Contents

Review article

1. Neuroanatomical effects of capsaicin on the primary afferent neurons (HIURA, A.)
   

Original articles

1. Ultrastructural and histochemical characterization of special muscle cells in the monkey small intestine (SHIMODA, H.)
   
2. In vitro studies on PGC or PGC-like cells in cultured yolk sac cells and embryonic stem cells of the mouse (NAKAGAWA, S.)
   
3. Impairment of glucokinase translocation in cultured hepatocytes from OLETF and GK rats, animal models of type 2 diabetes (TOYODA, Y.)
   
4. Postnatal morphodifferentiation of the subneural apparatuses of the posterior cricoarytenoid muscle in rats: a scanning electron microscopy study (YAMAGATA, T.)
   
5. Immunoreactivity to a monoclonal antibody (0S-3) is shared by osteoclasts and bicarbonate-secreting cells (IRIE, K.)
   
6. Effect of low temperatures on compound 48/80-induced intracellular Ca2+ changes and exocytosis of rat peritoneal mast cells (MORI, S.)
   
7. Localization of alkaline phosphatase and osteopontin during matrix mineralization in the developing cartilage of coccygeal vertebrae (SASAKI, T.)
   
8. The existence of Na+/K+-ATPase-immunoreactive cells in the pharyngeal villiform-papilla epithelium of the soft-shelled turtle, Trionyx sinensis japonicus (YOSHIE, S.)

Summary

Title
Neuroanatomical Effects of Capsaicin on the Primary Afferent Neurons

Author
Akio HIURA

Address
Department of Second Oral Anatomy, Tokushima University School of Dentistry, Tokushima, Japan

Summary
Studies by N. JANCSO and his associates in the 1970's established that capsaicin in paprika exerts selective damage on nociceptive primary sensory neurons. The physiological and pharmacological aspects of capsaicin's effect have been repeatedly reviewed, but no report seems available concerning the neuroanatomical changes caused by capsaicin. This paper first reviews the neuroanatomical aspect of the lesion caused by capsaicin. Special attention is paid to quantitative estimations made by our group and others on the loss of dorsal root ganglion (DRG) cells, dorsal root nerve fibers, the saphenous nerve, chorda tympani nerve, and pulp nerves after neonatal treatment with capsaicin. The degenerating process of DRG cells induced by capsaicin is discussed with respect to necrosis and apoptosis. The capsaicin receptors found recently are concisely introduced with reference to their action. A discrepancy between a marked loss of dorsal root C-fibers and an unexpectedly intact response to noxious heat in mice treated with capsaicin at neonate is discussed, and attension is given to nerves sprouting from capsaicin-resistant DRG cells in the superficial dorsal horn. In addition, the architecture of the synapses between the central endings of the capsaicin-sensitive primary afferent neurons and the intrinsic inhibitory interneurons is described and its possible significance considered in terms of the transmission of nociceptive information.

Dr. Akio HIURA
Department of Second Oral Anatomy
Tokushima University School of Dentistry
Tokusima, 770-8504 Japan
Fax: +81-88-633-7342
E-mail: hiuraaki@dent.tokushima-u.ac.jp

Title
Ultrastructural and Histochemical Characterization of Special Muscle Cells in the Monkey Small Intestine

Author
Hiroshi SHIMADA, Tetsuji KUDO, Yoshiaki TAKAHASHI and Seiji KATO

Address
Department of Anatomy, Oita Medical University, Oita, Japan.

Summary
The ultrastructure, three-dimensional arrangement, and histochemical features of special muscle cells in the monkey small intestine were investigated. The cells formed a special layer separated from the main part by a connective tissue space along the submucosal surface of the circular muscle coat. Scanning electron microscopy using alkali maceration demonstrated this inner sublayer to be a continuous thin sheet consisting of irregularly-shaped muscle cells equipped with many cytoplasmic projections and caveolae. Other ultrastructural features included direct contact with interstitial cells, due to their close association with nerve fibers of the deep muscular plexus. Histochemical examination revealed significant alkaline phosphatase activity and immunoreactivity for vascular smooth muscle alpha actin in these muscle cells, whereas the ordinary circular muscle cells were immunopositive for enteric smooth muscle gamma actin. These findings suggest that the special muscle cells play an important role in regulating the radial stretch of the monkey small intestinal wall.

