Cadmium stimulates prostaglandin E2 synthesis in osteoblast-like cells, MC3T3-E1

The effect of Cd on prostaglandin E2 production in osteoblasts was studied using cloned osteoblast-like cells, MC3T3-E1, which were established from new-born mouse calvaria. Treatment of the cells with Cd caused a dose- (0-10 microM) and time- (0-24 h) dependent increase in the release of prostaglandin E2 from the cells into the culture medium. A lag time of 4 h was required for the onset of the phenomenon. The release of [14C]arachidonic acid from prelabeled cell membrane was little influenced by the Cd treatment, while conversion of [14C]arachidonic acid to prostaglandin E2 by the homogenate of the cells treated with Cd was enhanced as compared to that by untreated cells. The stimulatory effect of Cd on prostaglandin E2 production was abolished in the presence of cycloheximide (100 ng/ml). By Western blot analysis with polyclonal rabbit anti-cyclooxygenase antibody, it was revealed that Cd treatment augmented the amount of immunoreactive cyclooxygenase in the cells. These results strongly suggest that Cd stimulates prostaglandin E2 production through the induction of cyclooxygenase in MC3T3-E1 cells. This effect of Cd may be involved in the mechanisms of Cd-induced bone injury.     

Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenases: the detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence.

Rabbit antiserum has been prepared against the prostaglandin endoperoxide-forming cyclooxygenase (EC 1.14.99.1) purified from sheep vesicular glands. Ouchterlony double diffusion and immunoelectrophoretic analyses indicate that the anti-cyclooxygenase serum is monospecific for the enzyme. The anti-cyclooxygenase serum reacts with both active and inactivated forms of the sheep vesicular gland (SVG) cyclooxygenase. Furthermore, the immune serum precipitates solubilized microsomal cyclooxygenases from each of six other tissues examined, including bovine seminal vesicle, rabbit kidney medulla, guinea pig lung, dog spleen, sheep uterus, and human platelets. Anti-SVG cyclooxygenase serum was used in combination with fluorescein isothiocyanate )FITC)-labeled goat anti-rabbit IgG to detect cyclooxygenases in cryostat sections from rat, rabbit and guinea pig kidneys by immunofluorescence. Highly prominent fluorescence was associated only with the epithelial cells lining the collecting ducts in rabbit and guinea pig kidneys, and except for the nucleus, was uniformly distributed within the interior of these cells. In rat kidney, fluorescence was detected not only in collecting tubules but also in the interstitial cells of the renal papilla. Our results are consistent with the emerging hypothesis that PGE2 produced intrarenally plays a physiological role in natriuresis.     

Signal transduction by tumor necrosis factor alpha is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3-E1.

Transmembrane signalling mechanisms of tumor necrosis factor alpha (TNF alpha) were examined with special reference to the involvement of G-protein, in intact and permeabilized murine osteoblast-like cells. TNF alpha stimulated the release of 3H radioactivity from intact cells labeled with [3H]arachidonic acid within 10 min in a dose dependent manner and the production of lyso forms of phospholipids, an event presumably mediated through the activation of phospholipase A2. Production of cAMP and inositol 1,4,5-trisphosphate was not affected by TNF alpha. Pretreatment of the cells with pertussis toxin inhibited the liberation of [3H]arachidonate. GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) reduced the binding affinity of [125I]TNF alpha to beta-escin-permeabilized cells. The addition of TNF alpha together with an unhydrolyzable analog of GTP, GTP gamma S, to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid led to a release of the 3H radioactivity. The production of prostaglandin E2 (PGE2) was markedly stimulated by TNF alpha in a dose over 100 ng/ml, with a latent time of about 3 h, and the stimulation was abolished by pretreatment with pertussis toxin. The time and dose requirements for this process differed from those for the possible activation of phospholipase A2, thereby indicating that other process(es) in addition to the activation of phospholipase A2 may be responsible for the enhanced production of PGE2. The activity of cyclooxygenase (i.e. the combined activities of prostaglandin endoperoxide syntase and PGH2-PGE2 isomerase) was stimulated by TNF alpha with much the same time and dose requirements as for the production of PGE2, and the activation was found to be due to the increased amount of the enzyme, as assessed by a Western blot analysis with anti-cyclooxygenase antibody. This process was also sensitive to pertussis toxin. Therefore, receptors for TNF alpha in MC3T3-E1 cells apparently couple to G-protein sensitive to pertussis toxin and the coupling regulates the activations of phospholipase A2 and the de novo synthesis of cyclooxygenase.     

Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum

Using fluorescent antibody staining, we have established the association of methionyl-tRNA synthetase with the endoplasmic reticulum in PtK2 cells. After Triton X-100 extraction, 70% of the recovered aminoacyl-tRNA synthetase activity was found in the detergent-insoluble fraction. This fraction of the enzyme remained localized with insoluble endoplasmic reticulum antigens and with ribosomes, which were stained with acridine orange. By both fluorescence microscopy and electron microscopy the organization of the detergent-insoluble residue was found to depend on the composition of the extracting solution. After extraction with a microtubule-stabilizing buffer containing EGTA, Triton X-100, and polyethylene glycol (Osburn, M., and K. Weber, 1977, Cell, 12:561-571) the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, sucrose, and CaCl2 (Fulton, A. B., K. M. Wang, and S. Penman, 1980, Cell, 20:849-857), the ribosomes were in small clusters and there were few morphologically recognizable membranes. In both cases the methionyl-tRNA synthetase and some endoplasmic reticulum antigens retained approximately their normal distribution in the cell. Double fluorochrome staining showed no morphological association of methionyl-tRNA synthetase with the microtubule, actin, or cytokeratin fiber systems of PtK2 cells. These observations demonstrate that detergent-insoluble cellular components, sometimes referred to as "cytoskeletal" preparations, contain significant amounts of nonfilamentous material including ribosomes, and membrane residue. Caution is required in speculating about intermolecular associations in such a complex cell fraction.     

Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenases: The detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence

Rabbit antiserum has been prepared against the prostaglandin endoperoxide-forming cyclooxygenase (EC 1.14.99.1) purified from sheep vesicular glands. Ouchterlony doùble diffusion and immunoelectrophoretic analyses indicate that the anti-cyclooxygenase serum is monospecific for the enzyme. The anti-cyclooxygenase serum reacts with both active and inactivated forms of the sheep vesicular gland (SVG) cyclooxygenase. Furthermore, the immune serum precipitates solubilized microsomal cyclooxygenase from each of six other tissues examined, including bovine seminal vesicles, rabbit kidney medulla, guinea pig lung, dog spleen, sheep uterus, and human platelets.Anti-SVG cyclooxygenase serum was used in combination with fluoresence isothiocyanate (FITC)-labelled goat anti-rabbit IgG to detect cyclooxygenase in cryostat sections from rat, rabbit and guinea pig kidneys by immunofluorescence. Highly prominent fluorescence was associated only with the epithelial cells lining the collecting ducts in rabbit and guinea pig kidneys, and except for the nucleus, was uniformly distributed within the interior of these cells. In rat kidney, fluorescence was detected not only in collecting tubules but also in the interstitial cells of the renal papilla. Our results are consistent with the emerging hypothesis that PGE₂ produced intrarenally plays a physiological role in natriuresis.     

Immunocytochemical localization of reserve protein in the endoplasmic reticulum of developing bean (Phaseolus vulgaris) cotyledons

The ultrastructure of the storage parenchyma cells of the cotyledons of developing bean (Phaseolus vulgaris L.) seeds was examined in ultrathin frozen sections of specimens fixed in a mixture of glutaraldehyde, formaldehyde and acrolein, infused with 1 M sucrose, and sectioned at-80° C. Ultrastructural preservation was excellent and the various subcellular organelles could readily be identified in sections which had been stained with uranyl acetate and embedded in Carbowax and methylcellulose. The cells contained large protein bodies, numerous long endoplasmic reticulum cisternae, mitochondria, dictyosomes, and electron-dense vesicles ranging in size from 0.2 to 1.0 m. Indirect immunolabelling using rabbit immunoglobulin G against purified phaseolin (7S reserve protein), and ferritin-conjugated goat immunoglobulin G against rabbit immunoglobulin G was used to localize phaseolin. With a concentration of 0.1 mg/ml of anti-phaseolin immunoglobin G, heavy labeling with ferritin particles was observed ober the protein bodies, the cisternae of the endoplasmic reticulum, and the vesicles. The same structures were lightly labeled when the concentration of the primary antigen was 0.02 mg/ml. Ferritin particles were also found over the Golgi bodies. The absence of ferritin particles from other organelles such as mitochondria and from areas of cytoplasm devoid of organelles indicated the specificity of the staining, especially at the lower concentration of anti-phaseolin immunoglobulin G.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G     

Does diaminobenzidine demonstrate prostaglandin synthetase? A study on polyunsaturated fatty acid-induced DAB oxidation in sheep vesicular glands and rabbit kidney medulla.

