Contents

Original articles:

1. The Pulmonary Neuroendocrine System: The Past Decade(A. VAN LOMMEL)
   
2. Collagen-Phagocytosing Ability of Periodontal Osteoblasts at the Bone Surface(Toshihiko YAJIMA)
   
3. Distribution of Gap Junction Protein Connexin 37 in Smooth Muscle Cells of the Rat Trachea and Pulmonary Artery(Kyoko NAKAMURA)
   
4. Histology of the Kidney and Urinary Bladder of Siphonops annulatus(Amphibia-Gymnophiona)(Elcinéa T. C.)
   
5. Imaging of Living Cultured Cells of an Epithelial Nature by Atomic Force Microscopy(Tatsuo USHIKI)
   
6. Postnatal Expression of Calretinin-Immunoreactivity in Periodontal Ruffini Endings in the Rat Incisor: A Comparison with Protein Gene Product 9.5 (PGP 9.5) Immunoreactivity(Toshikazu ASAHITO)
   
7. Perineuronal Nets of proteoglycans in the Adult Mouse Brain, with Special Reference to Their Reactions to Gömöri's Ammoniacal Silver and Ehrlich's Methylene Blue(Takuro MURAKAMI)
   
8. Roles of a Macrophage Receptor with Collagenous Structure (MARCO) in Host Defense and Heterogeneity of Splenic Marginal Zone Macrophages(Shigeo ITO)
   
9. The Existence of Merkel Cells in the Lingual Connective Tissue of the Surinam Caiman, Caiman crocodilus crocodilus (Order Crocodilia)(Sumio YOSHIE)
   

Summary

Title
The Pulmonary Neuroendocrine System: The Past Decade

Author
A. VAN LOMMEL, T. BOLLÉ, W. FANNES and J. M. LAUWERYNS

Address
Laboratory of Pathological Anatomy, Medical Faculty, Katholieke Universiteit te Leuven, Leuven, Belgium

Summary
The pulmonary neuroendocrine system consists of specialized airway endocrine epithelial cells, associated with nerve fibres. The epithelial cells, the pulmonary neuroendocrine cells (PNEC), can be solitary or clustered to form neuroepithelial bodies (NEB). During the last thirty years, the pulmonary neuroendocrine system has been intensively investigated and much knowledge of its function has been obtained. This text reviews work which dates from the last ten years. In this period, the picture of the pulmonary neuroendocrine system we previously had, has not fundamentally changed. The pulmonary neuroendocrine system is still regarded as an oxygen sensitive chemoreceptor with local and reflex-mediated regulatory functions, and as a regulator of airway growth and development. Continuing research has much more refined this picture. This text reviews several aspects of the pulmonary neuroendocrine system: phylogeny, the amine and peptide content of its epithelial cells, ontogeny and influence on lung development, the influence of hypoxia and nonhypoxic stimuli, immunomodulatory function, innervation and pathology. Among the discoveries of the past decade, three stand out prominently because of their great significance: additional proof that the neural component of the pulmonary neuroendocrine system is sensory, sound experimental evidence that PNEC stimulate airway epithelial cell differentiation and the discovery of a specific membrane oxygen receptor in the PNEC.

Prof. A. VAN LOMMEL
Laboratory of Pathological Anatomy
Medical Faculty
Katholieke Universiteit te Leuven
Minderbroedersstraat 12
3000 Leuven, Belgium
Tel: 32 16 33 66 04
Fax: 32 16 33 66 24
E-mail: alfons.vanLommel@uz.kuleuven.ac.be

Title
Collagen-Phagocytosing Ability of Periodontal Osteoblasts at the Bone Surface

Author
Toshihiko YAJIMA, Yasunori SAKAKURA, Eichi TSURUGA, Toshihiro HIRAI, Yasuhiro IKEDA, Shigehito FUJII and Noriyuki SHIDE

Address
Departments of Oral Anatomy and Removable Prosthodontics, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, Japan