Dr. Hiroshi SHIMODA
Department of Anatomy
Oita Medical University
1-1 Idaigaoka, Hasama-machi, Oita
879-5593 Japan
Tel/Fax: +81-97-586-5623
E-mail: hshimoda@oita-med.ac.jp

Title
In vitro Studies on PGC or PGC-Like Cells in Cultured Yolk Sac Cells and Embryonic Stem Cells of the Mouse

Author
Shin-ichiro NAKAGAWA, Sakura SABURI, Keitaro YAMANOUCHI, Hideaki TOJO and Chikashi TACHI

Address
Laboratory of Applied Genetics, Department of Animal Resource Sciences, School of Agriculture and Life Sciences, University of Tokyo, Tokyo; Molecular Biology Department, Nippon Shinyaku CO. Ltd., Sakura, Tsukuba; and Laboratory of Developmental and Reproductive Biotechnology, Department of Animal Resource Sciences, School of Veterinary Medicine and Life Sciences, Azabu University, Sagamihara, Japan

Summary
The present study aims: 1) to determine those conditions which promote the proliferation of primordial germ cells (PGCs) of the migratory phase in the yolk sac; and 2) to examine the effects of yolk sac cells as a feeder layer under the conditions mentioned above upon the embryonic stem (ES) cells (R1) with high potential for entering the germ line in vivo in chimeras. In murine yolk sac cells obtained on Day 10.5-11.5 of pregnancy and cultured in a modified Dulbecco's modified Eagle's medium (DMEM-plus/20: the postfix represents the concentration of FBS added in percentage), many cells exhibited strong immunoreactivities to the monoclonal antibodies 4C9 and 2C9 which are known to react with PGC specifically. Both the 4C9- and the 2C9-positive cells were sensitive to the treatment with busulfan added in vitro, supporting the supposition that they were PGCs. The respective numbers of the 4C9- and the 2C9-positive cells increased approximately 4 and 12 times when they were cultured in DMEM-plus/20 fortified with SCF, LIF, bFGF and TNF-alpha (DMEM-NT/20). When the R1 cells were cultured in the yolk sac-conditioned DMEM-NT/20 medium on the laminin substratum, the entire colonies were faintly stained with 4C9 but not with 2C9. At times solitary ES cells migrated out from the colonies, and reacted strongly with 4C9. When yolk sac cells and R1 cells were cultured on the two sides of a collagen-coated membrane, the yolk sac cells being feeder cells, some R1 cell colonies were intensely stained as a whole with either the 4C9 or the 2C9 antibody, suggesting that these colonies might be composed of cells clonally derived from stem cells which either had been destined to become the germ line cells or had already acquired cellular characteristics close to PGCs. It was tentatively concluded that the R1 cell population contained, as judged from the immunoreactivities, germ-cell-like cells, and that the yolk sac cells and/or their secretory products might facilitate the proliferation of, or the conversion of R1 cells to, the germ-cell-like cells.

Prof. Chikashi TACHI, Ph. D.
Laboratory of Developmental and
Reproductive Biotechnology
School of Veterinary Medicine and Life Sciences
Azabu University
1-17-71 Fuchinobe, Sagamihara-shi,
Kanagawa-ken, 229-8501 Japan.
Tel: +81-427-54-7111 ext 277 or 278
      +81-427-86-7567 (direct)
Fax: +81-427-86-7578
E-mail: tachi@azabu-u.ac.jp

Title
Impairment of Glucokinase Translocation in Cultured Hepatocytes from OLETF and GK Rats, Animal Models of Type 2 Diabetes

Author
Yukiyasu TOYODA, Yuki ITO, Keiichiro TANIGAWA and Ichitomo MIWA

Address
Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, Nagoya; Pharmacy of Nagoya Daini Red Cross Hospital, Nagoya; and Department of Clinical Nutrition, Faculty of Health Science, Suzuka University of Medical Science, Suzuka, Japan

Summary
We examined sugar-induced translocation of glucokinase in cultured hepatocytes from Otsuka Long-Evans Tokushima Fatty and Goto-Kakizaki rats, animal models of type 2 diabetes, and compared this with that in Long-Evans Tokushima Otsuka and Wistar rats, respectively, as control strains. When hepatocytes from the four strains were incubated with 5 mM glucose, glucokinase was present predominantly in the nuclei. Higher concentrations of glucose, 5 mM glucose plus 1 mM fructose, and 5 mM glucose plus 1 mM sorbitol all induced the translocation of glucokinase from the nucleus to the cytoplasm in hepatocytes from these rats. The extent of glucokinase translocation under these conditions, however, was less marked in both diabetic rat types than in the control rats. The extent of the phosphorylation of glucose as estimated by the release of 3H2O from [2- 3H] glucose is significantly lower in Goto-Kakizaki rats than in Wistar rats. The results indicate that the translocation of glucokinase is impaired in the hepatocytes of diabetic rats. They also suggest that the impaired translocation of glucokinase is associated with abnormal hepatic glucose metabolism in type 2 diabetes.

