Kinetic studies of glutaraldehyde binding in liver.

To study the kinetics of glutaraldehyde fixation, fresh rabbit liver cubes were immersed in 3% buffered 3H-glutaraldehyde for various periods of time. Following weighing and a brief rinse in water, the tissues were solubilized, and the radioactivity was measured in a scintillation counter. Binding of the isotope was half-maximal after approximately 4 h and a plateau was reached after approximately 20 h. We also investigated the reversibility of glutaraldehyde fixation. Fixed liver cubes were weighed and immersed in water for various periods of time, and after solubilization, the radioactivity was determined. After rinsing for 48 h, approximately 95% of the radioactivity was lost from the tissue specimens, indicating that fixation with glutaraldehyde is largely reversible. Light and electron microscopy of specimens rinsed for 1 and 48 h showed essentially similar morphology. Rinsing for 48 h restored some of the immunoreactivity that was absent after rinsing for only 1 h.

Detection and localization of H+-K+-ATPase isoforms in human kidney.

An H+-K+-ATPase contributes to hydrogen secretion and potassium reabsorption by the rat and rabbit collecting ducts. Transport of these ions appears to be accomplished by one or both of two isoforms of the H+-K+-ATPase, HKalpha(1) and HKalpha(2,) because both isoforms are found in the collecting ducts and transport of hydrogen and potassium is attenuated by exposure to inhibitors of these transport proteins. To evaluate whether an H+-K+-ATPase is present in the human kidney, immunohistochemical studies were performed using normal human renal tissue probed with antibodies directed against epitopes of three of the known isoforms of the H+-K+-ATPase , HKalpha(1), HKalpha(2), and HKalpha(4), and the V-type H+-ATPase. Cortical and medullary tissue probed with antibodies against HKalpha(1) showed cytoplasmic staining of intercalated cells that was less intense than that observed in the parietal cells of normal rat stomach stained with the same antibody. Also, weak immunoreactivity was detected in principal cells of the human collecting ducts. Cortical and medullary tissue probed with antibodies directed against HKalpha(4) revealed weak, diffuse staining of intercalated cells of the collecting ducts and occasional light staining of principal cells. Cortical and medullary tissue probed with antibodies directed against the H+-ATPase revealed staining of intercalated cells of the collecting ducts and some cells of the proximal convoluted tubules. By contrast, no discernible staining was noted with the use of the antibody against HKalpha(2). These data indicate that HKalpha(1) and HKalpha(4) are present in the collecting ducts of the human kidney. In this location, these isoforms might contribute to hydrogen and potassium transport by the kidney.

Formaldehyde prepared from paraformaldehyde is stable.

For critical histological investigations, tissue fixation is sometimes carried out in formaldehyde freshly prepared from paraformaldehyde by heating. The purity of formaldehyde produced in this way is superior to that of commercial stock solutions. We studied the stability of freshly prepared formaldehyde solutions by determination of pH and titration of acid, which reflect the formation of formic acid. It was found that very small amounts of acid are produced during the heating of paraformaldehyde. Prolonged heating or storage of freshly prepared formaldehyde for up to 8 days did not significantly increase the amount of acid. It was also found that heating of the paraformaldehyde is not necessary, since depolymerization may take place at room temperature. We conclude that formaldehyde prepared from paraformaldehyde remains stable for considerable periods of time, and it is therefore unnecessary to prepare it immediately prior to fixation. Also, in many cases, buffering of the fixative may be omitted, since only minor changes in the pH occur during fixation.

Localization of mRNA for the muscarinic M1 receptor in rat stomach.

Cholinergic stimulation of receptors in the oxyntic mucosa results in secretion of mucus, pepsinogen and hydrochloric acid. There has been speculation as to the cellular localization of these receptors in the mucosa and as to which subtype is present in the different cell types. In the present study, utilizing radioactive riboprobes for the M1 muscarinic receptor subtype, we carried out in situ hybridization to determine which cells of the gastric corpus transcribe mRNA for this receptor. The antisense M1 probe hybridized strongly on the zymogen cells and, to a lesser extent, on the surface mucous cells and the muscle layers. Control sections from brain also displayed specific hybridization.

Selective ligand-induced intracellular calcium changes in a population of rat isolated gastric endocrine cells.

