effect of pentagastrin on mouse gastric and small intestinal mucosa in vivo and in vitro

Cokp. Biochrvl. Physiol. Vol. 74A. No. I. pp. 37 to 44. 1983 Printed in Great Britain.

0300.9629!83:010037-08$03.00,0 0 19X3 Pergamon Press Ltd

EFFECT OF PENTAGASTRIN ON MOUSE GASTRIC AND SMALL INTESTINAL MUCOSA

ZN VII/O AND IN VITRO

J.-G. CHABOT,* N. PAYETt and J. S. HUGON$

Department of Anatomy and Cell Biology, Intestinal Biology Unit, Faculty of Medicine, Centre Hospitalier Universitaire de Sherbrooke,

Sherbrooke, P.Q. Canada, JlH 5N4

(Received 2 April 1982)

Abstract-l. Single administration of various doses (62.5, 125, 250, 500 pg/kg bodyweight) or multiple administration of a single dose (250&kg body wt) of pentagastrin to fasted mice do not alter the protein and DNA content, nor the DNA synthesis in fundus of stomach, distal duodenum, and proximal jejunum.

2. Pentagastrin has also been added at different doses into an organ culture of adult mouse duodenum or jejunum.

3. The explants were taken from animals fasted for 24 or 48 hr before sacrifice. 4. Protein and DNA content, protein and DNA synthesis, activities of several brush border enzymes

and morphology have not been modified. 5. Therefore, the drug does not seem to have a direct effect on the mouse intestinal mucosa. 6. These observations are compared to results obtained from other species.

INTRODUCTION

Several studies have suggested that gastrin, a gastrointestinal hormone, secreted by the antral mucosa of the stomach, or pentagastrin, a synthetic analogue, could have trophic action on the stomach, the small intestine and the colon (Johnson, 1980). These results have been obtained from in viuo studies in the rat (Johnson, 1980), in the dog (Ryan et al., 1978; Willems et al., 1972) and in human (Hansen et al., 1976; Mitz- negg et al., 1975; Mitznegg et al., 1976; Nylund et al., 1978). However, many of these works have used different approaches concerning the fasting period before the experiments, the doses used, the type of injections, and the length of the study. Moreover, recent works, in vitro (Black & Moog, 1978; Shields et al., 1980; Yek & Moog, 1978; Yeomans et al., 1976) have projected serious doubts about the real trophic effect of this hormone. We have therefore initiated a thorough study on the action of pentagastrin on the adult mouse gastro-intestinal mucosa, in vioo and in vitro. We have also measured the gastrin content of the serum in adult mouse.

MATERIALS AND METHODS

Three-month-old Swiss ICR male mice were used. They were subjected to an artificial light-day cycle with light from 6 a.m. to 6 p.m. During periods of fasting, animals

* The present work has been done as part of a fulfillment of a PhD thesis in the Department of Anatomy and Cell Biology. Mr. Chabot was a recipient of an MRC studentship.

t Dr. N. Payet is a recipient of an MRC fellowship. $This work has been supported by an MRC grant

(JSH). The requests for reprints should be sent to J. S. Hugon.

were housed in cages with wide mesh bottoms and allowed free access to water.

Experiments in vivo (I) Dose response study. The mice were fasted for 24 hr.

Half of the animals were injected intraperitoneally (i.p.) with 62.5, 125, 250 and 500 pg/kg bodyweight (body wt) of pentagastrin (Johnson, 1977; Johnson & Guthrie, 1974) and the other half received an equal volume of saline (0.1 ml). Mice were killed 12 hr after the injection by rapid cervical dislocation. This time was chosen according to the experiments made in the rat by Johnson & Guthrie (1974). Thirty minutes before sacrifice, each mouse was injected i.p. with 25 &i 6-3H-thymidine (sp. act. 20 Ci/mM: Amer- sham, England). The fundus of the stomach, the second half of the duodenum (4 cm from the pylorus) and a segment of proximal jejunum (11 cm from the pylorus) were rapidly excised, washed, weighed and frozen. The protein and the DNA contents and the incorporation of 6-3H- thymidine into DNA were assayed within 24 hr after the sacrifice.