A method histochemical localization of prostaglandin synthetase using DAB, potassium cyanide and polyunsaturated fatty acid has been revised. The arachidonic acid-induced DAB oxidation observed in the secretory epithelium of sheep vesicular glands and in collecting tubules as well as intersititial cells of rabbit kidney medulla was found to be insensitive to antiinflammatory cyclooxygenase (formerly referred as prostaglandin synthetase) inhibitors, such as indomethacin, aspirin, mefenamic acid and paracetamol, whereas aminotriazole caused complete inhibition of the reaction. Furthermore, DAB was oxidized in the presence of polyunsaturated fatty acids inconvertible to prostaglandins (linoleic and linolenic acid) as well as in the presence of H2O2--in the latter case reaction possessed identical features with that induced by fatty acids. Ultrastructurally, the reaction product was localized on the membranes of nuclear envelope and endoplasmic reticulum. On the ground of the results obtained a hypothesis is presented, that the polyunsaturated fatty acid-induced DAB oxidation is due to a peroxidatic activity of the investigated tissues. Possible relations between such peroxidatic activity and prostaglandin biosynthesis are discussed.     

Light and electron microscope immunocytochemical localization of 5- and 12-lipoxygenases and cyclooxygenase enzymes in human granulosa cells from preovulatory follicles

Cellular and subcellular distribution of 5- and 12-lipoxygenases and cyclooxygenase enzymes were investigated in human granulosa cells from preovulatory follicles using light and electron microscope immunocytochemistry. The results demonstrated that all three enzymes are present in granulosa cells but not in minor contaminating red blood cells. While the distribution of cyclooxygenase and 12-lipoxygenase was relatively uniform among the granulosa cells, 5-lipoxygenase was not uniformly distributed among these cells. All three enzymes are present in microvillus plasma membranes, rough endoplasmic reticulum, cytoplasm, nuclear membranes and chromatin. In summary, 5- and 12-lipoxygenases and cyclooxygenase enzymes, which catalyze the transformation of arachidonic acid into different eicosanoids, are present in several subcellular organelles including nuclei of granulosa cells from preovulatory follicles.     

Does diaminobenzidine demonstrate prostaglandin synthetase?

Summary A method histochemical localization of prostaglandin synthetase using DAB, potassium cyanide and polyunsaturated fatty acid has been revised. The arachidonic acid-induced DAB oxidation observed in the secretory epithelium of sheep vesicular glands and in collecting tubules as well as interstitial cells of rabbit kidney medulla was found to be insensitive to antiinflammatory cyclooxygenase (formerly referred as prostaglandin synthetase) inhibitors, such as indomethacin, aspirin, mefenamic acid and paracetamol, whereas aminotriazole caused complete inhibition of the reaction. Furthermore, DAB was oxidized in the presence of polyunsaturated fatty acids inconvertible to prostaglandins (linoleic and linolenic acid) as well as in the presence of H₂O₂ — in the latter case reaction possessed identical features with that induced by fatty acids. Ultrastructurally, the reaction product was localized on the membranes of nuclear envelope and endoplasmic reticulum. On the ground of the results obtained a hypothesis is presented, that the polyunsaturated fatty acid-induced DAB oxidation is due to a peroxidatic activity of the investigated tissues. Possible relations between such peroxidatic activity and prostaglandin biosynthesis are discussed.     

Detection of plasma cell immunoglobulins in tissue sections optimally fixed for ultrastructural immunocytochemistry

We describe the ultrastructural localization of plasma cell immunoglobulins in vibratome sections of popliteal lymph nodes. Fixation with glutaraldehyde-paraformaldehyde gave better tissue and antigen preservation than paraformaldehyde or periodic acid lysine-paraformaldehyde; biotinylated Fab fragments of sheep anti-mouse IgG-streptavidin-biotinylated horseradish peroxidase (HRP) or Fab-HRP conjugates gave similar results. With both immunoreagents, excellent tissue preservation and antigen detection was observed in the first layer of cells sectioned with the vibratome. Conjugates of anti-mouse IgG with HRP did not show any staining. Peroxidase stain was observed in the nuclear envelope, cisternae of the rough endoplasmic reticulum, and the Golgi apparatus complex. In the Golgi apparatus, staining was seen consistently in cisternae of the cis face and in adjacent vesicles; the trans cisternae showed weak or no stain, and adjacent vesicles, "coated" vesicles, and granules were not stained. This study shows that high quality of tissue preservation and antigen detection, by both light and ultrastructural immunocytochemistry, is feasible in tissue fixed with glutaraldehyde-paraformaldehyde followed by vibratome sectioning and immunostaining with Fab-biotin-streptavidin-biotin-HRP, or Fab-HRP.     

