We examined kinetics and characterization of regenerating proximal tubule (PT) cells after various degrees of tubular injury in S3 segments of PT and assessed label-retaining slow cycling cells in S3.

PT injury was induced by different doses of uranyl acetate (UA) injection into rats, and initially regenerating PTs were identified by in vivo bromodeoxyuridine (BrdU)-labelling before sacrifice or were examined on vimentin positivity. Next, the 3H-thymidine pulse/chase approach was applied to the early regenerating PTs identified by BrdU-labelling after UA injection.

Low-dose UA induced focal PT depletion and initial BrdU positivity in the proximal three-quarters of the S3 segment of PT. Autoradiography showed the increased number of label-retaining and label-diluted cells in the proximal three-quarters of S3 in rats treated with low-dose UA compared to normal rats. High-dose UA induced almost complete PT depletion in the proximal three-quarters of S3 and less PT depletion in the distal quarter of S3 and initial BrdU+ cells were restrictedly found in the distal quarter of S3. Label-retaining and label-diluted cells were increasingly found in the entire S3 at day 7, but only label-retaining cells remained in similar numbers in the distal quarter of S3 until day 42. Initially regenerating PT cells with any doses of UA expressed vimentin, suggesting dedifferentiated PT cells.

Initially regenerating cells after PT injury in S3 are dedifferentiated pre-existing PT cells, which may scatter throughout S3 and be responsible for focal repair of S3. Some initially regenerating PT cells in the distal S3 showed persistent label-retaining cells at day 42 after high-dose UA insult and contributed to renewal of the entire S3, thus they might be slow cycling cells with responsibility for S3 repair.

Subcutaneous administrations of monosodium glutamate (MSG) to neonatal animals result in obesity and induce the toxicity on the central nervous system, and furthermore, have an effect on entero-pancreatic hormone. The effect of MSG on the cell turnover of organs, especially the pancreas, has received little attention until now. This study was designed to examine the effect of MSG on pancreatic cell turnover by immunohistochemistry and [(3)H]thymidine autoradiography.

Male JcI-ICR strain mice were SC injected with MSG (2 mg/g body weight daily) for 5 days after birth, received 112 repeated injections of [(3)H]thymidine at 6-hour intervals for 28 days after birth, and then were killed immediately thereafter, or 30, 60, or 120 days after the last injection. Autoradiography was performed on sections immunostained for glucagon, insulin, and somatostatin.

After continuous labeling, most pancreatic cells were labeled, and thereafter, labeling of cells decreased in control and MSG-treated mice. The mean grain counts of acinar cells in MSG-treated mice decreased more slowly than those in control mice. On the other hand, those of islet cells, including glucagon, insulin, and somatostatin cells, decreased more rapidly in MSG-treated mice than those in control mice.

Cell turnover of acinar cells was decelerated and that of islet cells including glucagon, insulin, and somatostatin cells was accelerated in MSG-treated mice pancreas. MSG-induced hypothalamic lesions exert the contrary influences on the cell turnover of acinar and islet cells.

The source of new cells in the normal adult liver has been controversial. Some investigators have hypothesized the streaming liver model. On the other hand, others reject this hypothesis. We examined hepatic cell kinetics by a special labeling method with [3H]thymidine.

ICR mice received 112 repeated injections of [3H]thymidine at 6 h intervals for 28 days after birth and were killed immediately thereafter, or 100, 200 or 300 days after the last injection. Immediately after killing the animals, samples of the liver were taken and autoradiography was performed.

After continuous labeling, more than 90% of the cells in the liver were labeled. Mean grain counts of hepatocytes decreased to half over approximately 100 days. Those of bile duct cells decreased at a slower rate (50%) than hepatocytes. Mean grain counts of hepatocytes decreased over the regions, although those in perivenular region decreased more rapidly in comparison to those in periportal region.

The present study indicated that most cells in the liver arise postnatally. The changes in labeling of cells show that there is no special zone for proliferation of hepatocytes and they renew in all regions of the hepatic lobule, suggesting (i) that hepatocytes are supplied by postnatal replication and (ii) streaming of hepatocytes from periportal to pericentral regions does not occur in the adult mouse liver. The bile duct cells renewed more rapidly than hepatocytes.

Although cell kinetics of the gastrointestinal mucosa has been extensively examined, that of the pancreas has not been fully analyzed.

To determine the renewal rate of pancreatic cells directly.

Postnatal proliferative activity and cellular renewal of the parenchymal cells in ICR mouse pancreas were studied by immunohistochemistry and [(3)H]-thymidine autoradiography.

In the single labeling experiments, the proliferative activity of the parenchymal cells in pancreas showed peaks at a few days after birth, decreased thereafter, and reached a low level at 2 months after birth. Continuous labeling experiments revealed that, in the adult pancreas, the half lives of acinar cells, islet cells, and duct epithelial cells were approximately 70 days, 47 days, and 40 days, respectively. Moreover, in the exocrine pancreas, acinar cells of the peri-insular region proliferated more actively than those of the tele-insular region. The renewal rate of glucagon cells was more rapid than that of insulin cells or somatostatin cells. Large ducts showed a high rate of cell renewal in comparison with small ducts.