Summary
The collagen-phagocytosing activity of osteoblasts at the alveolar bone-ligament interface of rat mandibular first molars was investigated both histologically and histochemically. Alveolar bones of male Wistar rats (6 months old) were used in this study. Collagen-containing phagosomes appeared in cuboidal osteoblasts aligned on the bone surface. The 5.7% of the osteoblasts exhibiting alkaline phosphatase activity revealed collagen-containing phagosomes, and the collagen fibrils within the phagosomes were at various stages of degradation. In addition, acid phosphatase activity and the immunocytochemical distribution of cathepsin B were found in these collagen-containing phagosomes at similar locations. The presence of both enzymes in the phagosomes suggests that an intracellular degradation of collagen occurs. Therefore, in addition to the osteoblastic functions of synthesizing and secreting bone matrices, osteoblasts are also capable of phagocytosis and the intracellular disintegration of collagen. Our findings suggest that osteoblasts at the alveolar bone-periodontal ligament interface have a collagen-phagocytosing ability and play an important role in the physiological remodeling and metabolic breakdown of collagen fibrils of periodontal ligament without ostoclastic bone remodeling.

Prof. Toshihiko YAJIMA
Department of Oral Anatomy
School of Dentistry
Health Sciences University of Hokkaido 1757 Kanazawa, Ishikari-Tobetsu
Hokkaido, 061-0293 Japan
Tel: +81-1332-3-1211
Fax: +81-1332-3-1218
E-mail: tyajima@hoku-iryo-u.ac.jp

Title
Distribution of Gap Junction Protein Connexin 37 in Smooth Muscle Cells of the Rat Trachea and Pulmonary Artery

Author
Kyoko NAKAMURA, Tetsuichiro INAI, Keiichiro NAKAMURA and Yosaburo SHIBATA

Address
Department of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka, Japan

Summary
Connexin 37, one of the gap junction protein families, has been detected by Northern blotting in various organs and tissues, and found to be especially abundant in the lung. However, detailed information on the precise types of cells which express connexin 37 has not been previously published. We therefore prepared site-specific connexin 37 antibodies and examined the distribution of connexin 37 immunohistochemically. Connexin 37 was detected in endothelial cells, in the tunica media of both the pulmonary artery and the aorta, and in the smooth muscle layer of the trachea and bronchioles. In the tracheal smooth muscle layer, connexin 37 overlapped with desmin-positive areas, but was clearly segregated from vimentin- and von Willebrand factor-positive areas. These results suggest that connexin37 is expressed in smooth muscle cells in the trachea, but not in fibroblastic cells or endothelial cells. Connexin 37 was partially colocalized with connexin 43 in tracheal smooth muscle cells, and showed a gradual increase in expression during postnatal development. To our knowledge, this is the first report to be published regarding the expression of connexin 37 in smooth muscle cells.

Dr. Kyoko NAKAMURA
Department of Anatomy
Faculty of Medicine
Kyushu University
Fukuoka
812-8582 Japan
Tel: +81-92-642-6052
Fax: +81-92-642-6202
E-mail : knaka@ana2.med.kyushu-u.ac.jp

Title
Histology of the Kidney and Urinary Bladder of Siphonops annulatus (Amphibia-Gymnophiona)

Author
Elcinéa T. C. CARVALH0 and L. C. U. JUNQUEIRA

Address
Department of Biological Sciences, School of Pharmacy and Odontology of Alfenas, Minas Gerais ; and Cell Biology Laboratory, Department of Pathology, Medical School, University of São Paulo, Brazil

Summary
The histology of the kidney and urinary bladder of Siphonops annulatus was studied by light microscopy in semithin sections of tissue embedded in hydrophilic resin. The kidney's nephron comprises the renal corpuscle, neck segment, proximal tubule, intermediate segment, distal tubule and collecting tubule. Nephrostomes are present. This structure, the neck segment, and intermediate tubules present long cilia, and probably play important roles in the propulsion of the peritoneal fluid and glomerular filtrate. The proximal tubule cells possess loosely packed microvilli and contain abundant polymorphic granules and vesicles that assume the aspect of lysosomes in different stages of intracellular digestion. The distal tubules are characterized by large, vertically disposed mitochondria assuming the aspect of ions transporting cells. The urinary bladder is lined with a transitional epithelium, whose aspect varies according to the quantity of urine.

