Total counts of antral gastrin cells: a simple direct method

A simple technique has been developed to quantitate the gastrin cells (G-cells) from the pyloric antrum of the rat. The antrum was digested in pronase to suspend the epithelial cells. This cell suspension was counted and pelleted. The pellet was embedded in paraffin, sectioned, then labeled using the indirect immunofluorescence technique specific for gastrin. The percentage of G-cells was determined from photographs of fluorescing sections and total G-cell numbers were determined by relating these data to total epithelial cell counts. In 14 rats the average G-cell population totaled 1.03 +/- 0.21 X 10(5) G-cells/antrum. The technique is simple, time-saving and avoids the uncertainties inherent in previous procedures for the estimation of G-cell numbers.     

Total Counts of Antral Gastrin Cells: A Simple Direct Method

A simple technique has been developed to quantitate the gastrin cells (G-cells) from the pyloric antrum of the rat. The antrum was digested in pronase to suspend the epithelial cells. This cell suspension was counted and pelleted. The pellet was embedded in paraffin, sectioned, then labeled using the indirect immunofluorescence technique specific for gastrin. The percentage of G-cells was determined from photographs of fluorescing sections and total G-cell numbers were determined by relating these data to total epithelial cell counts. In 14 rats the average G-cell population totaled 1.03 ± 0.21 ± 10⁵ G-cells/antrum. The technique is simple, time-saving and avoids the uncertainties inherent in previous procedures for the estimation of G-cell numbers.     

Antral G-cell in gastrin and gastrin-cholecystokinin knockout animals

The antral hormone gastrin is the key regulator of gastric acid secretion, mucosal growth and differentiation. Gastrin is synthesized in the endocrine G-cells in the antroduodenal mucosa. We have now examined the way in which the loss of gastrin alone or gastrin plus cholecystokinin (CCK) affects the antral G-cell. Immunohistochemistry, radioimmunoassay and quantitative real-time polymerase chain reaction techniques were employed to examine the expression of genes belonging to the G-cell secretory pathway in gastrin and gastrin-CCK knockout mice. Transmission electron microscopy was used to examine the ultrastructure of the G-cells. The number of G-cells increased but the secretory granules were few and abnormally small in the G-cells of both mouse models compared with wildtypes. Thus, gastrin is not necessary for the formation of G-cells as such but the lack of gastrin reduces the number and size of their secretory granules suggesting that gastrin is vital for the formation and/or maintenance of secretory granules in G-cells. This work was supported by the Novo Nordic Foundation (L.F.-H.) and Swedish Research Council (grant no. 4499; F.S. and N.W.).     

Xenopsin immunoreactivity in antral G-cells may reside in the N-terminus of gastrin 17

The nature of xenopsin immunoreactivity in mammalian antral G-cells has been reassessed. Xenopsin immunostaining was most intense in human antral G-cells, present in those of the dog and pig and not detected in guinea pig or rat tissues. Rigorous specificity controls for ionic binding of immunoglobulins to antral G-cell granules indicated that this mechanism was not responsible for xenopsin immunostaining. Preincubation of the xenopsin antiserum with xenopsin, human gastrin 1-13 and gastrin 2-17 completely abolished immunostaining at similar molar concentrations. Gastrin 34 was ineffective at much higher concentrations. These results infer that xenopsin-immunoreactivity in antral G-cells resides in the N-terminal region of gastrin 17. Examination of the primary structures of xenopsin and the N-terminal regions of some mammalian gastrins reveals a hitherto unrecognized homology.     

The G-cells in the dog: a light and electron microscope immunocytochemical study

Summary An immunohistochemical study has been performed to analyse the distribution of gastrin cells in the gastrointestinal tract of the dog. This study revealed that G-cells immunoreactive for gastrin were almost exclusively present in the pyloric antral mucosa, mainly in the middle third of the pyloric mucosa. The calculated number of G-cells per surface unit area was 8.5×10³–1.2×10⁴ cells cm^–2. Some gastrin-immunopositive cells were found in the first 10 mm of the proximal duodenum, mainly in the villous region. The fundic area of the dog stomach, the oesophagus, small intestine, caecum, colon, rectum, salivary glands, liver and pancreas were all immunonegative for gastrin. At the ultrastructural level, three different types of granules (150–400 nm) were evident in G-cells: electron-dense, electron-lucent and intermediate forms. Most of them were located in the subnuclear region of the cell. The effect of fixation of the antral mucosa at different pH levels was studied. In samples fixed with acid solutions, most of the G-cell granules were of the electron-dense type and were stronly immunopositive for gastrin. Fixation of samples at a basic pH resulted in most of the gastrin granules losing their contents into the cytoplasm, and the positive reaction to gastrin was then located in the cytoplasm and at the periphery of the electron-lucent granules.     

