml. The bicarbonate concentration was measured by adding 0.5 ml of juice (0.2 ml if 15-min collection was less than 0.5 ml) to 1.0 ml of 0.1 N HCl and backtitrating the mixture to pH 7 with 0.2 N NaOH with an automatic titrator (Radiometer, Copenhagen) . In some experiments portions of two consecutive 15-min samples were combined, and their pepsin concentration was determined using radioiodinated serum albumin as substrate.• Sodium and potassium concentrations were determined by flame photometry (Zeiss, Germany) and chloride by a chloridometer (Buchler Instruments, Inc., New J ersey). Mate rials. Two preparations of gastrin were used, the pure polypeptide porcine gastrin I and a relatively crude preparation prepared from t he mucosa of the pyloric gland area of hogs by modification of the method of Gregory and Tracy• described by Gillespie and Grossman.• The dose of crude gastrin is expressed as grams of wet weight of mucosa from which the extract was obtained. Extracts of oxyntic gland mucosa and the proximal 3 feet of small
bowel of the hog were also prepared using the method for preparing crude gastrin."· • Two commercial preparations of secretin were used; a relatively crude product (Boots Pure Drug Company, Nottingham, England) and a preparation (Vitrum, Stockholm) which Jorpes and Mutt (personal communication) state is identical with an apparently pure polypeptide described by them.7..., Cecekin (Vitrum) was used as a source of pancreozymin. This preparation is not chemically homogeneous and contains cholecystokinin as well as some secretin-like material.10 Other test substances used were histamine dihydrochloride, bethanechol chloride (U recholine, Merck, Sharp and Dohme) and a proprietary dog food (Friskies, Carnation Company, California).
Results
Secretory Studies R esponse to a meal. When the animals were given 1 pound of dog food (Friskies) there was no secretory response. However, when each animal was allowed to eat as much as it desired (usually 3 to 4 pounds) a clear increase in volume occurTed. The response began within 15 min, reached a
14
FOOD
~
12
...__. OUTPUT UNITS/30' ._ ___ CONC. U/ml •·-·-• VOLUME ml
10
8
6
4
2
0+----.-----.----.----,r----.----.-----.----, 0
2 4 6 30 MINUTE COLLECTION PERIODS
8
FIG. 1. Pepsin and volume response of Brunner's gland pouches to a meal. Each point in this and subsequent figures, unless otherwise stated, is the mean of nine experiments in 3 dogs; the vertical bars are the standard error of the mean.
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COOKE AND GROSSMAN
2
0
f
I
10
20
I I 40
80
l 160
UNITS
SECRETIN (BOOTS)
FIG. 2. Responses to graded single intravenous doses of Boots secretin.
peak at 30 min, and was complete by 180 min (fig. 1). Pepsin concentration and output were significantly increased in response to food (fig. 1). Response to secretin. Rapid intravenous injections of Boots secretin evoked a dose~elated secretory response (fig. 2). In contrast Vitrum secretin produced no response (fig. 3). When a continuous intravenous infusion of histamine dihydrochloride (2.0 mg per hr) was given no secretory response was obtained. Similar results were found using 4.0 mg per hr.
Vol. 51, No.4
Response to crude gastrin and gastrin I. Crude hog gastrin given as a single intravenous injection (40 g) evoked a short-lived secretory response (fig. 4). When given as a continuous intravenous infusion (40 g per hr) a sustained secretory response was obtained (fig. 5). The pure polypeptide, gastrin I, was an ineffective stimulus (fig. 5). The same batch of pure gastrin was shown in other dogs with Heidenhain pouches to produce strong stimulation of acid secretion at this dose level (40 p.g per hr). Response to other preparations. Vitrum Cecekin (75 units) given as a single intravenous injection was found to be an effective stimulant of Brunner's glands as were fundic and jejunal mucosal extracts (fig. 4 and table 1). Response to bethanechol chloride. When the animals were given a continuous intravenous infusion of bethanechol chloride (Urecholine) in increasing doses ranging from 0.25 mg per hr to 4 mg per hr, there was no secretory response (fig. 6). A single subcutaneous injection (2.0 mg) of bethanechol also failed to increase secretory rate (six experiments in 3 dogs). In a further series of experiments (six experiments in 2 dogs) using a continuous intra150 U VITRUM
+
J
~
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e
w
SALINE
~
3
HISTAMINE DIHYDROCHLORIDE 2.0 mg/hr. I.V.
2
0
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15 MINUTE COLLECTION PERIODS
FIG. 3. Response to single intravenous injections of Boots and Vitrum secretin (upp er diagram) and a continuous infusion of histamine (lower diagram).
BRUNNER'S GLAND POUCHES
October 1966
CECEKIN VITRUM 75U
509
SECRETIN VITRUM 300 U
l
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FUNDIC GLAND
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JEJUNAL
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CRUDE GASTRIN(ANTRAL EXTRACT)
PERIODS
FIG. 4. Effects of single intravenous inj ections of Vitrum Cecekin, Vitrum secretin; oxyntic gland, pyloric gland, and jejunal mucosal extracts. Each block is the mean of six experiments in 3 dogs.
!
1
SALINE
CRUDE GASTRIN 40 G/hr. IY.
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GASTRIN I 40)Jg/hr. I.V.
