pouches and in Heidenhain pouches the responses to 2-deoxy-D-glucose and to food were established, as were also the responses to gastrin pentapeptide

Transcription

1 J. Physiol. (197), 27, pp With 9 text-ftgure8 Printed in Great Britain THE SECRETORY PATTERN OF THREE STOMACH PREPARATIONS IN THE RAT BY S. E. SVENSSON From the Institute of Physiology, University of Lund, Sweden (Received 11 August 1969) SUMMARY 1. In conscious rats provided with a gastric fistula, or a Pavlov pouch, or a Heidenhain pouch, the secretion of acid and pepsin was studied in the interdigestive state and in response to hog gastrin II. In Pavlov pouches and in Heidenhain pouches the responses to 2-deoxy-D-glucose and to food were established, as were also the responses to gastrin pentapeptide and methacholine in Heidenhain pouches. 2. Interdigestive secretion of acid and pepsin is substantial in the gastric fistula and the Pavlov pouch preparation, whereas in the Heidenhain pouch acid secretion is low or scanty, and the ratio pepsin/acid output is higher than in the innervated preparations. 3. Feeding evoked secretion of acid and pepsin in the Heidenhain and Pavlov pouches; the increase in pepsin secretion lasted much longer in the Heidenhain than in the Pavlov pouch. 4. Methacholine, infused in a threshold dose, augmented the acid output in response to food in the Heidenhain pouch, but did not affect the pepsin output. 5. Gastrin II (hog) evoked graded acid responses in the three stomach preparations, and the innervated preparations were more sensitive to gastrin II than the denervated one, whereas stimulation of pepsin secretion was apparent only in the Heidenhain pouch. 6. Methacholine, on infusion, in the Heidenhain pouch, gave a maximal acid secretory response, greater than twice that obtained by gastrin, and produced strong stimulation of pepsin. 7. Methacholine, infused in a threshold dose, accentuated the acid secretory response to graded doses of gastrin deoxy-D-glucose stimulated acid and pepsin secretion consistently in the Pavlov pouch, whereas in the Heidenhain pouch the response was scanty or absent. 9. A large dose of gastrin pentapeptide injected intravenously while a background secretion was maintained by gastrin II, produced in the

2 33 S. E. SVENSSON Heidenhain pouch triphasic changes in the sequence: stimulation, inhibition, stimulation. INTRODUCTION Gastric secretion in rats has mainly been studied under anaesthesia by perfusing the stomach via the oesophagus (Ghosh & Schild, 1958), or in conscious rats by ligating either the pylorus (Shay, Sun & Gruenstein, 1954) or also the oesophagus (Kim & Shore, 1963), with subsequent collection of gastric juice for a few hours only. The pylorus-ligated rat was originally used with the object of producing gastric ulceration (Shay, Komarov, Fels, Meranze, Gruenstein & Siplet, 1945). Secretory studies in the intact rat have been made by Thornton & Clifton (1959), employing the sucrose test meal of Hunt & Spurrell (1951), in which the time of observation by necessity is less than an hour. Lane, Ivy & Ivy (1957) employed a gastric fistula in conscious rats to record the secretory response to histamine. The Heidenhain pouch preparation was adapted to the rat by Alphin & Lin (1959), who in 1962 observed graded responses to food and histamine in this pouch. Finally, the Pavlov pouch operation has been performed in rats, and preliminary observations have been communicated to the Physiological Society (Svensson, 1969). Studies of gastric secretion in the rat appear now the more desirable, because in this species an association between acid secretion and changes in gastric mucosal histamine metabolism has been disclosed and extensively studied in this laboratory (see Rosengren & Svensson, 1969, for references). A further reason for studying gastric secretion in the rat lies in the fact that a standardized technique has now been evolved, which permits quantitative determinations of the gastric secretory responses to food and to individual secretory stimuli operating on feeding. METHODS Animal&. Male and female rats from a closed colony bred at the Institute of Physiology, Lund, were used throughout. The body weight of the males was 2-25 g and of the females g. The rats were fed on a standard pellet diet (type 142, Teknosan, Malmo, Sweden); they drank water freely and those provided with pouches had the choice of Tyrode solution. Drug&. One batch of hog gastrin II (a generous gift of Professor Gregory), gastrin pentapeptide (Peptavlon, ICI 5123, kindly supplied by Dr J. D. Fitzgerald) methacholine chloride (Fluka AG) and 2-deoxy-D-glucose (Sigma Chemical Co.) were used. Anaesthesia. Ether only was used in the surgical procedures. The vagus nerves were stimulated after chloralose, 1 mg/kg intravenously, preceded by ether.