The results of this study indicate that cell renewal rates of the pancreas are not homogeneous, but heterogeneous.

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.

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.

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.

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.

Entero-endocrine cells and the rare cells named caveolated or brush cells have been examined in light microscopic radioautographs of the mouse corpus after various periods of continuous 3H-thymidine infusion. Moreover a search for immature forms and mitoses of these cells was undertaken in the electron microscope. Entero-endocrine cells are present in the four regions of the epithelial units, but their number is low in the pit, intermediate in the isthmus and neck, and high in the base. The labeling pattern after continuous 3H-thymidine infusion indicates that these cells are produced in the isthmus from undifferentiated granule-free cells presumed to be the stem cells of the epithelium, and may retain a limited ability to divide. A few of the newly formed entero-endocrine cells migrate to the pit, but the majority goes to the neck and, from there, to the base where they are present in relatively high numbers. Little information is available on the dynamics of caveolated cells. Since immature forms are present in the isthmus and mature ones in the other regions, it is concluded that they arise in the isthmus and migrate away in both directions. Finally, concluding remarks are presented on the kinetics of each one of the cell lineages described in this and the four previous articles.

Administration of epidermal growth factor (EGF) stimulates DNA synthesis in gut epithelial cells and inhibits gastric acid secretion. A physiological role of EGF in cell kinetics of gastric epithelium, however, has not been fully understood. In mature male mice, large amounts of EGF are produced in the submandibular glands, and sialoadenectomy (removal of the submandibular glands) causes a marked reduction of plasma EGF levels. For the evaluation of a biophysical function of EGF, sialoadenectomized mice and sham-operated mice were injected with 3H-thymidine to compare the proliferative activity and the cell-turnover of gastric epithelium between the two groups using the autoradiographic analysis. When mice were killed 90 min after a single injection of 3H-thymidine, the percentages of fundic gland mucosal cells radiolabeled in sialoadenectomized and sham-operated mice were 27.3 +/- 5.0% and 26.3 +/- 5.8% (mean +/- SD), respectively. The difference was not significant (p > 0.05). Similarly, the labeling indices of pyloric gland mucosal cells were not different between the two groups (26.7 +/- 4.3% vs 27.8 +/- 3.7%, p > 0.05). In contrast, when mice were given 17 repeated injections of 3H-thymidine at 6 hr intervals and killed 48 hr after the last injection, labeling indices in sialoadenectomized mice were significantly lower than those in sham-operated mice (35.3 +/- 4.3% vs 52.8 +/- 1.1% in the fundic gland area; 41.0 +/- 6.2% vs 55.1 +/- 5.9% in the pyloric gland area, p < 0.001, respectively). Treatment of sialoadenectomized mice with EGF (5 mg/mouse per day) completely restored the percentages of the radiolabeled cells to control levels. These findings suggest that endogenous EGF plays a major role in maintaining biological cell-turnover of the mouse gastric epithelium.

We have tested the suggestion that the reported increase, in hypoxic rats, in the number of lung endocrine cells immunoreactive for the regulatory peptide CGRP is caused by an accumulation of peptide within the cells which renders them more detectable, rather than by a real increase in proliferation. The incorporation of continuously infused 5'-bromodeoxyuridine (BrdU) into nuclei of CGRP-containing cells was studied by immunohistochemistry in the airway and respiratory epithelium of rats kept in a hypoxic (10% O2), normobaric conditions for 7 days and in normoxic, normobaric controls. Some CGRP-immunoreactive cells could also be labelled for BrdU. However, the ratio of the number of cells labelled with both CGRP and BrdU to the number of cells labelled with CGRP alone did not differ significantly between hypoxic and normoxic rats (7.1 +/- 0.7 and 6.1 +/- 1.2, respectively; mean +/- SEM; P = 0.49). These data strongly suggest that CGRP-containing endocrine cells or their precursors do proliferate in adult rat lung, but that the proliferation is not increased significantly in hypoxia.

Time-related changes of serum gastrin levels, gastrin cell, and enterochromaffinlike cell densities, and proliferation kinetics of these cells have been examined in rats during treatment with the substituted benzimidazole BY 308 over a period of 73 days. Serum gastrin levels increased very rapidly from 74 +/- 6 pg/mL (controls) to 438 +/- 31 pg/mL (day 1) and 727 +/- 68 pg/mL (day 4). Thereafter, a steady increase was observed until day 70 (2097 +/- 208 pg/mL). Enterochromaffinlike cell density was unchanged until day 10, but then increased progressively without reaching a plateau (144% above control on day 73). The labeling index of these cells was enhanced shortly after drug application and remained on a constant elevated level from day 14 to day 73 (about 10-fold to 12-fold above controls from day 14 to day 70). The number of gastrin cells increased rapidly within the first week and reached a plateau after 17 days (96% increase above controls). In contrast to enterochromaffinlike cells, the labeling index did not change immediately but increased on day 7 by 37% and returned beneath control values after day 28. The results suggest that in drug-induced achlorhydria, the progressive increase of enterochromaffinlike cells is explained by an enhanced mitosis, whereas other factors in addition to proliferation are responsible for the augmentation of gastrin cells.

