Gastric fundic epithelial proliferation after pancreaticobiliary diversion. Studies with quantitative histological analysis and autoradiography in the hamster.

The effect of pancreaticobiliary diversion (PBD) with hypercholecystokininemia on the gastric fundic mucosa was studied in the Syrian golden hamster over 5 and 24 days. Sham-operated animals served as controls. Basal plasma gastrin concentrations were significantly decreased on days 5 and 24. Five days after PBD, there was a significant increase in the scintigraphically measured [3H]-thymidine incorporation into fundic tissue. Correspondingly, there was a significant increase in the number of cells with [3H]-thymidine-labeled DNA in the proliferative zone of the fundic mucosa. The total number of cells in the gastric pits, the number of cells in the proliferative zone and the proliferation index were also significantly increased 5 days after PBD. Although the mean values of all variables were higher after PBD than in the control group on day 24, these increases were not significant. It is concluded that PBD at least transiently stimulates gastric fundic epithelial proliferation in the hamster. Whether this is an effect of hypercholecystokininemia remains to be definitely proven in further studies.     

Growth hormone-releasing factor (somatocrinin) stimulates epithelial cell proliferation in the rat digestive tract

The possible influence of growth hormone-releasing factor (GHRF) on epithelial cell proliferation in the digestive tract was investigated. Fasted young rats received five hourly subcutaneous injections of either GHRF or saline. They were killed 6, 12, or 18 h after the initial injection and 45 min after [3H]thymidine pulse labeling. At the time of death, blood was taken to determine circulating growth hormone and gastrin levels. After radioautography, DNA synthetic and mitotic activities were estimated in the fundic, antral, duodenal, jejunal, and colonic mucosae. Growth hormone-releasing factor significantly increased labeling indices 6, 12, and 18 h after the initial injection in fundic mucosa, and 6 and 18 h after injection in antral and duodenal mucosae. Furthermore, GHRF significantly increased mitotic indices at 12 h in fundic mucosa and at 12 and 18 h in jejunal mucosa. No effect was seen in the colon. At the three checkpoint times, circulating growth hormone showed no change, but plasma gastrin was increased in the rats treated with GHRF as compared with controls. However, whether the reported stimulatory effect of the GHRF on target cells is direct or indirect remains to be determined.     

Effect of misoprostol and cimetidine on gastric cell labeling index

The effect of misoprostol and cimetidine on gastric cell turnover was studied. Endoscopic biopsy specimens of fundic and antral mucosa were obtained from duodenal ulcer patients before and after 4 wk of therapy with cimetidine 1.2 g/day or misoprostol 800 micrograms/day. Biopsy specimens were incubated with [3H]thymidine. Glandular column length and number of labeled cells were determined after autoradiography. There was no significant difference in column length of antral or fundic glands before or after therapy with cimetidine and misoprostol. The number of antral and fundic labeled cells was significantly decreased after misoprostol treatment (3.6 +/- 0.3 and 4.6 +/- 0.4, mean +/- SE), as opposed to their respective number before therapy (6.9 +/- 0.5 and 8.3 +/- 0.8) (p less than 0.01). On the other hand, after treatment with cimetidine, the number of antral and fundic labeled cells was significantly higher (11.8 +/- 0.9 and 7.5 +/- 1.0, respectively) as compared with their number before therapy (5.7 +/- 0.5 and 5.6 +/- 0.6, respectively). The decreased gastric cell turnover induced by misoprostol indicates that the trophic effect of prostanoids on gastric mucosa is not due to an increase in cellular kinetics. The increased gastric cell turnover induced by cimetidine may contribute to its therapeutic effect in peptic ulcer disease.     

The effect of chemical sympathectomy on the cell kinetics of gastric mucosa in golden hamsters

The effects of chemical sympathectomy on the differentiation of the generative cells, superficial epithelial cells and gastrin cells of the gastric mucosa of hamster were examined by 3H-thymidine autoradiography. The labeling indices of the generative cell zone in the gastric mucosa and antral gastrin cells showed a transient significant decrease after chemical sympathectomy, and then they were gradually restore with time. After 4 weeks onward, the labeling indices of the generative cells showed a slightly low value compared with those in controls. The renewal of superficial epithelial cells and gastrin cells was examined in the hamster sympathectomized for 4 weeks. The time required for the differentiation to PAS positive superficial epithelial cells or gastrin cells had a tendency to elogate after chemical sympathectomy. These results suggest that chemical sympathectomy played an inhibitive action on the proliferation and the differentiation of gastric mucosa.     