3 GASTRIC SECRETION IN CONSCIOUS RATS 331 Stomach preparations. Food was withheld for 15 hr before operations, except for the gastric fistula in which case access to food before and after the operation was allowed. The post-operative treatment is essential for survival rate and was the same as described in detail elsewhere (Rosengren & Svensson, 1969) for rats provided with a Heidenhain or Pavlov pouch. The gastric fistula was prepared by a technique, the principle of which has been described by Lane et at. (1957), and the Heidenhain pouch was prepared as devised by Alphin & Lin (1959). This pouch comprises a small part of the squamous portion of the stomach, in which an indwelling cannula is placed, and a glandular part along the greater curvature. At the operation all blood connexions of the pouch were cut except the right gastro-epiploic artery. ThelHeidenhain pouch is generally believed to be vagally denervated because the pouch is completely separated from the rest of the stomach. In the present work it was occasionally noticed that the interdigestive secretion of a Heidenhain pouch, which initially was at a low and steady rate, increased with the passing of time. The reason for this change is not known; possible explanations may be reestablishment of secretory innervation, or increased sensitivity to endogenous gastrin, or both. Rats in which augmented interdigestive secretion occurred were excluded from further use. The absence of visual motor response in the pouch, and its presence in the main stomach under electrical stimulation of the vagus nerves in the neck, was also taken as evidence of effective vagal denervation (Kahlson, Rosengren & Svensson, 1968). As the technique of providing a Pavlov pouch in the rat has not been described before, a description of the operative procedure appears justified. In the main the procedure employed is an adaptation of two successful methods for making a Pavlov pouch in the dog (Gregory, Hallenbeck & Code, 1942; Thomas, 1942). The abdomen is opened by a mid line incision and the stomach gently delivered into the wound, whilst the peritoneal cavity is protected by gauze packs. A small incision, about 5 mm long, is made in the glandular part of the stomach, parallel to and near the greater curvature, equidistant from the antrum and the rumen. Through this incision a portion of the glandular stomach is turned inside out, a delicate procedure. At this stage a rosette of mucosal tissue presents itself as described by Thomas (1942) for the dog. Two sutures are now fixed at the two points destined to be the fundi of the pouch. By traction on the sutures in opposite directions two parallel ridges of mucosa stretched between the sutures are obtained. With a blunt pair of scissors two incisions are made along the ridges through the mucosa meeting each other only at the place of the two sutures. At this stage of the operation bleeding may be troublesome, but can be minimized by wrapping the gauze packs more tightly around the stomach. A small cuff of mucous membrane is dissected free from the underlying muscle at the edges of the now oval incision. The nearest edges of the cuffs are joined by interrupted sutures so as to invert the mucosa into the main stomach, and the remaining two mucosal cuffs are inverted into the pouch. Next, the pouch is turned into the main stomach and the original gastric incision is closed by sutures around the lip of a plastic cannula. Finally, the wings of the cannula are fixed to the peritoneal surface of the abdominal wall on each side of the mid line, and the abdomen is closed around the cannula. After many initial trials and failures, fifteen Pavlov pouch operations were successful; of these three were later lost by perforation of the double mucosal fold, two of them spontaneously in the first week after the operation and the third by rupture on rinsing the pouch to remove blood clots. Administration of secretory stimulants. Before an actual experiment the rats were fasted for 16 hr, but they were allowed to drink water and Tyrode solution. During the period of observation, in some instances for 15 hr, the rat was kept unanaesthetized in a restraining cage of the Bollman type. Rats, in which the response to a meal was studied, had been trained to eat in the Bollman cage, and food was available for