BACKGROUND & AIMS: Peripheral regulation of acid secretion depends mainly on stimulation or inhibition of the three major gastric endocrine cells (enterochromaffin-like, gastrin, and somatostatin). The aim of this paper was to define physiological responses of enterochromaffin-like, gastrin, and somatostatin cells in a mixed endocrine cell population by measuring ligand-selective changes of intracellular calcium ([Ca2+]i) in individual cells. METHODS: Endocrine cells were enriched from a rat gastric cell suspension by elutriation, a density-gradient fractionation, and a 48-hour short-term culture. [Ca2+]i responses of individual cells to various ligands such as gastrin/carboxy-terminal cholecystokinin octapeptide and selective cholecystokinin antagonists, carbachol, and gastrin-releasing peptide were monitored using video imaging in a perfusion chamber. Characteristic [Ca2+]i changes distinguished the three cell types, confirmed by immunostaining. RESULTS: All enterochromaffin-like cells respond to cholecystokinin-B receptor stimulation, but only a few respond to carbachol. Gastrin cells respond to both gastrin-releasing peptide and carbachol but not to cholecystokinin-receptor agonists. Somatostatin cells have both stimulatory cholecystokinin-A and cholecystokinin-B receptors and inhibitory muscarinic receptors. All cells have inhibitory somatostatin receptors. CONCLUSIONS: Calcium-signaling responses of gastric endocrine cells are distinctive. This allows individual cell types in a mixed population to be characterized and permits an analysis of the hormones and transmitters that act directly on a specific cell type.

Kinetic studies of formaldehyde binding in tissue.

Specimens of rabbit liver were fixed for various periods up to 6 days in buffered 14C-formaldehyde. Binding of the isotope reached a plateau after fixation for approximately 24 hr; the half-maximal binding level was reached after approximately 100 min. Formaldehyde binding at 37 C was faster than at 25 C, and faster at pH 7.0 than at pH 4.0. During rinsing of the fixed tissue in water for up to 26 days there was a progressive decrease in isotope content to 10-20% of the pre-rinse level, indicating that formaldehyde fixation is a reversible process.

In situ hybridization of mRNA for the gastric H+,K(+)-ATPase in rat oxyntic mucosa.

The H+,K(+)-ATPase member of the phosphorylating ion motive ATPases is composed of two subunits, a large alpha-subunit composed of about 1030 amino acids and a smaller beta-subunit consisting of about 290 amino acids. By biochemical and immunological methods both subunits have been found in high abundance in the gastric parietal cell. In the present study in situ hybridization was used for localizing and comparing concentrations of the mRNA for the two subunits in the gastric epithelium. For this purpose 3H-labelled probes were preferred. Hybridization was detected only in the parietal cells. The older parietal cells in the bottom of the mucosa gave a weaker hybridization signal than the younger parietal cells closer to the surface. The margin of experimental ulcers, where the parietal cells are of low differentiation, showed very weak, if any, hybridization. The differences observed in hybridization densities may reflect differences in mRNA synthesis or stability. It is conceivable that older parietal cells, as well as parietal cells of low differentiation, produce relatively small amounts of H+,K(+)-ATPase.

Stereologic investigations of human gastric mucosa. II. Oxyntic mucosa from patients with Zollinger-Ellison syndrome.

Biopsy specimens from the oxyntic mucosa were obtained on 210 occasions from 76 patients with the Zollinger-Ellison syndrome (ZES) before and during omeprazole treatment. One-micrometer sections were examined by light microscopy, and in 5% linear hyperplasia of endocrine cells was observed. Morphometry was carried out in 91 of the specimens and showed a significant increase of the mean endocrine cell density in comparison with both young, healthy subjects and patients suffering from active peptic ulcer disease (PUD). No metaplasia, dysplasia, or neoplasia was detected in patients with ZES, and the mean mucosal thickness and parietal cell density remained normal. The parietal cells often displayed endosome-like structures, and occasionally there were lingulate cytoplasmic projections into the gland lumen. Electron microscopic morphometry was carried out in specimens from nine patients with ZES and did not show any significant differences in the parietal cells in comparison with healthy subjects.

Structure of mucosa in continent ileal reservoirs 15 to 19 years after construction.

Mucosa biopsy specimens were obtained from 12 patients with continent ileostomy reservoirs constructed 15 to 19 years previously. Biopsies from normal ileal mucosa, taken from six other patients with no apparent bowel disease, served as controls. The specimens were processed for light and electron microscopy. The reservoir mucosae showed an increased amount of inflammatory cells, but there were no signs of dysplasia. In the goblet cells, sialomucins dominated over sulfomucins; in this respect no difference was found between reservoirs and controls. Morphometric studies showed an increase of mucus-storing goblet cells in the reservoir mucosae, both with regard to relative number and to volume density. The mitotic index was higher than normal in the reservoirs, but the relative number of the Paneth cells and the height of the villus epithelial cells were similar in the reservoirs and the controls. In the reservoirs, the surface amplification factors due to villi and to microvilli (near the villus tips) were reduced by some 29% and 20%, respectively, indicating villus hypotrophy. It is concluded that only minor morphologic changes appear in the ileal reservoir mucosa 15 to 19 years after construction. Morphometry provides a sensitive tool to demonstrate such changes in intestinal morphology.

Alcohol consumption and synthesis of ethyl esters of fatty acids in adipose tissue.

