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M Asada-Kubota

Publications and source records attributed to M Asada-Kubota.

34 records · Page 2Linked to original sources

Receptor-mediated endocytosis of glucagon in isolated mouse hepatocytes.

The binding of glucagon to the cell surface and the pathway of intracellular transport of the hormone in isolated mouse hepatocytes were studied by autoradiography, colloidal gold-labeled glucagon (Au-glucagon), and biochemical methods. In cells incubated with 1251-glucagon at 4 degrees C, the label was mainly localized to the plasma membrane even after 60 min of incubation. At 20 degrees C, the labeled ligand was internalized by the cells and the amount of internalized ligand increased with time of incubation. At 37 degrees C, the ligand was rapidly internalized and found to be associated with coated or uncoated vesicles. Au-glucagon experiments revealed clearly the process of internalization of glucagon. Au-glucagon bound to the plasma membrane was transported to coated regions and then internalized into vesicles via coated pits. Biochemical results supported these findings from autoradiography and Au-glucagon experiments. Thus, glucagon is internalized by hepatocytes via receptor-mediated endocytosis.

Animals↗

Quantitative analysis of smooth endoplasmic reticulum proliferation in hepatocytes of early postnatal and adult mice treated with phenobarbital.

The smooth endoplasmic reticulum in hepatocytes of early postnatal and adult ddY male mice injected with phenobarbital was analyzed stereologically. The animals received daily intraperitoneal injections of phenobarbital and were killed about 24 h after the last injection. In early postnatal animals (injected with 35 mg/kg of the drug at days 1 and 2 after birth, or injected with 35 mg/kg at day 2 and with 50 mg/kg at days 3 and 4 after birth), the smooth endoplasmic reticulum proliferated both in perivenular and periportal hepatocytes; there was no significant difference in amount of smooth endoplasmic reticulum between the cells of the two zones. In adult animals, however, proliferation occurred only in perivenular cells after the administration of 35 mg/kg of phenobarbital for 2 days, and predominantly in perivenular cells even after the administration of 150 mg/kg of the drug for 7 days. These results suggest that the conspicuous proliferation of the smooth endoplasmic reticulum in perivenular hepatocytes in response to phenobarbital administration, which is characteristic of adult liver, does not occur in early postnatal mice but becomes manifest during postnatal development.

Aging↗

Development of structural heterogeneity of mitochondria in mouse hepatocytes.

The volume and shape (the axial ratio:length/diameter) of mitochondria in periportal and perihepatic hepatocytes from newborn, 5 days old, 10 days old and adult male ddY mice were analysed by quantitative electron microscopy. In newborn animals, the volume and relative histogram of the axial ratio were not significantly different between periportal and perihepatic cells. Thereafter, the volume did not change significantly in periportal cells, whereas it decreased between 5 days of age and the adult in perihepatic cells. In adult animals, the volume in perihepatic cells was 26% of that in periportal cells. The relative histogram of the axial ratio for periportal cells deviated significantly to smaller values than in that for perihepatic cells in 5 days old, 10 days old and adult animals. Thus, the structural heterogeneity of hepatocyte mitochondria, evident in adult animals, is not present in newborn animals but arises during postnatal development.

Animals↗

Effect of castration and testosterone replacement on high glucose 6-phosphatase activity in principal cells of the mouse epididymis.

Glucose 6-phosphatase activity is higher in the principal cell than in other cell types in the terminal segment and caudal half of the middle segment of the mouse epididymis. Effect of castration and testosterone replacement on the high enzyme activity in the principal cell was studied in the terminal segment and the caudal half of the middle segment (cytochemical study), and in the whole epididymis (biochemical study). Ten, 20, or 30 days after castration, the abundant amount of reaction product seen in principal cells from intact control animals decreased to the level in basal cells, halo cells, and smooth muscle cells. However, in animals treated with testosterone following castration, the reaction product in principal cells remained abundant. Changes in the biochemical activity after castration or testosterone administration following castration paralleled the cytochemical results. Thus, the high activity in the principal cell is under the control of testosterone.

