dopamine. pylorus ligation did not affect their argyrophilia or their ability to produce and store

Transcription

1 J. Physiol. (1982), 323, pp With 1 plate and 4 text-figures Printed in G~reat Britain GASTRIC ACID RESPONSE TO PYLORUS LIGATION IN RATS: IS GASTRIN OR HISTAMINE INVOLVED? BY J. ALUMETS, M. EKELUND, R. HAKANSON, J. HEDENBRO, J. F. REHFELD, F. SUNDLER AND S. VALLGREN From the Departments of Pharmacology, Histology and Surgery, University of Lund, Sweden and the Institute of Medical Biochemistry, University of Aarhus, Denmark (Received 23 November 198) SUMMARY 1. Pylorus ligation stimulated the acid output in vagally intact rats. The serum gastrin concentration and the gastric mucosal histamine content were not affected. The gastric histidine decarboxylase activity was initially slightly elevated and then greatly reduced (12-2 hr after ligation). 2. Pylorus ligation stimulated the acid output in chronically, but not in acutely, vagotomized rats. Chronic vagotomy raises the serum gastrin concentration, the gastric histamine content and histidine decarboxylase activity. The serum gastrin concentration was further raised by pylorus ligation. The histamine content was initially lowered but returned to preligation values after 2 hr. The histidine decarboxylase activity first decreased, but increased to very high levels 5-6 hr after ligation. Twelve hours after ligation it was lower than before ligation. 3. Following pylorus ligation pentagastrin and histamine stimulated the acid output in vagally intact and in acutely vagotomized but not in chronically vagotomized rats. By contrast, pentagastrin raised the histidine decarboxylase activity in vagally intact and in chronically vagotomized, but not in acutely vagotomized rats. 4. The two major populations of endocrine cells of the oxyntic gland area (ECL cells and A-like cells) are argyrophil, store histamine and are capable of taking up exogenous DOPA and of decarboxylating it to dopamine which is retained in the cytoplasm for several hours. As evidenced by light and fluorescence microscopy pylorus ligation did not affect their argyrophilia or their ability to produce and store dopamine. 5. Pylorus ligation caused ultrastructural changes in the gastrin cells of the pyloric gland area and in the histamine-storing ECL and A-like cells of the oxyntic gland area. The two endocrine cell types in the oxyntic gland area were enlarged by pylorus ligation, more so after 16 hr than after 4 hr. The size of the gastrin cells seemed unaffected. In all three cell types pylorus ligation reduced the number of cytoplasmic granules. There was no increase in the Golgi area or in the endoplasmic reticulum in any of the endocrine cell types of the oxyntic gland area. It appears unlikely that the ultrastructural changes of the ECL and A-like cells reflect an increased rate of histamine mobilization. 6. The acid response to pylorus ligation probably reflects neuronal reflex mechanisms exclusively. There is no evidence that gastrin or histamine released from gastric endocrine cells mediate the response.

2 146 J. ALUMETS AND OTHERS INTRODUCTION Acute pylorus ligation in the rat causes copious secretion of gastric acid (Shay, Komarov, Fels, Meranze, Gruenstein & Siplet, 1945; Shay, Sun & Gruenstein, 1954). This response is probably elicited by vago-vagal reflexes (Brodie, 1966; Brodie & Knapp, 1966; Hikanson, Hedenbro, Liedberg, Sundler & Vallgren, 198). The acid response is abolished immediately after vagal denervation but returns gradually. Still, 8 weeks after the denervation the response is only 75 % of that for innervated controls (Hakanson et al. 198). Recent observations suggest that in chronically vagotomized rats the acid reponse to pylorus ligation is caused by intramural reflexes activated by the gastric distension. Thus, entirely different nervous mechanisms seem to be responsible for the acid hypersecretion following pylorus ligation in innervated and denervated animals (Hakanson et al. 198). Histamine has been claimed to be the final common mediator for the stimulation of the parietal cells (Code, 1965; Kahlson, Rosengren, Svahn & Thunberg, 1964; Kahlson, Rosengren & Svensson, 1973). Accordingly, it has been suggested that histamine might be involved also in the hypersecretion of gastric juice following pylorus ligation (Bussolati & Monga, 1973; McAlpin, Moore & Lemmi, 1974). In the rat there is evidence that the bulk of gastric histamine (and histidine decarboxylase) is stored in endocrine cells which are argyrophil and of two ultrastructurally different types, ECL and A-like (Hakanson, Larsson, Liedberg & Sundler, 1976). McAlpin et al. (1974) reported an increased histidine decarboxylase activity following pylorus ligation and Bussolati & Monga (1973) described ultrastructural changes in the ECL cells which were regarded as signs of activation (partial degranulation, formation of pro-granules, increase in free ribosomes and enlargement of the rough endoplasmic reticulum). These observations were interpreted as favouring the view that histamine is involved in the hypersecretion following pylorus ligation. It is our aim to find out which chemical messengers are responsible for the stimulated acid output after pylorus ligation. Therefore we have now studied the effects of pylorus ligation on the mobilization of gastrin and on the activity of the histamine-storing endocrine cells in the rat stomach. METHODS Experimental procedure Male Sprague-Dawley rats, weighing approximately 2 g, were used. They were fed a standard diet of food pellets and tap water. One group was subjected to bilateral sectioning of the vagal trunks below the diaphragm as described by Shay, Komarov & Gruenstein (1949). A pyloroplasty was always performed at the same time in order to prevent food retention and fatal gastric dilatation. Unless otherwise stated the denervated rats were left for at least 6 weeks before they were used in experiments. Before the rats were killed they were placed in individual metabolic cages with wire-mesh bottom for 48 hr without access to food but with water ad libitum. In innervated rats fasting reduces the level of gastric histidine decarboxylase activity (cf. Kahlson et al. 1973; HAkanson et al. 1976). Laparotomy was performed through a midline incision of approximately 3 cm under light diethyl ether anaesthesia. The pyloric portion of the stomach was gently mobilized and occluded with a 4- silk ligature around the pyloric sphincter in the manner described by Shay et al. (1945). The incision was closed in two layers with 3- silk sutures. All the animals received 1 ml. 9 % saline subcutaneously prior to pylorus ligation in order to prevent dehydration. The rats were killed at various time intervals following the pylorus ligation but always so that they

3 ACID RESPONSE TO PYLORUS LIGATION 147 had been fasted for at least 48 hr at the time of death. In one study, pylorus-ligated rats received either pentagastrin (Peptavlong, ICI; 5,ag/kg) or histamine dihydrochloride (25 mg/kg) subcutaneously. These rats were killed 5 hr after the ligation, and 1 and 2 hr after injection of pentagastrin or histamine, respectively. In another study, unoperated rats and pylorus-ligated rats received L-DOPA (1 mg/kg, intraperitoneally) 11 hr before they were killed. In one series of experiments rats were fitted with chronic gastric fistulas implanted in the rumen (Lane, Ivy & Ivy, 1957; Bel, Leurat, Nesmoz & Girard, 1966). They were allowed to recover from the operation for at least 2 weeks. During this time they were familiarized with Bollman-type restraining cages. Before collection of gastric juice they were fasted for 48 hr and their stomachs were rinsed with warm 9 % saline. Gastric juice was collected as indicated in Results. Chemical analysts Radioimmunoassay of gastrin. Blood was drawn from the abdominal aorta. Serum was lyophilized and stored at -25 T until analysis. The concentration of gastrin in serum was determined radioimmunochemically using antiserum 264 (Rehfeld, Stadil & Rubin, 1972) as described in detail elsewhere (Stadil & Rehfeld, 1973; HAkanson, Kroesen, Liedberg, Oscarson, Rehfeld & Stadil, 1974). Volume and acidity of gastric content. The stomach was removed and its contents collected. The volume of the gastric content was measured and the acid output was determined by titration with -2 N-NaOH, using phenolphthalein as indicator. Determination of histidine decarboxylase activity. The mucosa of the oxyntic gland area was homogenized in ice-cold 1 M phosphate buffer, ph , to a final concentration of 1 mg (wet weight) per ml. Aliquots ( 5 ml.) of the homogenate were incubated with 4 x 1-4 M-1-14C-L-histidine (1-3 mc/m-mole; New England Nuclear) in the presence of 1-5 M-pyridoxal-5-phosphate and 5 X 1-4 M-reduced glutathione at 37 for 1 hr under nitrogen. Total reaction volume was 53 ml. The enzyme reaction was stopped by acidification and the amount of 142 produced was determined as described in detail elsewhere (HAkanson et al. 1974; Hakanson, Larsson, Liedberg, Rehfeld & Sundler, 1977). Enzyme activities are expressed as p-moles 14-2 produced per mg tissue (wet weight) per hour. Determination of histamine. The oxyntic mucosal homogenate (see above) was rehomogenized with an equal volume of 3 % trichloroacetic acid and stored in the refrigerator over-night after which precipitated proteins were spun down by centrifugation at low speed. The supernatant was diluted 1: 5 with redistilled water and 1 d1. was taken for fluorometric assay of