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Correspondence to: Kunio Fujiwara, Faculty of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8131, Japan..
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Summary |
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The polyamines (PAs) are ubiquitous polycationic metabolites in eukaryotic and prokaryotic cells and are believed to be intimately involved in the regulation of DNA, RNA, and protein biosynthesis, the exact function of which remains unclear, mainly because of a lack of knowledge of PA subcellular localization. In this study, using immunoelectron microscopy, we have demonstrated that PAs are predominantly located on free and attached ribosomes of the rough endoplasmic reticulum in the neurons of the lateral reticular nucleus of rat medulla oblongata. The nuclei, axons, and nerve endings were devoid of PA. This suggests that PAs are one of the components of biologically active ribosomes, being closely involved in the translation processes of protein biosynthesis. (J Histochem Cytochem 46:13211328, 1998)
Key Words: polyamines, spermine, spermidine, immunoelectron microscopy, monoclonal antibody, neuron, ribosome, protein biosynthesis, ribosomal subunit association
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Introduction |
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Virtually all organisms contain the polyamines (PAs) spermidine (Spd) and spermine (Spm) and their precursor putrescine (
The present study demonstrated that PAs are predominantly located in the ribosomes in the neurons of the central nervous system of rats. However, the possibility that PAs are located in sites other than the ribosomes in cells could not be excluded, because it is known that PAs have effects on membrane proteins such as ion channels and receptors (for reviews see
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Materials and Methods |
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Antibody
The monoclonal antibody (MAb) ASPM-29 (IgG1 subisotype) was produced against spermine conjugated to human serum albumin (HSA) using glutaraldehyde (GA) and sodium borohydride (-aminobutyric acid, or histamine (
Animals
Normal adult male Wistar rats (Otsubo Experimental Animals; Nagasaki, Japan), body weight 200250 g, were used in this study. They were housed in temperature- and light-controlled rooms (21 ± 1C and 12L:12D) and had free access to standard food and tapwater.
Immunocytochemistry (ICC)
Under sodium pentobarbital (Abbott Laboratories; North Chicago, IL) (60 mg/kg) anesthesia, 10 rats were perfused intracardially with PBS at 50 ml/min for 2 min at room temperature (RT), and then with a freshly prepared solution of 2.5% GA in 10 mM phosphate buffer at pH 7.2 for 6 min. Brains were quickly excised and immersed overnight at 4C in the same fixative. They were cut into 50-µm-thick sections with a Microslicer (Dosaka EM; Kyoto, Japan) through all levels of the lower parts of the medulla oblongata. The sections were treated with 0.2% NaBH4 for 10 min and incubated at 4C for 48 hr with primary MAb ASPM-29 at concentrations ranging from 10 to 50 ng/ml of 50 mM Tris-HCl buffer, pH 7.4, containing 0.86% NaCl (TBS) [the concentration of which was determined by the conventional "sandwich ELISA" using chromatographically purified mouse IgG (Zymed; San Francisco, CA) as a standard]. Sections were then incubated with a goat anti-mouse IgG/Fab' labeled with horseradish peroxidase (HRP) (MBL; Nagoya, Japan) 1:200 for 12 hr at 4C. After rinsing with TBS, the HRP was revealed for 510 min with diaminobenzidine and H2O2. The HRP substrate consisted of 10 mg of 3,3'-diaminobenzidine tetrachloride (Sigma; St Louis, MO) dissolved in 20 ml of 50 mM Tris buffer at pH 7.4 supplemented with 20 µl of 30% H2O2. The samples were rinsed first with Tris-HCl buffer, pH 7.4, and then with cacodylate buffer, pH 7.4, for 30 min at RT.
Electron Microscopy
The color-developed specimens prepared as above were then fixed with 1.0% osmium tetraoxide in 50 mM cacodylate buffer, pH 7.4, for 1 hr at RT and dehydrated in a series of graded ethanol solutions. After immersion in propylene oxide (Nacalai Tesque; Kyoto, Japan) (three times for 10 min each), the samples were immersed in a mixture (1:1) of propylene oxide and Epon 812 resin (Taab Laboratories; Reading, Berks, UK) overnight and embedded in Epon 812 resin in a routine way. The regions to be studied were cut with a 2-mm diameter punch, mounted on Epon blocks, and sectioned on a horizontal plane into ultrathin sections, which were then immediately observed in a 100 CX Jeol electron microscope.
For quantification, images of electron microscopic photographs (both sample and control specimens) taken under constant conditions were digitized into a computer through a video camera head (ITC-370M; OlympusIkegami, Tokyo, Japan) according to the method of
Control Experiments
In the PA immunocytochemistry study of the rat brain, the specificity of immunostaining was ascertained by incubating control sections with (a) the secondary antiserum alone, (b) type-matched MAb (IgG1) anti-penicillin (3080 ng/ml; Cosmo Biological, Tokyo, Japan), and (c) ASPM-29 MAb preabsorbed with Spm at a concentration of 50 µg/ml.