4 332 S. E. SVENSSON 3 min, during which time a hungry rat usually consumed 2-3 g of the pellet diet. Before any kind of stimulus was applied, interdigestive secretion was collected for 2-3 hr and collection of secretion was in 3 nin portions throughout. When the stimulant was administered by intravenous infusion, a vein was freed in the tail or the neck and a polyethylene tube was inserted, procedures which required ether anaesthesia for 5-1 min. The tube in the vein was connected to a motor-driven syringe, which delivered 9 % NaCl (w/v) at a rate of 3*5 ml./hr in order to maintain body fluid balance. In establishing dose-response curves, gastrin II or methacholine were infused in stepwise increasing doses, each dose for 9 min, and sequentially succeeded by twice the dose. When the dose-response curve for gastrin or feeding was super-. imposed on a background stimulation by methacholine, infusion of this stimulant was started 1 hr before the lowest dose of gastrin was infused or feeding was commenced. In testing inhibition by rapid injection ofgastrin pentapeptide, a submaximal plateau secretion was maintained by infusing gastrin or methacholine. Collection of gastric juice. Gastric juice was collected by direct drainage or by perfusion. In order to determine, by the simplest possible means, the interval between commencement of feeding and onset of the secretory reponse in a Pavlov pouch the drops of gastric juice falling from the cannula were recorded on a smoked drum. On the average there were 36 drops to 1 ml. water. In perfusing the pouch preparations a two-way plug was attached to the pouch cannula. One opening was connected via a polyethylene tube to the lower end of a narrow vertical glass tube. Into the upper open end of the glass tube 9 % NaCl was delivered at a rate of 3-5 ml./hr from a motor-driven syringe, and the perfusate was collected via another tube leading away from the pouch into a graduated centrifuge tube. A perfusing pressure of about 2 cm H2 was maintained by constant adjustment of the meniscus in the narrow glass tube. The flow was blocked occasionally, but this was easily recognized by the rising level of the meniscus, and the obstructing mucous clot could be removed by gentle suction. In the gastric fistula rats gastric juice was collected by direct drainage into graduated tubes, after the screw cap of the cannula had been removed; the stomach was repeatedly rinsed with warm -9 % NaCl for 45 min before collection of interdigestive secretion commenced. Determination of hydrochloric acid and pepsin. The volume of the fluid rinsing the pouch and of the gastric juice collected from the gastric fistula was determined and the peptic activity was estimated in 5 ml. of the perfusate and.1 ml. of the undiluted gastric juice. In the remainder, HCl was determined by titrating against.1 N-NaOH with phenol red as an indicator and the total amount of HCI secreted was expressed in,u-equiv/3 min. The peptic activity was determined bythe method of Hunt (1948), a gammaglobulin-free fraction being substituted for dehydrated human plasma of serum employed by Hunt. Pepsin output is expressed in jug, the activity of a commercial preparation (lot 95B-127, Sigma Chemical Co.) being the reference standard reconmmended by Bitsch (1966). For each new batch of substrate used, a standard curve was established, from which the concentration of pepsin in the gastrio juice could be read. Because of the large number of samples, even in an individual experiment only single estimations of pepsin were made. The accuracy of the procedure was tested in fifteen duplicate estimations with pepsin values in the range 12-36,ug/ml.; the mean difference between duplicate estimations was % (S.D.). RESULTS Interdigestive secretion. In the three stomach preparations employed in the present study, a substantial interdigestive acid secretion was regularly found in those with preserved vagal innervation. Interdigestive acid

5 GASTRIC SECRETION IN CONSCIOUS RATS 333 secretion was (s.e. of mean),t-equiv/3 min in the Heidenhain pouch, in the Pavlov pouch and in the gastric fistula preparation (Tables 1, 2 and 3). The interdigestive acid secretion in the Pavlov pouches was five to ten times higher than in the Heidenhain pouches, although the area of glandular mucosa was about equal in both preparations, as judged by inspection at autopsy. The interdigestive secretion of pepsin varied greatly between different Heidenhain pouches and also in the same pouch during different collection periods. The pepsin output in,ug during 3 min was (S.E. of mean) in the gastric fistula, in the Pavlov pouch, and 6 + 4*8 in the Heidenhain pouch (Table 4). The ratio between output of pepsin and acid secretion is much greater in the Heidenhain pouch than in the innervated preparations. Effect of gastrin infusion. The main object here was to determine the sensitivity to gastrin in innervated and denervated preparations. The smallest dose of gastrin which evoked a detectable increase in secretion was first determined. This was easy in the Heidenhain pouch with its low and stable interdigestive secretion, but is difficult in the gastric fistula and Pavlov pouch preparations, in which a great number of tests had to be done in order to find the smallest infusion of gastrin, which evoked a statistically significant increase in acid secretion. To assess the range of response pattern it was essential to obtain dose-response curves with intravenously infused gastrin over the full range of secretorily active doses. The results thus obtained are detailed in Tables 1, 2 and 3. Graded doses of gastrin gave graded acid responses in all preparations. A possible reduction in the response to supramaximal doses of gastrin, of which there were indications in some tests, was not systematically investigated. In order to make the dose-response curves for the pouch preparations comparable, the interdigestive secretion was deducted from the responses obtained on stimulation. Thereafter the maximal secretory response to gastrin obtained in individual pouches was designated 1 %, which was taken as the reference point in comparing the magnitude of response to various doses. In a few instances it was not certain whether the maximal secretory response to gastrin had been obtained and these pouches have been excluded in constructing the graphs of Fig. 1 (excluded: determinations detailed in row 6 of Table 1 and in rows 2 and 3 of Table 2). Excluded from Fig. 1 are also responses obtained after the maximal secretory responses had been attained. Figure 1 shows that the Pavlov pouch is more sensitive to gastrin than the Heidenhain pouch. This is strikingly apparent at the lower dose levels. It is apparent from Table 3 that the gastric fistula preparation is the least satisfactory for quantitative secretory studies, a circumstance which will be commented on in the Discussion.