histamine as described by HAkanson & R6nnberg (1974). Fluorescence and light microscopy The following experimental groups were examined: unoperated rats; pylorus ligated, 4 hr; pylorus ligated, 16 hr (three animals in each group). Pieces from the oxyntic portion of the stomach of L-DOPA-injected rats were frozen to the temperature of liquid nitrogen in a propane-propylene mixture. After freeze-drying, the specimens were exposed to gaseous formaldehyde for 1 hr at 8 according to the Falck-Hillarp technique (Bj6rklund, Falck & Owman, 1972) for the demonstration of catecholamines and 5-hydroxytryptamine The tissue blocks were then embedded in paraffin in vacuo and sectioned at 6 or 3,sm thickness. Some sections (6,um) were mounted in Entellan (Merck) and examined in a fluorescence microscope equipped with an HBO 2 high pressure mercury lamp. Schott BG 12 was used as excitation filter and Schott OG 4 as barrier filter. Other sections (3 jam) were examined for argyrophil cells using a modification of the Grimelius (1968) technique. Following incubation in a fresh solution of 2-4 % silver nitrate in -2 M-sodium acetate buffer, ph 4-5, at 5 for 48 hr, the sections were exposed to a solution of 1 % hydroquinone and 5 % sodium sulphite in distilled water at 4 for 2 min. Finally, they were washed in distilled water, dehydrated and mounted in Canada balsam. Electron microscopy The following experimental groups were examined: unoperated rats; pylorus ligated, 4 hr; pylorus ligated, 16 hr (at least five animals in each group). Fasted animals were anaesthetized with diethyl ether and exsanguinated via the abdominal aorta. The stomach was rapidly dissected out. Small pieces were taken from the mucosa of the oxyntic gland area and of the pyloric gland area and immersed in a 2-5 % solution ofglutaraldehyde in 75 M-phosphate buffer overnight (16-2 hr). The material from each experimental group was pooled, antrum and oxyntic gland area separately. All specimens were post-fixed for 1 hr in 1 % osmium tetroxide in -75 M-phosphate buffer.

4 148 J. AL UMETS AND OTHERS dehydrated in graded ethanol solutions, contrasted en bloc in a mixture of 1 % phosphotungstic acid and -5% uranyl acetate in ethanol and embedded in Epon. Ultrathin sections (6-8 nm) were contrasted with uranyl acetate and lead citrate and examined and photographed in an electron microscope. At least two hundred sections, randomly selected, were examined for gastrin cells in specimens from the pyloric gland area and for the two predominant endocrine cell types (ECL and A-like) in the oxyntic gland area. The cells, which were identified by their characteristic cytoplasmic granules, were photographed and used for morphometry only if sectioned in such a way that the nucleus was visible. The photographs were reproduced in magnification 3 x Morphometric analysis was performed using the point counting technique (multipurpose test system) described by Weibel & Bolender (1973). The results are expressed as #um2 cell area or cytoplasm area. The error of this method has been estimated in several previous publications (Alumets, El Munshid, HAkanson, Liedberg, Oscarson, Rehfeld & Sundler, 1979; Alumets, El Munshid, HAkanson, Hedenbro, Liedberg, Oscarson, Rehfeld, Sundler & Vallgren, 198). It varies from 1-6 to 3-2 %. The cytoplasmic granules in each cell section were counted. Granule size was estimated by measuring the diameter of all granule profiles in eight to eighteen cells of each type from each experimental group. Because only those endocrine cells which showed their nuclei were photographed, the cell size, the nuclear volume density, the Golgi area and the volume density of the endoplasmic reticulum were systematically over-estimated. An error is introduced also in the values for granule size and granule volume density, because the sections had a finite thickness (Holmes effect). In addition, volume changes and/or distortion of the tissue during processing for electron microscopy contribute certain errors. However, as the changes induced by the pylorus ligation were of greater interest to us than the values in absolute terms we decided to present the experimental data uncorrected. RESULTS Effect of pylorus ligation on gastric functions Acid output. In a previous study we reported that following pylorus occlusion the acid output and the volume of the gastric content increased progressively until about 6-8 hr after ligation and that chronically vagotomized rats responded to pylorus ligation much like the control rats (Hakanson et al. 198). Fig. 1 illustrates that pylorus ligation induces a true stimulation of the acid output rather than causing merely an accumulation of basal gastric secretion in the occluded stomach (cf. Ridley, Groves, Schlosser & Massenberg, 