Radioautographic study on the localization of an anti-allergic agent, tranilast, in the rat liver

In order to demonstrate the localization associated with metabolism of an anti-allergic agent, Tranilast, in the liver, light microscopic radioautography of the liver was performed. Rats were administrated orally with 3H-Tranilast, and were sacrificed at 15 minutes to 24 hours after the administration. The livers were taken out and fixed, embedded and processed for light microscopic radioautography. 3H-Tranilast was absorbed rapidly, and the radioactivity in the liver increased and decreased within several hours. The number of radioautographic silver grains reached a maximum 3 hours after the administration. From 1 to 6 hours after the administration, the silver grains decreased from the portal area toward the central area. Seventy to 80% of all silver grains on the hepatocytes were retained in the cytoplasms of the hepatocytes at any experimental period. From these results, it was concluded that the localization of radioautographic silver grains was associated with Tranilast uptake of hepatocytes in each hepatic lobular compartment and that the metabolic process from uptake to excretion of Tranilast took part in the hepatocytes in each hepatic lobular compartment.     

Electron microscopic radioautographic study of the localization of an anti-allergic agent, Tranilast, in rat mast cells.

We have previously reported that Tranilast, an anti-allergic agent, was rapidly taken into the cytoplasm of rat mast cells in vitro by means of light microscopic radioautography. The present study was performed at the electron microscopic level to elucidate the fine localization of this agent in the mast cells. The results revealed that the number of radioautographic silver grains in the cells increased by the incubation with 3H-labelled Tranilast for 0 to 60 min. and that many silver grains were localized on the specific granules, especially on the perigranular membranes. These results suggest that the mode of inhibitory action of mast cell degranulation by Tranilast is related to the specific localization of this agent on the perigranular membranes.     

Localization of axonally transported 125I-wheat germ agglutinin beneath the plasma membrane of chick retinal ganglion cells

The distribution of 125I-wheat germ agglutinin (WGA) transported by axons of chick retinal ganglion cells to layer d of the optic tectum was studied by electron microscopic autoradiography. We found that 52% of the radioactivity was located in axons and axon terminals in the contralateral optic tectum 22 h after intravitreal injection of affinity-purified 125I-WGA. Axons comprised 43% of the volume of layer d. Dendrites, glial cells, and neuron cell bodies contained 20%, 17%, and 3% of the label, whereas these structures comprised 24%, 21%, and 2% of the tissue volume, respectively. We also measured the distances between the autoradiographic silver grains and the plasma membranes of these profiles, and compared observed distributions of grains to theoretical distributions computed for band-shaped sources at various distances from the plasma membranes. This analysis revealed that the radioactive source within axons was distributed in a band of cytoplasm extending in from the plasma membrane a distance of 63 nm. Because WGA is known to bind to specific membrane glycoconjugates, we infer that at least some glycoconjugates may be concentrated within an annular region of cytoplasm just beneath the axonal plasma membrane after axoplasmic transport from the neuron cell body.     

RADIOAUTOGRAPHIC DEMONSTRATION OF 5-HYDROXYTRYPTAMINE-3H UPTAKE BY PULMONARY ENDOTHELIAL CELLS

The lung is able to rapidly remove 5-hydroxytryptamme (5-HT) from the circulation by a Na⁺-dependent transport mechanism. In order to identify the sites of uptake, radioautographic studies were done on rat lungs which had been isolated and perfused with 5-HT-³H and 0 5 mM iproniazid, a monoamine oxidase inhibitor. In control experiments 10^-4 M imipramine was added to the perfusate to inhibit the membrane transport of 5-HT At the light microscope level, silver grains were seen concentrated near capillaries and in the endothelium of large vessels From electron microscope radioautographs a semiquantitative grain count was made and 90% of the silver grains were observed over capillary endothelial cells. The grains were found over the nucleus and cytoplasm of the cell and shewed no preferential association with any particular cytoplasmic inclusion bodies, organelles, or vesicles Other cell types were unlabeled except for a few mast cells, certain vascular smooth muscle cells, and one nerve ending. This radioautographic demonstration of the cell type responsible for the rapid removal of 5-HT from the lung circulation clearly establishes the existence of a new metabolic role for pulmonary endothelial cells.     