6 334 S. E. SVENSSON o oa 1 to CC + X S C:~ 12m 1 P t- 11* CO aq 11o 2 ~~~~~~~1 X I I I I ~~~~~~~+l R-.2 q 2 4 ; O co q t-. 11 *NI CO cq +1 C cs+l n CO co w-4~ to 4-D ~ 24 +l COG +l. *. CO z co di *; M +1+ x * 3 d ec O ~~~~~~+1 s _, co t'lc O t Ci o C -{ Ct S IOC: c. C C 1 ~1M 1 t. eq Ho e r- P to r- o +l +l +l +l +l +l +l +l +l +l X 1C5 CO X t 4 o d=4 o CZO CO 1 CO o +l +l +l +l +l +l +l +l +l +l oo 1 ci> d.-.o *o 14 C9 o +l 12'O4CO1DCqO +1 +l > ~.~~~~~~~4 m O _ ' "4~ Cr =CZ2 *q t- - a CO w 15. +I +I +l +I +I +I +I +I +I +I ~ *s "E D U o d 2$ g; 3 d; ~1 o CQ. o +l CSC C~I CO -1 a O6 o +1 s P- m cq q -4 m P- o +1 u: 6 o r t- 1 +I +I +I +I +I +I +I P- -I Ci Ci Ci _- _- _-Cq 1,o f S >o> -P +l+i o + +l +l - o dci t o - C3O r- CO 4P4 qrim -4c I -. q.. 4 q 1'-; +1I+1 t - cc e to O CO c- a 1lf *4 r1 - d 4 +1 t- Cq a r 1 O CW... C ,- +l +l +l +l +l +l +l +l +l +l +l ee oc co o = t- _- a) I _- _- c q cli es "4r- 4,D - -, -., - -,,- Ca aq "-4 P-4 P-4 "-4 P-4 "

7 GASTRIC SECRETION IN CONSCIOUS RATS 335 o q o CD4 n X OC -* <z) - It.o o AA +l o (D o t- m 14 U:e r4m -4 o cc +l aq C) C _1C S HC ~4 ;,S:;~~~~~~~ C, co cq oo 4 _- o +l +l +l +l +l +l - C R - -. ~~~~~~~c a o- q=aqm -4o -a ---- ;,W*S, Ct aq Ci q_q P- _C _C We O -4.4x C) mc e - ~W- - d O. ~ *_*~~~~~~ to L- lo L- = P- r- aq m e o g~~~~~~~~ aq. W ;l O- P*co.,-I~ ~~~~+I+I ~ ~ +I +I +I +I a;>x X~~~ m to = = aq mq -Q r _ O M m t CO CO N CA X r_ 4 _ o Eq; >' ~~~~+I+I +I +I +I +lil+ + l+ X; o _~~~~~~ t- CNt t-<;ca -'S O = t- oo= ao r 4 X C e : m co m.,o +I +1+ I+ GQ D o4-1 o Ev +1

8 336 S. E. SVENSSON Pepsin secretion is excited by gastrin under certain circumstances only. Surprisingly, gastrin increases pepsin secretion in the Heidenhain pouch, but not in the innervated preparations, as shown in Table 4. In the Heidenhain pouch, the highest rate of gastrin infusion about doubled pepsin secretion, an effect which apparently is the result of genuine stimulation. 1 9 s C ml 7 C Z- 6 (U -a 5 E E24 T,, Gastrin II (ug/hr) Fig. 1. Acid secretory responses to intravenous infusion of gastrin II in rats provided with a Pavlov pouch (@ * ) or a Heidenhain pouch ( ). The acid responses are expressed as percentage of the maximal response to gastrin after deduction of the interdigestive secretion. The vertical bars represent the S.E. of the mean and the individual observations summarized in these graphs are detailed in Tables 1 and 2. Responses to gastrin and methacholine in the Heidenhain pouch. First, dose-response curves for acid and pepsin after gastrin in the dose range Itg/hr, and also for methacholine in the dose range 1-64,ug/hr, were established in each of seven Heidenhain pouches. Next, a doseresponse curve for gastrin was obtained, but now superimposed on a background infusion of methacholine in the dose 1,tg/hr. The resulting doseresponse curves are shown in Figs. 2 and 3, from which it is seen that the background infusion of methacholine throughout augments the acid responses to gastrin at infusion rates of ,tg/hr (P <.5). At the