(II) Repeated doses. The mice were randomly divided into two groups receiving during their 48 hr of fasting, 6 equally spaced i.p. injections of saline or of 250 pg/kg/body wt of pentagastrin (Johnson, 1977). All animals were killed by cervical dislocation, 8 hr after the last injection. Thirty minutes before sacrifice, they were injected i.p. with 25 PCi of 6-3H-thymidine. The sampling and the assays were similar to those of the dose response study.

Experiments in vitro The mice were fasted for 24 or 48 hr before sacrifice. The

explants were taken from the second half of the duodenum or proximal jejunum (see: Dose response study). The organ culture was made according to the technique described by Ferland & Hugon (1979) using a medium composed of DMEM and NCTC 135 (Gibco, Grand Island, NY) with 10% fetal bovine serum and several antibiotics added. The medium was renewed every 24 hr. In each culture. 60 explants from the same intestinal segment and from the same animal were taken. Twenty were used to test the

37

38 J.-G. CHABOT et al.

proteins, thi DNA and the enzymatic contents, at the start of the culture: Forty others were cultured in presence or absehce of pentagagtrin. The hormone was added to the medium from the Btart of the culture at the following concentration: 0.5 pg/ml medium (Shields et al., 1980; Hoff- man & Kuksis, 1980; Litchenberger et al., 1973; Miller et al., 1973), 2.5 kg/ml medium (Reiss & Williams, 1979) and 5.0 &/ii11 medium. At the end of the culture, after a tho;&gh washing, the explants were weighed and homogenized. The proteins, the DNA and the enzymatic contents as well as the protein and DNA synthesis were determined.

Biochekcal methods The intestinal or gastric tissues and the explants were

homogenized for 2 min in bidistilled water with an Omni- mixer equipped with a microattachment. Proteins were determined according to Lowry et al. (1951) using bovine serum albumin as standard. DNA was measured by the method of Burton (1956) modified by Giles & Myers (1965) using DNA calf thymus as standard. Sucrase, maltase and trehalase were measured according to the modification of Lloyd & Whelan (1969) of the Dahlqvist method (1964), glucoamylase, using soluble starch as substrate, according to Shlegel-Haueter et al. (1972) and alkaline phosphatase according to Eichholz (1967).

The media were dialyzed against 3 x 2 1. saline for 3 days in order to remove glucose interfering with the gluco- sidase assays. The media were then centrifuged at 45,OOOg for 1 hr. The pellet was resuspended and centrifuged another hour at 45,000 9. The final pellet was called P, or particulate fraction of the media. The supernatant, S, was the soluble fraction of the media (Berteloot et al., 1981).

Protein synthesis. In these experiments we have analyzed the specific activity of the amino acid pool and the rate of protein synthesis in the intestinal explants cultured in presence or absence of pentagastrin. A quantity of 5 &i/ml of 14C amino acid mixture (sp. act. 0.1 mCi/ml NEN) was added to the medium for 3 hr (Kagnoff el al., 1972) after various times of culture in a cold medium (0, 6, 21, 30, 45 hr). At the end of the pulse, the explants were homogenized. A quantity of 0.5 ml of homogenate was precipitated with 0.5 ml of 15% TCA at 4°C and centrifuged at 12,000 9 for 15 min; the pellet was resuspended in 0.5 ml of 15% TCA and centrifuged at 12,000 9 for another 15 min.

The tissue amino acid content was determined on the pooled supernatant of the two centrifugations by the method of Rosen (1957) using leucine as a standard. The use of a mixture of radioactive amino acids instead of a single labelled species allow a legitimate estimation of the

overall amino acid specific activity by relating radioactivity to the amount of acid-soluble ninhydrin-positive material.