Quantitative radioautographic study of intracellular localization of calmodulin antagonist, W-7, in Chinese-hamster ovary cells

The purpose of the present study was to analyse quantitatively the localization of calmodulin antagonist, n-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) in CHO-Kl cells. The cultured CHO-Kl cells were labelled with 1 (16.7 microM), 2 (33.4 microM), 5 (83.5 microM) and 10 microCi/ml (167 microM) tritiated W-7. Some cells were preincubated in 10, 50 and 100 microM unlabelled W-7 for 30 min and then labelled with 2 or 5 microCi/ml tritiated W-7 for 1 h. The cells were doubly fixed in glutaraldehyde and osmium-tetroxide solution, and embedded in Epon. For light-microscopic radioautography, 2 micron-thick sections were wet mounted with radioautographic emulsion and exposed for 1 month. The radioautograms showed that large numbers of silver grains were mainly localized in the cytoplasm as well as in the nucleus. Quantitative analysis demonstrated that, in both the cytoplasm and nucleus, the number of silver grains was dependent on the concentration of the administered tritiated W-7 and the number was dramatically decreased by the pretreatment of unlabelled W-7. These results show that, in CHO-Kl cells, the W-7 binding sites are saturable. It is concluded that W-7 may get into CHO-Kl cells and be bound to a specific protein that may be calmodulin protein.     

Light microscopic radioautographic study of the localization of an anti-allergic agent, Tranilast, in rat mast cells

In order to demonstrate the localization of an anti-allergic agent, Tranilast, in the mast cells, light microscopic radioautography was performed. The mast cells collected from rat peritoneal cavity were incubated for 0 to 60 min in a medium containing 3H-Tranilast. After the incubation, they were fixed, embedded and processed for light microscopic radioautography. The radioautographic silver grains were frequently localized around and over the cytoplasmic granules and their number increased according to the prolongation of incubation time. From the results obtained at present it was demonstrated that Tranilast was rapidly taken into the cytoplasm of mast cells. This phenomenon may suggest an important role of this agent in the inhibition of allergic reactions of mast cells.     

Phosphoprotein synthesis and secretion by odontoblasts in rat incisors as revealed by electron microscopic radioautography

The secretory pathway of dentin phosphoproteins in rat incisors was studied by electron microscopic radioautography after the injection of 3H-serine, and the results were compared with those using 3H-proline as a tracer. Five min after injection of 3H-serine, radioactivity was found in the rough endoplasmic reticulum. At 10 min, silver grains were observed over the spherical portions of the cisface of the Golgi apparatus. At 20 min after injection, silver grains were seen over the cylindrical portions of the transface of the Golgi apparatus. The secretory granules showed the strongest reaction from 20 min to 1 hr. At 45 min, a significant labeled band appeared at the mineralization front. At 1 hr, the labeling at the mineralization front began to appear in the mineralized dentin, and after 12 hr this labeled band was located within the mineralized dentin. The pathway of 3H-proline was essentially the same as that of 3H-serine, but 3H-proline moved more slowly than 3H-serine, especially in transit from the rough endoplasmic reticulum to the Golgi apparatus. Secretory granules were heavily labeled from 30 min to 1 hr after injection of 3H-proline; no labeling was found at the mineralization front at 45 min. The labeling seen initially over the predentin was over the mineralized dentin no earlier than 6 hr after injection. The labeling pattern with 3H-serine is closely related to the localization of phosphoproteins, whereas the pattern with 3H-proline reflects the production of collagen rather than of phosphoproteins. The present radioautographic results indicate that dentin phosphoproteins are related to secretory granules and are secreted by odontoblasts at the mineralization front and also that phosphoproteins are involved in the process of mineralization of the circumpulpal dentin.     

Different phosphatidylinositol 3-phosphate asymmetries in yeast and mammalian autophagosomes revealed by a new electron microscopy technique

Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.     