1973). The ph of the acid juice (4-5 hr after pylorus ligation) ranged from 1-1 to 1-5 in control rats and from 2- to 3-4 in denervated rats. As illustrated in Fig. 2 the chronically vagotomized rats responded with acid secretion to pylorus ligation, whereas the acutely vagotomized rats did not. Both pentagastrin and histamine stimulated the acid output in innervated and acutely vagotomized but not in chronically vagotomized rats. In a follow-up study it was found that acutely vagotomized rats responded to pylorus ligation with acid secretion at 1 day after the vagotomy. At this stage the acid output (collected 5 hr after pylorus ligation) was 172 gtequiv H (S.E.M., n = 7); this value is not significantly different from the one observed in chronically denervated rats ( , n = 12 in one series, , n = 6 in another). Gastrin mobilization. Pylorus ligation did not raise the serum gastrin concentration in the innervated rats (Fig. 3). In the chronically vagotomized rats the serum gastrin concentration was elevated; it was further elevated by the ligation, reaching a maximum after about 6 hr. Histamine mobilization. The gastric mucosal histamine content of innervated rats was not significantly affected by pylorus ligation. The gastric histidine decarboxylase activity, however, was slightly raised 4 hr after ligation, followed by a progressive

5 ACID RESPONSE TO PYLORUS LIGATION 149 A..54B. ZL3 3 2 ' L-k Time (hr) Time (hr) Fig. 1. Pylorus ligation induces gastric acid hypersecretion as illustrated in this study which compares the accumulated acid output in conscious gastric fistula rats (filled columns) over different periods of time with that of age-matched pylorus-ligated rats (open columns). The rats were vagally intact (A) or vagally denervated (B). Denervation was made 2 months before the experiment. Eighteen to forty-three vagally intact rats and five to ten vagally denervated rats in each group. Vertical bars give S.E.M. * for p < 5, ** 5 > p > 1, *** for p < * A '- o < NaCI Pentagastrin Histamine Fig. 2. Effect of pentagastrin (5,ag/kg) and histamine dihydrochloride (25 mg/kg) on the gastric acid response to pylorus ligation (5 hr) in innervated (A) and acutely (B) or chronically (C) vagotomized rats. Controls received saline. The number of animals in each experimental group is indicated below the columns. Significant difference between stimulated and control rats are given by asterisks (p <.5,*; p < 1,**; p < 1,***: Student's t test).

6 15 7 E *, 6 - ) U) I 5 cm CL Q J. ALUMETS AND OTHERS A 7 1 -c E 6- X 5- E L B ) C 3 - C XW 2 - m E 1 X On C._ C 3 - (U x E.. 2 Q, 1 C o 'a O -i r 5 1 Time (hr) Time (hr) = 75 C a) can5 P C" c 2 E 4-) s Cu w Ie I I T I Time (hr)....l 2 Fig. 3. Serum gastrin concentration (A), gastric histidine decarboxylase activity (B) and gastric mucosal histamine concentration (C) at various time intervals following pylorus ligation of innervated () and chronically vagotomized () rats. Six to twenty-three (198). Mean+s.E.M. animals in each group. Same animals as in HAkansoja et al. reduction to very low activities 12-2 hr after ligation (Fig. 3). Chronic vagotomy is known to raise not only the serum gastrin concentration, but also the gastric histidine decarboxylase activity and histamine concentration (Hakanson et at. 1974). In the chronically vagotomized rats there was a transient reduction of the histamine concentration, most pronounced 6 hr after the ligation; the histamine concentration returned to the pre-ligation level about 12 hr after ligation. There was an initial drop in enzyme activity (1-2 hr after ligation) followed first by a return to the normal level 4-8 hr after ligation) and then by a decline to quite low levels (12-2 hr after ligation)

7 ACID RESPONSE TO PYLORUS LIGATION 151 (Fig. 3). Pentagastrin was found to raise the histidine decarboxylase activity in vagally intact rats and in chronically, but not in acutely vagotomized, pylorus-ligated rats (Fig. 4). The acutely vagotomized rats differed from the chronically vagotomized rats also in that the enzyme activity was low. In a follow-up study it was found that the high enzyme activity, which was characteristic of chronically vagotomized rats, was established at about 4 days after the vagotomy. At this stage the enzyme activity in the saline-injected rats was (s.e.m., n = 1); this is significantly higher 2 ~1,5 E decaryvagly intact Acute vagotomy irwesk ligated, pentagastrin-inj rats 4 days after vagotomy.agoom was eioo I S S..5~~~~~~~~~~~~~~~~ns Fig. 4. Effect of pentagastrin (5hig/kg hatched columns) on the gastric histidine decarboxylase activity in pylorus ligated (5 hr) rats, with or without vagal innervation. These are the same animals as in Fig. 2. Open columns indicate controls, which received