Effect of Cimetidine on Gastric Mucosal Cell Proliferation in Man

Seven duodenal ulcer patients were treated for 3 months with cimetidine. Before and after treatment endoscopic biopsy specimens were taken for autoradiographic estimation of cell proliferation in the gastric mucosa in the antral and fundic part of the stomach and from the duodenum. In all three areas the estimated labeling index was increased during medication with cimetidine. The increase in epithelial cell renewal may participate in the ulcer healing effect of cimetidine.     

Effect of cimetidine on gastric mucosal cell proliferation in man

Seven duodenal ulcer patients were treated for 3 months with cimetidine. Before and after treatment endoscopic biopsy specimens were taken for autoradiographic estimation of cell proliferation in the gastric mucosa in the antral and fundic part of the stomach and from the duodenum. In all three areas the estimated labeling index was increased during medication with cimetidine. The increase in epithelial cell renewal may participate in the ulcer healing effect of cimetidine.     

Degradation of elastin in experimental elastase-induced emphysema measured by a radioimmunoassay for desmosine

Experimental emphysema, produced by a single intratracheal injection of elastase in hamsters, progresses in severity over months. To investigate whether this progression is due to continuous elastolysis, we measured the urinary excretion of desmosine by radioimmunoassay (RIA) as a measure of elastin catabolism in vivo. Normal hamster excreted 1.6 microgram of desmosine, equivalent to a daily turnover of approximately 0.4 mg of elastin. During the first 24 hr after injection of 25 units of elastase, excretion of desmosine was increased threefold, rapidly returning to normal over several days. Desmosine excretion was normal after 6 days. Homogenates of lungs from elastase-injected hamsters were incubated in vitro, and the release of soluble desmosine was followed by RIA as a measure of the active elastase in the tissue. The method was sufficiently sensitive to detect 0.1 microgram of enzyme bound to elastin. Desmosine solubilized in vitro from lungs removed at intervals after elastase injection was 10-fold that of control at 1 hr and slightly elevated at 48 hr, but equaled control levels at 7 days. These results indicate that the late progression of elastase-induced emphysema is not accompanied by increased elastolysis.     

Light and electron microscopic and autoradiographic studies onN-methyl-N-amylnitrosamine-induced rat esophageal carcinogenesis

The purpose of this study was to investigate the histogenesis of experimental tumors in the rat esophagus. Thirty rats received 0.0015% N-methyl-N-amylnitrosamine (MNAN) in the drinking water for 12 weeks. Another 30 rats received tap water. All rats then received tap water until sacrifice. Rats from each group were sacrificed immediately after MNAN administration, four weeks after, and eight weeks after. One hour before sacrifice, [³H]TdR was injected by tail vein to label proliferating cells. The entire esophagus and stomach were removed and processed for light and electron microscopy and autoradiography. The overall frequency of esophageal tumors after MNAN was 83% and did not differ significantly among the three experimental groups. Tumors were primarily papillomas and squamous cell carcinomas and occurred with equal frequency in the upper, middle, and lower thirds of the esophagus. No tumors were found in the squamous-lined forestomach. Electron microscopy revealed abundant tonofilaments, free ribosomes, and mitochondria accompanied by vacuoles. By autoradiography, esophageal epithelial proliferation was markedly stimulated in nontumorous mucosa from all three experimental groups. We conclude that MNAN ingestion for 12 weeks reliably produces papillomas and squamous cell carcinomas throughout the rat esophagus, but not in the squamouslined forestomach, and that MNAN stimulated marked epithelial proliferation which is accompanied by thickening of the epithelium in nontumorus esophageal mucosa.     