Different phosphatidylinositol 3-phosphate asymmetries in yeast and mammalian autophagosomes revealed by a new electron microscopy technique

Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.     

ABSOLUTE SENSITIVITY OF ELECTRON MICROSCOPE RADIOAUTOGRAPHY

A calibration method is described for measuring absolute radioautographic sensitivities under various experimental conditions. Sensitivities to ³H and ³⁵S radiation, i.e. ratio of developed grains to radioactive decays in the specimen, were determined with Ilford L4 and Kodak NTE emulsions. The highest values obtained in monolayers of emulsion were ⅛ for ³H and 1/21 for ³⁵S. The influence of various experimental parameters on sensitivity is described, and the possibilities for quantitative electron microscope radioautography are discussed.     

High resolution analysis of the secretory pathway in mammotrophs of the rat anterior pituitary

The secretory process in pituitary mammotrophs was analyzed by quantitative electron microscope autoradiography. Dispersed pituitary cells from estrogen-treated female rats were subjected to pulse- labeling with [3H]leucine (5 min) followed by a chase incubation of up to 4 h. Autoradiograms were prepared using fine-grained emulsion (Kodak 129-01), and analyzed using a three-step "mask analysis' procedure: (a) the distribution of autoradiographic grains is determined as in a simple grain density analysis; (b) masks (transparent overlays) are used to generate expected grains from assumed sources; and (c) a computer program compares these two distributions and varies the expected distribution to match the observed distribution, thereby identifying the radioactive sources in the tissue. The overall route of intracellular transport of prolactin from rough endoplasmic reticulum (ER) leads to Golgi complex leads to immature secretory granules leads to mature secretory granules was as established in previous studies. However, by use of the high resolution emulsion and method of analysis, the precision with which label could be localized within individual source compartments was much greater and the time resolution was much sharper than achieved previously using Ilford L4 emulsion and simple grain density analysis. The main new findings were as follows: (a) the ER was essentially drained of radioactivity by 30 min, the Golgi complex by 1 h, and the immature secretory granules by 2h postpulse. This indicates that the secretory product (prolactin) is rapidly and efficiently transported out of these compartments. (b) approximately 30% of the total radioactivity remains located in the ground cytoplasm over the entire postpulse period examined (up to 4 h), and by 30 min postpulse the grain density in the ground cytoplasm exceeded that of the ER. This indicates the ability to resolve ER-associated label (presumably associated mainly with secretory products) from the cytoplasmic label (presumably associated with nonsecretory proteins). (c) the specific activity of immature secretory granules was much greater than previously appreciated; at 1 h postpulse it was greater than 200 times that of the adjacent Golgi complex cisternae. This large dynamic range in observed grain density demonstrates the ability to effectively correct for radiation spread and thus to detect with great accuracy high concentration of label even from very small structures (20-100 nm) which constitute a small percentage (less than 1%) of the total cell area.     

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. II. Observations in tissues other than liver

Biochemical evidence from the preceding paper indicated that [3H]N- acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N- acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.     

BETA GRANULE FORMATION IN ISOLATED ISLETS OF LANGERHANS : A Study By Electron Microscopic Radioautography

The distribution of radioautographic grains over organelles within the beta cells of rat islets of Langerhans was investigated at various times after pulse labeling of the isolated islets with tritium-labeled amino acids. Ten minutes after the start of labeling most of the grains were situated over the endoplasmic reticulum and cytoplasm; by contrast, 60 min from the start of labeling the majority of the grains were associated with the beta granules. At 20, 30, and 45 minutes after pulse labeling the proportion of grains associated with the Golgi complex was increased two- to three-fold over the 10- or 60-minute values. The distribution of radioautographic grains over granules in the intact cells did not suggest that the electron-lucent type of secretory granules were precursors of the electron-opaque granules. Furthermore, studies of the pattern of grains over granules isolated by centrifugation 60 min after pulse labeling showed no preferential labeling of the electron-lucent type of granule. It is concluded that labeled amino acids are incorporated initially in the endoplasmic reticulum, and that the label subsequently appears in the beta granules. The Golgi complex participates either in the formation of the beta granule or in the translocation of the granule through the cytoplasm of the cell.     