The specific activity of the tissue-free amino acid pool was determined on 0.5 ml of the pool supernatant of the two centrifugations. The results were expressed as dpm/pg of amino acids. This pool was considered a precursor pool of protein synthesis as demonstrated in in uiuo studies (Fern & Garlick. 1974: McNurlan et al., 1979). ’ The incorporaiion of amino acids into proteins was ana- lvzed on the final nellet resusDended in 0.2 ml of 70% PCA and 0.4ml of 30% H20,, heated at 55°C for complete solubilization and mixed with 1Oml of Aquasol-2. The results were expressed as dpm/g of proteins/hr.

The rate of protein synthesis was evaluated as the ratio multiplied by 100 between the total protein activity of acid precipitable tissue protein and the total radioactivity of the tissue-free amino acid pool.

DNA synthesis. To evaluate the DNA synthesis in organ culture, 2 $i of 6-3H-thymidine (sp. act. 22 Ci/mmol) per ml of medium were added for 1 hr at different time periods. For all the experiments in vivo and in vitro, 0.5 ml of homogenate was precipitated with 0.5 ml of 20% TCA at 4°C and centrifuged at 1650 g for 10 min. The pellet was resuspended in 1 ml of 10% TCA at 4°C and recentrifuged for 10 min. The final pellet was suspended in 2 ml of 5% TCA, heated at 100°C for 30 min and centrifuged. Ten milliliters of Aquasol- was mixed with 0.5 ml of the supernatant. All the results were expressed as dpm/pg of DNA.

Radioactivity was measured in a Packard Tricarb liquid scintillation spectrophotometer. Quench correction was made by external standardization.

Morphological methods. Explants were fixed at several time periods, in 3.5% cacodylate buffered glutaraldehyde for 2 hr, postfixed in 2% buffered Os04, dehydrated and embedded in Epon. Thin sections were stained with uranyl- acetate and lead citrate.

Statistical significance of differences was determined by Student’s t-test.

Gnstrin assays. Mouse serum gastrin and the gastrin content of the culture media before and after the culture have been measured with the radioimmunoassay kit [lz51] from Becton-Dickinson. The method used was described by Yalow & Berson (1971).

RESULTS In vivo

(1) Biochemical data. Neither protein nor DNA content of fundus (Table I), duodenum and jejunum (data

Table 1. Protein content, DNA content and DNA synthesis in the mouse fundus stomach. Effect of pentagastrin

Single injection Multiple injections (N = 4) (N = 7)

C P C P

Protein 139 + 7’ 149 + 3 130+ 7 135 + 6

(;k$ tissue) 5.27 + 0.15 5.53 + 0.21 5.42 + 0.21 5.99 + 0.36 (mg/g tissue) DNA synthesis 48 + 9 44+7 73 + 13 59 + 12 Wm/~g DNA)

Single injection: The mice were fasted for 24 hr and were then injected i.p. with ;250 @kg body wt of pentagastrin (P) or with an equal volume of saline (C). Mice were ‘killed 12 hr after injection.

Multiple’injections: During the 48 hr fasting, mice received 6 equally spaced i.p. ‘injections of saline (C) or pentagastrin (P) (250 yg/kg body wt). All animals were killed 8 hr after ihe last injection.

*Mean +SEM.

Effect of pentagastrin on mouse gastric and small intestine mucosa

3 3 3 7 1

12h 12h 48h

Fig. 1. DNA synthesis of the duodenal mucosa of imice receiving a single injection of 62.5, 125, 250 or 500 pg/k/body wt of pentagastrin, after 24 hr fasting and killed 12 hr later or receiving 6 x 250 pg/k/ body wt of pentagastrin during 48 hr fasting. columns represent controls receiving an equal

as mean + SEM.

not shown) were modified by single or repeated injections of pentagastrin.

(2) DNA synthesis. The levels of DNA synthesis in the fundus (Table l), duodenum (Fig. 1) and jejunum (data not shown) of the pentagastrin-treated mice (single or repeated injections) were similar to those observed in the controls.

In vitro The measurements have been made after 24 or

48 hr of culture on explants taken from animals fasted for 24 hr and after 48 hr of culture on explants taken from animals fasted for 48 hr.