Isolation and separation of highly enriched fractions of viable mouse gastric parietal cells by velocity sedimentation

Methods of tissue dissociation and cell separation have been modified to obtain highly enriched fractions of mouse gastric parietal cells. Suspension of gastric mucosal cells are prepared by pronase digestion of the glandular portion of the stomach from adult mice. By utilizing the velocity sedimentation technique to separate cells of different sizes it is possible to recovery parietal cells, which are larger than the other cell types, in fractions with purity of 75-95%. The homogeneity of cell fractions has been assessed by light and electron microscopy. The ability of the isolated cells to exclude the dye trypan blue, to incorporate labeled substrate, to consume oxygen, and to retain their structural integrity indicates that they are viable and still capable of functional activity.     

Co-localization of xenopsin and gastrin immunoreactivity in gastric antral G-cells

Studies indicating evidence for the presence of the amphibian octapeptide xenopsin in gastric mucosa of mammals prompted us to investigate the cellular localization of this peptide. Using the peroxidase-antiperoxidase method and a specific antiserum to xenopsin (Xen-7) on paraffin and adjacent semithin sections of gastric antral mucosa from man, dog, and Tupaia belangeri, we found numerous epithelial cells showing a specific positive immunoreaction. These cells were of typical pyramidal shape and could be classified as of the "open" type. Cell quantification in serial sections processed for xenopsin and gastrin immunoreactivity, respectively, revealed an identical number of cells per section and an identical distribution of these cells in the middle zone of the antral mucosa. Furthermore, adjacent semithin sections demonstrated the colocalization of xenopsin and gastrin immunoreactivity within the same G-cell. The xenopsin antiserum could be completely absorbed with synthetic xenopsin but not with gastrin. Preabsorption tests with neurotensin, a xenopsin related peptide, or with somatostatin, glucagon, and enkephalins gave no evidence for crossreactivity of the xenopsin antiserum with these peptides. It is concluded that gastric antral G-cells in addition to gastrin also contain the amphibian peptide xenopsin.     

The effect of total parenteral nutrition (TPN) on gastrin release in the rat

The effect of short-term (7 days) total parenteral nutrition (TPN) on gastrin release was studied in vivo and in the isolated vascularly perfused rat stomach. The daily plasma gastrin concentration of parenterally fed rats was significantly lower than in ad lib fed control animals (53 +/- 17 pg/ml vs 159 +/- 32 pg/ml, P less than 0.05) as early as day 2 and a similar pattern was observed on days 4 and 6. The fasting plasma gastrin concentration of control animals was 2-fold greater than of the parenterally fed group (P less than 0.05). Following oral peptone, the gastrin response of TPN and control animals doubled although peak gastrin levels were greatly reduced in TPN rats. Basal gastrin release from the perfused stomachs of control rats was 2-fold greater than from TPN rats (P less than 0.05). Electrical stimulation of the vagal trunks resulted in a significantly greater elevation in gastrin secretion from control stomachs compared to TPN animals (4-fold vs. 2.4-fold increase, P less than 0.05). Quantification of the antral G-cell population revealed a significant reduction in the number of G-cell of TPN rats compared to controls (97 +/- 8 cells/mm vs 76 +/- 6 cells/mm, P less than 0.05). These results indicate that luminal nutrient stimulation is necessary for the maintenance of normal G-cell secretory activity in vivo and from the in vitro stomach. G-cell hypoplasia appears to be partially responsible for reduced gastrin output to basal and stimulated conditions after TPN.     