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......__,......<-L--''--.........,_L_,....._..._..,_~-L.z-L-,
15 MINUTE COLLECTION PERIODS
FIG. 5. Response to continuous intravenous infusion of crude hog gastrin and gastrin I. Each block is the mean of nine experiments in 3 dogs (crude gastrin) and six experiments in 3 dogs (gastrin I); the vertical bars are the standard error of the mean.
venous infusion of bethanechol (2.0 mg per hr), juice was collected for pepsin estimation. Bethanechol caused no increase in pepsin concentration or output. (Mean pepsin output before bethanechol was 4.9
units per 30 min; after bethanechol 5.2 units per 30 min.) Electrolyte content of Brunner's glands juice. Thirty samples of juice (basal and following feeding) were examined for elec-
510 T ABLE
Vol. 51, No.4
COOKE AND GROSSMAN 1. The effect of vm·ious agents on sec1·etion and moti li ty %
Agent
Dose
% No. of change, change, secre- motility tests tion -- -
Secretin (Boots) . .. Secretin (Vitru m) . Cru de gastrin . ... . Gastri n I. . . . . . ... Cecekin (Vitrum). Oxyntic gland extract ......... . . . J ej unal extract . ... Bethanechol ... . ... Food ..... . ..... . . .
u u
40 l'g/hr 75 u
132 12 160 14 106
50 g 50 g 2 mg/ hr 3 lb
68 120 - 13 150
95 75
40 g/ hr
- -200 6 -10 5 150 6 2 2 70 3 50 95 78 95
3 3 3 5
given as a continuous intravenous infusion caused a mean incr ease of 78% in motility which continued t hroughout the infusion but had no effect on secret ory rate. In three experiments in 1 dog which had both a pouch fistula of the first part of the duodenum (Brunner's gland pouch) as well as a pouch fistula of the fourth part of the duodenum, feeding produced a motility response from the Brunner's gland pouch but not from the pouch of the fourth part of t he duodenum (fi g. 9). Discussion
trolyte content. The concentrat ions in milliequivalents per liter were : sodium, mean 145 (range 136 to 150) ; potassium, mean 6.3 (range 4.5 to 8.0) ; chloride, mean 136 (range 130 to 140) ; bicarbonate, mean 17 (range 14 to 22). The sum of cations was approximately equal t o the sum of anions. Relationship between vo lume and bicarbonate concentration. The bicarbonate concentration was found to range between 16 and 42 mEq per liter in response to a variety of stimuli (food, crude gastrin , and Boots secretin) and was unrelated t o t he volume rate of secretion (fig. 7) .
Combined Motility and Secre tory Studies All agents that caused an increase in secretion were associated with an increase in motility (table 1). Gastrin I and Vitrum secretin caused neither an increase in secretion nor motility. Typical motility patterns are seen in figure 8. Bethanechol
It has been clearly established that the duodenal glands of Brunner have a hormonal mechanism of control but the n ature of this hormone is unknown. 1 • 11 • 12 Florey and Harding 2 as well as Fogelson and Bachrach 13 have shown t hat impure intestinal extracts containing secretin or secretin-like material will stimulate a flow of juice from these glands. Sonnenschein and co-workers/ 2 using a highly purified secretin extract, showed t hat it was without significant effe ct on Brunner's glands. However, it was not shown in those experiments that the preparation used was in fact an active pancreatic stimulant. In the present study we have confirmed the findings of Fogelson and Bachrach 13 that a relatively crude extract of secretin will stimulate Brunner's glands and that this effect is dose related (fig. 2). We have also shown that fundi c, antral as well as jejunal mucosal extracts are effective stimulants (figs. 4 and 5, table 1). Secretin Vitrum, however, from a batch shown to be an effective stimulus to pancreatic se-
URECHOLINE
1.0 mg/hr.
2.0 mg/hr.
4.0 mg/hr.
15 MINUTE COLLECTION PERIODS
Fw. 6. R esponse to increasing doses of bethanechol chloride (Urecholine).
October 1966
511
BRUN N ER'S GLA N D POUCHES 50
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0.0
0.4
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1.6
2.0
2.4
2.8
3.2
VOLUME ml/15 min.
FIG. 7. Scatter diagram of bicarbonate concentration of juice from Brunner's glands and volume rate of secretion. E ach point represents one estimation. BOOTS SECRETIN 95 U
VITRUM SECRETIN 75 U
~
~1~~.L.lltlLl~JJJ~c~,~-~~~~.J}JJ!I!WJAlui~U~l,l~•ltutWlhlluJJ--1UJ)JJJUJllillJJiliiJJJlli.JJJwAJ~~~lJJJJ. BETHANECHOL
CHLORIDE 2
mg/hr
CECEKIN 75 U
llo~Jl;wJJJWI~~~.~~ FUNDIC EXTRACT 50 11
.J_LWJ.wli1.~~JlJ~~Uwull~JilltWllWlhllwlllk~u~ FIG. 8. Effect of various agents on the motility of Brunner's gland pouches.
cretion in other dogs in our laboratory, 14 did not stimulate Brunner's glands (figs. 3 and 4). Further evidence that secretin is not the hormone regulating Brunner's glands was obtained from experiments with histamine (fig. 3). Infusion of this drug in a dose adequate to stimulate gastric se-
cretion maximally produced no response from Brunner's gland pouches. Since acid from the stomach had free passage into the duodenum and jejunum it can be reasonably assumed that endogenous secretin was · released. 15 In dogs with pancreatic fistulas stimulation of gastric se-
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Vol. 51, No .4
COOKE AND GROSSMAN FOOD
~
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Fra. 9. Motility response to feeding of a pouch of Brunner's glands (upper tracing) and a pouch of the fourth part of the duodenum in the same animal (lower tracing).