Mitochondrial function in diaphragm of emphysematous hamsters after treatment with nandrolone

Respiratory failure in patients with COPD may be caused by insufficient force production or insufficient endurance capacity of the respiratory muscles. Anabolic steroids may improve respiratory muscle function in COPD. The effect of anabolic steroids on mitochondrial function in the diaphragm in emphysema is unknown. In an emphysematous male hamster model, we investigated whether administration of the anabolic steroid nandrolone decanoate (ND) altered the activity of mitochondrial respiratory chain complexes in the diaphragm. The bodyweight of hamsters treated with ND was decreased after treatment compared with initial values, and serum testosterone levels were significantly lower in hamsters treated with ND than in control hamsters. No difference in the activity of mitochondrial respiratory chain complexes in the diaphragm between normal and emphysematous hamsters was observed. Treatment with ND did not change the activity of mitochondrial respiratory chain complexes in the diaphragm of both normal and emphysematous hamsters. In emphysematous hamsters, administration of ND decreased the activity of succinate:cytochrome c oxidoreductase compared with ND treatment in normal hamsters. We conclude that anabolic steroids have negative effects on the activity of succinate:cytochrome c oxidoreductase and anabolic status in this emphysematous hamster model.     

Cell kinetics of slow renewing cell populations in mice stomach

BACKGROUND: The renewal rates of parietal and chief cells in the gastric mucosa and smooth muscle cells of muscularis propria have not been examined as precisely as superficial epithelial cells. To examine cell renewal of these cells, continuous labeling with tritiated ([3H])-thymidine was performed. METHODS: Mice received 112 repeated injections of [3H]-thymidine at 6-hour intervals for 28 days after birth and were killed immediately thereafter, or 60, 120, 200 or 300 days after the last injection. RESULTS: After continuous labeling, most cells in the stomach were labeled. At 60 days, unlabeled parietal cells in the neck area of the gland and unlabeled chief cells in the middle part of the gland appeared. Thereafter, the area of unlabeled cells expanded downwards to the bottom of the gland. Times required for labeling of total cell populations of parietal and chief cells to half were less than 60 days and more than 200 days, respectively. At 300 days, most parietal cells and about half of the chief cells remained labeled in the bottom of the gland. The labeling index of smooth muscle cells was about 100% for 300 days. CONCLUSIONS: The time required for the newly formed parietal and chief cells to reach the lower end of the gland was more than 300 days. As a total cell population, the renewal rate of parietal cells was more rapid than that of chief cells. However, in terms of the downward migrating cell population, the renewal rate of parietal cells was a little slower than that of chief cells. Smooth muscle cells showed almost no renewal.     

Cell renewal in non-specific duodenitis, ulcer-related duodenitis and duodenal ulcer. Experiments in Mastomys (Praomys natalensis)

Cell renewal in the duodenal mucosa of Mastomys was studied by autoradiography 1 and 24 h after intraperitoneal injection of tritiated thymidine. Non-specific duodenitis and duodenal ulceration were produced with a continuous infusion of histamine. Mucosal renewal in the duodenum of 30 control Mastomys was compared with 30 which received histamine dihydrochloride for 5 days. No abnormality developed in the controls, but 11 of the experimental group developed non-specific duodenitis and 12 duodenal ulcers. The size of the proliferative zone was increased in the Mastomys sacrificed 1 h after injection of tritiated thymidine which received histamine, compared with controls (p = 0.004). The number of labelled nuclei (p = 0.0003) and the size of the columns of labelled nuclei (p = 0.001) were increased in the Mastomys receiving histamine and sacrificed 24 h after injection of tritiated thymidine, compared with controls. The number of labelled nuclei (p = 0.004) and the size of the columns of labelled nuclei (p = 0.01) were increased in the Mastomys with ulcers compared with the other Mastomys which had received histamine. Cimetidine prevented duodenitis and ulceration, normalising the pattern of cell renewal. There was correlation between the severity of non-specific and ulcer-related duodenitis as judged by Lance's system and the number of labelled nuclei (Spearman rank correlation coefficient 0.771, p less than 0.002). Cell renewal increased as duodenitis became more severe and duodenal ulcers were found in duodenal mucosa where cell renewal was fastest.     