Animals↗

Observation of cytochemical alkaline phosphatase activity on the plasma membrane of cultured rat hepatocytes by backscattered electron imaging.

Distribution of alkaline phosphatase (ALPase) activity on the plasma membrane of rat hepatocytes in primary monolayer culture was observed using the backscattered electron image (BEI) mode. Apparent reaction product was seen in hepatocytes cultured for 48 or 72 hours in a concentration of 8 X 10(5) cells per ml, and those cultured for 72 or 96 hours in a concentration of 6 or 4 X 10(5) cells per ml. The reaction product was observed as scattered fine dots, as spots, and as bands along the cell edge, with high contrast in flattened polyhedral hepatocytes forming cell trabeculae. The deposition of the reaction product was generally more abundant at the periphery of the cell trabeculae. X-ray microanalysis revealed that the reaction product contains both lead and phosphorus as real ALPase reaction product. In transmission electron microscopy, the reaction product was exclusively localized on the external surface of the plasma membrane. However, the plasma membrane adjoining the culture substratum was devoid of the reaction product. Further, the highest biochemical ALPase activity appeared in earlier culture stages when the density of cultured cells was larger. Thus, during a few days of culture, ALPase activity increases on the plasma membrane predominantly at the periphery of the hepatocyte trabeculae. This increase in the activity might be related to the mechanism of preserving the cell shape.

Alkaline Phosphatase↗

Cytochemical glucose-6-phosphatase activity in the cells of mouse pancreas and submandibular gland.

Ultrastructural localization of glucose-6-phosphatase activity was studied in the cells of the pancreas and submandibular gland of the mouse using a incubation medium modified from that of Wachstein & Meisel (1956). In pancreatic acinar cells, the reaction product for the enzyme activity was not found even after 90 min of incubation with three changes of the medium. However, the reaction product was localized in the endoplasmic reticulum and nuclear envelope of all other cell types composing the pancreas and submandibular gland. The reaction product appeared in moderate to abundant amounts in acinar cells and striated duct cells of the submandibular gland, and in the B cells, A and D cells of the pancreatic islet, but it was scarce in other cell types.

Animals↗

Binding and internalization of 125I-glucagon in hepatocytes of intact mouse liver. An autoradiographic study.

Localization of labeled material in hepatocytes of intact mouse liver injected via the portal vein with 125I-glucagon was studied. At 3 minutes after pulse injection of the labeled glucagon, most grains were localized associated with the plasma membrane. At 10 min after the injection, the grains were distributed throughout the cytoplasm and did not appear localized exclusively in any specific cell organelles. At 20 min after the injection, the labeled material appeared decreased, and was not yet localized exclusively in any specific cell organelles. Thus, in hepatocytes of intact liver, labeled glucagon is internalized more rapidly than in freshly isolated hepatocytes (Barazzone et al. 1980), and there appear to be no cell organelles in which internalized glucagon is preferentially localized.

Animals↗

Development of ultrastructural heterogeneity among hepatocytes in the mouse.

Ultrastructural stereological analyses of periportal and centrilobular hepatocytes of newborn, 5- and 10-day-old, and adult male ddY mice were carried out to study the postnatal development of the morphologic heterogeneity among hepatocytes. In newborn animals, the periportal and centrilobular cells did not differ in the volume densities of the smooth and rough endoplasmic reticulum; in the volume and numerical densities of the mitochondria, lysosomes, peroxisomes, and lipid droplets; or in the shape (the axial ratio) of the mitochondria. In 5-day-old animals, the volume densities of the mitochondria and rough endoplasmic reticulum were greater in periportal cells than centrilobular cells, and the volume density of the smooth endoplasmic reticulum was greater in centrilobular cells than periportal cells. In 10-day-old animals, a further difference was seen in the numerical density of the mitochondria, which was greater in centrilobular cells than periportal cells. Adult hepatocytes showed also a difference in the axial ratio of the mitochondria, which was greater in centrilobular than periportal cells; there was no difference in the volume density of the rough endoplasmic reticulum. When the data were expressed as volume and number per hepatocyte, the patterns of sublobular distributions of these organelles differed from the patterns seen in the volume and numerical density data, mainly in adult animals. This difference was caused by the marked increase in hepatocyte volume between 10 days of age and adulthood, especially in centrilobular cells. The results show that, in general, the ultrastructural heterogeneity among hepatocytes, evident in adult animals, is not present in newborn animals but arises during postnatal development, and suggest the occurrence of a lobular gradient in postnatal development of hepatocyte functions.