9 GASTRIC SECRETION IN CONSCIOUS RATS 337 O O.S~~~C + +1 CO C)~~~~~ o-x.d *c +1 S o5o CO D +I 1 t'- 1 lo +I _6 r o _ O Cb Co 'a)~ ~ ~ bo o 1 C3 C3o ~~,bow1 +1 t o&. 1C) CO CO o co CO CO w P ;4 e+ E-4O d t ; 4Z ho Ca~~C + bo~ 1 $ Q V.'sOD m@m s CO +l +l.4 4 r1 *O C +l +l C) e

10 338 S. E. SVENSSON maximum dose level of -64,g/hr, an augmentation is apparent also, although statistically of low significance (P >.5). The acid secretory responses to methacholine alone is noteworthy, in that at the highest infusion rates this agent produced an acid response, which usually was more than twice as large as the maximal response after gastrin alone, and E, 6 o' Z- 5 *r * *28 Gastrin II (#ag/hr) Methacholine (,ug/hr) Fig. 2. Acid secretory responses of seven Heidenhain pouches to intravenous infusions of gastrin II (A A), methacholine (A *), and gastrin II in combination with methacholine (1 #ug/hr) (---- ). Each point represents the mean of determinations at the second and third 3 min period at each dose level with not less than one infusion in each rat. The vertical bars represent the S.E. of the mean. even larger than the combined responses of gastrin and the background dose of methacholine (P < 1). It was not investigated whether a higher background infusion of methacholine would enhance further the secretory responses to gastrin. Pepsin secretion was also stimulated by methacholine infusion, as seen in Fig. 3. However, the pattern of stimulatory response after methacholine was peculiar at every dose level; with each dose the initial stepwise increase was followed by a decrease in secretion,

11 GASTRIC SECRETION IN CONSCIOUS RATS 339 which was overcome by the next step of increased dose, which again produced the phasic pattern of response. The output of pepsin was maximal at 16 jtg methacholine/hr, and decreased at higher dose levels. Except at these levels, methacholine was more effective than gastrin as a stimulant of pepsin secretion, although accurate comparison was difficult because of the complex pattern of the response to methacholine. From Fig. 3 the %7 I % 1 15 Z l Gastrin~~~~~~~~ II (/hr l l l Methacholine (,ccg/hr) Fig. 3. Pepsin secretory responses of seven Heidenhain pouches to infuasions of ga-strin II (A *A), methacholine ( ),and ga-strin II in -- combination with methacholine (1 /ug/hr) ( ). Each point represents the mean of determinations at the first, second and third 3 min period at each dose level with not less than one infusion in each rat. The vertical bars represent the S.Em. of the mean. pepsin secretion after gastrin superimposed on a background methacholine infusion appears higher than with gastrin alone, but the difference is not statistically significant. Food-induced gastric secretion in the Pavlov pouch. In one type of experiment the volume of gastric juice was registered by manual operation of a device which indicated on a revolving smoked drum the numbers of drops falling from the tip of the cannula. The object was to assess the latency