saline. Number of animals in each group is indicated below the columns. than in acutely denervated rats (p < 1). The enzyme activity in the pylorusligated, pentagastrin-injected rats 4 days after vagotomy was 55-3+±7.4 (S.E.M., n = 11); this is not significantly higher than in the saline-injected controls. Not even 8 days after vagotomy was the enzyme activity in pylorus ligated, pentagastrintreated rats significantly higher than in saline-treated: (8) vs (6). Hence, the capacity in these animals to respond to pentagastrin with enzyme activation develops slowly. Effect of pylorus ligation on the fluorescence and light microscopic appearance of gastric endocrine cells The two predominant endocrine cell types of the oxyntic mucosa, the ECL cells and the A-like cells, are known to be able to take up and decarboxylate L-DOPA and to store the resulting dopamine in the cytoplasm for several hours. This ability makes it possible to demonstrate the cells in the fluorescence microscope by the Falck-Hillarp technique. Plate 1 shows that pylorus ligation had no overt effect on the number and fluorescence intensity of the dopamine-containing endocrine cells of the gastric

8 152 J. ALUMETS AND OTHERS X qe *! ce bi&> I., N CII CtS c1 C * o * C * (:;, * d, 4 N cqi M *_ *_ V +V =V t- w 2 C) PC 1-1) " 9 a) -o 2 a)~ C3) - C) 2.-- pp._ w._ ~ 2 ea - z - W& 4a t E2 C 2Q o-qao) 4) a~a e el' O V N co 114 N ~I* ts r- d> N 1 * - *r. *,. * u1 * N- *d- * - O M o *,4 *- 4 C*t *qt *N *. *. *= * *C *1J * IF4* - *o *o *6, *C;: *,:: *,,, V co cc ec r- P-4 1- V-4 V- 6 * * - * -N *_C + _ - aq 1 aq 1* * *'. *_ *N.* lb~*cb. 1 - e 1 14 t- co r- CC 1 ~ 1- o Co * _ Mo - Czb * C C*N - Pa om o No O - 1-~ m a) 2 _ *s O O 2 ce O C 4 O E-l V P1V O V.4. 4 u 4)

9 ACID RESPONSE TO PYLORUS LIGATION 153 mucosa. It also shows that the argyrophilia of these cells was unaffected by pylorus ligation. Effect of pylorus ligation on the ultramorphology of gastric endocrine cells The gastrin cells in the pyloric gland area and the ECL and A-like cells in the oxyntic gland area were analysed by ultrastructural morphometry. The two endocrine cell types in the oxyntic gland area were enlarged by pylorus ligation, more so after 16 hr than after 4 hr (Table 1). This enlargement affected nucleus and cytoplasm alike. The size of the gastrin cells was unaffected. In all three cell types pylorus ligation TABLE 2. Effect of pylorus ligation on granule profile diameter (nm) in the gastric endocrine cells. Treatment G cells ECL cells A-like cells No operation (2149) (873) (1263) Pylorus ligation, 4hr 265-(113) 253-(115) 18(147) Pylorus ligation, 16 hr (995) 274 (126) 177±+5 (192) Mean+ S.E. of mean. Number of granules in parenthesis. Each group comprised granules from eight to eighteen cells. Analysis of variance was performed. For the A-like cells the difference between unoperated rats and pylorus-ligated rats was statistically significant. *** for P < -1. reduced the number of granules and the granule volume density, i.e. the proportion of the cytoplasm made up of granules (Table 1). Pylorus ligation affected the granule profile diameter only in the A-like cells (Table 2); the mean granule size was increased. There was no increase in the Golgi area or in the endoplasmic reticulum in any of the endocrine cell types of the oxyntic gland area after pylorus ligation; on the contrary there was a reduction in the endoplasmic reticulum of the A-like cells (Table 1). The gastrin cells displayed an enlarged endoplasmic reticulum 4 hr after pylorus ligation; after 16 hr the increase was less marked and not statistically significant (Table 1). DISCUSSION Pylorus ligation stimulates acid output over a time period of 4-6 hr. After that time there is no evidence of stimulated acid secretion (Hakanson et al. 198). The acid response to pylorus ligation in innervated rats is probably elicited by vago-vagal reflexes activated by pressure receptors located in the pyloric gland area (cf. Brodie, 1966). The response is inhibited by acute vagotomy, by atropine and by the ganglionic blocking agent chlorisondamine but not very effectively by the histamine H2-receptor antagonist metiamide (Hakanson et al. 198). Conceivably, therefore, cholinergic and/or other nervous mechanisms are involved rather than histaminergic. This view is supported also by the finding that the hypersecretion does not seem to be accompanied by an increased mobilization of gastric histamine (see also Aures & Thompson, 1972). From the results of the present study it further appears that gastrin is not responsible for the hypersecretion since the serum gastrin concentration was not raised by pylorus ligation. This observation agrees with a previous report by Nagy, Mozsik, Tarnok, Szalai, Poth & Javor (1978).

