Evaluation of the effects of pentagastrin, gastrin and pancreatic glucagon on cell proliferation in the rat gastrointestinal tract

The effects of six injections of a range of doses (100-1000 micrograms/kg bodyweight) of pentagrastrin and single injection of a range of doses of porcine gastrin (10-40 micrograms/kg bodyweight) and pancreatic glucagon (25-100 micrograms/kg bodyweight) on cell proliferation in the intestine of fasted rats has been investigated. The end-point employed included the measurement of 14C leucine incorporation and thymidine-derived tritium content of the body of the stomach, duodenum, jejunum, ileum and colon. The carbon 14 and tritium content per microgram of tissue in triplicate samples of fifty individually dissected crypts of glands were determined. From these data and the wet weight of the washed, blotted, intestinal segments, values for crypts/micrograms tissue and crypts/segment were calculated. The results demonstrated that pentagastrin at physiological doses decreased cell proliferation slightly in stomach, while gastrin and glucagon were without effect. In the small intestine, pentagastrin and gastrin were without significant effect with the exception that they increased the weight of the duodenum. In contrast, a high physiological dose of glucagon increased DNA and protein synthesis throughout the small bowel, but particularly in the ileum. Pharmacological doses of pentagastrin and all doses of gastrin appeared to increase cell proliferation in the colon although the possibility could not be excluded that this was due to stimulation of precursor uptake. Gastrin also increased colonic weight. Glucagon had no effects in the colon. These observations are compatible with the hypothesis that (i) the primary effects of gastrin and pentagastrin on the proximal intestine are as secretogogues and effects on cell proliferation may be secondary, (ii) gastrin and pentagastrin at physiological levels do not stimulate small intestinal cell proliferation, however glucagon does, and (iii) gastrin at physiological levels and pentagastrin at pharmacological levels may stimulate cell proliferation in the colon.     

Changes in thymidine uptake in the gastrointestinal tract of the rat following treatment with 16,16-dimethyl prostaglandin E2

Thymidine uptake in the organs of the gastrointestinal tract of the rat was studied to determine if cell synthesis was involved in the increases in weight of the stomach, small intestine and colon which result from treatment with 16,16-dimethyl prostaglandin E₂ (16,16-dimethyl PGE₂). Animals were treated for 2 days with 16,16-dimethyl PGE₂. They were injected with the ³H-thymidine, sacrificed and the organs of interest were removed. The total amount of tritium in the stomach, duodenum, jejunum, ileum, and colon was determined.Thymidine uptake was significantly increased in the duodenum (1.50 times), jejunum (1.53 times), and colon (1.40 times) but not in the stomach and ileum. The increases were dose related in the duodenum and jejunum. The colon showed a similar dose response pattern but the changes with dose did not reach significance.These results confirm and extend a previous report that 16,16-dimethyl PGE₂ increased thymidine uptake in the duodenum but not the stomach (1). This is different from gastrin which has been shown by others to increase thymidine uptake in the stomach, duodenum, ileum and colon (2,3).     

The distribution of endocrine cells along the mouse intestine: a quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30-60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the bottom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3-4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

The distribution of endocrine cells along the mouse intestine: A quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30–60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the botom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3–4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

The effect of dibutyril cyclic guanosine monophosphate,a competitive antagonist to cholecystokinin-pancreozymin,on gastric acid secretion in the isolated mouse stomach

Dibutyryl cyclic guanosine monophosphate (dbcGMP), a specific competitive inhibitor of the gastrin, cholecystokinin-pancreozymin (CCK-PZ) family of peptides in pancreas, gallbladder and ileum, had no effect on basal acid secretion in the isolated mouse stomach nor on secretion stimulated by bethanechol or histamine. Secretion evoked by low doses of pentagastrin were likewise unaffected by dbcGMP but responses to high doses of pentagastrin were augmented. CCK-PZ and glucagon each inhibited acid secretion evoked by pentagastrin. DbcGMP blocked CCK-PZ-mediated inhibition but was without effect on inhibition by glucagon. These observations suggest that in the gastric glands there exist two receptors with different affinities for gastrin and CCK-PZ which mediate excitation and inhibition respectively.     