Prof. L. C. U. JUNQUEIRA Santiago, 99
Poç)os de Caldas-MG
37701-373 Brazil
Tel: +55-035-7142984
Fax: +55-035-7142951

Title
Imaging of Living Cultured Cells of an Epithelial Nature by Atomic Force Microscopy

Author
Tatsuo USHIKI, Jiro HIT0MI, Takeshi UMEMOT0, Susumu YAMAMOTO, Hiroaki KANAZAWA and Masatsugu SHIGENO

Address
Department of Anatomy, Niigata University School of Medicine, Niigata; and Takatsuka Unit, Seiko Instruments Inc., Matsudo, Chiba, Japan

Summary
The present paper describes the applicability of atomic force microscopy (AFM) to the observation of living cultured cells of an epithelial nature (human esophageal squamous cell carcinoma cells, or C7 subclone of KESC2 cells) in a culture medium. For this purpose, we made a fluid chamber system which allows a constant-speed perfusion of fluid at a regulated temperature in the chamber. Using this system, AFM images of living cells were successfully obtained for over one hour at time intervals of 2-4 min during continuous perfusion of the fresh culture medium. A series of these AFM images proved useful for examining the movements of cellular processes in relation to subcellular cytoskeletal elements. Time-lapse movie records produced by sequential AFM images further verify the reality of the cellular dynamics.

Prof. Tatsuo USHIKI
Department of Anatomy
Niigata University School of Medicine 1, Asahimachi-dori, Niigata
951-8510 Japan
Tel: +81-25-227-2058
Fax: +81-25-224-1767
E-mail : t-ushiki@med.niigata-u.ac.jp

Title
Postnatal Expression of Calretinin-Immunoreactivity in Periodontal Ruffini Endings in the Rat Incisor: A Comparison with Protein Gene Product 9.5 (PGP 9.5) Immunoreactivity

Author
Toshikazu ASAHITO, Hayato OHSHIMA, Kooji HANADA, Satoshi WAKISAKA and Takeyasu MAEDA

Address
Departments of Oral Anatomy and Othodontics Niigata University School of Dentistry, Niigata; and Department of Oral Anatomy and Developmental Biology, Osaka University Faculty of Dentistry, Suita, Japan

Summary
The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis, At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized. Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP9.5-immunohistochemistry, while calretinin.immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered oft without forming the Ruffini type endings. A small number of Ruffini endings showing calretinin. immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction, The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction.

Prof. Takeyasu MAEDA
Department of Oral Anatomy
Niigata University School of Dentistry
2-5274, Gakkocho-dori, Niigata
951-8514 Japan
Tel: +81-25-227-2815
Fax: +81-25-223-6499
E-mail: maedat@dent.niigata-u.ac.jp

Title
Perineuronal Nets of proteoglycans in the Adult Mouse Brain, with Special Reference to Their Reactions to Gömöri's Ammoniacal Silver and Ehrlich's Methylene Blue

Author
Takuro MURAKAMI, Tetsuro MURAKAMI, Hiroyuki SATO, Wafaa Alaa El-din MUBARAK, Aiji OHTSUKA and Koji ABE

Address
Departments of Anatomy and Neurology, Okayama University Medical School, Okayama, Japan; and Department of Anatomy, Faculty of Medicine, Uiversity of Assiut, Assiut, Egypt

Summary
As our previous studies have indicated, mainy subsets of neurons in the vertebrate brain possess a sulfated proteoglycan surface coat which reacts to cationic iron colloid and aldehyde fuchsin, The present study demonstrated that this surface coat is supravitally stained with Ehrlich's methylene blue, and doubly with this blue and aldehyde fuchsin, a finding suggesting its being identical to Cajal's superficial reticulum (red superficial) and to Golgi's reticular coating (revètement réticulare).

The perineuronal surface coat was further stained with Gömöri's ammoniacal silver, and doubly with this silver and cationic iron colloid. These neurons with such a proteoglycan surface coat usually expressed cell surface glycoproteins which were labeled with lectin Wisteria floribunda agglutinin. Hyaluronidase digestion did not interfere with this lectin labeling of the glycoproteins, methylene blue and Gömöri's ammoniacal silver staining of the surface coat, while it erased the cationic iron colloid and aldehyde fuchsin staining of the surface coat.

These findings suggest that the perineuronal proteoglycan surface coat is associated with some additional molecules which are resistant to hyaluronidase digestion and stainable with methylene blue and Gömöri's ammoniacal silver. The possibility is suggested that these molecules might represent "ligand proteoglycans" connecting the perineuronal proteoglycans and cell surface glycoproteins.