cretion by histamine has been shown to cause release of secretin when the gastric acid is allowed to fiovv into the duodenum. 16 It would seem likely from this evidence that secretin is not t he hormone regulating Brunner's glands. Furthermore, the pure polypeptide gastrin I was also an ineffective stimu!ant of Brunner's glands (fig. 5). Thus gastrin is not the hormone regulating Brunner's glands. Cecekin stimulated a flow of juice from Brunner 's gland pouches (fig. 4, table 1). However, t his preparation is too impure to implicate pancreozymin or cholecystokinin as the hormone regulating Brunner's glands. Whether the activity of the various crude mucosal extracts is due to their containing t he hormone for Brunner's glands or to nonspecific stimulants cannot be resolved at present. We do not know which mucosal region or regions release stimulants of Brunner's glands in response to feeding. Fogelson and Bachrach13 suggested that secretion was secondary to an increase in motility since t hey were unable to separate a secretory response from a motor one. Blickenstaff and co-workers3 also studied secretion and motility and postulated from their work and that of previous investigators that t here may be two factors; one stimulating secretion directly, the other stimulating motility. In the present investigations all substances that stimulated secretion were associated with a increase in motility (table 1). Bethanechol chloride (Urecholine), however, increased motility but not secretion when given intravenously (fig. 8 and table 1) . Subcutaneously administered bethanechol (2 .0 mg) also had no effect on secretion. These findin gs wit h bethanechol differ from those of Blicken-
staff et al., who found t hat a single subcutaneous injection (2.0 mg) stimulated secretion as well as motility. We cannot explain this discrepancy. The results of our investigation show that a stimulus for motility does not necessarily cause a secondary increase in secretion. However, whenever secretion was increased an increase in motility occurred. The question of two hormonal factors (motor and secretory) raised by Blickenstaff et aJ.3 has not been resolved by these studies. In the experiments with two duodenal pouches the Brunner's gland pouch responded to food but the pouch of the fourth part of the duodenum did not (fig. 9) . This study provides further evidence that Brunner's glands behave different ly from other parts of the small intestine since it is generally agreed that loops of small intestine show neither an increase in secretion nor motility following a meal.H Although a number of early workers 18 - 22 found a variety of enzymes present in juice from Brunner's gland pouches, careful work by Wright et al.U discounted these findin gs. The results of the present study confirm the presence of pepsin in low concentration, a finding first described by Ponomarev. 18 P epsin output increased in response to feeding (fig. 1) , a finding recently confirmed by Preshaw and Knauf (unpublished data). Seijffers et al. 23 have shown that pepsinogen I (the precursor of pepsin I) is present in duodenal mucosal extracts from human subjects. Whether t he tissue from which these extracts were made contained Brunner's glands is not stat e(:!. · It is of interest to compare the pyloric gland area of the stomach with the duodenal glands of Brunner. Bot h secrete
TABLE
513
BRUNNER'S GLAND POUCHES
October 1966
2. Comparison of electrolyte composition of alkaline juice obtained from oxyntic gland area, pyloric glands, and B1·unner's glands•
Secretions
Oxyntic alkaline Pyloric glands Brunner's glands
Sodium
Potassium
Chloride
Bicarbonate
133 (114-148) 151 (135-162) 145 (136- 150)
4.4 (2 .5-6 . 9) 9.2 (5.1- 12.5) 6 . 3 (4.5-8.0)
120 (96-144) 145 (133-158) 136 (130-140)
13.1 (6.5- 29.8) 8.0 (3.3-12.1) 17.0 (14-22)
Reference
27 26 Present study
• The values are the mean (and range) in milliequivalents per liter.
small amounts of pepsin even though pyloric gland areas of stomach and from neither has chief cells. 4 • 24 In contrast to duodenum and jejunum stimulated secrethe pyloric glands, Brunner's glands show tion and motility. Neither the pure an increase in pepsin output in response polypeptide gastrin I, nor exogenous nor to food (fig. 1). Bethanechol chloride did endogenous secretin had any effect on not increase volume rate of secretion or motility or secretion. Bethanechol produced pepsin output in either the pyloric glands 4 a motility response but not a secretory reor Brunner's glands. sponse. These studies indicate that neither There have been no previous studies on the electrolyte composition of Brunner's gastrin nor secretin is the hormone regulatgland juice other than bicarbonate. Sodium ing Brunner's gland and that a secretory concentration was similar to that of plasma response is not secondary to primary motor but chloride and potassium concentra- stimulation. The electrolyte composition of Brunner's tions were higher. Bicarbonate concentration was unrelated to the volume rate of gland juice was examined. Sodium was secretion (fig. 7), thus differing from the found in concentration similar to blood pancreatic secretion. 25 Table 2 compares plasma; potassium and chloride concentrathe electrolyte concentrations of Brunner's tions were higher than blood plasma and gland juice from the present study with bicarbonate concentration was lower. those of pyloric gland juice and nonacid Bicarbonate concentration was unrelated oxyntic gland juice from previous studies. to secretory rate. Pepsin was present in low concentrations Juice from the pyloric glands 26 had concentrations of sodium and chloride slightly in the juice from Brunner's glands. higher than those present in Brunner's REFERENCES the mean bicarbonate concentration was 1. Florey, H. W., and H. E. Harding. 1935. A also greater in pyloric gland juice but humoral control of secretion of Brunner's the mean bicarbonate cocentration was glands. Proc. Roy. Soc. (Bioi.) 117: 68-77. significantly lower than values obtained 2. Florey, H. W., and H. E. Harding. 1935. The in Brunner's gland juice. In studies with nature of the hormone controlling Brunalkaline canine mucus from Heidenhain ner's glands. Quart. J. Exp. Physiol. '25: 27 pouches, Hollander found concentrations 329-339. of sodium, potassium, and chloride that were 3. Blickenstaff, D., M. I. Grossman, and A. C. all consistently lower than the values obIvy. 1949. Stimulating effect of sugar, fat tained from antral pouches and Brunner's and meat meals on duodenal secretion in the dog. Amer. J. Physiol. 158: 122-128. gland pouches; mean bicarbonate concentra4. Grossman, M. I., and I. N. Marks. 1960. Setion was intermediate between Brunner's cretion of pepsinogen by the pyloric glands gland and pyloric gland juice. Summary
Motility and secretion were studied in dogs with innervated pouches of Brunner's glands. Mucosal extracts from oxyntic and
of the dog, with some observations on the histology of the gastric mucosa. Gastroenterology 38: 343-352. 5. Gregory, R. A., and H. J. Tracy. 1961. The preparation and properties of gastrin. J. Physiol. 156: 523-543.
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6. Gillespie, I. E., and M. I. Grossman. 1963.