(1) Protein content. Pentagastrin (0.5 pg, 2.5 pg/ml) was added to the culture medium of duodenal explants. No statistical differences were registered in the amount of proteins per mg tissue after the culture in the presence or absence of pentagastrin (data not shown). The addition of pentagastrin (0.5 pg/ml) to the medium of jejunal explants did not modify either the protein content during the 48 hr of culture (data not shown).

(2) DNA content. A quantity of 0.5 and 2.5 pg/ml pentagastrin were added to the culture medium of duodenal explants. Then 0.5, 2.5 and 5 pg/ml pentagastrin were added to the jejunal explants. The amount of DNA/g tissue was not modified in the duodenum (data not shown) or jejunum (Table 2) by the presence of pentagastrin.

Table 2. DNA content and DNA synthesis in control and pentagastrin treated jejunal explants

DNA content DNA synthesis Pentagastrin (mg/g tissue) (dpm/vg DNA)

None 7.34 + 0.07 50.54 + 0.98 2.5 pg/rnl 7.68 + 0.21 57.39 + 0.40; 5.0 pg/ml 7.43 + 0.20 55.36 + 4.07

The explants were cultured for 23 hr before the introduction of 2.5 or 5 pg of pentagastrin/ml. The culture was continued for 12 hr before the pulse of thymidine.

Mean * g~h4 for 3 experiments. *P < 0.05 compared to control culture.

\ (3) Enzymatic activities. For these experiments, 0.5 pg/ml pentagastrin was added to the culture medium. The wet weight of the cultured explants, treated or not by pentagastrin being the same (data not shown), we have used the International Units to express the enzymatic activities.

The activities of sucrase (Fig. 2) maltase, trehalase. glucoamylase and alkaline phosphatase (Fig. 2) were not modified by the presence of pentagastrin in the duodenal and jejunal explants, taken from animals * fasted for 24 or 48 hr. The amount of enzymes secreted during the culture was also very similar..

(4) Protein synthesis.

(a) Controls The amino acid content of the jejunal explants

remained stable during the culture (data not shown). The specific activity of the tissue free amino acid pool decreased slowly but not significantly (data not’ shown). The incorporation of amino acids into the proteins was also decreasing slowly by not significantly but the rate of protein synthesis remained stable during the 48 hr of culture (Fig. 3).

(b) Pentagustrin No significant difference was found for .the same

parameters between the control and the pentagastrin treated duodenal and jejunal explants during the 48 hr of culture (Fig. 3).

(5) DNA synthesis. In the duodenum, the DNA synthesis measured by a I hr 6-3H-thymidine pulse at the.. end of the culture was not increased in presence of pentagastrin at any of the 3 concentrations used (data not shown). In the jejunum, the resuhs were identjcal between the controls and the treated explants if the pentagastrin was added at the start of’the culture (Fig. 4) after 6 or 23 hr (Table 2) or ,if it remained m *. contact during 13, 16, 18 or 25 hr. A Significant difference was nevertheless obtained whrmb’2.5,ng/ml of pentagastrin was added to the medium for 13 hr after 23 hr of culture (Table 2).

(6) ,Vforphological studies-Fig. 5. NO uftrdStrUC-

tural alterations of the absorptive cells or the undiffer-

J.-G. CHABOT et al.

SUCttASE ALKALINE PtiOSPtiAlASE

EO MC % EC Ep E. MC Mp EC Ep

Fig. 2. Sucrase and alkaline phosphatase activities in duodenal explants and in the medium after a 48 hr organ culture. The animals were fasted for 24 hr before the sacrifice. EO: enzymatic activity in the explants at the start of the culture. EC: control explants after 48 hr of culture. EP: pentagastrin-treated explants after 48 hr of culture. MC: control medium. MP: pentagastrin treated medium. The stripped bars indicate the enzymatic activity present in the P, fraction of the medium, the clear bars, the S, fraction. Results are expressed in total IU in which 1 IU is equivalent to 1 PM substrate hydrolyzed/min.

Data presented are the mean + SEM of 5 experiments.