Prof. Takuro MURAKAMI
Department of Anatomy
Faculty of Medicine
Okayama University Medical School 2-5-1 Shikata-cho, Okayama
700-8558 Japan
Tel: +81-86-235-7088
Fax: +81-86-235-7095
E-mail: em2kai@med.okayama-u.ac.jp

Title
Roles of a Macrophage Receptor with Collagenous Structure (MARCO) in Host Defense and Heterogeneity of Splenic Marginal Zone Macrophages

Author
Shigeo ITO, Makoto NAITO, Yoshiaki KOBAYASHI, Hisakazu TAKATSUKA, Shuying JIANG, Hiroyuki USUDA, Hajime UMEZU, Go HASEGAWA, Masaaki ARAKAWA, Leonard D. SHULTZ, Outi ELOMAA and Karl TRYGGVASON

Address
Second Departments of Pathology and of Internal Medicine, Niigata University School of Medicine, Niigata, Japan; The Jackson Laboratory Bar Harbor, ME, USA; and Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Sweden

Summary
Class A type I and type II macrophage scavenger receptors (MSR-A) and a macrophage receptor with collagenous structure (MARCO) are trimeric membrane glycoproteins mediating the uptake of chemically modified low density lipoproteins. MSR-A is expressed constitutively in several tissue macrophages and in liver sinusoidal endothelial cells, whereas MARCO is expressed constitutively in splenic marginal zone macrophages and in macrophages and endothelial cells in the lymphatic medullary sinuses of lymph nodes. The administration of LPS, zymosan, BCG, or L. monocytogenes to mice resulted in marked and transient MARCO expression and in the upregulation of MSR-A expression in the liver and spleen. In osteopetrotic (op) mutant mice defective in the production on M-CSF, ER-TR9-positive marginal zone macrophages and MOMA-1-positive marginal metallophilic macrophages were absent, whereas MARCO-expressing marginal zone macrophages were present, indicating the heterogeneity of marginal zone macrophages. Intravenous administration of BCG resulted in marked accumulation of BCG bacilli in the both marginal zone macrophages and marginal metallophilic macrophages in littermate control mice. In contrast, BCG bacilli were incorporated almost exclusively by MARCO-expressing marginal zone macrophages in op/op mice. These results indicate that MARCO is not only expressed constitutively in specific macrophage subpopulations but is also induced by various bacterial antigens and plays a role in host defense against bacteria.

Prof. Makoto NAITO, M. D.
Second Department of Pathology
Niigata University School of Medicine
Asahimachi-dori 1, Niigata
951-8510 Japan
Fax: +81-25-227-0761
Tel: +81-25-227-2102
E-mail: 2byori@med.niigata-u.ac.jp

Title
The Existence of Merkel Cells in the Lingual Connective Tissue of the Surinam Caiman, Caiman crocodilus crocodilus (Order Crocodilia)

Author
Sumio YOSHIE, Hiroyuki YOKOSUKA, Hiroaki KANAZAWA and Tsuneo FUJITA

Address
Department of Histology, Nippon Dental University School of Dentistry at Niigata, Niigata; and Department of Radiological Technology, Nagoya University School of Health Sciences, Nagoya, Japan

Summary
The tongue of the Surinam caiman (a reptilian species) was studied by light microscopy including immunohistochemistry for protein gene product 9.5 (PGP 9.5), and transmission electron microscopy. The connective tissue immediately under taste buds housed a cluster of cells immunoreactive for PGP 9.5. These cells synapsed on nerves, and their cytoplasm contained characteristic granules of 90 nm in the mean diameter, glycogen particles, and bundles of intermediate filaments. In light of these ultrastructural features, they were identified as Merkel cells. The Merkel cells were also surrounded by Schwann cells. These findings indicate that the present Merkel cell-neurite-Schwann cell complex is comparable to the avian Merkel corpuscle. On the basis of the granule localization in the cytoplasm, the caiman Merkel cell was presumed to be involved in not only mechanoreception but also endocrine or paracrine functions.

Dr. Sumio YOSHIE
Department of Histology
Nippon Dental University
School of Dentistry at Niigata 1-8 Hamaura-cho, Niigata
951-8580 Japan
Tel: +81-25-267-1500
Fax: +81-25-230-7361
E-mail: yoshie@ngt.ndu.ac.jp