Osamu Amano

Immunohistochemical and functional characterization of pH-dependent intestinal absorption of weak organic acids by the monocarboxylic acid transporter MCT1

The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues.

Immunolocalization and pharmacological relevance of oligopeptide transporter PepT1 in intestinal absorption of β-lactam antibiotics

A polyclonal antibody (anti‐PepT1/C) was raised against the rabbit intestinal H+‐coupled oligopeptide transporter, PepT1. Anti‐PepT1/C detected 70–80‐kDa protein in crude membranes obtained from rabbit duodenum, jejunum and ileum. PepT1 was localized in the brush‐border of the absorptive epithelial cells by subcellular fractionation of membranes on a sucrose density gradient and by immunohistochemistry using light and electron microscopy. Transport activity for cephalosporins and dipeptide expressed in Xenopus laevis oocytes injected with total mRNA obtained from rabbit small intestine was eliminated completely by prehybridization of the mRNA with antisense oligonucleotide against the 5′‐coding region of rabbit PepT1 cDNA.

Expression and Localization of Hepatocyte Growth Factor in Rat Submandibular Gland

By combination of in situ hybridization and immunohistochemical techniques, the expression of hepatocyte growth factor (HGF) was demonstrated in the submandibular gland of rats. Both the mRNA signal and immunoreactivity for HGF were localized exclusively to the epithelial cells of granular convoluted tubules, whereas they were absent from the other components of the submandibular gland. In the granular convoluted tubule cells, HGF-immunoreactivity was localized to the apical secretory granules, which was further substantiated by immunoelectron microscopy. These results added HGF to the list of many growth factors that are produced in the rat submandibular gland and secreted into the saliva.

Expression and localization of intestinal 15 kDa protein in the rat

Rat intestinal 15 kDa protein (I-15P) is highly homologous to porcine gastrotropin. We studied the occurrence, distribution and subcellular localization of I-15P in the entire rat body, using the immunocytochemistry to localize protein andin situ hybridization to localize mRNA. Both techniques demonstrated the expression of I-15P in the enterocytes of ileum, luteal cells of ovary and a subpopulation of steroid-endocrine cells of adrenal gland. Immuno-electron microscopy further demonstrated that I-15P is localized in both the cytoplasmic and nuclear matrix regions of these cells. The present results suggest roles of I-15P not only in the transport of bile salts but also in the metabolisms of certain steroid hormones.

Basic fibroblast growth factor in rat salivary glands

We studied the occurrence and localization of basic fibroblast growth factor (bFGF) in rat salivary glands using a specific monoclonal antibody. It was shown that the extract of rat salivary glands has a pronounced stimulatory activity on the growth of bovine capillary endothelial cells, which is blocked by the addition of an antibody against bFGF. The concentration of bFGF in the submandibular/sublingual gland, as determined by radioimmunoassay, was ∼80% that in the brain. Immunocytochemistry revealed bFGF-immunoreactivity localized primarily in the epithelial cells lining the striated ducts and excretory ducts of the parotid, sublingual and submandibular glands. In addition, intense bFGF-immunoreactivity was observed in the granular convoluted tubule of the submandibular gland, localized predominantly in the agranular pillar cells, which lay in small numbers among the majority of weakly immunostained cells containing many apical secretory granules. At the electron-microscopic level, the immunoreactive material was distributed diffusely in the cytoplasmic matrix and nuclei of all immunoreactive cells, whereas it was absent from all cytoplasmic organelles including the secretory granules. These results indicate that bFGF is localized in different cellular and subcellular compartments from those of other growth factors in the duct system of rat salivary glands.

Downregulation of Vascular Endothelial Growth Factor and Its Receptors in the Kidney in Rats with Puromycin Aminonucleoside Nephrosis

Aim: We aimed to examine the possible involvement of vascular endothelial growth factor (VEGF) in the pathogenesis of puromycin aminonucleoside nephrosis (PAN). Methods: The expression and localization of the mRNA of VEGF and its receptors, flt-1 and flk-1, were analyzed in the kidneys of puromycin aminonucleoside-injected rats by use of Northern blotting and in situ hybridization. Results: In association with the induction of proteinuria, VEGF mRNA underwent decrease in amount from 3 days after the injection, reaching the minimum level at 7 days, followed by a gradual recovery by 28 days. The levels of flk-1 and flt-1 mRNA showed similar transient decrease in PAN kidney, whereas the mRNA of von Willebrand factor, a marker of endothelial cells, showed no change in amount. In the normal rat kidney, VEGF mRNA was localized primarily to podocytes, and flk-1 mRNA was localized exclusively to endothelial cells with much higher intensity in glomeruli than in peritubular capillaries. In PAN kidney, the intensities of both VEGF and flk-1 signals in podocytes and glomerular endothelial cells, respectively, appeared much lower at 7 days than in normal kidney. Conclusion: These results indicate that the VEGF-VEGF receptor system is downregulated in PAN, implying that it is not involved in the mechanism of proteinuria in PAN.