Indomethacin inhibits cell proliferation and increases cell losses in rat gastrointestinal epithelium

Gastrointestinal cell proliferation was estimated in histological sections of rats treated with low and high doses of parenteral indomethacin for 3 to 60 days. Mitoses were arrested with vincristine and cells in S phase were labeled with tritiated thymidine. Short-term, low-dose treatments reduced the mitotic activity in the oxyntic and small intestinal epithelium, whereas moderate doses restored the mitotic index and high doses increased the proliferative activity and produced epithelial hyperplasia. Long-term, low-dose treatments increased cell proliferation in the small intestine and reduced the number of villous cells. Indomethacin did not affect the proliferative response elicited by refeeding in the oxyntic mucosa, but the simultaneous administration of prostaglandin E₂ analog increased the number of arrested mitoses. The turnover of labeled cells was accelerated by indomethacin, particularly in the small intestine. These findings indicate that prostaglandins are regulators of the cell kinetics of the gastrointestinal epithelium but, at the same time, they disclose the presence of trophic mechanisms that are independent of the synthesis of endogenous prostaglandins.     

Importance of uterine cell death, renewal, and their hormonal regulation in hamsters that show progesterone-dependent implantation

This study was initiated to investigate the significance of uterine cell death and proliferation during the estrous cycle and early pregnancy and their correlation with sex steroids in hamsters where blastocyst implantation occurs in only progesterone-primed uteri. The results obtained in hamsters were also compared with mice where blastocyst implantation occurs in progesterone-primed uteri if estrogen is provided. Apoptotic cells in the uterus were detected by using terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique. Uterine cell proliferation was determined by 5-bromo-2'-deoxyuridine labeling followed by immunohistochemistry and methyl-tritiated [(3)H]thymidine labeling. Active caspase-3, an executor protein of cell death, expression was assayed by immunohistochemistry/immunofluorescence. Our results demonstrate that epithelial proliferation on the second day after mating marks the initiation of pregnancy-related uterine changes in both species despite their differences in hormonal requirements. Hamsters and mice showed subtle differences in uterine proliferative and apoptotic patterns during early pregnancy and in response to steroids. There existed almost a direct correlation between apoptosis and caspase-3 expression, suggesting uterine cell death mostly involves the caspase pathway. Consistent with these findings, we showed, for the first time, that execution of uterine epithelial cell apoptosis by caspase-3 is important for blastocyst implantation because a caspsase-3 inhibitor N-acetyl-DEVD-CHO when instilled inside the uterine lumen on d 3 of pregnancy inhibits implantation in hamsters and mice. The overall results indicate that uterine cell apoptosis and proliferation patterns are highly ordered cell-specific phenomena that play an important role in maintaining the sexual cycle and pregnancy-associated uterine changes.     

Indomethacin accelerates clearance of labeled cells and increases DNA synthesis in gastrointestinal mucosa of the rat

Sprague-Dawley rats treated with placebo, parenteral indomethacin, or oral prostaglandin E₂ for six days were given an intraperitoneal injection of [³H]methyl-thymidine and killed at 45 min and 96 hr after labeling. Treatments were continued, until death. The dpm/DNA index was determined in mucosal scrapings of the stomach, duodenum, and jejunum and used to estimate DNA synthesis (45 min) and the clearance of labeled cells (96 hr). Indomethacin increased the DNA synthesis in both the duodenal and jejunal mucosa (P<0.05). In comparison to the controls, the clearance of labeled cells from the antral, duodenal, and jejunal mucosa was accelerated by indomethacin treatment, whereas the elimination of labeled cells from the antral and jejunal mucosa was slowed by PGE₂ treatment (P<0.05). DNA synthesis of the antral mucosa was significantly reduced by PGE₂ (P<0.05). The cyclooxygenase blocker did not affect the cell kinetic parameters of the oxyntic mucosa. The plasma levels of somatostatin were significantly higher both in PGE₂- and indomethacin-treated rats than in controls (P<0.05). It is concluded that indomethacin treatment increases the cell losses from the epithelial surface, which in turn trigger a compensatory trophic reaction. It is suggested that an important physiological action of endogenous prostaglandins is to regulate the outflow of cells from the superficial zones of the epithelium. Finally, this study disclosed the presence of hitherto unknown regulatory mechanism that promote cell proliferation in the gastrointestinal, mucosa despite inhibition of the synthesis of endogenous prostaglandins.     