Isolated brush cells of the rat stomach retain their structural polarity

Summary The brush cells (BC) are highly polarized elements occurring in epithelia of endodermal origin. They have a preferential topographical distribution in the organs in which they reside. In the stomach of the rat, BC prevail near the transitional zone separating the forestomach from the glandular stomach. Thus, a method was developed to isolate and recover BC from this organ with the aim of investigating the changes they may undergo after dissociation. Strips of the rat stomach were severed from the very proximal border of the glandular region and incubated in Hanks' balanced salt solution containing pronase. After sedimentation of the dissociated cells (crude sediment containing all stomach epithelial cell types) two successive cell fractions were prepared on preformed Percoll gradient in an attempt to enrich BC in a defined layer. BC were recovered in a fraction at a density close to 1.03 g/ml where they represented about 2% of all cells. The isolated BC changed their form from columnar to pear-shaped; however, they maintained their structural polarity over 2 h as demonstrated by light microscopy, transmission-and scanning-electron microscopy. The fine structure of BC was always satisfactorily preserved. Maintenance of the structural polarity of isolated BC is contrary to the general rule according to which all conventional epithelial cells examined to date lose their polarity after isolation. This result is discussed in relation to morphological findings in isolated sensory cells (hair cells, photoreceptor cells) leading to the suggestion that BC are more similar to these than to conventional epithelial cells.     

Association of glycoproteins and pepsinogen in the secretory granules of fundic epithelial cells isolated from guinea pig stomach

Epithelial cells were isolated from the fundic portion of the guinea pig stomach. Cells were separated by velocity sedimentation at unit gravity in a Ficoll 70 gradient and pooled in three fractions. By morphological and biochemical criteria, each fraction was characterized as a population highly enriched in one of the three main functional types: oxyntic cell; chief cell and mucus-secreting cell. Measure of the pepsinogen content and specific stainings of the secretory granules for light and electron microscopy led to the definition of two types of mucus-secreting cells in nearly equal quantity; mucous cells with smaller secretory granules entirely glycoproteic in nature and muco-peptic cells containing larger heterogeneous secretory granules. These granules were made of a proteic core containing pepsinogen surrounded by a thin membrane and a voluminous cap, both containing carbohydrates. The cap appeared as if built of orderly packed layers of glycoproteins. Secretory granules of chief cells were also surrounded by a membrane containing glycoproteins and occasionally a small glycoproteic cap. Pepsinogen content was estimated to be three times higher in a single chief cell than in a muco-peptic cell.     

Gastrin release in obese Zucker rats

In this study, gastrin release in the obese Zucker rat was investigated by in vivo and in vitro experiments. Obese rats exhibited normal plasma gastrin levels at 3 weeks (preobese), were moderately hypergastrinemic at 3 months and severely hypergastrinemic at 5 months, compared to lean littermates. Following oral peptone, plasma gastrin levels doubled in both lean and obese rats. Basal and vagally stimulated gastrin release from perfused stomachs was greater in obese compared to lean rats and atropine had no effect on basal gastrin release in either group. Basal somatostatin release from the perfused stomach was found not to differ in both groups of animals. Morphological studies revealed an increase in the number of gastrin-containing G-cells in adult obese rats compared to lean littermates, but not in 3-week-old pups compared to lean littermates, indicating a strong correlation between cell number and plasma gastrin levels. These data indicate that the obese Zucker rat exhibits fasting hypergastrinemia in vivo, a condition which appears after weaning and increases in severity with age. Gastrin hypersecretion persists from the vascularly perfused stomach preparation. The basal hypergastrinemia of the obese Zucker rat is independent of a hyperactive postganglionic cholinergic drive but is associated with and probably causally related to an increase in antral G-cell numbers.     

Prostaglandin E2 output by isolated rat gastric parietal cells and non-parietal epithelial cells

Prostaglandins have acid antisecretory and cytoprotective effects in gastric mucosa when given exogenously. This study's purpose was to isolate preparations of parietal and non-parietal cells from rat stomachs and to compare prostaglandin output by these cells. Gastric epithelial cells were isolated from rat stomachs using pronase. Cells from different incubation times were collected separately and enriched by discontinuous Percoll gradient. Cell types were identified by hematoxylin and eosin stain, succinic dehydrogenase activity (parietal cells), periodic acid Schiff staining (mucous cells), Bowie staining (chief cells) and electron microscopy. Prostaglandin E2 activity was measured by radio-immunoassay. Parietal cells were purified to over 90% while the non-parietal preparation contained 67% chief cells and over 31% mucous cells. By electron-microscopy, cell integrity was seen to be maintained. The parietal cell enriched fraction contained two and one-half times the amount of prostaglandin E2 that the non-parietal chief cell enriched fraction did, p less than 0.01. These results raise the question as to whether output of PGE2 by parietal cells could play a role in modulating gastric acid secretion directly by parietal cells as well as in protecting the deeper layers of gastric mucosa against damaging agents in-vivo.     

