An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. II. Intracellular fates of the 125I- ligands

Electron microscope autoradiographic and biochemical methods were used to study the intracellular fates of several 125I-glycoproteins, known to be specifically bound and internalized by the different cell types in the liver. At the earliest times examined (1--2 min), 125I-glycoproteins (ASGP) were localized predominantly along the sinusoidal front of hepatocytes. Analysis of the distribution of autoradiographic grains indicated that: (a) approximately 40--60% of the 125I-ligand could be ascribed to the plasmalemma; (b) a significant fraction had already been internalized; yet (c) very little 125I-ligand was present in the lysosome-Golgi region. Between 4 and 15 min after administration of 125I-ASGPs, there was a dramatic redistribution of autoradiographic grains from regions of the plasmalemma and peripheral cytoplasm (30% decrease) to the lysosome-Golgi region (30% increase). At longer times (30 min), there was continued drainage of 125I-ASGP into this region. The grain density over secondary lysosomes was 60--90 times higher than that over recognizable Golgi elements, clearly indicating that lysosomes were the ultimate destination of the 125I-ASGP. However, no more than 60% of the total 125I-ligand could be localized to lysosome-rich regions of the hepatocyte, with the remaining 40% primarily in the intermediate cytoplasm. Biochemical evidence for proteolysis of the internalized 125I-ASGP (presumably within lysosomes) was obtained when [125I]-mono-iodotyrosine was found in the liver (i.e., hepatocytes) at times later than 15 min. The temporal redistribution observed for mannose and N-acetylglucosamine-terminated glycoproteins (ahexosamino-orosomucoid and agalacto-orosomucoid, respectively) in endothelial cells indicated that the 125I-ligands resided in macropinocytic vesicles (1--15 min) before their ultimate residence in dense bodies (15 min). The same 125I-ligands were also localized to structures resembling secondary lysosomes in Kupffer cells. The lysosomal nature of "these organelles" was implied from the appearance of [125I]mono-iodotyrosine in the liver at later times. 125I-beta-glucuronidase followed the same intracellular pathway in both cell types but was not degraded.     

An electron microscope autoradiographic study of DOC conversion to corticosterone in the rat adrenal cortex

Summary Seven thymuses from children between 1 and 12 years were examined by electron microscopy. Biopsies had been taken during surgical correction of congenital heart defects.In all cases we found interdigitating reticulum cells (IRC) in the medulla and inner cortex. These cells resembled the IRC which have been described previously in the thymus-dependent regions of the spleen and lymph node. They were characterized by an irregularly shaped nucleus, narrow cisterns of rough endoplasmic reticulum, and widespread interdigitation and invagination of the cell membrane. The surfaces of the IRC were in close contact with those of small lymphocytes, sometimes polysomal lymphatic cells, epithelial cells, and occasionally with those of lymphatic cells containing ergastoplasm.The IRC is apparently a specific cell of thymus-dependent regions. It may be that the IRC in the thymus, lymph node, and spleen contribute to the microenvironment needed for the differentiation of T-cells.Supported by the Deutsche Forschungsgemeinschaft, SFB 111/CII and III.—We wish to thank Miss M. Neubert and Mrs. R. Köpke for their technical assistance and Mrs. M. Soehring for her help with the translation.     

An electron microscope study of basophile substances of frozen-dried rat liver

Small pieces of liver from rats subjected to different dietary regimes were fixed by freeze-drying, and postfixed by in vacuo heating and denaturation with alcohol. Specimens were digested with ribo- or deoxyribonuclease, and stained with gallocyanin-chromalum, azure II, the Feulgen procedure or alcoholic platinic tetrabromide. Some specimens were reserved as controls of the effects of enzyme treatment. Stained and unstained specimens were embedded in methacrylate and examined by light and electron microscopy. Basophilic and Feulgen-positive substances, after contact with watery reagents, were found by electron microscopy to exist as small dense granules embedded in a less dense homogeneous matrix, forming the walls of submicroscopic vacuoles. These granules were absent after digestion with nucleodepolymerases. In specimens (unstained, or stained with platinic tetrabromide) which had not passed through water, the dense (basophile) substances in nuclei and cytoplasm were found to exist, not as granules, but as ill defined submicroscopic concentrates which blended imperceptibly into the homogeneous matrix of the vacuolar walls. Objections to the use of stains for improving contrast conditions in electron microscopy of tissues are discussed, and it is concluded that the reagents do not necessarily produce the observed increases in contrast by selectively stabilizing certain structures. The concept of microsomes as pre-existing distinct morphological entities in intact (unhomogenized) cells is thought to be inconsistent with the distribution of basophile substances in frozen-dried liver.     