Induction of cyclooxygenase-1 in a human megakaryoblastic cell line (CMK) differentiated by phorbol ester

Human megakaryoblastic cells (CMK line) are known to differentiate to mature megakaryocyte-like cells by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). There are two isozymes of prostaglandin-forming cyclooxygenase enzyme. Constitutive cyclooxygenase-1 and inducible cyclooxygenase-2 were followed during differentiation of CMK cells. Treatment of the cells with 0.1 μM TPA for 4 days resulted in a 5–20-fold increase in cyclooxygenase activity. Northern and Western blot analyses revealed that cyclooxygenase-1 mRNA and protein increased in parallel with the enzyme activity. In contrast, cyclooxygenase-2 mRNA was detected only at 3 h. Furthermore, most of the increased cyclooxygenase activity was immunoprecipitated with anti-cyclooxygenase-1 antibody, and was not affected by a cyclooxygenase-2-specific inhibitor, NS-398. These results indicated that cyclooxygenase-1 rather than cyclooxygenase-2 was predominantly induced depending on TPA. The enzyme thus induced was localized by immunoelectron microscopy in nuclear envelope and endoplasmic reticulum of the CMK cells.     

cAMP-dependent induction of fatty acid cyclooxygenase mRNA in mouse osteoblastic cells (MC3T3-E1).

In an osteoblastic cell line, MC3T3-E1, cloned from mouse calvaria, epinephrine stimulated the production of prostaglandin E2 as an essentially sole arachidonate metabolite (Kusaka, M., Oshima, T., Yokota, K., Yamamoto, S., and Kumegawa, M. (1988) Biochim. Biophys. Acta. 972, 339-346). Western and Northern blot analyses showed increases in the enzyme protein and mRNA of fatty acid cyclooxygenase in the epinephrine-treated cells. A rapid cAMP production caused by epinephrine was followed by increases in the activity and mRNA of cyclooxygenase. Both dibutyryl cAMP and 8-bromo-cAMP also increased the level of the cyclooxygenase activity and mRNA. These results suggest that cAMP produced by beta-adrenergic stimulation was responsible for the increased cyclooxygenase mRNA level leading to induction of the cyclooxygenase enzyme. Furthermore, the addition of prostaglandin E2 (the final arachidonate metabolite in the MC3T3-E1 cells) brought about a rapid synthesis of intracellular cAMP followed by increases in the enzyme protein and mRNA of cyclooxygenase.     

Subcellular localization of prostaglandin H synthase-2 in a human amnion cell line: implications for nuclear localized prostaglandin signaling pathways

We have determined that prostaglandin H synthase-2 localises strongly to the nuclear membrane as well as being found in the endoplasmic reticulum in human amnion-derived WISH cells which have been stimulated with interleukin 1beta and phorbol ester. This is consistent with findings in cells of non-reproductive origin. There is strong evidence that prostaglandin J2 derivatives, which in other tissues exhibit tumour suppressing, antiproliferative and/or differentiation promoting activities, act through binding of intracellular receptors which then enter the nucleus. In addition, some arachidonic acid derivatives are clearly generated by enzymes at the nuclear envelope and localise to sites in nuclei or bind sites in nuclei. The WISH cell line will make an excellent system for studying these perinuclear intracellular prostanoid signaling mechanisms.     

Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach

Summary A method involving rapid freezing followed by substitution fixation was developed, using acrolein as a fixative. This was then applied to several cytochemical stainings, and showed well preserved and clear cell structures. Membranes were apparently negatively stained and the ultrastructure of mitochondria, rough endoplasmic reticulum and Golgi apparatus was clearly discernible. The mitochondrial and cytoplasmic matrices were stained rather densely compared with routine chemically fixed preparations, implying a good preservation of matrix substrances. Periodic acid-thiocarbohydrazide-silver proteinate staining was applied to the present method. The mucous granules of surface covering epithelial cells indicated fine staining of bipartite structure and the Golgi apparatus of mucous cells showed clear staining differences based on polarity. Postembedding lectin-ferritin and immunocytochemical stainings were applicable to the present preparations and stable stainings of secretory granules were obtained. A low temperature embedding material, Lowicryl K4M, was also examined. The cell preservation of these samples was not as good as those embedded in Epon, but the rough endoplasmic reticulum and Golgi apparatus of chief cells were stained with anti-pepsinogen antibody as were the secretory granules. The present method was also applicable to light microscopy.     