Dr. Ichitomo MIWA
Department of Pathobiochemistry
Faculty of Pharmacy
Meijo University
Tempaku-ku, Nagoya
468-8503 Japan
Fax: +81-52-834-8780
E-mail: miwaichi@meijo-n.ac.jp

Title
Postnatal Morphodifferentiation of the Subneural Apparatuses of the Posterior Cricoarytenoid Muscle in Rats: A Scanning Electron Microscopy Study

Author
Takahiko YAMAGATA, Seiji KAWAKITA, Masamitsu HYODO and Junzo DESAKI

Address
Department of Otolaryngology and Anatomy, Ehime University School of Medicine, Shigenobu, Ehime, Japan

Summary
The subneural apparatus, i. e., the post-synaptic component of the neuromuscular junction, in the posterior cricoarytenoid muscle of the rat was studied by scanning electron microscopy, with special attention given to its postnatal differentiation along with the functional development of the muscle. Primitive synaptic troughs observed in the first postnatal week consisted of single cup-like depressions 5-6 microm in diameter. On the 7th day, low sarcoplasmic ridges appeared in the trough. In the second postnatal week, muscle fibers could be classified into two groups: large (10-15 microm in diameter) and small (less than 10 microm in diameter). In the large muscle fibers, many low ridges became circular and protruded to transform the single trough into numerous cup-like depressions (2-5 microm in diameter). In contrast, the subneural apparatus in the small muscle fibers consisted of a small number of cup-like depressions. The two types of subneural apparatus differentiated into adult forms by the 28th postnatal day, although they remained smaller in size than those of adults. In the large muscle fibers, the number of pit-like or elongated invaginations increased and gradually transformed into slit-like junctional folds by the 28th postnatal day, while the small muscle fibers still possessed a few pit-like or elongated junctional folds at this point in time. The two types of morphodifferentiation of the subneural apparatus are thought to reflect the two types of muscle fibers in the rat posterior cricoarytenoid muscle.

Dr. Takahiko YAMAGATA
Department of Otolaryngology
Ehime University School of Medicine
Shigenobu-cho, Onsen-gun, Ehime
791-0295 Japan

Title
Immunoreactivity to a Monoclonal Antibody (OS-3) Is Shared by Osteoclasts and Bicarbonate-Secreting Cells

Author
Kazuharu IRIE, Michiaki ORIKASA, Yasunori SAKAKURA, Eichi TSURUGA, Toshihiko IWANAGA, and Toshihiko YAJIMA

Address
Department of Oral Anatomy, Health Sciences University of Hokkaido School of Dentistry, Ishikari-Tobetsu, Hokkaido; College of Biomedical Technology, Niigata University, Niigata; and Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Summary
OS-3, a monoclonal antibody raised against macrophagic cells derived from cultured rat glomeruli, reacts with the plasma membrane of various bicarbonate-secreting cells such as epithelial cells of the pancreatic excretory duct and type B intercalated cells of the kidney, suggesting that the antigenic molecule of OS-3 is involved in bicarbonate production and/or secretion. Since osteoclasts must vigorously extrude bicarbonate to maintain cytoplasmic pH in a physiologic range during proton secretion, we examined the localization of OS-3 immunoreactivity in the bone tissue to determine the involvement of the detected molecule in the transmembrane transport of bicarbonate in osteoclasts. The OS-3 selectively stained the basolateral plasma membrane of osteoclasts. Ultrastructurally, the immunoreactivity with OS-3 was associated with small cytoplasmic projections and microplicae of the basolateral plasma membrane. This finding suggests that osteoclasts express the molecule common to bicarbonate-secreting cells to utilize it for bicarbonate transport during bone resorption.

Kazuharu IRIE, D. D. S., Ph. D.
Anatomy/Dentistry
Health Sciences University of Hokkaido
Ishikari-Tobetsu, Hokkaido
061-0293 Japan
Tel & Fax: +81-1332-3-1218
E-mail: irie@hoku-iryo-u.ac.jp

Title
Effect of Low Temperatures on Compound 48/80-Induced Intracellular Ca2+ changes and Exocytosis of Rat Peritoneal Mast Cells

Author
Shiho MORI, Tomoyuki SAINO and Yoh-ichi SATOH

Address
Department of Cell Biology and Neuroanatomy, Iwate Medical University, Morioka, Japan