NORADRENALINE IN THE GUINEA PIG ALIMENTARY CANAL: REGIONAL DISTRIBUTION AND SENSITIVITY TO DENERVATION AND RESERPINE

The concentrations of noradrenaline, dopamine and 5-hydroxytryptamine were studied in the stomach, duodenum, ileum, caecum (taenia coli), colon and rectum of the guinea pig. The wall of the alimentary canal was dissected into two fractions, one formed by the longitudinal muscle and myenteric plexus, the other formed by circular muscle, submucous plexus, submucosa and mucosa. In the longitudinal muscle-myenteric plexus, the amount of noradrenaline was lower in the ileum (0.56 μg/g of fresh tissue), higher in the duodenum and caecum (0.75 μg/g and 0.82 μg/g respectively) and even higher in the stomach and rectum (0.85 μg/g and 0.91 μg/g). The highest value was found in the colon (1.33 μg/g), probably related to the occurrence of intramural adrenergic neurons. When extrinsic nerves to the ileum were damaged, the amount of noradrenaline in the corresponding ileal segment was reduced to less than 5 per cent within 3 days in both fractions of the wall. 6-Hydroxydopamine (35 mg/kg intravenously) reduced to approx. one-third the amount of noradrenaline in longitudinal muscle-myenteric plexus of ileum and colon. Reserpine (1 mg/kg, subcutaneously injected 72 h-earlier) reduced the amount of noradrenaline in the longitudinal muscle-myenteric plexus of both ileum and colon to 0.01 μg/g. Doses of reserpine as small as 0.02 mg/kg were still effective in causing a great reduction in noradrenaline concentration. No difference in the effects on ileum and colon was observed.     

Stimulating effect of pentagastrin on cancer cell proliferation kinetics in chemically induced colon cancer in rats

Pharmacological doses of pentagastrin or gastrin are known to stimulate cell proliferation in normal colonic epithelium but the growth-promoting effect of gastrin on colon carcinoma is still controversial. In this study morphological parameters were measured to study the effect of pentagastrin (240 micrograms/kg) on the cell proliferation kinetics in experimental tumours. Colon cancer was produced in rats by weekly injections (20 mg/kg b.wt.) of 1.2-dimethylhydrazine for 24 weeks. Tritiated thymidine was given after administration of pentagastrin or the control solution to the animals. 75% of the animals from the pentagastrin group and 66% of the controls had at least one colon cancer. Autoradiographs of the colonic tumors were performed and the percentage of labeled cells in the cancer cell population was determined after counting 4000 to 16,000 cancer cells per tumor. The labeling index for cancer cells in the pentagastrin-treated group (21.49 +/- 1.76%) was higher (P less than 0.01) than in the control group (14.76 +/- 0.66%). In a second study vincristine sulphate (1 mg/kg) was given to the animals 20 h after administering pentagastrin or the control solution. The percentage of arrested metaphases in the tumours was determined after counting 10,000 to 24,000 cancer cells per histological section. Pentagastrin increased (P less than 0.01) the mean metaphase index by 108% (4.9 +/- 0.44% vs 2.35 +/- 0.32%). These data indicate that cell cycle manipulation of colon cancer is possible with hormonal peptides.     

Epithelial cell proliferation and uptake of radiolabeled urogastrone in the intestinal tissues following abdominal irradiation in the mouse

We have evaluated the rate of crypt cell production and uptake of radiolabeled recombinant human urogastrone (125I-rhUG) in the intestinal tissues of the mouse at 3, 5, 7, 9, and 12 days following irradiation of the abdomen with 9 Gy. At autopsy, the animals were injected intraperitoneally with 1 microgram/g body weight of the metaphase arrest agent, vincristine sulfate, and 25 muCi of 125I-rhUG (specific activity 1.7 muCi/micrograms) to quantify the rate of crypt cell production and uptake of radiolabeled urogastrone, respectively. The results indicated that the rate of crypt cell production was increased significantly in the irradiated animals compared to the unirradiated animals and showed a peak on the 3rd and 5th postirradiation days in small intestine and colon, respectively. The uptake of 125I-rhUG was increased significantly on the 3rd postirradiation day in the intestinal tissues but showed a bimodal pattern with peaks on the 3rd and 9th postirradiation days. These results suggest that there may be a close association between epithelial cell proliferation and uptake of 125I-rhUG, particularly in the early part of recovery of intestinal mucosa following irradiation. However, these data do not discriminate whether the increased uptake of 125I-rhUG is the cause or the effect of proliferation induced by an irradiation stimulus. Further analysis also revealed that there was no relationship between crypt depth and 125I-rhUG uptake. However, crypt depth was inversely correlated with villus height in the proximal small intestine but not in the ileum. Villus height was correlated inversely with 125I-rhUG uptake in the ileum and jejunum but not the duodenum. The rate of crypt cell production was strongly correlated with crypt depth throughout the intestine and inversely correlated with villus height. This suggests that villus-to-crypt inhibitory feedback may be a primary regulator of cellular proliferation in the crypts and the association of 125I-rhUG uptake with proliferation indirectly reflects this interaction.     