Effects of postpubertal oestrogen injections on mitotic activity of vaginal and uterine epithelial cells in mice treated neonatally with oestrogen.

Cell cycles of vaginal and uterine epithelial cells were studies using [3H]thymidine autoradiography in adult ovariectomized mice given oestrogen injections neonatally. The mice were in a 'persistent-oestrous' state, showing ovary-independent, continued proliferation and cornification of the vaginal epithelium. The duration of different stages of the cell cycle could not be assessed in such mice, since the percentage of labelled mitoses failed to rise to 100%. In neonatally oestrogenized, adult mice the vaginal epithelium appeared to contain a mixed population of cells. After an oestrogen injection, almost all mitoses of vaginal epithelial cells became labelled, with a generation time of about 17 h. By contrast, the generation time was about 15 h in vaginal epithelial cells of ovariectomized 'normal' mice injected with oestrogen when adult. The uterine epithelium of neonatally oestrogenized, ovariectomized mice also consisted of a mixed population of cells. A single oestrogen injection produced an increase in both the mitotic rate and cell number in the vaginal and uterine epithelium of ovariectomized 'normal' adults but not in neonatally oestrogenized, ovariectomized adults. These studies show that in mice given oestrogen neonatally, uterine and vaginal epithelial cells were not responsive to oestrogen or at least less sensitive to oestrogen than ovariectomized 'normal' controls.     

Electrical effects of histamine on monolayers formed in culture from enriched canine gastric chief cells.

To develop techniques for studying transport properties and secretory function of selected cell types in the gastric mucosa, separated fractions of dispersed canine fundic mucosal cells were placed in short-term culture to form epithelial monolayers. Cell fractions enriched in either chief, parietal, or mucous cells were prepared by using counterflow centrifugation and were plated on type I collagen. An epithelial monolayer formed by approximately equal to 36 hr. Immunofluorescence with an antipepsinogen I antibody revealed pepsinogen-containing granules in greater than 95% of the cells, regardless of whether the monolayers were formed from the mucous, chief, or parietal cell-enriched fractions. Upon achieving confluency, chief cell monolayers were mounted in Ussing chambers to study their electrical properties. Under basal conditions, monolayers (n = 6) had a spontaneous potential difference (PD) (+/- SEM) of 26 +/- 4 mV (apical surface negative), a short-circuit current (Isc) (+/- SEM) of 16 +/- 2 microA/cm2, and a transepithelial resistance (R) (+/- SEM) of 1,480 +/- 210 omega X cm2. Histamine increased the short-circuit current, an effect blocked by an H2-receptor antagonist. Seventy percent of the spontaneous PD was amiloride sensitive, suggesting sodium absorption accounted for a major component of the PD. These preparative techniques yield highly enriched chief cell monolayers, which maintain morphological and functional cellular differentiation for greater than 48 hr in culture, thus allowing study of oriented functions of a selected cell type. The present studies indicate that an H2 receptor enhances electrogenic ion transport in chief cell monolayers, indicating that histamine can act on fundic mucosal cells other than just parietal cells.     

Steroidogenesis in rabbit preimplantation embryos.