12 34 S. E. SVENSSON of response to feeding. With this simple technique the shortest latency was 1.1 min and the longest 441 min. There was an inverse relationship between latency and interdigestive secretion. The mean latency of seven determinations in two Pavlov pouches was min (S.D.). In another type of experiment designed to describe the time course of the secretory responses to a meal, the Pavlov pouch was perfused as described under Methods. Typical results obtained in a Pavlov pouch are depicted in Fig. 4a and the mean values recorded in fourteen experiments on ten pouches are represented in Fig. 4b. In response to food, the increase in acid and pepsin secretion is rapid during the first 3 min of feeding. Thereafter pepsin secretion declines, whilst acid secretion proceeds at high rates for 3-4 hr. Effect of 2-deoxy-D-glucose on gastric secretion in the Pavlov pouch. This compound, which is alleged to produce central vagal stimulation (Hirschowitz & Sachs, 1965), was used because an adequate technique for shamfeeding in rats has not been evolved. A subcutaneous injection of 2 mg/ kg of this stimulant evoked secretion of acid and pepsin at high rates which were sustained for 3-4 hr. The responses in an individual Pavlov pouch and the mean responses obtained in a single test in each of ten pouches are summarized in the Fig. 5a and b. Effect of 2-deoxy-D-glucose on gastric secretion in the Heidenhain pouch. In the eleven Heidenhain pouches investigated, a subcutaneous injection of 2 mg/kg of this compound, a dose which in the Pavlov pouch was secretorily very effective, produced a small but definite acid response in seven of the pouches. The highest rate of acid secretion noted after this compound was 13 /t-equiv/3 min in a pouch which secreted a maximum of 93 #t-equiv/3 min in response to food. Pepsin secretion was increased, but slightly, for a brief period only, in six of the eleven pouches. These results are summarized in Fig. 6. Response to food with and without methacholine in the Heidenhain pouch. Feeding produced a pattern of response which differs from that described by others in the Heidenhain preparation in the dog. As shown in Fig. 7 (continuous line), the rate of acid secretion ascends steeply to a plateau value which is maintained for 2-3 hr, and then falls abruptly to the interdigestive value. A characteristic feature of the response in the rat is the rapidity with which maximum secretion is attained; this indicates rapid and effective recruitment of humoral stimulating agent(s) on feeding. The pepsin secretion in response to food is initially very high, presumably in part due to a washing-out effect known to occur in other species, and subsequently pepsin secretion proceeds for 14 hr (Table 5). A background infusion of methacholine in threshold dose enhanced the secretion of acid in response to food, but not that of pepsin (Fig. 7 and Table 5).

13 GASTRIC SECRETION IN CONSCIOUS RATS 341 a 12 6 Food W._ '6 3. > b 7 ~~~Food o 6 3'' 5 4 2~ 3 E Time. (min) Fig. 4. (a) Acid (continuous line) and pepsin (interrupted line) secretion in a Pavlov pouch in response to a meal of pellets. The rat was allowed to eat for 3 min after the interdigestive secretion had been determined for 2 hr. (b) Acid (continuous line) and pepsin (interrupted line) secretion in response to food in fourteen experiments in ten Pavlov pouches. The vertical bars represent the S.E. of the mean.

14 342 S. E. SVENSSON 6 'f 2-Deoxy-D-glucose c- 5 E.> 4 cr a' 3 u 'v b.' 5- E *' 4-_1-2 C E C> 'I 2-Deoxy- D-gludose ' IV 3-._ CL ' I I I I I I I I I I I I I Time (min) Fig. 5. (a) Acid (continuous line) and pepsin (interrupted line) secretion in a Pavlov pouch in response to a subcutaneous injection of 2-deoxy-Dglucose, 2 mg/kg. (b) Acid (continuous line) and pepsin (interrupted line) secretion in ten experiments in ten Pavlov pouches after a subcutaneous injection of 2- deoxy-d-glucose, 2 mg/kg. The vertical bars represent the S.E. of the mean.

15 .E- 5.. SC r._, 4-.* a, * i- 1 - GASTRIC SECRETION IN CONSCIOUS RATS I 2-Deoxy-D.g1ucose bo 4L 4-1 I I I I I I I I I I I I Time (min) Fig. 6. Acid (continuous line) and pepsin (interrupted line) output in response to a subcutaneous injection of 2-deoxy-D-glucose, 2 mg/kg. Each point is the mean of eleven experiments in eleven Heidenhain pouches and the vertical bars represent the S.E. of the mean I 9 Food _ 8_ so a7 _ 6 co Time (min) Fig. 7. Response of a Heidenhain pouch to a meal of pellets. Continuous line represents values obtained during infusion of 9 % NaCl solution and interrupted line during an infusion of methacholine, 1,ug/hr, which started 1 hr before onset of feeding. Each point is the mean of two determinations.

16 344 S. E. SVENSSON o d o t QO d O - ~~~~~~+I V I +1 G to e~~~~~~~~~~~ m xo Il4 B 4 on.se m Pn Ox o r4- C &4d Q oc,;d u Q > +l~~~+i +I +I +I +1 m +l +l +l c;;~~~~~~~~~~~~~~- "- Co e -4 s CDC O A,~~~~~~~~~~~~~~~~~- r- > r-- bn r 4 *e~~~~~~~~~~~~~~~ to o Co *>c~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~- B-4 r tb. t gt W U> X e b n O o. ee~~~~~ e t dtz U n ~~~~~~+I +l +l +l t- +I +l +l +l *> ~~~~~~~~~~ o o "- o :~~~~~~~~~~~~~~~~~~~a -*;4 t ~~~~ixs5 5 Us:~~~~~~~-4r- > 4-D v A