SITES OF GLYCOGEN SYNTHESIS IN RAT LIVER CELLS AS SHOWN BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY AFTER ADMINISTRATION OF GLUCOSE-H3

Glycogen synthesis was investigated by giving tritium (H³)-labeled glucose with carrier to fasted rats in vivo or incubating liver slices from fasted rats in vitro using a glucose-H³-containing medium. After 15 min or 1 hr, pieces of liver were fixed and radioautographed for light and electron microscopy. In vivo and in vitro, radioautographic reactions appeared over "glycogen areas" and over zones transitional between these areas and ergastoplasm. Treatment of sections by alpha amylase removed all but about 5% of the radioactivity, so that about 95% of it consisted of glycogen (synthesized during the 15 min or 1 hr elapsing after administration of glucose-H³). Within glycogen areas and transitional zones, most silver grains were over or very close to glycogen granules and smooth (or partly smooth) vesicles. Presumably, much of the label was added onto growing glycogen granules, in accord with the biochemical view that glycogen may serve as substrate for further glycogen synthesis. The few silver grains located far from glycogen granules—15% at the 15 min interval in vivo—approximated smooth (or partly smooth) vesicles of endoplasmic reticulum. This observation raised the possibility that smooth membranes play a role in glucose uptake at an early stage in de novo formation of glycogen granules.     

LIPID SYNTHESIS, INTRACELLULAR TRANSPORT, AND STORAGE : III. Electron Microscopic Radioautographic Study of the Rat Heart Perfused with Tritiated Oleic Acid

Rat hearts pulse-labeled by perfusion in vitro with 9,10-oleic acid-³H for 15 or 30 sec were shown to take up the fatty acid extensively. In hearts postperfused with unlabeled medium for 15 sec or more, 90% of the radioactivity was recovered in esterified lipids. The radioautographic reaction was localized initially over elements of the sarcoplasmic reticulum and mitochondria. After longer periods of postperfusion (2–20 min), there was concentration of silver grains over lipid droplets. In mitochondria and sarcoplasmic reticulum isolated from hearts postperfused for 1 min or more, most of the esterified lipid was in the form of triglyceride. The ratio of the specific activity of isolated sarcoplasmic reticulum triglyceride to mitochondrial triglyceride changed from a value of 3.2 to 1.3 during 5 min of postperfusion. Under conditions of hypothermia, considerable uptake of free fatty acid occurred. The radioactivity recovered in the heart was mostly in the form of free fatty acid, and the radioautographic reaction was seen over sarcoplasmic reticulum and mitochondria, but not over lipid droplets or myofibrils. The results are interpreted to show that intracellular transport of free fatty acid, which occurs also when esterification is repressed, proceeds through intracellular channels, i.e. the sarcoplasmic reticulum. Esterification of fatty acid into triglycerides occurs mostly in the sarcoplasmic reticulum, especially in the region of the dyad, in the vicinity of which lipid is stored in the form of droplets.     

Electron microscopic radioautographic study on the incorporation of 3H-proline by mouse decidual cells

A qualitative radioautographic analysis showed that mature decidual cells of the mouse are able to incorporate 3H-proline. After 1 hour silver grains are concentrated mainly over these cells although some of them can be found over collagen fibrils. After 2,6 and 24 hours there is a progressive increase of silver grains on the extracellular space most of them concentrated over thick collagen fibrils. These results strongly indicate that mature decidual cells of the mouse produce collagen and that they conserve a behavior of the fibroblast from which they originated.     

A radioautographic study on RNA synthesis in aging mouse spleen after 3H-uridine labeling in vitro.