10 154 J. ALUMETS AND OTHERS The acid response to pylorus ligation in chronically vagotomized rats is probably elicited by intramural reflexes activated by distension of the stomach. This response is blocked by atropine, chlorisondamine and metiamide (Hakanson et al. 198). In the present study we found that in vagotomized rats pylorus ligation raised the serum gastrin concentration, mobilized gastric histamine and activated gastric histidine decarboxylase. Hence, these results do not exclude the possibility that in vagotomized rats gastrin and/or histamine are responsible for the acid response to pylorus ligation or that gastrin acts with histamine as the mediator. On the other hand, pentagastrin and histamine stimulated the acid response to pylorus ligation in innervated and acutely vagotomized but not in chronically vagotomized rats. These results suggest that chronic vagotomy lowers the sensitivity of the parietal cells to both gastrin and histamine. Our observations therefore do not favour the view that either gastrin or histamine bring about the acid response to pylorus ligation in chronically vagotomized rats. By contrast, the endocrine cells that contain histidine decarboxylase responded to pentagastrin by enzyme activation in the vagally intact and chronically vagotomized rats but not in the acutely vagotomized rats. Thus, the response to gastrin of the parietal cells and the response of the endocrine cells were differently affected by the denervation. This finding is difficult to reconcile with the view that the parietal cells lack gastrin receptors (Bergqvist & Obrink, 1979) and that histamine-storing endocrine cells act as intermediaries in conveying to the parietal cells the message carried by gastrin (Kahlson et al. 1973). Bussolati & Monga (1973) reported a reduced number of argyrophil cells in the oxyntic mucosa and a reduced dopamine storage after L-DOPA administration in the histamine-containing ECL cells of the pylorus-ligated rat. They also described marked ultrastructural changes in the ECL cells, but not in the G cells and A-like cells, following pylorus ligation. These changes, which included reduced number of cytoplasmic granules and increased amount of endoplasmic reticulum, were thought to support the view that the histamine of the ECL cells is important for bringing about the acid response to pylorus ligation. In the present study we have failed to corroborate these findings. Also, our attempt to quantitate the ultrastructural changes in the various gastric endocrine cells only partly confirms the report by Bussolati & Monga (1973). The granule content of the ECL cells was reduced but we did not find an increased amount of endoplasmic reticulum and no enlargement of the Golgi area. Further, the ultrastructural changes in the ECL cells were manifest not only after 4 hr, when the acid output was high, but also after 16 hr, when the acid output seems to be low. The A-like cells displayed progressively decreased endoplasmic reticulum with time after pylorus ligation. On the whole, the ultrastructural changes in the gastric endocrine cells are difficult to interpret. We found both the ECL cells and the A-like cells to be enlarged and to display a reduced number of granules at 4 and 16 hr after pylorus ligation. Together these two features may suggest activation of the cells. However, the findings regarding the endoplasmic reticulum and the Golgi area speak against such an interpretation, as does the low activity of histidine decarboxylase, which has previously proved to be a good marker for ECL cell activity. Also, the increased cell size may be due to altered fluid transport across the cell membrane secondary to ligation-induced damages to the mucosa. Analogous events seem to take place in gastrin cells. Although they were not increased in size, the number of granules was greatly reduced and there was a tendency to