Ethyl esters of fatty acids (EEFA) have been found to be formed during ethanol metabolism. Human adipose tissue contains high concentrations of free fatty acids, the substrate for EEFA synthesis, and might therefore be a tissue with great potential for EEFA formation. In order to explore their potential usefulness as markers of alcohol abuse, the EEFA concentration and the activity of EEFA-synthesizing enzyme were therefore determined in adipose tissue from men belonging to the following categories: teetotalers, social drinkers, alcoholics under treatment, or established alcoholics found to have died as a result of alcohol intoxication. In order to estimate the half-life of EEFA and the synthase activity induction, the alcoholics were examined after different time periods of abstinence from alcohol. Comparisons were also made with several established markers of alcohol abuse. EEFA were not found in teetotalers, and were found in low concentrations in some of the social drinkers. EEFA were found in several alcoholics, and the forensic cases had high concentrations. EEFA-synthesizing enzyme activity was found in all subjects, increasing from teetotalers to social drinkers, and being 2-fold higher in alcoholics and 5-fold higher in dead alcoholics. The induction of the enzyme after abstinence appeared to have a half-life of the order of several weeks. Correlations were found between EEFA synthase activity and previously established markers of alcohol abuse known to remain for a long time period after abstinence, such as mean erythrocyte corpuscular volume. This preliminary study suggests the possibility that EEFA synthase induction in adipose tissue might have a longer half-life than previously used markers of alcohol abuse. It is therefore suggested that the induction of EEFA synthase might be a potentially useful new marker for alcohol abuse because of its apparent proportionality to alcohol intake over a prolonged time period, its presumed specificity, and long-term elevation after alcohol abstinence. This potential marker should be analysed further.

Histology of gastric biopsies from peptic ulcer patients before and after short-term treatment with omeprazole or H2-receptor antagonists.

Sixty patients with duodenal or prepyloric ulcers were given omeprazole (30 or 40 mg o.m.; average period of treatment: 2.9 weeks) or histamine H2-receptor antagonists (cimetidine 400 mg b.i.d. or ranitidine 150 mg b.i.d.; average period of treatment; 3.5 weeks) for a single period ranging between 2 and 6 weeks. At the end of the treatment period fasting plasma gastrin levels were moderately increased in both groups in comparison with the pretreatment values. Endoscopic biopsies were taken from the oxyntic mucosa at the beginning and at the end of the treatment period. Light microscopy of the biopsies was aimed particularly at determining the number of endocrine cells. In addition, the mucosal thickness and the volume densities of the parietal cells, the lamina propria and the gland lumina were measured. There were no significant differences in the endocrine or parietal cell populations, between biopsies taken from the patients before and after treatment with omeprazole or histamine H2-receptor antagonists. The mucosal thickness and the densities of the lamina propria and of the gland lumina remained unaffected by the treatment.

Immunocytochemical studies of gastric H+,K+-ATPase in the developing rat.

The appearance of the enzyme H+,K+-ATPase was studied in the gastric mucosa of rats during the perinatal period. By means of monoclonal antibodies against the enzyme, immunoreactivity was regularly detected in parietal cells 1 day before birth. The intensity of the staining and the frequency of stained cells increased up to 10-12 days after birth, when adult levels were approached. Electron microscopy showed that the initial staining occurred at the apical surface of the parietal cells, with only faint or no staining at the secretory canaliculi. From 5 days after birth immunoreactivity was observed also at the tubulovesicular membranes.

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size.

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.

Studies on the rate of dehydration of histological specimens.

Pieces of liver, kidney and urinary bladder were fixed in 10% formalin. In order to study the velocity of the dehydration process tissue specimens of standardized size were rinsed and equilibrated with water containing 3H2O. The specimens were then put into vials with 100% ethanol or acetone; the vials were either shaken continuously or left stationary. The concentration of 3H in the dehydration medium was determined at frequent intervals. If the vials were shaken, steady concentrations of 3H were reached within about 50 min when ethanol was used, and about 30 min with acetone, indicating that dehydration was complete. With the vials left standing still the corresponding times exceeded 12 h. In other experiments dehydration was carried out with intermittent shaking in rising concentrations of ethanol or acetone; in these cases about 4 h were required.

Thickness variations within individual paraffin and glycol methacrylate sections.

The object of this study was to investigate whether there are intra-section thickness variations in individual paraffin and glycol methacrylate (GMA) sections. Using steel or glass knives sections were cut from liver and urinary bladder. Section thickness variations were measured with an interference microscope and amounted to 1-3 microns within individual paraffin sections and 0.3 microns within GMA sections. The results were confirmed by observations on sections which had been re-embedded and re-sectioned. Some of the variations within the paraffin sections were associated with the cell nuclei. It is concluded that GMA sections are much smoother than paraffin sections and thus more suitable for quantitative histological studies.