Enhancement by hepatocyte growth factor of bone and cartilage formation during embryonic mouse mandibular development in vitro.

To elucidate the possible roles of hepatocyte growth factor (HGF) in the early development of mouse mandible, HGF was applied to an organ-culture system with chemically defined media. Mandibular arches microdissected from mouse embryos at the 10th day of gestation were cultured for 10 days with or without HGF, HGF plus HGF-receptor (c-met) antisense oligodeoxyribonucleotide, or HGF plus c-met sense oligodeoxyribonucleotide in the media. The cultured mandibles were then analysed, histologically in serial paraffin sections. In the absence of HGF, the tooth organs of bud stage, Meckels cartilage and the tongue were formed, whereas only a slight amount of bone tissue was formed in the cultured mandible. The expression of intrinsic HGF and c-met in the cultured mandibles was confirmed by reverse transcriptase-polymerase chain reaction. Furthermore, immunohistochemistry demonstrated that both HGF and c-met were localized in areas of the mesenchymal tissue forming bone and cartilage. With HGF in the medium, the volume of both bone and cartilage increased significantly and dose-dependently. HGF also increased the rate of proliferation of osteogenic cells and chondrocytes. Addition of c-met antisense oligodeoxyribonucleotide partially inhibited the HGF-induced enhancement of bone and cartilage formation, whereas addition of c-met sense oligodeoxyribonucleotide had no effect. These results revealed that exogenous HGF enhances bone and cartilage morphogenesis in the cultured mandibles, suggesting physiological roles for intrinsic HGF in the early development of mouse mandible.

Expression, localization and developmental regulation of insulin-like growth factor I mRNA in rat submandibular gland

The mRNA for insulin-like growth factor I (IGF-I) in the submandibular gland of mature and developing rats was examined by Northern blotting and in situ hybridization with an oligonucleotide probe. In the mature adult rat, IGF-I mRNA was expressed at a higher level in the submandibular gland than in the liver, and was localized primarily in the granular convoluted tubule (GCT) cells. A 4.7-kb mRNA on Northern blots, which was expressed only slightly in the liver, proved to be the predominant size species of IGF-I transcripts in the GCT cells, and its level increased progressively with the postnatal development of GCTs in the gland. In addition, a 1.8-kb mRNA for IGF-I was also expressed at a much lower level throughout the acinar and duct systems, irrespective of age. These results have shed a light on the status of IGF-I as one of the many biologically active polypeptides that are produced in the rodent submandibular gland.

Constitutive expression of the 27-kDa heat-shock protein in neurons and satellite cells in the peripheral nervous system of the rat

By use of reverse transcriptase‐polymerase chain reaction, abundant expression of the mRNA of 27 kDa heat shock protein (Hsp27) was revealed in the sympathetic and parasympathetic ganglia as well as in the sensory ganglia of unstressed adult rats. In situ hybridization and immunohistochemistry further localized Hsp27 mRNA and protein to both neurons and satellite cells in all types of ganglia examined. Schwann cells in the ganglia and peripheral nerve fibers were devoid of Hsp27 signal. These results suggested that Hsp27 is constitutively expressed in neurons and satellite cells in the entire peripheral nervous system of the rat. Anat Rec 262:213–220, 2001.

The role of cyclic AMP response element-binding protein in testosterone-induced differentiation of granular convoluted tubule cells in the rat submandibular gland.

The postnatal development of granular convoluted tubules (GCT) in the duct system of the rodent submandibular gland is known to be androgen-dependent, but the underlying molecular mechanism is unclear. To test the possible role of the transcription factor, cyclic AMP response element-binding protein (CREB), in the androgen-induced differentiation of GCT, the effect of testosterone on the expression and localization of epidermal growth factor (EGF), a marker of GCT cells, and of CREB was examined in the submandibular glands of immature 3-week-old rats. Northern blotting demonstrated increases in both EGF and CREB mRNA 1-4 days after testosterone administration. Immunoprecipitation also indicated that CREB protein was increased in amount with testosterone administration, and that induced CREB was phosphorylated at the serine residue as in the active form of CREB. In situ hybridization demonstrated that cells with CREB mRNA signal first appeared in the distal portions of striated ducts at 1 day and had increased in number by 4 days after giving testosterone, when cells with EGF mRNA signal became evident in the same duct portions. Immunohistochemistry also showed the occurrence of CREB protein in the nuclei of duct epithelial cells before their differentiation into EGF-positive GCT cells. Finally, pieces of submandibular gland from immature rats were cultured in vitro and their expression of EGF mRNA analysed by the reverse transcriptase-polymerase chain reaction. Testosterone in the medium caused a marked enhancement of EGF expression in the gland in 1-4 days, which was attenuated by simultaneous administration of the antisense oligonucleotide for CREB as well as that for the androgen receptor. These results suggest the CREB is upregulated by androgen and has a crucial role in androgen-induced differentiation of GCT in the duct system of the rat submandibular gland.