11 ACID RESPONSE TO PYLORUS LIGATION 155 enlargement of the endoplasmic reticulum and the Golgi area 4 hr after pylorus ligation. However, it is notable that the low serum gastrin concentration following pylorus ligation does not indicate activation of the gastrin cells. Such a conclusion is also supported by experiments which demonstrate a lack of influence of antrectomy on the acid response to pylorus ligation, suggesting a non-hormonal mechanism for this type of hypersecretion (Brodie, 1966). On the whole therefore, the ultrastructural changes induced by pylorus ligation in various gastric endocrine cells do not seem to reflect cellular activation. More specifically, the gastrin cells do not seem to have an increased rate of gastrin release, and the ECL cells and the A-like cells do not seem to have an increased rate of histamine mobilization. It is difficult, therefore, to avoid the conclusion that the acid response to pylorus ligation involves neuronal reflex mechanisms exclusively and proceeds independently of gastrin and of histamine released from gastric endocrine cells. Conceivably, cholinergic and/or peptidergic neuronal pathways are involved. Grant support from the Danish and Swedish Medical Research Councils (4X-17) and from A. PAhIsson's foundation. REFERENCES ALUMETS, J., EL MUNSHID, H. D., HAKANSON, R., LIEDBERG, G., OSCARSON, J., REHFELD, J. F. & SUNDLER, F. (1979). Effect of antrum exclusion on endocrine cells of rat stomach. J. Physiol. 286, ALUMETS, J., EL MUNSHID, H. A., HAKANSON, R., HEDENBRO, J., LIEDBERG, G., OSCARSON, J., REHFELD, J. F., SUNDLER, F. & VALLGREN, S. (198). Gastrin cell proliferation after chronic stimulation: effect of vagal denervation or gastric surgery in the rat. J. Physiol. 298, AURES, D. & THOMPSON, J. H. (1972). Simultaneous collection of gastric secretion and gastric mucosal biogenic amines and enzymes, and their comparison between pylorus-ligated and sham-operated rats. Europ. J. Pharmac. 18, BEL, A., LEURAT, R., NESMOZ, J. & GIRARD, M. (1966). Nouvelle technique de fistula gastrique chez le rat. J. med. Lyon Special Issue, October, BERGQVIST, E. & OBRINK, K. J. (1979). Gastrin-histamine as a normal sequence in gastric acid stimulation in the rabbit. Upsala J. med. Sci. 84, BJORKLUND, A., FALCK, B. & OWMAN, Ch. (1972). Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In Method of Investigative and Diagnostic Endocrinology, ed. BERSON, S. A., vol. 1, The Thyroid and Biogenic Amines, ed. RALL, J. E. & KOPIN, I. J., pp Amsterdam: North-Holland. BRODIE, D. A. (1966). The mechanism of gastric hyperacidity produced by pylorus ligation in the rat. Am. J. dig. Dis. 11, BRODIE, D. A. & KNAPP, P. G. (1966). The mechanism ofthe inhibition ofgastric secretion produced by esophageal ligation in the pylorus-ligated rat. Gastroenterology 5, BussoLATI, G. & MONGA, G. (1973). Histochemical and ultrastructural investigation of the endocrine cells of the stomach in hypersecreting (pylorus-ligated) rats. Virchows Arch. Abt. B. Zellpath. 13, DE, C. F. (1965). Histamine and gastric secretion: a later look Fedn Proc. 24, GRIMELIUS, L. (1968). A silver nitrate stain for a2 cells in human pancreatic islets. Acta soc. med. upsal. 73, HAKANSON, R., HEDENBRO, J., LIEDBERG, G., SUNDLER, F. & VALLGREN, S. (198). Mechanisms ofgastric acid secretion after pylorus and oesophagus ligation in the rat. J. Physiol. 35, HAKANSON, R., KROESEN, J. H., LIEDBERG, G., OSCARSON, J., REHFELD, J. F. & STADIL, F. (1974). Correlation between serum gastrin concentration and rat stomach histidine decarboxylase activity. J. Physiol. 243,