EM radioautographic study on RNA synthesis in aging mouse spleen was conducted after 3H-uridine labeling in vitro. The localization of radiolabelled precursor was used to determine the site of RNA synthesis. The site of the radiolabelled uridine uptake was localized in the haematopoietic cells, particularly in the lymphoblasts. In the labelled cells, most of the silver grains were localized in the nucleus, specifically in the euchromatin. Few cytoplasmic organelles such as the mitochondria and endoplasmic reticulum were labelled with 3H-uridine. Silver grains were also observed over the nucleoli. The labeling index was expressed as the percentage of labelled cells over the total number of cells counted. The labeling index increased from day one after birth and progressively until the 14th day. Thereafter, the labeling index decreased gradually until the 10th month. A significant difference of p less than 0.05 was noted. In all the EMRAG analyzed, it was observed that the number of silver grains per cell increased proportionally with the labeling index. The result of the quantitation of the changes in RNA synthesis correlated well with the maturational development/aging of the animal.     

Negative-staining autoradiography: a new technique for ultracryotomy utilizing an interposed film

A new radiocytochemical technique is reported for ultrastructural localization of diffusible substances, using negatively stained ultra-cryostat sections. A sheet of film interposed between the cryostat section and the emulsion layer has rendered negative-staining autoradiography (NSA) practical. The rationale of NSA is that the film completely shields the section from all moisture-producing autoradiographic processes, so that phosphotungstic acid (PTA) can stain the section either before or after autoradiography (ARG), without the possibility of ultrastructural damage by alkaline solutions, interference between PTA and photoprocessing compounds, and superimposed images of a gelatin layer stained with PTA. As a model to demonstrate the newly developed procedure of NSA, rat brains were labeled with [125I]-triiodothyronine, fixed with tannic fixative, immersed in a cryoprotectant, frozen in liquefied propane, and cryostat sectioned. The resulting higher yield of radioactivity (85%) on the section was confirmed by a radiation counter. The retention rate was approximately 20% greater than that of conventional sections. Developed silver grains were found on synaptic vesicles and mitochondria in the polymorphic layer of the dentate gyrus. In this report we will also discuss the problems associated with cryostat sectioning of fresh tissues, the concept of ARG resolution, the distribution pattern of developed silver grains, and the possible applications of NSA.     

Characteristics of ribosomal RNA synthesis in the blood lymphocytes of chronic lympholeukemia patients

A study was made of the number of silver grains over the nucleoli and of the content of ribosomes in the lymphocyte cytoplasm for six healthy persons and for 20 patients with chronic lymphatic leukemia. Besides ratios of compact, nucleolonemic and ring types of nucleoli were evaluated in addition to counts of the specific radioactivity of mature 28S rRNA in lymphocytes. In the majority of cases examined, cells with 1 or 2 nucleoli of compact and nucleolonemic types were seen dominating. The number of silver grains over the nucleoli in the control healthy persons did not differ from that in patients who did not receive any treatment, which contrasted with high value grain counts in the treated patients. The lymphocyte ribosome contents varied within the normal and decreased values in both the patient groups. The specific radioactivity in 28S rRNA leukemic lymphocytes was significantly lower in groups of patients with low ribosome contents than in those with the normal ones. The data suggest that in the leukemic cells with a high or unaltered activity of ribosome cistrons and low ribosome levels rRNA processing is broken.     

Localization of sulfated glycosaminoglycans within cell nuclei by high-resolution autoradiography

Skin fibroblasts cultured from patients with Sanfilippo's disease type B and Hurler's disease, preincubated with [³⁵S]sulfate, were subjected to high resolution autoradiography. A 4-day pulse followed by a 24 h chase resulted in a labelling of 61% of the cell nuclei by silver grains, thus indicating the existence of ethanol-insoluble [³⁵S]sulfate-containing material within the nuclei, especially within their chromatin-rich peripheral zone. A computer-calculated statistical evaluation of the autoradiographic results showed that the silver grains overlying the cell nuclei originated from [³⁵S]radioactivity within the nuclei and not from an overall background or cross fire effects of cytoplasmic radiation sources. Chemical analyses of chloroform/methanol extracts and pronase digests of the [³⁵S]labelled cells provided evidence that neither [³⁵S]sulfatides nor [³⁵S]glycopeptides contribute substantially to the cellular [³⁵S]radioactivity. The results strongly suggest the association of sulfated glycosaminoglycans with cell nuclei.