Co-localization of xenopsin and gastrin immunoreactivity in gastric antral G-cells

Summary Studies indicating evidence for the presence of the amphibian octapeptide xenopsin in gastric mucosa of mammals prompted us to investigate the cellular localization of this peptide. Using the peroxidase-antiperoxidase method and a specific antiserum to xenopsin (Xen-7) on paraffin and adjacent semithin sections of gastric antral mucosa from man, dog, and Tupaia belangeri, we found numerous epithelial cells showing a specific positive immunoreaction. These cells were of a typical pyramidal shape and could be classified as of the open type. Cell quantification in serial sections processed for xenopsin and gastrin immunoreactivity, respectively, revealed an identical number of cells per section and an identical distribution of these cells in the middle zone of the antral mucosa. Furthermore, adjacent semithin sections demonstrated the colocalization of xenopsin and gastrin immunoreactivity within the same G-cell. The xenopsin antiserum could be completely absorbed with synthetic xenopsin but not with gastrin. Preabsorption tests with neurotensin, a xenopsin related peptide, or with somatostatin, glucagon, and enkephalins gave no evidence for crossreactivity of the xenopsin antiserum with these peptides.It is concluded that gastric antral G-cells in addition to gastrin also contain the amphibian peptide xenopsin.     

The antrat gastrin-producing G-cell: Biochemical and ultrastructural responses to feeding

Summary The effect of feeding on serum and antral immunoreactive gastrin (IRG) concentrations and on the ultrastructural appearance of antral G-cell granules has been examined. Serum and tissue IRG concentrations were dependent upon the length of time (12 or 48 h) the rats had been fasted before receiving food; IRG release was biphasic; the first peak was more pronounced in rats fasted 12h. Antral tissue IRG content increased significantly postprandially. An initial depletion of antral IRG was seen in rats fasted 48 h. Examination of the subcellular distribution of antral IRG revealed more of the 5–15 min postprandal total IRG in the cytoplasm and less in the secretory granules.Ultrastructurally, G-cells from fasting rats contained mainly electron-dense granules. Five minutes postprandially numerous electron-lucent granules were observed. More electron dense granules were apparent 60 and 120 min postprandially. Fasting rats had the highest G-cell granule density index; a significantly lower index was observed 5 min postprandially. Indices at 60 and 120 min postprandially increased but were still lower than the fasting index. These studies indicate that gastrin biosynthesis is necessary for food stimulated gastrin release and that the electron density of the G-cells' granules is not an accurate reflection of the G-cell gastrin content.The authors thank Elisabeth Bothe, Heidi Dörler and Heide Karl for technical assistance and the Deutsche Forschungsgemeinschaft (Bonn-Bad Godesberg, Grant Cr 20/7), the Atkinson Charitable Foundation and the Canadian MRC for financial support     

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.     

Occurrence of met-enkephalin,met-enkephalin-Arg6-Phe7 and met-enkephalin-Arg6-Gly7-Leu8 in gastrin cells of hog antral mucosa

Summary Region-specific antisera to three enkephalins: met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸, together with four region specific antisera to progastrin: C-terminal G17 specific, N-terminal G34 specific, cryptic peptides A- and B-specific, were used in immunohistochemical studies of hog antral mucosa. A sub-population (6–10%) of the gastrin-containing endocrine cells (G-cells) was found to react with antisera to met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸. About 30% of all the enkephalin-containing cells were identified as G-cells. The results indicate that a fraction of G-cells produces both enkephalin-like peptides and gastrin.     

