Yosaburo Shibata

Immunolocalization of GLUT1 and connexin 26 in the rat placenta

Abstract.Interhemal membrane in the rat placenta is composed of three trophoblastic layers and endothelial cells. GLUT1, an isoform of the facilitated-diffusion glucose transporter, is abundant in the cells of the placental barrier, i.e., syncytiotrophoblastic layers I and II. GLUT1 is localized at the plasma membranes of the maternal-blood side of syncytiotrophoblastic layer I, and of the fetal-blood side of syncytiotrophoblastic layer II. Double-immunofluorescence microscopy has shown that connexin 26 is present between these GLUT1-positive sites, i.e., between syncytiotrophoblastic layers I and II. Immunogold electron microscopy has revealed that connexin 26 is localized in the gap junctions connecting the two layers. Connexin 26 in these layers therefore makes them functionally a single syncytial layer for the transfer of small molecules such as glucose in the rat placental barrier. These results suggest that glucose transfer in the rat placental barrier is carried out as follows: GLUT1 is used for the entry of glucose into the cytoplasm of syncytiotrophoblastic layer I, connexin 26 for the transfer of glucose from syncytiotrophoblastic layer I to syncytiotrophoblastic layer II, and GLUT1 for the exit of glucose to the fetal circulation.

SPECIFIC LOCALIZATION OF GAP JUNCTION PROTEIN, CONNEXIN45, IN THE DEEP MUSCULAR PLEXUS OF DOG AND RAT SMALL INTESTINE

Abstract Cellular networks of pacemaker activity in intestinal movements are still a matter of debate. Because gap-junctional intercellular communication in the intestinal wall may provide important clues for understanding regulatory mechanisms of intestinal movements, we have attempted to clarify the distribution patterns of three types of gap junction proteins. Using antibodies for connexin40, connexin43, connexin45, smooth muscle actin, and vimentin, immunocytochemical observations were made with the confocal laser scanning microscope on cryosections of fresh-frozen small intestine and colon of the dog and rat. Connexin 45 was localized along the deep muscular plexus of the small intestine in both dog and rat. Double labeling studies revealed that connexin45 overlapped with vimentin –, but not actin-positive areas, indicating the fibroblast-like nature of the cells, rather than their being smooth muscle-like. Connexin43 immunoreactivity appeared along the smooth muscle cell surface in the outer circular layer of the small intestine of both animals. Connexin 40 immunoreactivity was not observed in the muscle layer other than in the wall of large blood vessels. It is suggested that connexin45-expressing cells along the deep muscular plexus of dog and rat small intestine are likely to act as a constituent of a pacemaker system, which may include a conductive system, by forming a cellular network operating via specific types of gap junctions.

Epidermal mast cells in atopic dermatitis

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Identification of rab12 as a Secretory Granule Associated Small GTP-Binding Protein in Atrial Myocytes

A subfamily of small GTP-binding proteins, rab, has been shown to be involved in regulation of vesicular traffic in eukaryotic cells. The goal of this study was to identify the rab proteins associated with atrial secretory granules. A [32P]GTP-overlay assay showed the presence of multiple small GTP-binding proteins on the atrial granules. By biochemical analysis, we have demonstrated that one of the small GTP-binding proteins associated with the atrial granules is a rab12 protein (rab12p), one of the rab proteins that are most closely related to a Sec4 protein of yeast. Association of rab12p with the atrial granules was confirmed by immunogold electron microscopy. Immunoprecipitation followed by immunoblot analysis with anti-rab12 antibody showed that in addition to atria, rab12p was expressed in multiple other organs and cell lines. These results suggest that rab12p may function in vesicular traffic in multiple diverse types of cells.