A quantitative electron microscope autoradiographic study on 125I-human choriogonadotropin internalization in bovine luteal slices

Very few silver grains were seen on the cell surface and none intracellularly after incubation for 2 h at 4 degrees C. However, numerous grains were seen in various subcellular organelles when the tissues were incubated for 2 h at 22 degrees or 38 degrees C. The grain distribution was qualitatively similar, but quantitatively, there were fewer grains at 22 degrees than at 38 degrees C. Co-incubation of 125I-hCG with excess unlabelled hCG resulted in the virtual disappearance of silver grains from all the subcellular organelles. Excess unlabelled human luteinizing hormone (but not follicle-stimulating hormone or prolactin) inhibited the appearance of silver grains in luteal tissue. There were no silver grains in bovine liver slices incubated with 125I-hCG. The plasma membrane-associated grains progressively decreased, while intracellular organelle-associated grains increased with time at 38 degrees C. There were no grains in nuclei at 5 min, but they appeared at 10 min and increased until 120 min. After correction for radiation spread by three-step mask analysis, smooth endoplasmic reticulum and mitochondria did not contain any grains. The grain density was the highest in Golgi, followed by lysosomes, rough endoplasmic reticulum, nuclei, and plasma membranes after incubation for 2 h at 38 degrees C. Thus, the electron microscope autoradiography approach confirmed our biochemical data in the preceding paper (Chegini et al., Exp cell res 151 (1984) 466 [5]) on time, temperature dependency and specificity of 125I-hCG internalization, association of internalized hormone with a variety of intracellular organelles, and the highest uptake in Golgi.     

Localization of 125I-atrial natriuretic factor (ANF)-binding sites in rat renal medulla. A light and electron microscope autoradiographic study

Using light and electron microscope autoradiography in vivo, the localization of 125I-(Arg 101-Tyr 126) atrial natriuretic factor (ANF)-binding sites was studied in the renal medulla of rats. At the light microscopic level, the autoradiographic reaction was mainly distributed in patches in the outer medulla, and followed the tubular architecture in the innermost part of the inner medulla. At the electron microscopic level, binding sites were mainly found in the outer medullary descending vasa recta and inner medullary collecting ducts. These results suggest that, in rats, the renal medulla may participate in the natriuresis and diuresis produced by ANF through vascular and tubular effects; the former by changing medullary blood flow at the level of descending vasa recta and the latter by acting on electrolyte and water transport at the level of collecting ducts.     

Intracellular distribution of GABA in the rat anterior pituitary. An electron microscopic autoradiographic study

We studied the internalization and intracellular distribution of [3H] GABA in rat anterior pituitary cells. Electron microscopic autoradiography of anterior pituitary fragments or dispersed pituitary cells incubated with [3H] GABA showed that lactotrophs and, to a lesser extent, somatotrophs were the only cells that contained radioactive grains. Grain density analysis performed on dispersed pituitary cells after a pulse-chase experiment (10 min pulse and then change to a medium without radioactive GABA for various periods up to 2 h) revealed that GABA internalized by lactotrophs was distributed in various intracellular membranous organelles. Of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria and secretory granules had different time-dependent labeling patterns. The highest grain density values were associated with plasma membrane (at the first chase time) and the Golgi apparatus. Mitochondria and secretory granules also showed significant grain density values. A similar pattern of distribution was observed when fragments of prolactin-secreting pituitary adenomas were incubated with [3H] GABA. These results provide morphological data on the cellular specificity and intracellular distribution of GABA in anterior pituitary cells.     

Transport of 125I-poly I : poly C incorporated in liposomes from the enteric cavity to the small intestine mucosa. An electron microscopic autoradiographic study

Transport of 125I-poly(I) : poly(C) incorporated into liposomes trough the small intestine mucose was investigated by electron microscopic autoradiography. With the migration of liposomes into the mucous layer on the luminal surface of the intestine up to the glycocalix level of microvilli these undergo degradation with the formation of monolayer liposomes from which polynucleotide is released. Later on the poly(I) : poly(C) or its fragments transported through the enterocytes to be accumulated in cells of the connective tissue stroma of the small intestine mucose. Part of polynucleotide was incorporated up to the arterial and lymphoid capillary level. Apparently, on the way of its transport the polynucleotide is affected by pancreatic and tissue nucleases. The accumulation of polynucleotide in macrophages, fibroblasts, lymphocytes, plasma cells and smooth muscle cells was traced. It is supposed that the polynucleotide accumulated in stroma of the small intestine mucose may preserve its interferon inducing activity.     

Scanning electron microscope study of the liver microcirculation

During the experimental investigation performed in dogs and rats, by means of scanning electron microscopy of corrosive anatomical preparations, the spatial organization of all parts of the hepatic vascular bed (arterial, venous and lymphatic) has been studied, specific features of their components construction have been described. Within the limits of one hepatic lobule the number of vessels included in the portal vein system exceeds that of the arterial ones, originating from the proper hepatic artery system. In every part of the vascular bed the gradient of the form, orientation and pronouncement of the nuclei-containing zones in endotheliocytes and myocytes has been established. Various appliances participating in the blood and lymph stream regulation in different parts of the vascular bed have been revealed. As initial elements of the lymph bed, closed digital or loop-like capillaries should be regarded, they localize in the organ's connective tissue framework. Around the portal and hepatic veins and their branches, as well as around the biliary ducts, well developed plexuses of the lymphatic and blood capillaries and vessels localize, they are the main draining pathways of the organ. The degree of development and pronouncement of these plexuses depends on the lumen size in the formation they accompany.     

Distribution of phosphatidylinositol biosynthetic activities among cell fractions from rat liver.

The distribution of activities for synthesis of phosphatidylinositol among cell fractions from rat liver was determined. Activity was concentrated in endoplasmic reticulum; rough and smooth fractions were nearly equal. Golgi apparatus exhibited a biosynthetic rate 44% that of endoplasmic reticulum. Plasma membranes and mitochondrial fractions were only 6% as active as endoplasmic reticulum. Thus, endoplasmic reticulum and Golgi apparatus fractions from rat liver catalyze the net synthesis of phosphatidylinositol in vitro, whereas plasma membrane and mitochondrial fractions do not.