17. Gregory, R. A. 1962. Secretory mechanisms of
Inhibition of gastric secretion by extracts containing gastrin . Gastroenterology 44 :
the gastrointestinal tract. Edward Arnold, Ltd., London. Ponomarev, S. J . 1902. Physiology of Brunner's area of tbe duodenum. Thesis, St. Petersburg. Cited in B. P. Babkin. 1928. Die aussere Sekretion der Verdauungsdri.isen. J. Springer, Berlin. Pavlov, I. P ., and S. W. Parastschuk. 1904. Uber die und demselben Eiweisfermente zukommende proteolytische und milchkoagulierende Wirkung verschiedener Verdauungssafte. Hoppe Seyler Z. Physiol. Chern. 42: 415-452. Abderhalden, E., and P. Rona. 1906. Zur Kenntnis des proteolytischen Fermentes des Pylorus und des Duodenalsaftes. Hoppe Seyler Z. Physiol. Chern. 47: 359-365. Agren, G. 1942. The secretion of aminopolypeptidases by the pyloric and duodenal mucosa of the cat after injection of crystalline secretin. Arkiv Kemi 16: 1-10. Agren, G. 1942. The secretion of proteolytic enzymes into the duodenum of the cat after secretin injections. Enzymologia 10: 161-
301-310. 7. Jorpes, J. E., and V. Mutt. 1962. The gastro-
intestinal hormones secretin and cholecystokinin, p. 150-164. In A. V. S. de Reuck and M. P. Cameron [ed.], Ciba Foundation Symposium on the Exocrine Pancreas. J . and A. Churchill, Ltd., London. 8. Jorpes, J. E., and V. Mutt. 1961. On the bio. logical activity and amino acid composition of secretin . Acta Chern. Scand. 15:
18.
19.
1790-1791.
9. Jorpes, J. E., V. Mutt, S. Magnuson, and B. B. Steele. 1962. Amino acid composition and N-terminal amino acid sequence of porcine secretin. Biochem. Biophys. Res. Commun.
20.
9: 275-279. 10. Dhariwal, A. P . S., A. V. Schally, J. Meyer,
21.
D. C. H. Sun, J. E. Jorpes, and V. Mutt. 1963. Purification of pancreozymin. Gastroenterology 44: 316-321. 11. Wright, R. D., M. A. J ennings, H. W. Florey, and R. Lium. 1940. The influence of nerves and drugs on secretion by the small intestine and an investigation of the enzymes in intestinal juice. Quart. J . Exp. Physiol. 30: 73-120. 12. Sonnenschein, R. R., M. I. Grossman, and A. C. Ivy. 1947. The humoral regulation of
Brunner's glands. Acta Med. Scand. (Suppl.) 196: 296-307. 13·. Fogelson, S. J ., and W. H. Bachrach. 1939.
Response of Brunner's glands to secretin. Amer. J. Physiol. 128 : 121-123. 14. Preshaw, R. M., and M. I. Grossman. 1965. Stimulation of pancreatic secretion by extracts of the pyloric gland area of the stomach. Gastroenterology 48: 36-44. 151 Wang, C. C., and M. I. Grossman. 1951. Physiological determination of release of secretin and pancreozymin from the intestine of dogs with transplanted pancreas. Amer. J. Physiol. 164: 527-545. 16. Preshaw, R. M., A. R. Cooke, and M . I. Grossman. 1966. Quantitative aspects of the response of the canine pancreas to duodenal acidification. Amer. J. Physiol. 210: 629-634.
22.
164. 23. Seijffers, M. J., H. L. Segal, and L. L. Miller. 1963. Separation of pepsin I, pepsin IIA,
pepsin liB and pepsin III from human gastric mucosa. Amer. J. Physiol. 205 : 10991105. 24. Bensley, R. R. 1903. The structure of the
glands of Brunner. Decennial Publication, University of Chicago 10: 279-329. 25. Bra-Rasmussen, F., S.-A. Killman, and J. H. Thaysen. 1956. The composition of pancreatic juice as compared to sweat, parotid saliva and tears. Acta Physiol. Scand. 37: 97-113. 26. Grossman, M. I. 1959. The secretion of the pyloric glands of the dog, p. 226-228. In
Symposia and Special Lectures. XXI International Congress of Physiological Sciences, Buenos Aires. 27. Hollander, F. 1963. The electrolyte pattern of gastric mucinous secretions; its implication for cystic fibrosis. Ann. N. Y. Acad. Sci. 106: 757-766.
Vol. 51, No.4 Printed in U.S.A .
Copyright © 1966 by The Williams & Wilkins Co.
STUDIES ON THE SECRETION AND MOTILITY OF BRUNNER'S GLAND POUCHES A. R. CooKE, M.R .A.C.P.,
AND
M. I. GRossMAN, M .D., PH .D.
Resea1'ch, Veterans Administ1'ation Center and Department of Physiology, University of Califomia Cente1' fo 1' the H ealth Sciences, Los Angeles, California
In 1935 Florey and Harding1 showed that the secretory activity of Brunner's glands was controlled by a humoral mechanism, but the hormone has not yet been identified. Florey and Harding 2 believed the agent to be secretin but subsequently Blickenstaff et aJ.3 presented evidence to show that there were possibly two humoral factors-one stimulating secretion directly and the other increasing secretion indirectly by stimulating motility. The purpose of this study was to investigate further the nature of the hormonal agent or agents regulating Brunner's glands using various mucosal extracts, including preparations of gastrin, secretin, and pancreozymin.