O-3H s-on 2l-24ti 30-33 H 45-u Ii

Fig. 3. Rate of protein synthesis during the culture. Results expressed as the ratio multiplied by 100 between the total radioactivity of acid precipitable tissue protein and the total activity of the tissue-free amino acid pool. Animals have been fasted for 24 hr before the sacrifice. The 14C amino acid mixture (5 &i/ml) was added at the start of the culture and after 6,21, 30 and 45 hr in the presence or absence of pentagastrin (0.5 pg/ml) for a 3 hr pulse. The squared columns: control explants. Dotted columns:

pentagastrin-treated explants. Data shown are the mean k SEM of 3 experiments.

100

ii 9 60

Q

20

13 h 25 h

Fig. 4. DNA synthesis in the jejunal explants taken from animals fasted for 24 hr before the sacrifice and cultured for 13 or 25 hr in presence or not of pentagastrin. 6-3H-thymidine was added during the last hours of culture. 13 hr: first clear column: controls. Diamond shaped columns: 0.5 pg/ml pentagastrin. Black dot columns: 2.5 pgg/ml pentagastrin. 25 hr: first two columns: controls and 0.5 pg/ml pentagastrin. Second two columns: controls and 2.5 pg/ml pentagastrin. Third two columns: controls and 5.0 pg/ml

pentagastrin. In brackets: the number of cultures analyzed. Data shown as mean + SEM.

Effect of pentagastrin on mouse gastric and small intestine mucosa 41

Fig. 5. Absorbing cells of the villus from an explant cultured for 48 hr in presence of 0.5 pg/rnl pentagastrin. Ultrastructural cells are not modified in comparison with controls.


entiated cells of the, duodenum or the jejunum have been recorded at any time and with any doses used.

(7) Gastrin content. The amount of gastrin present in the serum of fed mice (N = 9) reaches 182.06 k l-2.26 pg/ml. After 24 hr of fasting (N = 9) a decrease of 16% (152.77 f 11.78 pg/ml) was observed and’after 48 hr of fasting (N = 11) a very significant decrease of 41% (107.85 k 12.09 pg/ml, P < 0.001) was noted. In the fetal bovine serum (Gibco) 28 pg/ml of gast& was measured. The amount of gastrin present in the total culture medium before and after the culture was not detectable.

DISCUSSION :

The in uivo experiments in the rat have shown a stimulation of protein synthesis (Johnson, 1980) but have not analyzed the state of the amino acid precur- sorpool which has been shown to be rather stable in uiuo. However, in organ culture of intestine, the proteins content can vary and we thought essential to measure the specific activity of the amino acid pool at different time periods in presence or not of pentagastrin to ascertain that the rate of protein synthesis remained constant.

In our study, exogenous administration of pentagastrin has not modified the protein and DNA content nor thymidine incorporation into DNA of the stomach’s fundus, of the distal duodenum and of the proximal jejunum of fasted mice. In addition, pentagastrin has not modified in vitro, the protein and DNA content nor the enzymatic activities nor the protein gnd DNA synthesis nor the ultrastructural morphology of duodenal and jejunal explants of adult mouse. For the DNA synthesis, only one time of contact (13 hr) after 23 hr of culture gives a significant difference. Nevertheless, an increase was not observed after 16, 18 and 25 hr of culture and this observation remained therefore unaccountable.

These results are at variance with other observations made in vivo (Johnson, 1980; Ryan et al., 1978; Willems et al., 1972; Hansen et al., 1976) and in vitro but in different experimental conditions, as in isolated intestinal cells in culture (Hoffman Jr Kuksis, 1980; Litchenberg et al., 1973), in gastric mucosal cells (Miller et al., 1973; Sutton & Donaldson, 1975). in carcinoma of the colon (Murakami & Masui, 1980) and in the colonic mucosa (Reiss & Williams, 1979). However, in the mouse, Willems & Lehy (1975) have only detected a small increase in DNA synthesis in the fundic gland after 6 days of chronic administration of supraphysiological doses of gastrin. Moreover, other workers have seriously questioned the trophic effect of gastrin on the small intestine. In fed rats, no modification of the labelling index was recorded in duodenum and colon (Mak & Chang, 1976). In fasting rats receiving a large range of gastrin or pentagastrin injections, Fatemi et al. (1980) have not observed an increase in DNA synthesis in the intestinal mucosa. In fasting rats, high levels of gastrinemia did not prevent the decrease of DNA content or modify the DNA synthesis in the small intestine (Oscarson et al., 1977). Injections of pentagastrin did not modify the ‘protein synthesis in the duodenum of fasted humans (Mitznegg et al., 1975; Mitznegg et al., 1976) nor the disaccharidase activities in the jejunum of