Animals↗

The pattern of glycogen withdrawal within the liver acinus during fasting.

Intraacinar regionality in glycogen withdrawal during fasting was studied in the liver of the mouse. When animals were fasted from 10.00 a.m. onwards, glycogen withdrawal began in zone 1, spreading gradually to zone 3, and after 12 h glycogen disappeared throughout the acinus. After 24 h new glycogen accumulated in various portions of the acinus. However, when animals were fasted from 10.00 p.m. or 12 midnight to the following morning, glycogen was withdrawn evenly throughout the lobule. After 24 h glycogen disappeared throughout the acinus. These results show that there is a heterogeneity in the hepatocyte with respect to glycogen withdrawal during fasting by day but not by night. Therefore, the discrepancy in reported results on the intraacinar distribution of glycogen during fasting is probably due to the time of sacrificing animals.

Animals↗

The heterogeneity of hepatocytes during the postnatal development of the mouse.

Development of the hepatocyte heterogeneity was studied histochemically during the postnatal period. At birth ornithine carbamoyltransferase (OCT). succinate dehydrogenase (SDH) and NADH dehydrogenase (NADHDH) activities were evenly distributed throughout the liver acinus. Slightly uneven distribution within the acinus appeared at 3 days after birth in SDH and at 4 days after birth in OCT and NADHDH, changing to that of adult type at 10 or 12 days after birth which is characterized by a marked difference in the activities between zone 1 and 3. However, in animals of all age groups studied, glycogen was decreased mainly in zone 1 and 2 after 6 or 10 h of fasting and glucose 6-phosphatase activity was markedly reduced or disappeared in zone 3 and often in zone 2 after carbon tetrachloride administration. The results show that so-called "functional and structural heterogeneity among hepatocytes" consists of at least two different components, that formed gradually during the postnatal development and that existing already at birth.

Age Factors↗

Culture of marrow stromal cells derived from bone marrow specimens formed at fracture site of human long bone.

To study the process of the internal callus formation, we cultivated marrow stromal cells derived from bone marrow specimens formed at fracture sites of human long bone in alpha-modified Eagle's medium containing 10% fetal calf serum. After 2 weeks of culture, the cells formed two types of colonies; one consisted of spindle cells, and the other comprised of polygonal cells. The two types of colonies were separated and cultured further. The spindle and polygonal cells proliferated to confluence within 3 weeks and after 4 weeks, respectively, after the separation. Both the spindle and polygonal cells showed on the plasma membrane moderate intensity of staining reaction of alkaline phosphatase (ALPase) activity before the period of confluence and strong intensity during the period of confluence. Then, the spindle cells did not produce calcified matrix, but detached from the dish after 6-8 weeks of culture. In the colonies of polygonal cells, however, dense nodules were formed after 9 weeks of culture, which became visible to the naked eye as white aggregates after 11-12 weeks. Electron microscopic studies on the polygonal cells demonstrated matrix vesicles in the intercellular ground substance after 6 weeks of culture, and electron-dense needle-like crystals on the matrix vesicles after 8-10 weeks of culture. On the basis of infrared spectroscopic analysis, the aggregates were composed of hydroxyapatite. Thus, stromal cells derived from bone marrow specimens formed at fracture site of human long bone differentiated to spindle and polygonal cells containing high ALPase activity (a marker for osteogenic capacity) during culture, and the polygonal cells but not spindle cells produced the calcified matrix.

Adult↗