Methods Three mongrel dogs were used . Under sodium pentobarbital anesthesia the abdominal cavity was opened and a double-walled mucosal septum was made at the pylorus. The duodenum was divided just proximal to the entry of the bile duct and the distal end of the first part of the duodenum was brought through a separate small incision in the abdominal wall as the pouch fistula. The duodenal stump was closed and intestinal continuity was reestablished by a side-to-side gastroduodenostomy. For another series of experiments, 1 animal was Received April 11, 1966. Accepted June 7, report of this work has been published in Gastroenterology 48 : 864, 1965. Address requests for reprints to: Dr. M. I. Grossman, Veterans Administration Center, Los Angeles, California 90073. This work was supported in part by National Science Foundation Grant GB 667. The authors wish to thank Professor R. A. Gregory, Liverpool, for his gift of gastrin I, and Ray Lichter and John Washington for technical assistance. 1966. A preliminary
506
prepared with the same kind of pouch of Brunner's glands plus a pouch of the fourth part of the duodenum. Experiments were not started until 4 to 6 weeks after surgery. Before each experiment, the animals were fasted for at least 18 hr. During tests the dogs stood in a sling harness. The juice was collected by a plastic funnel which led to a graduated centrifuge tube . The funnel surrounded but did not touch the e;<;posed mucosa of the opening of the pouch. Basal secretion was collected for four 15-min periods and then the test agents were given either by a single intravenous injection or as a constant intravenous infusion. The test substances were dissolved in 0.15 M sodium chloride and a constant infusion of 30 ml per hr was maintained by means of a peristaltic pump (Harvard Apparatus Company, Dover, Mass.). Combined motility and secretory studies were done in a number of instances. A waterfilled balloon (10 mm by 5 mm) was inserted into the pouch and was connected to a strain gauge (Statham, P23BB) by means of a waterfilled polythene tube. The contractions were recorded on a recording potentiometer (Sargent and Company, Chicago, Ill.) set at a paper speed of Y2 inch per min. The balloon was kept in position by attaching the polythene tube to the collecting funnel. A motility index was determined by multiplying the height by the numbed of contractions during three separate 5min periods before and after the test agent was given. The average of t he before and after periods was calculated and expressed as t he mean motility index (MI). Percentage change of motility was expressed as follows: o/o change of motility [(Mia - Mib) j Mib] X 100; Mia mean motility index after test drug; Mib mean motility index before test drug. Percentage change of secretion was determined using the formula : o/o change of secretion = [ (Va - Vb) j Vb] X 100;
=
= =
507
BRUNNER'S GLAND POUCHES
October 1966
= =
Va mean volume during 15-min period after giving the test agent; Vb mean volume during 15-min period before giving the test agent. The volume of juice collected every 15 min was recorded to the nearest 0.1 ml. The bicarbonate concentration was measured by adding 0.5 ml of juice (0.2 ml if 15-min collection was less than 0.5 ml) to 1.0 ml of 0.1 N HCl and backtitrating the mixture to pH 7 with 0.2 N NaOH with an automatic titrator (Radiometer, Copenhagen) . In some experiments portions of two consecutive 15-min samples were combined, and their pepsin concentration was determined using radioiodinated serum albumin as substrate.• Sodium and potassium concentrations were determined by flame photometry (Zeiss, Germany) and chloride by a chloridometer (Buchler Instruments, Inc., New J ersey). Mate rials. Two preparations of gastrin were used, the pure polypeptide porcine gastrin I and a relatively crude preparation prepared from t he mucosa of the pyloric gland area of hogs by modification of the method of Gregory and Tracy• described by Gillespie and Grossman.• The dose of crude gastrin is expressed as grams of wet weight of mucosa from which the extract was obtained. Extracts of oxyntic gland mucosa and the proximal 3 feet of small
bowel of the hog were also prepared using the method for preparing crude gastrin."· • Two commercial preparations of secretin were used; a relatively crude product (Boots Pure Drug Company, Nottingham, England) and a preparation (Vitrum, Stockholm) which Jorpes and Mutt (personal communication) state is identical with an apparently pure polypeptide described by them.7..., Cecekin (Vitrum) was used as a source of pancreozymin. This preparation is not chemically homogeneous and contains cholecystokinin as well as some secretin-like material.10 Other test substances used were histamine dihydrochloride, bethanechol chloride (U recholine, Merck, Sharp and Dohme) and a proprietary dog food (Friskies, Carnation Company, California).
Results
Secretory Studies R esponse to a meal. When the animals were given 1 pound of dog food (Friskies) there was no secretory response. However, when each animal was allowed to eat as much as it desired (usually 3 to 4 pounds) a clear increase in volume occurTed. The response began within 15 min, reached a
14
FOOD
~
12
...__. OUTPUT UNITS/30' ._ ___ CONC. U/ml •·-·-• VOLUME ml
10
8
6
4
2
0+----.-----.----.----,r----.----.-----.----, 0
2 4 6 30 MINUTE COLLECTION PERIODS
8
FIG. 1. Pepsin and volume response of Brunner's gland pouches to a meal. Each point in this and subsequent figures, unless otherwise stated, is the mean of nine experiments in 3 dogs; the vertical bars are the standard error of the mean.
508
COOKE AND GROSSMAN
2
0
f
I
10
20
I I 40
80
l 160
UNITS
SECRETIN (BOOTS)
FIG. 2. Responses to graded single intravenous doses of Boots secretin.
peak at 30 min, and was complete by 180 min (fig. 1). Pepsin concentration and output were significantly increased in response to food (fig. 1). Response to secretin. Rapid intravenous injections of Boots secretin evoked a dose~elated secretory response (fig. 2). In contrast Vitrum secretin produced no response (fig. 3). When a continuous intravenous infusion of histamine dihydrochloride (2.0 mg per hr) was given no secretory response was obtained. Similar results were found using 4.0 mg per hr.
Vol. 51, No.4
Response to crude gastrin and gastrin I. Crude hog gastrin given as a single intravenous injection (40 g) evoked a short-lived secretory response (fig. 4). When given as a continuous intravenous infusion (40 g per hr) a sustained secretory response was obtained (fig. 5). The pure polypeptide, gastrin I, was an ineffective stimulus (fig. 5). The same batch of pure gastrin was shown in other dogs with Heidenhain pouches to produce strong stimulation of acid secretion at this dose level (40 p.g per hr). Response to other preparations. Vitrum Cecekin (75 units) given as a single intravenous injection was found to be an effective stimulant of Brunner's glands as were fundic and jejunal mucosal extracts (fig. 4 and table 1). Response to bethanechol chloride. When the animals were given a continuous intravenous infusion of bethanechol chloride (Urecholine) in increasing doses ranging from 0.25 mg per hr to 4 mg per hr, there was no secretory response (fig. 6). A single subcutaneous injection (2.0 mg) of bethanechol also failed to increase secretory rate (six experiments in 3 dogs). In a further series of experiments (six experiments in 2 dogs) using a continuous intra150 U VITRUM
+
J
~
~------------------------------------------------~
.....
e
w
SALINE
~
3
HISTAMINE DIHYDROCHLORIDE 2.0 mg/hr. I.V.
2
0
>
15 MINUTE COLLECTION PERIODS
FIG. 3. Response to single intravenous injections of Boots and Vitrum secretin (upp er diagram) and a continuous infusion of histamine (lower diagram).