Role of protein kinase C and phosphorylation in bombesin-evoked gastrin release from isolated perfused rat stomach

We have recently reported that bombesin (BBS)-stimulated gastrin release is principally dependent on a Ca2+/calmodulin intracellular pathway, and that it is independent of the cyclic AMP-mediated pathway. Recently it was demonstrated that stimulation of protein kinase C (PK-C) resulted in increased gastrin release from the isolated canine G-cells in cultures. The role of PK-C in the BBS-evoked gastrin release, however, remains unexamined. In this study we examined a possible role of PK-C in the secretion of BBS-stimulated gastrin from isolated perfused rat stomach. The effect of phosphorylation on gastrin release, in response to BBS, was also determined. Administration of phorbol ester (PMA 10-100 nM, a PK-C activator) alone significantly provoked gastrin release, but markedly inhibited the BBS (1 nM) stimulated gastrin secretion in a dose-dependent manner. Molybdic acid (phosphatase inhibitor), caused an enhancement of BBS-evoked gastrin response at doses of 5 or greater than 5 mM. These results suggest that: (1) diacylglycerol/PK-C pathway may exert a negative feedback control over BBS-induced gastrin release; (2) phosphorylation step is required for gastrin secretion in response to BBS.     

Preparation of suspensions of pancreatic islet cells: a comparison of methods

A comparative study for preparation of cell suspensions from pancreatic islets has been performed using mechanical or enzymatic dissociation with proteolytic enzymes such as trypsin, dispase, and pronase. Treatment of isolated pancreatic islets from neonatal rats with these enzymes proved to be superior to a mechanical dissociation method. The enzymatic dissociation was performed by fractionated treatment of pancreatic islets with low concentration of enzymes in Hanks' solution for 2-3 min at room temperature. With the exception of trypsin the percentage of single cells was consistently higher with dispase and pronase treatment, being 83-92%. Cell viability (dye exclusion) was more than 90%. Mechanical disintegration of pancreatic islets resulted in a low yield of single cells, and cell viability was considerably reduced in comparison with the enzymatic methods. Labeling of islet cells with Na2 51CrO4 and measurement of the basal 51Cr-release demonstrated superior membrane preservation after pronase or dispase treatment. Islet cells isolated either by fractionated dispase or pronase treatment were found to be well preserved and very suitable for the detection of circulating cell surface antibodies and their cytotoxic effects to islet cells.     

Mixed-function oxidase activity in enriched populations of ciliated cells freshly isolated from rabbit tracheas

A method is described for the preparation of enriched populations of ciliated cells from rabbit tracheas. Following protease digestion of tracheal lumen tissue, cells were subjected to centrifugal elutriation. This produced two cell fractions of interest: an 8 µm diameter fraction believed to be composed largely of basal cells, and a 15 µm diameter fraction containing a mixture of ciliated cells and Clara cells. Further treatment of the 15 µm cells with a dextran/polyethylene glycol/phosphate buffer system resulted in separation of a highly enriched ciliated cell fraction (84.3 ± 2.7% ciliated cells with 6.5 ± 1.5% Clara cells) from a fraction containing both ciliated cells (42.0 ± 2.1%) and Clara cells (27.0 ± 3.5%). The yield of cells in the enriched ciliated cell fraction was 0.68 ± 0.09 × 10⁶ cells/ trachea. Analysis of mixed-function oxidase activity in tracheal cells showed 7-ethoxycoumarin deethylase and coumarin hydroxylase activities to be present in the 8 µm cells as well as in ciliated cells and Clara cells. Enzyme activities measured in the ciliated cells (152 ± 66 pmol/ min/ mg protein or 51.2 ± 20.5 pmol/ min/ 10⁶ cells for 7-ethoxycoumarin deethylase and 31.7 ± 15.4 pmol/ min/ mg protein or 10.5 ± 4.8 pmol/ min/ 10⁶ cells for coumarin hydroxylase) were not attributable to contamination with Clara cells.Abbreviations CD cell digest - DNase deoxyribonuclease I - E-1 first elutriator fraction - E-2 second elutriator fraction - E-3 third elutriator fraction - 7-Ec 7-ethoxycoumarin - FCS fetal calf serum - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - HpBS HEPES-buffered salt solution - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PEG Carbowax polyethylene glycol 6000     

大鼠附睾上皮细胞的识别

本文利用免疫荧光技术,用角蛋白和波形蛋白抗体作为组化探针,对不同年龄大鼠的整体和离体培养的附睾上皮细胞进行鉴定,区分出结缔组织成分。并证明用0.05%胰蛋白酶和0.1%胶原酶相继消化附睾管可得到较丰富的上皮细胞,体外培养能维持10d 以上,在培养过程中显示其形态特征。因此本文所用的分离、培养方法所得到的附睾上皮细胞可作为深入研究其功能的一种模型。