Influence of obestatin on the histological development of the small intestine in piglets during the first week of postnatal life

Obestatin is a gastrointestinal peptide having wide-ranging effects on cell proliferation; however, its mechanism of action remains poorly understood. Thus, the aim of the study was to elucidate the effect of exogenous obestatin on the postnatal structural development of the small intestine. Seven-day-old piglets with an average BW of 1.56 ± 0.23 kg were divided into four groups (n = 10) that received intragastrically obestatin (2, 10 or 15 μg/kg BW) or vehicle. After a 6-day experimental period, morphological analysis of gastrointestinal tract and small intestine wall (mitosis and apoptosis indexes, histomorphometry of mucosa and muscularis layers) was performed. The study revealed a seemingly incoherent pattern of the histological structure of the small intestine among the experimental groups, suggesting that the effect of obestatin is both intestinal segment specific and dose dependent. Histomorphometric analysis of the small intestine showed that higher doses of obestatin seem to promote the structural development of the duodenum while simultaneously hindering the maturation of more distal parts of the intestine. Intragastric administration of obestatin increased the crypt mitotic index in all segments of the small intestine with the strongest pro-mitotic activity following the administration of obestatin at a dose of 10 and 15 μg/kg BW. The significant differences in the number of apoptotic cells in the intestinal villi among the groups were observed only in proximal jejunum and ileum. In conclusion, it seems that obestatin shows a broad-spectrum of activity in the gastrointestinal tract of newborn piglets, being able to accelerate its structural development. However, the varied effect depending on the intestinal segment or the concentration of exogenous obestatin causes that further research is needed to clarify the exact mechanism of this phenomenon.     

Cell migration pathway in the intestinal epithelium: an in situ marker system using mouse aggregation chimeras

The cell migration pathway in the intestinal epithelium of DDK in equilibrium C57BL/6JLac mouse chimeras is demonstrated using Dolichos biflorus agglutinin-peroxidase as strain-specific marker. Cell sheets of one genotype extend in relatively straight lines from crypt to villus apex. Narrow sheets are mostly interrupted in the distal two-thirds of duodenal but not ileal villi, suggesting that in the duodenum cell loss occurs below the apical extrusion zone. These differences between duodenum and ileum correspond to differences in villus shape. The pattern of cell migration in Peyer's patch epithelium is consistent with that of the duodenum. In chimeric colon, sharply demarcated territories of crypts with a narrow cuff of surface epithelium represent the counterpart of the villus/crypt unit of the small intestine.     

Effects of reduced food intake on morphometry and cell production in the small intestine of the rat

The present study examined the effects of a 60% reduction in food intake on kinetic and morphometric parameters in the small intestine of adult male Lewis rats. We observed that, after 20 days, the wet weight of jejunum and ileum, thickness of muscularis externa of duodenum, crypt depth throughout the intestine, and DPM/mg and DPM/crypt in ileum were decreased in animals on reduced food intake when compared to their paired, normally fed controls. These results demonstrated that reduced food intake caused distinctive effects focused primarily in the ileum, thus opening to question the use of the technique of pair feeding as a control for studies of intestinal cell proliferation in which manipulations of the animals result in altered food intake or body weight.     

A METHOD FOR QUANTITATION OF PROLIFERATIVE INTESTINAL MUCOSAL CELLS ON A WEIGHT BASIS: SOME VALUES FOR C57BL/6

A method for determining the number of intestinal mucosal crypts, S cells, and total proliferative cells, on a weight basis has been presented. The number of crypts was obtained (following injection of tritiated thymidine) by dividing the disintegrations per minute (dpm) per mg intestine by the dpm per crypt. Multiplication of the number of crypts per mg by the number of labeled cells per crypt (determined radioautographically) resulted in the number of S cells per mg intestine. Division of the number of S cells per mg by the fraction of proliferative cells in S (obtained by cell cycle analysis) resulted in the number of proliferative cells per mg intestine. Values for duodenum, jejunum, and ileum of male C57BL/6 mice are given.     