BRUNNER'S GLAND POUCHES
October 1966
CECEKIN VITRUM 75U
509
SECRETIN VITRUM 300 U
l
l 2
c:
.E o -'-----------------==:......._j I{) .....
E ~
FUNDIC GLAND
:
~
15 MINUTE
JEJUNAL
COLLECTION
CRUDE GASTRIN(ANTRAL EXTRACT)
PERIODS
FIG. 4. Effects of single intravenous inj ections of Vitrum Cecekin, Vitrum secretin; oxyntic gland, pyloric gland, and jejunal mucosal extracts. Each block is the mean of six experiments in 3 dogs.
!
1
SALINE
CRUDE GASTRIN 40 G/hr. IY.
c]~
·e
10
::::::
e ILl
:E
3
~
2
l
I
SALINE
_L -
GASTRIN I 40)Jg/hr. I.V.
T
-
0 · '~
80)Jg/hr. I.V.
......__,......<-L--''--.........,_L_,....._..._..,_~-L.z-L-,
15 MINUTE COLLECTION PERIODS
FIG. 5. Response to continuous intravenous infusion of crude hog gastrin and gastrin I. Each block is the mean of nine experiments in 3 dogs (crude gastrin) and six experiments in 3 dogs (gastrin I); the vertical bars are the standard error of the mean.
venous infusion of bethanechol (2.0 mg per hr), juice was collected for pepsin estimation. Bethanechol caused no increase in pepsin concentration or output. (Mean pepsin output before bethanechol was 4.9
units per 30 min; after bethanechol 5.2 units per 30 min.) Electrolyte content of Brunner's glands juice. Thirty samples of juice (basal and following feeding) were examined for elec-
510 T ABLE
Vol. 51, No.4
COOKE AND GROSSMAN 1. The effect of vm·ious agents on sec1·etion and moti li ty %
Agent
Dose
% No. of change, change, secre- motility tests tion -- -
Secretin (Boots) . .. Secretin (Vitru m) . Cru de gastrin . ... . Gastri n I. . . . . . ... Cecekin (Vitrum). Oxyntic gland extract ......... . . . J ej unal extract . ... Bethanechol ... . ... Food ..... . ..... . . .
u u
40 l'g/hr 75 u
132 12 160 14 106
50 g 50 g 2 mg/ hr 3 lb
68 120 - 13 150
95 75
40 g/ hr
- -200 6 -10 5 150 6 2 2 70 3 50 95 78 95
3 3 3 5
given as a continuous intravenous infusion caused a mean incr ease of 78% in motility which continued t hroughout the infusion but had no effect on secret ory rate. In three experiments in 1 dog which had both a pouch fistula of the first part of the duodenum (Brunner's gland pouch) as well as a pouch fistula of the fourth part of the duodenum, feeding produced a motility response from the Brunner's gland pouch but not from the pouch of the fourth part of t he duodenum (fi g. 9). Discussion
trolyte content. The concentrat ions in milliequivalents per liter were : sodium, mean 145 (range 136 to 150) ; potassium, mean 6.3 (range 4.5 to 8.0) ; chloride, mean 136 (range 130 to 140) ; bicarbonate, mean 17 (range 14 to 22). The sum of cations was approximately equal t o the sum of anions. Relationship between vo lume and bicarbonate concentration. The bicarbonate concentration was found to range between 16 and 42 mEq per liter in response to a variety of stimuli (food, crude gastrin , and Boots secretin) and was unrelated t o t he volume rate of secretion (fig. 7) .
Combined Motility and Secre tory Studies All agents that caused an increase in secretion were associated with an increase in motility (table 1). Gastrin I and Vitrum secretin caused neither an increase in secretion nor motility. Typical motility patterns are seen in figure 8. Bethanechol
It has been clearly established that the duodenal glands of Brunner have a hormonal mechanism of control but the n ature of this hormone is unknown. 1 • 11 • 12 Florey and Harding 2 as well as Fogelson and Bachrach 13 have shown t hat impure intestinal extracts containing secretin or secretin-like material will stimulate a flow of juice from these glands. Sonnenschein and co-workers/ 2 using a highly purified secretin extract, showed t hat it was without significant effe ct on Brunner's glands. However, it was not shown in those experiments that the preparation used was in fact an active pancreatic stimulant. In the present study we have confirmed the findings of Fogelson and Bachrach 13 that a relatively crude extract of secretin will stimulate Brunner's glands and that this effect is dose related (fig. 2). We have also shown that fundi c, antral as well as jejunal mucosal extracts are effective stimulants (figs. 4 and 5, table 1). Secretin Vitrum, however, from a batch shown to be an effective stimulus to pancreatic se-
URECHOLINE
1.0 mg/hr.
2.0 mg/hr.
4.0 mg/hr.
15 MINUTE COLLECTION PERIODS
Fw. 6. R esponse to increasing doses of bethanechol chloride (Urecholine).
October 1966
511
BRUN N ER'S GLA N D POUCHES 50
..
__J
& w E
40
... I
<.)
6
30
.: I
:
I
••
(.)
w ~ z
20
0
(D
0::
;3
10
(D
0+----.----.---~---.----.----,----,---~
0.0
0.4
0.8
1.2
1.6
2.0
2.4
2.8
3.2
VOLUME ml/15 min.
FIG. 7. Scatter diagram of bicarbonate concentration of juice from Brunner's glands and volume rate of secretion. E ach point represents one estimation. BOOTS SECRETIN 95 U
VITRUM SECRETIN 75 U
~
~1~~.L.lltlLl~JJJ~c~,~-~~~~.J}JJ!I!WJAlui~U~l,l~•ltutWlhlluJJ--1UJ)JJJUJllillJJiliiJJJlli.JJJwAJ~~~lJJJJ. BETHANECHOL
CHLORIDE 2
mg/hr
CECEKIN 75 U
llo~Jl;wJJJWI~~~.~~ FUNDIC EXTRACT 50 11
.J_LWJ.wli1.~~JlJ~~Uwull~JilltWllWlhllwlllk~u~ FIG. 8. Effect of various agents on the motility of Brunner's gland pouches.
cretion in other dogs in our laboratory, 14 did not stimulate Brunner's glands (figs. 3 and 4). Further evidence that secretin is not the hormone regulating Brunner's glands was obtained from experiments with histamine (fig. 3). Infusion of this drug in a dose adequate to stimulate gastric se-
cretion maximally produced no response from Brunner's gland pouches. Since acid from the stomach had free passage into the duodenum and jejunum it can be reasonably assumed that endogenous secretin was · released. 15 In dogs with pancreatic fistulas stimulation of gastric se-
512
Vol. 51, No .4
COOKE AND GROSSMAN FOOD
~
,........ lm in.