Rabbit preimplantation embryos were flushed from the reproductive tract at 24 hr (1- to 2-cell stage), 48 hr (morula), 72 hr (morula), 96 hr (blastocyst), 120 hr (blastocyst), and 144 hr (blastocyst) post coitum. At 168 hr (early postimplantation period), gestation sacs were excised, frozen, and sectioned in a cryostat. Delta5-3beta-Hydroxysteroid dehydrogenase [3(or 17)beta-hydroxysteroid:NAD(P) oxidoreductase, EC 1.1.1.51] activity was determined histochemically in whole preimplantation embryos and in sectioned postimplantation embryos. 3beta-Hydroxysteroid dehydrogenase activity began at 48 hr and was sustained through the late blastocyst stage (144 hr), with the exception of a brief drop, possibly cessation, of activity at 72 hr. There was no activity at 168 hr. Since 3 beta-hydroxysteroid dehydrogenase is a key enzyme in the metabolism of steroid hormones, its presence is strong evidence for steroidogenesis. Only 144-hr preimplantation embryos were used to determine 17 beta-hydroxysteroid dehydrogenase (estradiol-17beta:NAD 17-oxidoreductase, EC 1.1.1.62) activity, which was present, suggesting synthesis of estrogen. By means of radioimmunoassay, 144-hr preimplantation embryos were found to contain estradiol-17beta. Other authors have shown that rabbit blastocysts contain progesterone and other steroids, and these embryos can synthesize steroids from non-steroid and steroid precursors. Therefore, our results plus those of others prove that rabbit preimplantation embryos synthesize steroid hormones. Our present and previous results (with rats, hamsters, and mice) suggest that the steroid hormones synthesized by the embryo are critical for preimplantation embryogenesis and for implantation of the lbastocyst.     

Effects of gastric fundectomy and antrectomy on the colonic mucosa in the hamster.

The effects of gastric fundectomy and antrectomy on the colonic mucosa were studied in hamsters over 5 and 25 days. Sham-operated animals served as controls. Basal plasma gastrin concentrations were significantly increased after fundectomy and significantly decreased after antrectomy. Five days after fundectomy, there was a significant increase in scintigraphically determined colonic tissue [3H]-thymidine uptake and [3H]-thymidine labeling index of goblet cells, both of which were reduced 5 days after antrectomy. After fundectomy, the labeling index was maximal in differentiating-proliferative cells in the midportion of the colonic crypts, whereas the labeling index of the immature proliferative cells at the base of the crypts did not differ from that in the controls. On day 25, the crypt size and the number and percentage of goblet cells in the crypts were significantly increased in fundectomized animals. The number and percentage of goblet cells in antrectomized animals were significantly reduced on day 25. It is concluded that fundectomy in the hamster induces colonic mucosal hyperplasia with goblet cell proliferation, whereas antrectomy leads to retardation of colonic goblet cell proliferation.     

Effects of progestins and glucocorticoids on deoxyribonucleic acid synthesis in the uterus of the neonatal mouse

The effects of progestins and glucocorticoids on cellular proliferation were examined in the uterus of 5-day-old mice by monitoring either the labeling index (LI) after exposure to [methyl-3H]thymidine ([3H]TdR) in vivo or the mitotic index (MI) after colchicine-induced arrest of cells in metaphase. In untreated 5-day-old mice, epithelial LI was 31%, and stromal LI was 15%. Eighteen hours after a single ip injection of 40 mg/kg progesterone, epithelial LI was reduced to 2.3% and remained low for 48 h. Stromal LI increased transiently, reaching a zenith (40%) 18 h after administration of progesterone and returning to control levels by 24 h. When mitotic activity was assessed 24 h after progesterone treatment, epithelial MI was decreased (control, 3.1%; progesterone, 0.23%) and stromal MI was increased (control, 0.60%; progesterone, 2.1%). Thus, the measured effects on LI were indicative of altered proliferative activity of the tissues. Glucocorticoids also inhibited epithelial LI, but had no effect on stromal LI. Eighteen hours after a single ip injection, dexamethasone inhibited epithelial LI to the same extent as progesterone treatment. Corticosterone did not significantly decrease epithelial LI, while cortisol produced an intermediate inhibitory response. To determine whether the high baseline LI in uterine epithelium of neonatal mice was estrogen dependent, uteri of 1-day-old mice were grafted under the kidney capsule of ovariectomized adult mice. Eight days later, the hosts were treated with either progesterone or vehicle and then killed 18 h later. After labeling the tissue with [methyl-3H]thymidine in vitro, the mean LI of the epithelium of the grafted uteri was 11.5%, while that of the vehicle-treated hosts was 0.10%. Progesterone treatment reduced the LI of the grafted uterine epithelium to 1.0%. These data demonstrate that uterine tissues of the neonatal mouse proliferate rapidly in the absence of gonadal steroids. Progestins and glucocorticoids specifically inhibit this estrogen-independent DNA synthesis of uterine epithelium.