Mechanism of hypocalcemia induced by intraperitoneal, intragastric, and intravenous administration of ethanol to the rat

Acute hypocalcemic effects of intraperitoneal administration of 3 and 5 g ethanol/kg body weight; intragastric administration of 3, 5, and 7 g ethanol/kg body weight; and intravenous administration of 2.5 a ethanol/kg body weight were investigated in 20 h fasted female Wistar rats. Dose-dependent hypocalcemia was similarly induced by intraperitoneal and intragastric routes of administration. Net calcium efflux from plasma, as indicated by the plasma 45Ca activity, was unaffected by 3 g ethanol/kg body weight but was delayed at higher doses of ethanol. Intragastric, but not intraperitoneal, administration of ethanol increased the gastrointestinal luminal calcium content partly by enhancing calcium secretion. Significantly increased tissue 45Ca content 30 min after ethanol administration was evident in the duodenum (31%), jejunum (27%), and colon (33%) in the intragastric ethanol-treated group and in the duodenum (40%), jejunum (38%), ileum (45%), colon (39%), and liver (25%) in the intraperitoneal ethanol-treated group. Thus, the hypocalcemia induced by both intraperitoneal and intragastric administration of ethanol could be partly accounted for by the suppression of calcium efflux from some soft tissues. In contrast, intravenous administration of ethanol was found to enhance the calcium efflux from plasma without affecting the net 45Ca content in the soft tissues. The mechanism(s) by which ethanol affects calcium transport has yet to be elucidated.     

Influence of high-calorie (cafeteria) diets on the population of Paneth cells in the small intestine of the rat

A high-calorie (cafeteria) diet is known to cause changes in the intestinal morphology and functioning that seem to be related to calorie overfeeding. Among the cell lineages found in the small intestine epithelium, the Paneth cell (PC) population is known to be influenced by factors related mainly to the intestinal microbiota. The role of PCs in the intestinal cell concert remains unclear, because experimental evidence suggests PC involvement in local processes other than protection against pathogens. Participation of PC in digestive mechanisms has been proposed on this basis. We have analyzed the effect of high-carbohydrate (HC) and high-fat (HF) cafeteria diets on the PC population in the small intestine of the adult rat. For 8 weeks, both HC and HF diets caused a gain in body weight, but whereas the HC-fed rats showed reduced counts of intestinal crypts per 5-mum section, the HF-fed group showed the opposite. In control rats, the number of crypts per section showed a slight tendency to decrease along the duodenum - ileum axis, whereas the number of PCs per crypt was increased towards the ileum. As a result, the number of PCs per section (calculated from these data) remained constant along the three segments of the intestine. The hypercaloric diets did not modify the general tendencies seen in the crypt and PC counts, but reduced the number of PCs per section in the duodenum by 50%. HC-fed, but not HF-fed, rats showed a similar reduction in jejunum also. These changes do not correlate particularly with any of the predictable effects of diet composition, so that a multifactorial control of PC density is proposed.     

Partial characterization of the gastrointestinal weight changes produced in the female rat by 16,16-dimethylprostaglandin E2

Oral and subcutaneous administration of 16,16-dimethylprostaglandin E2 (16,16-dimethyl PGE2) resulted in an increase in the dry weight of the stomach and small intestine of the female rat. This weight response was rapid, controlled rather than continuously progressing, dose dependent and reversible. The dry weight of the colon also increased but this was not studied in detail. Two-day treatment with 16,16-dimethyl PGE2 caused an increase in the incorporation of 3H-thymidine into the duodenum, jejunum and colon suggesting an increase in cell number. Incorporation into the stomach and ileum was not changed. The number of goblet cells per crypt was increased by prostaglandin treatment in all parts of the small intestine. Since these are mucus producing cells, the small intestine may have increased in cell number and mucus production. Both anti-secretory and cytoprotective doses of 16,16-dimethyl PGE2 caused weight increases in the stomach and small intestine. However, the weight gain by itself was not sufficient to protect the stomach or small intestine from necrotic agents after the prostaglandin was discontinued.     

Partial characterization of the gastrointestinal weight changes produced in the female rat by 16,16-dimethylprostaglandin E2

Oral and subcutaneous administration of 16,16-dimethylprostaglandin E₂ (16,16-dimethyl PGE₂) resulted in an increase in the dry weight of the stomach and small intestine of the female rat. This weight response was rapid, controlled rather than continuously progressing, dose dependent and reversible. The dry weight of the colon also increased but this was not studied in detail.Two-day treatment with 16,16-dimethyl PGE₂ caused an increase in the incorporation of ³H-thymidine into the duodenum, jejunum and colon suggesting an increase in cell number. Incorporation into the stomach and ileum was not changed.The number of goblet cells per crypt was increased by prostaglandin treatment in all parts of the small intestine. Since these are mucus producing cells, the small intestine may have increased in cell number and mucus production.Both anti-secretory and cytoprotective doses of 16,16-dimethyl PGE₂ caused weight increases in the stomach and small intestine. However, the weight gain by itself was not sufficient to protect the stomach or small intestine from necrotic agents after the prostaglandin was discontinued.     