~~~\,__,..l~......._,j,_JJwJ.tJI~\.o....,..,.,'"""-"V~h\..c._jJ...LJ
l.wl.
. .,
o
, .. ,.
!....~
Fra. 9. Motility response to feeding of a pouch of Brunner's glands (upper tracing) and a pouch of the fourth part of the duodenum in the same animal (lower tracing).
cretion by histamine has been shown to cause release of secretin when the gastric acid is allowed to fiovv into the duodenum. 16 It would seem likely from this evidence that secretin is not t he hormone regulating Brunner's glands. Furthermore, the pure polypeptide gastrin I was also an ineffective stimu!ant of Brunner's glands (fig. 5). Thus gastrin is not the hormone regulating Brunner's glands. Cecekin stimulated a flow of juice from Brunner 's gland pouches (fig. 4, table 1). However, t his preparation is too impure to implicate pancreozymin or cholecystokinin as the hormone regulating Brunner's glands. Whether the activity of the various crude mucosal extracts is due to their containing t he hormone for Brunner's glands or to nonspecific stimulants cannot be resolved at present. We do not know which mucosal region or regions release stimulants of Brunner's glands in response to feeding. Fogelson and Bachrach13 suggested that secretion was secondary to an increase in motility since t hey were unable to separate a secretory response from a motor one. Blickenstaff and co-workers3 also studied secretion and motility and postulated from their work and that of previous investigators that t here may be two factors; one stimulating secretion directly, the other stimulating motility. In the present investigations all substances that stimulated secretion were associated with a increase in motility (table 1). Bethanechol chloride (Urecholine), however, increased motility but not secretion when given intravenously (fig. 8 and table 1) . Subcutaneously administered bethanechol (2 .0 mg) also had no effect on secretion. These findin gs wit h bethanechol differ from those of Blicken-
staff et al., who found t hat a single subcutaneous injection (2.0 mg) stimulated secretion as well as motility. We cannot explain this discrepancy. The results of our investigation show that a stimulus for motility does not necessarily cause a secondary increase in secretion. However, whenever secretion was increased an increase in motility occurred. The question of two hormonal factors (motor and secretory) raised by Blickenstaff et aJ.3 has not been resolved by these studies. In the experiments with two duodenal pouches the Brunner's gland pouch responded to food but the pouch of the fourth part of the duodenum did not (fig. 9) . This study provides further evidence that Brunner's glands behave different ly from other parts of the small intestine since it is generally agreed that loops of small intestine show neither an increase in secretion nor motility following a meal.H Although a number of early workers 18 - 22 found a variety of enzymes present in juice from Brunner's gland pouches, careful work by Wright et al.U discounted these findin gs. The results of the present study confirm the presence of pepsin in low concentration, a finding first described by Ponomarev. 18 P epsin output increased in response to feeding (fig. 1) , a finding recently confirmed by Preshaw and Knauf (unpublished data). Seijffers et al. 23 have shown that pepsinogen I (the precursor of pepsin I) is present in duodenal mucosal extracts from human subjects. Whether t he tissue from which these extracts were made contained Brunner's glands is not stat e(:!. · It is of interest to compare the pyloric gland area of the stomach with the duodenal glands of Brunner. Bot h secrete
TABLE
513
BRUNNER'S GLAND POUCHES
October 1966
2. Comparison of electrolyte composition of alkaline juice obtained from oxyntic gland area, pyloric glands, and B1·unner's glands•
Secretions
Oxyntic alkaline Pyloric glands Brunner's glands
Sodium
Potassium
Chloride
Bicarbonate
133 (114-148) 151 (135-162) 145 (136- 150)
4.4 (2 .5-6 . 9) 9.2 (5.1- 12.5) 6 . 3 (4.5-8.0)
120 (96-144) 145 (133-158) 136 (130-140)
13.1 (6.5- 29.8) 8.0 (3.3-12.1) 17.0 (14-22)
Reference
27 26 Present study
• The values are the mean (and range) in milliequivalents per liter.