Summary
It has been well documented that compound 48/80-induced exocytosis of mast cells is accompanied by changes in intracellular Ca2+ concentration ([Ca2+]i) showing a biphasic pattern: an initial phase which constitutes an abrupt increase, followed by a plateau phase. The former is caused by Ca2+ release from intracellular Ca2+ stores, and the latter is the result of secondary Ca2+ influx. Low temperatures lead to the inhibition of exocytosis, but the precise mechanism remains unclear. The present study aims to reveal whether [Ca2+]i changes are affected by the environmental temperature. To this end, we developed a novel imaging method to record [Ca2+]i changes and exocytotic processes simultaneously. Rat peritoneal mast cells were loaded by Indo-1/AM or Fluo-3/AM for measuring [Ca2+]i, and the exocytosed granule matrices were stained by sulforhodamine-B. Cells were stimulated by compound 48/80, and [Ca2+]i changes and exocytosis were recorded by means of a real-time confocal microscope. At 37 degrees C, [Ca2+]i changes in stimulated mast cells showed a sustained plateau phase. Granule discharge was observed at the cell surface, and, in addition, most of the intracellular granule matrices were involved in compound exocytosis. The granule discharge and compound exocytosis proceeded over a period of a few minutes. At 4 degrees C, the plateau phase of [Ca2+]i changes declined rapidly, although the initial phase was not suppressed. Granule discharge occurred at the cell surface, but compound exocytosis ceased within a few minutes. These findings indicate that a low temperature inhibits compound exocytosis which can be caused by Ca2+ influx. The present imaging method represents a powerful tool for investigating the stimulus-secretion coupling of mast cells.

Dr. Yoh-ichi SATOH
Department of Cell Biology and Neuroanatomy
Iwate Medical University
Morioka, 020-8505 Japan
Tel: +81-19-651-5111
Fax: +81-19-651-5605
E-mail: yisatoh@iwate-med.ac.jp

Title
Localization of Alkaline Phosphatase and Osteopontin during Matrix Mineralization in the Developing Cartilage of Coccygeal Vertebrae

Author
Tomoyo SASAKI, Norio AMIZUKA, Kazuharu IRIE, Sadakazu EJIRI and Hidehiro OZAWA

Address
Department of Oral Anatomy, Niigata University Faculty of Dentistry, Niigata; and Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Ishikari, Tobetsu, Japan

Summary
We observed the manner in which alkaline phosphatase (ALPase) and osteopontin were localized in the cartilage and intramembranous bone of coccygeal vertebrae during matrix mineralization, shedding considerable light on the manner in which they develop. In the cartilage matrix of coccygeal vertebrae, we observed the localization of ALPase activity in the boundary of the proliferative and the hypertrophic zones. Granular nodules of mineralization were consistently found in the boundary of both zones, and increased in size when close to the hypertrophic zone. While osteopontin was rarely present in the early stages of mineralization, its localization along the margins of mineralized matrices in the hypertrophic zone was prominent. In contrast to cartilage, mineralized nodules in the intramembranous bone in the mid-portion of the vertebra displayed osteopontin-immunoreactivity, indicating its early synthesis and subsequent accumulation to early-stage mineralized nodules. When blood vessels, accompanied by osteoblastic and osteoclastic cell populations, invaded the cartilage, osteopontin was localized in the lower region of the hypertrophic zone, despite its maintaining the localization of ALPase and early-stage mineralization. Thus, our investigation demonstrated ALPase activity consistent with early-stage mineralization in the cartilage matrix. However, the fact that osteopontin-localization could not be pinpointed might account for its multifunctionality as concerns both the regulation of mineralization and the attachment of migrating osteogenic and osteoclastic cells to the mineralized matrix.

Prof. Hidehiro OZAWA
Department of Oral Anatomy
Niigata University Faculty of Dentistry
5274, 2-Bancho, Gakko-cho Dori
Niigata, 951-8514 Japan
Tel: +81-25-227-2814
Fax: +81-25-227-0804
E-mail: ozawa@dent.niigata-u.ac.jp

Title
The Existence of Na+/K+-ATPase-Immunoreactive Cells in the Pharyngeal Villiform-Papilla Epithelium of the Soft-Shelled Turtle, Trionyx sinensis japonicus

Author
Sumio YOSHIE, Hiroyuki YOKOSUKA, Toyoji KANEKO and Tsuneo FUJITA

Address
Department of Histology Nippon Dental University School of Dentistry at Niigata, Niigata; and Center for International Cooperation, Ocean Research Institute, University of Tokyo, Tokyo, Japan

Summary
The pharyngeal villiform processes of the hibernating soft-shelled turtle, Trionyx sinensis japonicus, were studied by immunohistochemistry for Na+/K+-ATPase in combination with a mitochondrion staining. Mitochondria-rich cells were recognized in the epithelium constituting the distal part of most processes, and exclusively showed the Na+/K+-ATPase immunoreactivity. These cells tended to attract each other to form clusters. When considering the physiological and histological data previously obtained in corresponding cells in the fish gill epithelium, the mitochondria-rich cells in the hibernating turtle were suggested to be involved in the electrolyte (Na+) uptake from the aquatic habitat.

Dr. Sumio YOSIIIE
Department of Histology
Nippon Dental University
1-8 Hamauracho, Niigata
951-8580 Japan
Tel: +81-25-267-1500
Fax: +81-25-230-7367
E-mail: yoshie@ngt.ndu.ac.jp