Sex- and Age-Related Temporal Variations in Intestinal-Epithelium Proliferation in the Suckling Mouse

Intestinal-crypt enterocytes are a cell population undergoing constant renewal in the mouse. Both adult and 28 d old animals have been shown to exhibit circadian rhythms in cell proliferative indices, but there are only scant data on the 24 h mitotic activity in the small and large intestine of younger mice. The present studies were thus undertaken in order to characterize the proliferative pattern of enterocytes in the duodenum and colon of 7 and 14 d old males and females of the C3H/S strain. Animals of each sex and from each age group were sacrificed every 4 h during a 24 h span, with each animal receiving an injection of colchicine 4 h before sacrifice. Samples of duodenum and colon were removed and processed for hematoxylin–eosin staining. Twenty longitudinally sectioned crypts within each sample were analyzed, and the mitotic indices of both cell populations from each animal were estimated. The arithmetic mean±SEM for each experimental group were then calculated and the statistical significance of differences between the means assessed by ANOVA and Student t-tests. We observed a greater daily mitotic activity in the duodenum than the colon, and moreover enterocytic proliferation in both those regions was greater in 14 than 7 d old animals. Twenty-four hour variations in mitotic activity occurred in all the experimental groups and tissues except for the large intestine of 7 d old females. Finally, the temporal profile of epithelium proliferation in the suckling mouse varied with age, sex, and site of the intestine studied.     

Histological characteristics of the intestinal mucosa of the rat during the first year of life

In spite of the widespread use of rats in gastrointestinal research, there is a lack of information on the qualitative and quantitative histological characteristics. Therefore, a study was performed in 69 male Wistar rats with ages ranging from one day to one year old. The features studied included: height and number of villi in the duodenum, jejunum and ileum, and depth and number of crypts in the duodenum, jejunum, ileum, colon and rectum. Morphometric observations were expressed in a mathematical logarithmic curve that showed a normal, pattern of intestinal growth for each intestinal level. The number of villi in the small intestine decreased from 1 to 35 days of age, whereas the other intestinal parameters all increased during the same period. After 35 days the rates of increase or decrease were lower. The quantification of these intestinal changes provides a new complementary pattern as a reference for research as indicators of normality or malfunction in the rat intestine.     

Reovirus serotype 1 intestinal infection: a novel replicative cycle with ileal disease.

After oral inoculation, reovirus serotype 1 strain Lang was shown to specifically infect the epithelial cells of the ileum, while sparing the epithelial cells in the duodenum, jejunum, and colon. The initial site of replication was localized in cells of the crypts of Lieberkühn adjacent to Peyer's patches. Virus was subsequently found by immunoperoxidase staining in cells migrating up the crypt-villus complex throughout the ileum. The severity of the pathological changes in the ileum was proportional to the concentration of the viral inoculum. This site-specific infection of the ileum by reovirus may provide a model for diseases that are restricted to specific sites in the intestine.     

Cellular Proliferation of Intestinal Epithelia in the Rat Two Months after Partial Resection of the Ileum

Sprague-Dawley rats subjected 2 months previously to partial resection (10 per cent) of the small intestine and their controls were injected with tritiated thymidine and sacrificed at 2 and 23 hours. Segments of the duodenum, jejunum, and ileum were autoradiographed, and the migration of the labelled cells during the period between 2 and 23 hours was measured with an eyepiece micrometer. The cells had migrated 35, 42, and 34 per cent of the total distance from the crypts to the tips of the villi in the control segments of duodenum, ileum, and jejunum respectively, and 43, 90, and 82 per cent, respectively, in similar segments from resected animals. The rate of migration in the portion of the intestine remaining after resection was approximately three times the normal rate in the ileum, twice the normal rate in the jejunum, and showed an increase of one-third in the duodenum. These results demonstrate that the rate of cell renewal is considerably greater in the remaining portion of the intestine of resected animals than in normal intestine. The increased rate of migration after resection, together with the increase in the height of the villi, resulted in an increase in the rate of cell renewal amounting to 141 per cent in the ileum, 114 per cent in the jejunum, and 23 per cent in the duodenum when compared with control segments.