Annexin V is localized in association with Z-line of rat cardiac myocytes

OBJECTIVE The aim of this study was to characterize a 33-kDa protein (p33) isolated from bovine liver and to determine the subcellular localization of the protein in rat cardiocytes as well as in non-cardiac tissues. METHODS Cycles of calcium-induced precipitation coupled with EGTA-resolubilization were used to isolate crude annexins from bovine and rat tissues. Column chromatography was performed to purify the p33 from the crude annexins. The protein was identified as annexin V by partial amino acid sequence determination. Specificity of anti-annexin V antibody was examined by using Western blotting after one- or two-dimensional electrophoresis. For characterization of the protein, immunofluorescence microscopy and actin-binding assays were carried out. RESULTS Immunofluorescence microscopy showed that annexin V was stained as a striated pattern along myofibrils on frozen sections of both atria and ventricles of adult rats. The striations were seen more clearly in cultured rat atrial myocytes. Examination of doubly stained cardiocytes with anti-annexin V and anti-alpha-actinin by a confocal laser scanning microscope suggests that annexin V is localized in association with the Z-line of rat cardiac myocytes. We also found that annexin V was stained intensely in actin-rich regions of non-cardiac tissues such as bile canaliculi of rat liver and brush border-terminal web region in the epithelial cells of both small intestine and kidney proximal tubular cells. F-actin binding experiments revealed that annexin V failed to bind to F-actin directly in vitro. CONCLUSION Our results suggest that annexin V is a new component of the Z-line in rat cardiocytes and might be involved in regulation of its organization.

Classification of ion channels in the luminal and abluminal membranes of guinea-pig endocardial endothelial cells.

1. The ion channels on both the luminal and abluminal membranes of endocardial endothelial (EE) cells were separately recorded using the patch clamp technique in the guinea‐pig heart. 2. The major population consisted of two types of non‐selective cation channels, which showed open probabilities of 0.21 and 0.33 at the resting potential, and conductances of 36 and 11 pS, respectively. 3. The next major class was Cl‐ channels with an ohmic conductance of 409 pS. The channel was quiescent in the cell‐attached mode but was activated by strong depolarization after excising the patch membrane. 4. The channels activated by intracellular Ca2+ were mainly K+ channels showing a 34 pS slope conductance and, less frequently, Ca(2+)‐dependent K+ channels having a large conductance (210 pS). The inward rectifier K+ channel (32 pS) was also observed. 5. The non‐selective cation channels were recorded on the luminal membrane, but scarcely on the abluminal membrane, suggesting an active transport of K+ and Na+ across the endocardium. 6. The resting membrane conductance of the EE cells may be provided mostly by non‐selective cation channels and 34 pS Ca(2+)‐dependent K+ channels.

Expression and localization of annexin V and annexin VI during limb bud formation in the rat fetus

Abstract We have investigated the expression and the localization of annexin V and annexin VI during the development of rat fetal limb buds by immunoblot and immunocytochemical analysis. Neither annexin V nor annexin VI was detectable in undifferentiated mesenchymal cells in limb buds of the day-13–day-16 rat fetus. Skeletal muscles, whose progenitor cells migrate from the somites and appeared in the limb buds at day 14, dramatically expressed annexin VI on the cell surface after differentiation from mononucleated myogenic cells into multinucleated myotubes. At day 16 both annexin V and annexin VI were found to be expressed in differentiated chondrocytes as well as in the perichondrium, a precursor of chondrocytes, whereas the compact layer of mesenchymal cells surrounding a chondrification center (precartilage) did not show any immunoreactivity for either of these proteins. The results suggest a close relationship between the expression of these annexins and cell differentiation of chondrocytes and skeletal muscles during limb but development.

Gap junction formation and regulation in cultured adult rat and guinea pig cardiac muscle cells

Formations and regulations of gap junction communications were examined in adult atrial muscle cell primary cultures by immunolabeling, freeze fracture replicas and dye spreading. Cocultures of adult atrial and neonatal ventricular cardiomyocytes were also examined.

Characterization of isolated guinea pig liver gap junctions

Guinea pig liver gap junctions are unique in that their major protein constituent is connexin (C×) 26. By use of electron microscopy and connexin-specific antibodies, we have characterized the isolated guinea pig liver gap junctions and made a comparison with those from rat liver.