small amounts of pepsin even though pyloric gland areas of stomach and from neither has chief cells. 4 • 24 In contrast to duodenum and jejunum stimulated secrethe pyloric glands, Brunner's glands show tion and motility. Neither the pure an increase in pepsin output in response polypeptide gastrin I, nor exogenous nor to food (fig. 1). Bethanechol chloride did endogenous secretin had any effect on not increase volume rate of secretion or motility or secretion. Bethanechol produced pepsin output in either the pyloric glands 4 a motility response but not a secretory reor Brunner's glands. sponse. These studies indicate that neither There have been no previous studies on the electrolyte composition of Brunner's gastrin nor secretin is the hormone regulatgland juice other than bicarbonate. Sodium ing Brunner's gland and that a secretory concentration was similar to that of plasma response is not secondary to primary motor but chloride and potassium concentra- stimulation. The electrolyte composition of Brunner's tions were higher. Bicarbonate concentration was unrelated to the volume rate of gland juice was examined. Sodium was secretion (fig. 7), thus differing from the found in concentration similar to blood pancreatic secretion. 25 Table 2 compares plasma; potassium and chloride concentrathe electrolyte concentrations of Brunner's tions were higher than blood plasma and gland juice from the present study with bicarbonate concentration was lower. those of pyloric gland juice and nonacid Bicarbonate concentration was unrelated oxyntic gland juice from previous studies. to secretory rate. Pepsin was present in low concentrations Juice from the pyloric glands 26 had concentrations of sodium and chloride slightly in the juice from Brunner's glands. higher than those present in Brunner's REFERENCES the mean bicarbonate concentration was 1. Florey, H. W., and H. E. Harding. 1935. A also greater in pyloric gland juice but humoral control of secretion of Brunner's the mean bicarbonate cocentration was glands. Proc. Roy. Soc. (Bioi.) 117: 68-77. significantly lower than values obtained 2. Florey, H. W., and H. E. Harding. 1935. The in Brunner's gland juice. In studies with nature of the hormone controlling Brunalkaline canine mucus from Heidenhain ner's glands. Quart. J. Exp. Physiol. '25: 27 pouches, Hollander found concentrations 329-339. of sodium, potassium, and chloride that were 3. Blickenstaff, D., M. I. Grossman, and A. C. all consistently lower than the values obIvy. 1949. Stimulating effect of sugar, fat tained from antral pouches and Brunner's and meat meals on duodenal secretion in the dog. Amer. J. Physiol. 158: 122-128. gland pouches; mean bicarbonate concentra4. Grossman, M. I., and I. N. Marks. 1960. Setion was intermediate between Brunner's cretion of pepsinogen by the pyloric glands gland and pyloric gland juice. Summary
Motility and secretion were studied in dogs with innervated pouches of Brunner's glands. Mucosal extracts from oxyntic and
of the dog, with some observations on the histology of the gastric mucosa. Gastroenterology 38: 343-352. 5. Gregory, R. A., and H. J. Tracy. 1961. The preparation and properties of gastrin. J. Physiol. 156: 523-543.
514
COOKE AND GROSSMAN
Vol. 51, No.4
6. Gillespie, I. E., and M. I. Grossman. 1963.
17. Gregory, R. A. 1962. Secretory mechanisms of
Inhibition of gastric secretion by extracts containing gastrin . Gastroenterology 44 :
the gastrointestinal tract. Edward Arnold, Ltd., London. Ponomarev, S. J . 1902. Physiology of Brunner's area of tbe duodenum. Thesis, St. Petersburg. Cited in B. P. Babkin. 1928. Die aussere Sekretion der Verdauungsdri.isen. J. Springer, Berlin. Pavlov, I. P ., and S. W. Parastschuk. 1904. Uber die und demselben Eiweisfermente zukommende proteolytische und milchkoagulierende Wirkung verschiedener Verdauungssafte. Hoppe Seyler Z. Physiol. Chern. 42: 415-452. Abderhalden, E., and P. Rona. 1906. Zur Kenntnis des proteolytischen Fermentes des Pylorus und des Duodenalsaftes. Hoppe Seyler Z. Physiol. Chern. 47: 359-365. Agren, G. 1942. The secretion of aminopolypeptidases by the pyloric and duodenal mucosa of the cat after injection of crystalline secretin. Arkiv Kemi 16: 1-10. Agren, G. 1942. The secretion of proteolytic enzymes into the duodenum of the cat after secretin injections. Enzymologia 10: 161-
301-310. 7. Jorpes, J. E., and V. Mutt. 1962. The gastro-
intestinal hormones secretin and cholecystokinin, p. 150-164. In A. V. S. de Reuck and M. P. Cameron [ed.], Ciba Foundation Symposium on the Exocrine Pancreas. J . and A. Churchill, Ltd., London. 8. Jorpes, J. E., and V. Mutt. 1961. On the bio. logical activity and amino acid composition of secretin . Acta Chern. Scand. 15:
18.
19.
1790-1791.
9. Jorpes, J. E., V. Mutt, S. Magnuson, and B. B. Steele. 1962. Amino acid composition and N-terminal amino acid sequence of porcine secretin. Biochem. Biophys. Res. Commun.
20.
9: 275-279. 10. Dhariwal, A. P . S., A. V. Schally, J. Meyer,
21.
D. C. H. Sun, J. E. Jorpes, and V. Mutt. 1963. Purification of pancreozymin. Gastroenterology 44: 316-321. 11. Wright, R. D., M. A. J ennings, H. W. Florey, and R. Lium. 1940. The influence of nerves and drugs on secretion by the small intestine and an investigation of the enzymes in intestinal juice. Quart. J . Exp. Physiol. 30: 73-120. 12. Sonnenschein, R. R., M. I. Grossman, and A. C. Ivy. 1947. The humoral regulation of
Brunner's glands. Acta Med. Scand. (Suppl.) 196: 296-307. 13·. Fogelson, S. J ., and W. H. Bachrach. 1939.
Response of Brunner's glands to secretin. Amer. J. Physiol. 128 : 121-123. 14. Preshaw, R. M., and M. I. Grossman. 1965. Stimulation of pancreatic secretion by extracts of the pyloric gland area of the stomach. Gastroenterology 48: 36-44. 151 Wang, C. C., and M. I. Grossman. 1951. Physiological determination of release of secretin and pancreozymin from the intestine of dogs with transplanted pancreas. Amer. J. Physiol. 164: 527-545. 16. Preshaw, R. M., A. R. Cooke, and M . I. Grossman. 1966. Quantitative aspects of the response of the canine pancreas to duodenal acidification. Amer. J. Physiol. 210: 629-634.
22.
164. 23. Seijffers, M. J., H. L. Segal, and L. L. Miller. 1963. Separation of pepsin I, pepsin IIA,
pepsin liB and pepsin III from human gastric mucosa. Amer. J. Physiol. 205 : 10991105. 24. Bensley, R. R. 1903. The structure of the
glands of Brunner. Decennial Publication, University of Chicago 10: 279-329. 25. Bra-Rasmussen, F., S.-A. Killman, and J. H. Thaysen. 1956. The composition of pancreatic juice as compared to sweat, parotid saliva and tears. Acta Physiol. Scand. 37: 97-113. 26. Grossman, M. I. 1959. The secretion of the pyloric glands of the dog, p. 226-228. In
Symposia and Special Lectures. XXI International Congress of Physiological Sciences, Buenos Aires. 27. Hollander, F. 1963. The electrolyte pattern of gastric mucinous secretions; its implication for cystic fibrosis. Ann. N. Y. Acad. Sci. 106: 757-766.