normal or diseased humans (Nylund et al., 1978). In neonatal rats, parenterally injected pentagastrin did not provoke a duodenal hyperplasia nor an alteration of the brush border enzymatic activities (Yek & Moog, 1978). Morin & Ling (1978) have shown, in resected and intravenously fed rats, that the trophic effect of pentagastrin was limited to the most proximal portion of the duodenum. Shields et al. (1980) have been unable to detect any stimulation of the protein synthesis in the duodenum, jejunum and ileum of adult rabbit in organ culture. In the embryonic chick duodenum organ culture, pentagastrin did not modify brush border enzyme activities (Black & Moog, 1978).

Several explanations could be proposed for the lack of trophic effect of pentagastrin in our experiments.

(1) The mouse may not respond to gastrin in the same manner as the rat even if gastrin receptors seem to be present in the mouse duodenal cells (Payet & Hugon, unpublished results). The same situation (presence of receptors and lack of effect) has already been demonstrated in cultured rat pituitary cells for EGF (Johnson, 1980). On the other hand, a rapid inactivation of pentagastrin seems unlikely (Temper- ley et al., 1971).

(2) As the number of gastrin receptors is regulated by circulating gastrin (Takeuchi et al., 1980), the lack of response in vivo and in vitro could be due to a decrease in the number of receptors. However, in vivo, repeated doses of pentagastrin, maintaining a high level of gastrinemia and therefore a normal level of receptors, have not provoked a trophic effect. In vitro, Forgue-Lafitte et a/. (1980) have shown that EGF receptors in the rat intestine decreased dramatically during a pre-incubation at 20 or 30°C. As the start of organ culture takes place about 30min after the beginning of the dissection process, we could specu- late that some receptors have already been interna- lized and are perhaps not renewed thereafter.

(3) The dose injected could be too low or the time of sampling, too short. However, the doses used were similar to those employed by other authors in viuo (Johnson, 1980) and in vitro (Shields et al., 1980; Hoff- man & Kuksis, 1980; Litchenberger et al., 1973; Miller et al., 1973; Reiss & Williams, 1979). The sampling was extended in vitro to 24 hr after the first contact.

(4) In the literature, fasting periods preceding the injection of gastrin or pentagastrin varies from nothing to 72 hr. Our experiments started in uioo during a fasting of 48 hr or after a fasting of 24 hr and in oitro, after a fasting of 24 or 48 hr.

(5) The saturation of the receptors by the gastrin present in the fetal bovine serum which is a com- ponent of the medium seems very unlikely. The gastrin concentration in several samples of fetal bovine serum never exceeded 28 pg/ml or 2.8 pg/ml in the final culture medium. In uivo, the concentration of gastrin in the serum of 48 hr fasting mice reached 107 pgjml. It is therefore very unlikely that the gastrin receptors were already saturated before the addition of pentagastrin.

In conclusion, in our experimental conditions, the pentagastrin does not seem to have a direct trophic effect on the gastronintestinal mucosa of the mouse in vivo and in vitro.

Effect of pentagastrin on mouse gastric and small intestine mucosa 43

Acknowledgement-This work has been supported by an MRC grant (JSH). J.-G. Chabot was the recipient of an MRC studentship. The authors are greatly indebted to Mrs. E. Hugon and Mr. D. Martel for their skilful techni- cal help and to Mrs. J. Paquet for her secretarial help.

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effect of pentagastrin on mouse gastric and small intestinal mucosa in vivo and in vitro

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