Fine-Structure Locations of α-Glucan Phosphorylase, As Shown by Lead Precipitation and Electron Microscopy

Sites of glucan phosphorylase activity in fine structures, as shown by the lead precipitation method (Hori, Stain Techn., 39: 275, 1964) were studied by electron microscopy. Rat livers were fixed 2 hr at 0 C in buffered 2.5% glutaraldehyde, frozen-sections cut and incubated in the medium containing glucose-1-phosphate, 2.7 mM; NaF, 20 mM; acetate buffer, pH 5.8, 80 mM; Pb(NO₃)₂, 4.2 mM; and sucrose, 0.44 M; refixed in buffered 1% OsO₄, dehydrated and embedded in Epon 812 as usual. The reaction product was found in close association with endoplasmic reticulum, but not in mitochondria, nuclear membrane and the cisternae of endoplasmic reticulum. The possibility of demonstrating by the present method the indirect hydrolysis of glucose-1-phosphate through the phosphoglucomutase-glucose-6-phosphatase system was ruled out by inhibiting glucose-6-phosphatase with fluoride and ethanol.     

PEROXISOMES IN INNER ADRENOCORTICAL CELLS OF FETAL AND ADULT GUINEA PIGS

Abundant membrane-bounded granules, 0.1–0.45 µm in diameter, occur among the elements of the smooth-surfaced endoplasmic reticulum in zona fasciculata and zona reticularis adrenocortical cells of guinea pigs. Acid phosphatase cannot be cytochemically demonstrated in them, and they are therefore distinct from lysosomes. Incubation in medium containing 3,3'-diaminobenzidine results in dense staining of the granules, identifying them as peroxisomes. These small peroxisomes increase in number as fetal adrenocortical cells differentiate, and they appear to arise from dilated regions of endoplasmic reticulum. They maintain interconnections with the smooth endoplasmic reticulum and with one another.     

Penetration of Toxoplasma gondii into host cells induces changes in the distribution of the mitochondria and the endoplasmic reticulum.

Fluorescence microscopy, using dyes which specifically label mitochondria, endoplasmic reticulum and the Golgi complex, and transmission electron microscopy, were used to analyze the changes which occur in the organization of these structures during interaction of Toxoplasma gondii with host cells. In uninfected cells the mitochondria are long filamentous structures which radiate from the nuclear region toward the cell periphery. After parasite penetration they become shorter and tend to concentrate around the parasite-containing vacuole (parasitophorous vacuole) located in the cytoplasm of the host cell. The mitochondria of extracellular parasites, but not of those located within the parasitophorous vacuole, were also stained by rhodamine 123. Labeling with DiOC6, which binds to elements of the endoplasmic reticulum, in association with transmission electron microscopy, revealed a concentration of this structure around the parasitophorous vacuole. The membrane lining this vacuole was also stained, suggesting that components of the endoplasmic reticulum are also incorporated into this membrane. The Golgi complex, as revealed by staining with NBD-ceramide and electron microscopy, maintains its perinuclear position throughout the evolution of the intracellular parasitism.     

Silver Staining of Ultrathin Sections of Tissues Embedded in Polyester Resin

Specimens 1 mm³ from rat liver and kidney were fixed for 50 min in cold (0-2° C) 1% OsO₄ in veronal-acetate buffer, pH 7.7, and containing 0.1% MgCl₂; then dehydrated and embedded in Vestopal-W. Sections were cut in two ranges, 0.1-2 µ and 60-90 mµ thick, and attached to slides by floating on water and drying at 60° C. The thicker ones, for light microscopy, were soaked in acetone 1.5-3 hr; the thinner, for electron microscopy, 20-30 min. Both kinds were stained by Wilder's (1935) method for reticulum. Those for light microscopy were finished by dehydrating, clearing and covering in the customary manner; those for electron microscopy, by coating with 1% parlodion, drying, cutting the film about 2 mm² around the section, and freeing the section by soaking in water. The section was then mounted on a grid. The structures stained are: nuclei, basement membrane of capillaries, reticulum fibers of the liver and kidney, and in addition, the basement membrane of the kidney tubules. The mitochondria, vesicles, endoplasmic reticulum and cell membranes were not defined.