The origin and renewal of secretin cells in the duodenum were investigated using the unlabeled antibody peroxidase-antiperoxidase technique and radioautography in rats killed at various times after single or multiple injections of [3H]thymidine. Secretin cells were spatially distributed from the upper crypt to the villus tip, being particularly numerous in the upper two-thirds of the duodenal villi. After a single injection of [3H]thymidine, there were no labeled secretin cells, indicating a lack of self-replicating activity. After repeated injections of the isotope, labeled secretin cells appeared and increased in number. They first occurred at the upper part of the crypt and the lower part of the villus, and later at the villus tip. All these cells were found to be labeled after continuous labeling for 120 h, which is considered to be the renewal time for this cell population.

Intestinal metaplasia of the stomach was grouped into 3 subtypes (A, B and C) according to the degree of pyloric gland involution which was judged from patterns of paradoxical Concanavalin A staining after Katsuyama and Spicer. The appearance of endocrine cells was investigated with immunohistochemical and silver methods. Type A metaplasia with slightly to moderately atrophic pyloric glands corresponded to the incomplete type in the previous classification, while Type C showing complete disappearance of pyloric glands corresponded to the complete type. Type B with severely atrophic pyloric glands was an intermediate. This subtyping reflects the cell kinetics in the intestinalized mucosa well. Regarding the endocrine cells, their total number varied in the order Type A greater than Type B greater than Type C. The selective populations of the endocrine cells including glicentin-containing cells, Grimelius-positive argyrophil cells without argentaffinity and intestinal-type enterochromaffin cells frequently formed hyperplastic foci in the intestinalized areas, where the other gut-type and proper gastric-type endocrine cells were scarcely noted. Immunoreactivity of glucagon or bovine pancreatic polypeptide were occasionally identified in a subpopulation of the glicentin-containing cells.

Diethylnitrosamine is known to cause squamous cell carcinoma and adenocarcinoma of the lung in Syrian golden hamsters. Sections of lungs obtained from hamsters treated with the systemic carcinogen diethylnitrosamine revealed a significant increase in the number of argyrophilic cells of neuroepithelial bodies. These affected cells also exhibited enhanced survival in vitro. After 7 days in culture, argyrophilia, dense-core vesicles, and corticotropin-like immunoreactivity were observed in many of the cells derived from the lungs of carcinogen-exposed hamsters by dissociation with pronase. In addition, nuclei of argyrophilic cells in neuroepithelial bodies of the exposed hamsters were labeled at 60 min following administration of [3H]thymidine. This suggests that the carcinogen stimulates the pulmonary neuroendocrine-like cells to divide. Normally, the component cells of neuroepithelial bodies may originate from nonargyrophilic precursor cells in the surrounding epithelium, as in control hamsters the argyrophilic cells of neuroepithelial bodies appeared labeled only at 8 days after the administration of thymidine. The relationship of the diethylnitrosamine-induced reactions to bronchial carcinoid tumors or small-cell carcinomas of the lung remains to be established.

The possibility that antral somatostatin cells have a self-replicating activity has been studied in three species of rodents: mice, rats and guinea-pigs, after a flash tritiated thymidine injection. The immunocytochemical staining of somatostatin cells, using specific antiserum, was combined with radioautographic procedures. The labelling index for somatostatin cells--and for gastrin cells identified on serial sections--was established after counting a large number of cells at the optical microscope level, on parallel tissue strips removed throughout the entire antrum. A significant percentage of the somatostatin cell population synthesized DNA. Values were similar for the three species of rodents ranging from 0.8 to 1.1%, that is slightly higher than the percentage of labelled gastrin cells, which was 0.67-0.7%. After a 36-hr continuous infusion of radioactive precursor in one rat, the labelling index observed remained low; 2.33% for somatostatin cells and 1.68% for gastrin cells. Colchicine injection in mice allowed the observation of mitotic figures in well differentiated somatostatin cells. Four hours after that injection, the mitotic index was estimated roughly at 0.3%. Thus, evidence has been presented that in rodents a fraction of the antral somatostatin cell population is capable of dividing, similar to the situation in gastrin cells.

A mucosal defect was produced by cryosurgery in the antral and the fundic wall of the rat stomach, and regeneration of gastric endocrine cells was studied 50, 100 and 200 days after operation. Fifty days after the operation, the mucosal defect was completely covered with regenerated epithelium. The regenerated mucosa both in the antral and in the fundic region consisted of mucinous glandular structures. The regenerated mucosa in the corpus remained pseudopyloric in type even 200 days after operation. Regardless of the time after operation, regeneration of endocrine cells was always observed. We could identify G cells and EC cells in the regenerated mucosa of the antrum, and EC cells, A cells and AL cells in the regenerated mucosa of the corpus, respectively. By electron microscopy, endocrine-exocrine cells were frequently encountered. These cells had two different types of intra-cytoplasmic granules; one was an endocrine-specific, small electron-dense granule, and the other a large, lucent mucin droplet-like granule. These findings indicate that the endocrine cells of the stomach are formed from endodermal precursor cells.