12 156 J. ALUMETS AND OTHERS HAKANSON, R., LARSSON, L. I., LIEDBERG, G., REHFELD, J. F. & SUNDLER, F. (1977). Suppression of rat stomach histidine decarboxylase activity by histamine: H2-receptor-mediated feed-back. J. Physiol. 269, HXKANSON, R., LARSSON, L.-I., LIEDBERG, G. & SUNDLER, F. (1976). The histamine-storing enterochromaffin-like cells of the rat stomach. In Chromaffin, Enterochromaffin and Related Cells, ed. UPLAND, R. E. & FuJITA, T. pp Amsterdam: Elsevier. HAKANSON, R. & R6NNBERG, A. L. (1974). Improved fluorometric assay of histamine: condensation with o-phthalaldehyde at -2 'C. Analyt. Biochem. 6, KAHLSON, G., RoSENGREN, E., SVAHN, D. & THUNBERG, R. (1964). Mobilization and formation of histamine in the gastric mucosa as related to acid secretion. J. Physiol. 174, KAHLSON, G., ROSENGREN, E. & SVENSSON, S. E. (1973). Histamine and gastric acid secretion with special reference to the rat. In Pharmacology of Gastrointestinal Motility and Secretion, vol. 1., ed. HOLTON, P. pp Oxford: Pergamon Press. LANE, A., Ivy, A. C. & Ivy, E. K. (1957). Response of the chronic gastric fistula rat to histamine. Am. J. Physiol. 19, McALPIN, C. D., MOORE, T. C. & LEMMI, C. A. E. (1974). Gastric histamine metabolism after acute pyloric ligation in the Shay rat. Arch. Surg., Chicago 19, NAGY, L., MdzsIK, Gy., TARNOK, F., SZALAI, M., P6TH, I. & JJAVOR, T. (1978). Interrelationships between the gastric secretary responses, prostaglandin E2 inhibition and serum level of immunoreactive gastrin in pylorus-ligated and antrectomized rats. Pharmacology 16, REHFELD, J. F., STADIL, F. & RUBIN, B. (1972). Production and evaluation of antibodies for the radioimmunoassay of gastrin. Scand. J. clin. Lab. Invest. 3, RIDLEY, P. T., GROVES, W. G., SCHLOSSER, J. H. & MASSENBERG, J. S. (1973). H2-Antagonist action on interdigestive gastric acid secretion and motility in the rat. In International Symposium on Histamine H2-Receptor Antagonists, ed. WOOD, C. J. & SIMKINS, M. A., pp Welwyn Garden City: Smith, Kline & French. SHAY, H., KOMAROV, S. A., FELS, S. S., MERANZE, D., GRUENSTEIN, M. & SIPLET, H. (1945). A simple method for the uniform production of gastric ulceration in the rat. Gastroenterology 5, SHAY, H., KOMAROV, S. A. & GRUENSTEIN, M. (1949). Effects of vagotomy in the rat. Arch. Surg., Chicago 59, SHAY, H., SUN, D. C. H. & GRUENSTEIN, M. (1954). A quantitative method for measuring spontaneous gastric secretion in the rat. Gastroenterology 26, 96f-913. STADIL, F. & REHFELD, J. F. (1973). Determination of gastrin in serum. An evaluation of the reliability of a radioimmunoassay. Scand. J. Gastroenterol. 8, WEIBEL, E. P. & BOLENDER, R. P. (1973). Stereological techniques for electron microscopic morphometry. In Principles and Techniques of Electron Microscopy. Biological Applications, vol. 3, ed. HAYAT, M. A., pp New York: Van Nostrand Reinhold. EXPLANATION OF PLATE PLATE 1 (A-C) Formaldehyde-induced dopamine fluorescence in endocrine cells of the oxyntic mucosa 11 hr after the intraperitoneal administration of L-DOPA (1 mg/kg). A, unoperated rat; B, pylorusligated rat, 4 hr; C, pylorus-ligated rat, 16 hr. x 15. (D-E) Argyrophil staining of the endocrine cells. D, unoperated rat; E, pylorus-ligated rat, 4 hr; F, pylorus-ligated rat, 16 hr. x 225. All rats were fasted for 48 hr before killing. The lack of effect of pylorus ligation disagrees with the previous findings of Bussolati & Monga (1973).

13 The Journal of Physiology, Vol. 323 Plate 1 t< St-j -.;r9}sr4 1*6;u. : *9 I"., -'... w:.*..,x 'r :*s: < ; 2;4S~~~~~~~~~ a A i,~ ~.ov ~~.>S;v %INIM. V.,'-,..1 r VY a.S@ X..~ I;6w, +Sr...s...,. E -p F rs.. '4 C *: :4.s dip I A f*\#44j 1L NjSt}k? t.. q r4) 4 'Av 1' - i_.'.c. lee' \* a'4.... "r A-^ it4-.:. } J. ALUMETS AND OTHERS (Facing p. 156)