17 GASTRIC SECRETION IN CONSCIOUS RATS 345 Inhibition of gastric secretion by injection of gastrin pentapeptide. In the present investigations it was often seen that a plateau secretion of acid was not maintained on infusing gastrin intravenously at constant rates but declined gradually or rapidly. To see whether an inhibitory component was involved in these instances a high rate of acid secretion was evoked by intravenous infusion of gastrin in conscious rats with a gastric fistula. In this preparation large doses of gastrin injected intravenously Gastrin II.2,pg/hr 7 7 I 6 6 Pentapeptide f I jig 1 ag i E 5 E So_ 5 1 j'g " r ~3 3~ -o L 1 1 L. J Time (min) Fig. 8. Effects of rapid injections of gastrin pentapeptide on acid (continuous line) and pepsin (interrupted line) secretion maintained by gastrin II, 2,ug/hr intravenously, in a Heidenhain pouch. did not cause a lowering of the plateau secretion. It was then investigated whether gastrin would produce inhibition in the denervated Heidenhain pouch. The spontaneous decline in the plateau secretion, which is a conspicuous finding in the denervated preparation, was partly obviated by employing small doses of gastrin, with which a plateau was maintained. In order to economize with the precious supply of gastrin II, gastrin pentapeptide was used as a substitute in testing inhibition. This compound was injected rapidly in doses of 1, 1 and 1,tg when a plateau of acid secretion had been reached by infusion of gastrin. A typical experiment is depicted in Fig. 8. No clear inhibition was noted. With 1,ug there was a phasic change, an initial increase in secretion, as seen with the smaller doses of pentapeptide, followed by a period of inhibition and finally a phase of increased stimulation. The transient inhibitory effect produced by the high dose of gastrin pentapeptide appeared worthy of further analysis. To

18 346 S. E. SVENSSON this end a plateau acid secretion was produced by infusion of either gastrin *2,tg/hr or methacholine in doses of 5 and 2 pg/hr, and an intravenous injection of 1,g gastrin pentapeptide was superimposed. On the gastrin-evoked plateau a triphasic change similar to that already described was noted. In contrast, the plateau secretion maintained by methacholine was in no way inhibited by gastrin pentapeptide and the combination of these two stimulants produced the most copious secretion of acid ever observed in any of the Heidenhain pouches investigated. Pepsin secretion, whether maintained by gastrin or by methacholine, was increased to about the same degree by injection of gastrin pentapeptide, but for 3 min only. The pertinent results are summarized in Fig. 9. DISCUSSION The main object of studies of gastric secretion is to describe the secretory response to food and to the individual components involved in the complex stimulus of feeding, and, more recently, to explore at the cellular level the mechanism by which the secretory cells are brought into action. To achieve the first part of these aims investigations in unanaesthetized animals are required. This prerequisite has so far been realized mainly in the dog. The present work shows that practically every type of experiment pertaining to gastric secretion, which hitherto has been carried out in unanaesthetized dogs, can be done in unanaesthetized rats. In this field of study the rat appears preferable in the following respects: large numbers of co-operative rats can easily be obtained; intravenous infusions can be instituted over periods up to 15 hr; consistent and graded secretory responses to various stimulants are obtained repeatedly in one and the same rat; relatively small doses of scarce gastrin preparations are required; a large number of rats can be studied simultaneously; and in investigations at the cellular level striking associations between secretory state and gastric inucosal histamine metabolism have been disclosed and studied more extensively in the rat than in other species. The present experiments are concerned with classical problems and also with problems where controversies exist about results obtained mainly in studies in the dog. Studies of gastric secretion in the rat would be of more general significance if the pattern of secretory responses were similar to those known to occur in the dog and man. In the present study this has in the main been the case. In the rat the Pavlov pouch and the Heidenhain pouch preparations responded readily and in a typical way to food intake. In the latter preparation, assumed to be vagally denervated, there is a rapid onset of secretion reaching a plateau value within 3 min. This would imply pro-

19 GASTRIC SECRETION IN CONSCIOUS RATS 347 2,cfg/hr Methacholine 5 fig/hr Gastrin 1122ug/hr 19 _ I Pentapeptide _1fig V 15 c 14 E _%, 11-1 Z _ c5 7 1II"I A I I I _ 6 5 ~Z4 c3 ~~~~~~~~~~..ib (- 2 / '----- a 1.. NjA Time (min) Fig. 9. The effect of a rapid injection of gastrin pentapeptide, 1 4ug, on acid and pepsin secretion maintained by infusion of gastrin II -2 /etgfhr ( x ~x), and of methacholine 5 sag/hr ( ----) and 2 #tg/hr (. ) in five Heidenhain pouches. Each point is the mean of five determinations.