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Results |
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The polyamine immunoelectron microscopic study was developed with the pre-embedding method using 50-µm Microslicer sections of the lower parts of the medulla oblongata. In the present PA immunocytochemistry using 2.5% GA as a fixative no proteolytic pretreatment of the sections was needed before the PA immunoreaction, in contrast to our previous studies using tissues other than central neural tissues (
By light microscopy PA immunoreactivity was seen in the cytoplasm and dendrites of all the nerve cells in the lower part of the medulla oblongata at all levels, whereas the nuclei and axons were devoid of staining. Strong PA immunostaining was noticed in the large nerve cells of the hypoglossal nucleus, nucleus ambiguus, accessorial nucleus, and dorsal motor nucleus of the vagus, and in the reticular nuclei including the gigantocellular reticular nucleus, lateral paragigantocellular nucleus, lateral reticular nucleus, and medullary reticular nucleus (Figure 1A). The patterns of PA immunostaining were either clustered masses or block-like (Figure 1B). Immunoreactivity was seen to a lesser degree in the small neurons of the inferior olive nuclei, in the nucleus of the solitary tract, in the gracile nucleus, and in the spinal trigeminal nucleus (Figure 1A). Very weak staining or no staining at all was observed in the glial cells and nerve fibers passing through the white matter and entering into the gray matter. No immunoreaction occurred with the ASPM-29 MAb preabsorbed with Spm (Figure 1C), with anti-penicillin MAb, or with the secondary antiserum alone.
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Immunoelectron microscopic examination of the neurons in the lateral reticular nucleus showed that PA was unevenly distributed in the cytoplasm and dendrites of the neurons. Variations in the amount of deposit were found from section to section and even within the same ultrathin section. The PA immunoreactivity was specifically and intensely located in the ribosomes attached to the membranes (rER), in the free ribosomes (polysomes) clustered between cisternae, and in the polysomes widely spread throughout the entire cytoplasm (Figure 2A and Figure 2B). In addition, cell organelles at the terminal and in the middle of short dendrites of the neurons were all strongly labeled (Figure 3B and Figure 3C). The nucleus, nucleolus, Golgi cisternae, Golgi vesicles, lysosomes, and mitochondria in the cytoplasm of the perikaryon were devoid of PA staining (Figure 2A and Figure 2B). Among the glial cell types, the oligodendrocytes were immunoreactive with the ASPM-29 MAb, staining the ribosomes (polysomes) in the cytoplasm (Figure 5A). The axons, both myelinated and unmyelinated, and the synaptic vesicles in the nerve endings around the neurons were completely PA-negative, suggesting that PAs are not axonally transported. The sections stained with the preabsorbed ASPM-29 MAb or the anti-penicillin MAb did not show any PA immunoreaction (Figure 3A and Figure 5B). Electron microscopic photographs of sample and control specimens of subcellular organelles in a neuron (Figure 2A and Figure 3A) were quantitatively analyzed by a computer and revealed that the rER was significantly labeled, in contrast to the nucleolus and nucleus (Figure 4).
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Discussion |
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Since the discovery of Spm phosphate complexes by Leeuwenhoek in 1678, the subcellular localization of PAs has not been previously elucidated.
The present immunoelectron microscopic findings that PAs are predominantly located on free (polysomes) and attached ribosomes of the rough endoplasmic reticulum (Nissl bodies) in the cytoplasm, but not in the nuclei, axons, and nerve endings of neurons of the central nervous system, suggests that PAs are closely involved in the translation processes of protein biosynthesis. PAs may be located close to the sites rich in proteins within the ribosomes because, in our previous study of polyamine immunocytochemistry, protease treatment was indispensable before the first immunoreaction (
The present light microscopic findings in the medulla oblongata, i.e., that the large neurons with efferent fibers and in the reticular nuclei possessed high amounts of PAs but the small neurons possessed low amounts of PAs (Figure 1A), appear to be in agreement with the commonly accepted idea that Nissl bodies (rER) are larger and more abundant in large neurons than in small ones. The patterns of PA immunostaining were either clustered masses or block-like, just like those of the Nissl bodies (Figure 1B).
Many reports, using extracts derived from a number of cell types, indicate that, in the cell-free systems PAs stimulate a variety of processes of protein biosynthesis, such as stabilization and activation of tRNA (
Because the present immunoelectron microscopic findings point out a coincidence between ribosomal subunit association in the maturation processes of the ribosomes and the presence of PAs, further polyamine immunocytochemistry and biochemistry are needed to determine whether or not PAs play a role in maturation processes (
We had previously identified the light microscopic localization of PAs and had found that the PAs are abundant in the cytoplasm of neoplastic cells, active protein- or peptide-secreting cells, including exocrine and endocrine cell types, central and peripheral nerve cells, and rapidly proliferative cells in the gastrointestinal epithelia (
Because many recent reports have also shown that PAs have effects on membrane proteins such as ion channels and receptors (
The present study suggests that PAs are one of the components of biologically active ribosomes in neurons, thus closely associating with protein biosynthesis. This may indicate an important role of PAs in cell proliferation and growth (
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Acknowledgments |
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We are very grateful to Dr M. Shin for the computer-assisted image analysis, Mr T. Suematsu for technical assistance with electron microscopy, and Dr Y. Inoue for valuable discussions throughout this study.
Received for publication March 27, 1998; accepted July 14, 1998.
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