20 348 S. E. SVENSSON vision of endogenous gastrin released under the influence of central vagal excitation. In the classical Heidenhain pouch preparation in the dog, the onset of secretion in response to food intake is slower than in the rat, unless the pouch is prepared as devised by Gregory (1958), in which the response pattern has been extensively studied by Gregory & Tracy (196). It is noteworthy that in the present study the Heidenhain pouch displays a substantial secretion of pepsin in response to food, in contrast to the situation in the dog (Schofield, 1957). This result in the rat cannot be explained by reinnervation of the Heidenhain pouch, because vagal excitation by injection of 2-deoxy-D-glucose failed to excite pepsin secretion in the pouch. The most likely assumption is that in the rat endogenous gastrin provokes pepsin stimulation in the denervated preparation. This assumption is supported by the observation that infusion of gastrin stimulated pepsin secretion in the Heidenhain pouch. The secretory patterns to food in Pavlov pouches in the rat and the dog are in the main similar. There is a minor dissimilarity in that in the dog there is a parallel secretion of acid and pepsin (Schofield, 1957), whereas in the rat pepsin secretion ceases within about 3 min, whilst secretion of acid proceeds for hours. Incidentally this would imply that in the rat the type of central vagal excitation produced by feeding is not achieved with 2-deoxy-D-glucose, because with this compound the secretion of acid and pepsin run parallel. The innervated pouch is more sensitive to the acid secretory action of gastrin than the denervated pouch. The sensitivity of the gastric fistula preparation cannot be strictly compared with that of the pouches. In the present study the sensitivity is judged by the amount of secretion obtained on stimulation. This amount cannot be accurately ascertained in the gastric fistula preparation because of the escape of some of the secretion into the duodenum and its unavoidable contamination with saliva. It has been ascertained in various studies in the dog and cat that vagal excitation, or its substitute provided by administration of vagomimetic drugs such as the one used in the present study, augment the acid secretory response to endogenous or exogenous gastrin (see Grossman, 1967, for references). The same situation has been shown to exist in the rat, in which a background infusion of methacholine accentuated the rate of acid secretion evoked by infusion of gastrin. The acid secretion in response to food in the Heidenhain pouch was also augmented by methacholine, an action for which there are two possible explanations. Methacholine may render the parietal cell more sensitive to the action of endogenous gastrin, or it may increase the release of endogenous gastrin. The latter explanation appears less likely because methacholine does not accelerate gastric mucosal histamine formation as gastrin does (Rosengren & Svensson,

21 GASTRIC SECRETION IN CONSCIOUS RATS ), nor does it augment pepsin secretion in response to food in the Heidenhain pouch. The present results with methacholine require further brief comments. It is striking that the maximum acid response with methacholine in the Heidenhain pouch is much larger than the maximum response to gastrin and even larger than the responses seen after gastrin superimposed on a background infusion of methacholine in a threshold dose. In the rat methacholine does not produce systemic side effects which may be adverse to its secretory stimulatory effect, as in the dog, in which for this reason the maximum secretory response cannot be attained. Although, as stated above, gastrin release by methacholine has so far not been demonstrated in the rat, the conspicuously high secretory response to methacholine should perhaps be considered as resulting from the combined effect of this drug and a small background release of endogenous gastrin. Regarding the stimulatory effect of gastrin and methacholine on pepsin secretion no evidence was found that in the rat the combination of these stimuli potentiate the response. On the controversial problem of inhibition of gastric secretion by intravenous injections of gastrin or its analogues the present study provides some new information. In the dog (see Gregory, 1968, for references) and in special circumstances in the anaesthetized rat (Barrett, Raventos & Siddall, 1966), a rapid intravenous injection of gastrin produces a transitory inhibition of acid secretion maintained by infusion of gastrin. In a Heidenhain pouch stimulated by an intravenous infusion of hog gastrin II, a rapid intravenous injection of a supramaximal dose of gastrin pentapeptide produced phasic changes in secretion, an initial increase, followed by a decrease, which finally was overtaken by a stimulatory phase. The transient inhibitory effect was obtained only with a dose which should be considered pharmacological rather than physiological, and on this ground the present study provides no clear indication of an inhibitory action of gastrin on acid secretion. There was no evidence of inhibition by gastrin pentapeptide of acid secretion maintained by infusion of methacholine, a fact which perhaps adds to the view held in this laboratory that the mode of action of methacholine and gastrin at the cellular level is not the same (Rosengren & Svensson, 1969). I should like to thank Professor Georg Kahlson for encouragement and advice. This work was supported by grants from the medical faculty of the University of Lund and from Albert PAisson's Foundation.

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