Biomedical subjects

M Yamamoto-Hino

Publications and source records attributed to M Yamamoto-Hino.

10 recordsLinked to original sources

PubMed1998

Apical vesicles bearing inositol 1,4,5-trisphosphate receptors in the Ca2+ initiation site of ductal epithelium of submandibular gland.

In polarized epithelial cells, agonists trigger Ca2+ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools into specific regions. We have investigated the relationship between the distribution of IP3 receptors (IP3Rs) and the spatiotemporal pattern of Ca2+ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP3Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP3R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca2+ spike occurred in the apical region. Subsequently, repetitive Ca2+ spikes spread from the apical to the middle cytoplasm. These apical Ca2+ initiation sites were found only in some "pioneer cells," rather than in all duct cells. We performed both Ca2+ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP3R2 in the Ca2+ initiation sites of the pioneer cells. The subcellular localization and expression level of IP3Rs correlated strongly with the spatiotemporal nature of the intracellular Ca2+ signal and distinct Ca2+ responses among the rat SMG duct cells.

Animals↗

PubMed1998

Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) serve as intracellular calcium release channels involved in signal transduction of various hormones in the kidney. Molecular cloning studies have shown that there are three types of IP3R, designated type 1, type 2, and type 3. To characterize their localizations in the rat kidney, we employed immunohistochemical studies using type-specific monoclonal antibodies that were raised against the 15 C-terminal amino acids of each type of IP3R. Type 1 was detected in glomerular mesangial cells and vascular smooth muscle cells. Type 2 was expressed exclusively in intercalated cells of collecting ducts from the cortex to the inner medulla. Type 3 was expressed in vascular smooth muscle cells, glomerular mesangial cells, and some cells of cortical collecting ducts, probably principal cells. As to the subcellular distribution, type 1 and type 2 showed a homogenous distribution in the cytoplasm, whereas type 3 was present mainly in the basolateral portion of the cytoplasm. These results indicate that IP3R isoforms were expressed in a cell-specific manner. The heterogeneous subcellular localizations among the IP3R types suggests compartmentalization of distinct IP3-sensitive Ca2+ pools.

Animals↗

PubMed1998

[IP3 receptor].

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Adenosine Triphosphate↗

PubMed1996

Subtype-specific expression patterns of inositol 1,4,5-trisphosphate receptors in rat airway epithelial cells.

We investigated the immunohistochemical localization of inositol 1,4,5-trisphosphate receptor (IP3R) Types 1, 2, and 3 in rat airway epithelium using the monoclonal antibodies KM1112, KM1083, and KM1082 specific for each type of IP3R. The epithelium from trachea to distal intrapulmonary airways (bronchioles) showed positive immunoreactivity for all types of IP3R. However, cell type as well as subcellular site immunoreactivity for each type of IP3R varied. IP3R Type 1 was found only in the apical thin cytoplasmic area of ciliated cells throughout all airway levels. IP3R Type 2 was exclusively localized to the entire cytoplasm of ciliated cells from the trachea to bronchioles. IP3R Type 3 was expressed mainly in the supranuclear cytoplasm not only of ciliated cells at all airway levels but also in Clara cells of the bronchiolar epithelium. Double fluorescent staining using combinations of KM1083 and Wisteria floribunda lectin or anti-rat 10-KD Clara cell-specific protein antibody confirmed that the IP3R Type 2-positive cells were neither seromucous cells nor Clara cells. These results indicate that the expression of three types of IP3Rs in different cell types and subcellular sites may reflect diverse physiological functions of IP3Rs within airway epithelial cells. The double staining studies suggested that the anti-IP3R Type 2 monoclonal antibody KM1083 would be a specific cell marker for ciliated cells of the airway epithelium.

Animals↗

PubMed1995

Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits.

The inositol 1,4,5-trisphosphate receptor (IP3R) exists as a tetrameric complex to form a functional inositol 1,4,5-trisphosphate-gated Ca2+ channel. Molecular cloning studies have shown that there are at least three types of IP3R subunits, designated type 1, type 2, and type 3. The levels of expression of IP3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids of each IP3R subunit. We found that all the three types of IP3R subunits were expressed in each cell line examined, but their levels of expression varied. To determine whether IP3Rs form heterotetramers, we employed immunoprecipitation experiments using Chinese hamster ovary cells (CHO-K1 cells), in which all three types are abundantly expressed. Each type-specific antibody immunoprecipitated not only the respective cognate type but also the other two types. This result suggests that distinct types of IP3R subunits assemble to form heterotetramers in CHO-K1 cells. We also detected heterotetramers in rat liver, in which IP3R type 1 and type 2 are expressed abundantly. Previous studies have shown some functional differences among IP3R types, suggesting the possibility that various compositions of subunits show distinct channel properties. The diversity of IP3R channels may be further increased by the co-assembly of different IP3R subunits to form homo- or heterotetramers.

Amino Acid Sequence↗

PubMed1995

The calmodulin-binding domain in the mouse type 1 inositol 1,4,5-trisphosphate receptor.

We determined the amino acid sequence responsible for the calmodulin (CaM)-binding ability of mouse type 1 Ins(1,4,5)P3 receptor (IP3R1). We expressed various parts of IP3R1 from deleted cDNA and examined their CaM-binding ability. It was shown that the sequence stretching from Lys-1564 to Arg-1585 is necessary for the binding. The full-length IP3R1 with replacement of Trp-1576 by Ala lost its CaM-binding ability. Antibody against residues 1564-1585 of IP3R1 inhibited cerebellar IP3R1 from binding CaM. The fluorescence spectrum of the peptide that corresponds to residues 1564-1585 shifted when Ca(2+)-CaM was added. From the change in the fluorescence spectrum, we estimated the dissociation constant (KD) between the peptide and CaM to be 0.7 microM. The submicromolar value of KD suggests an actual interaction between CaM and IP3R1 within cells. The CaM-binding ability of other types of IP3Rs was also examined. A part of the type 2IP3R, including the region showing sequence identity with the CaM-binding domain of IP3R1, also bound CaM, while the expressed full-length type 3 IP3R did not.

Amino Acid Sequence↗

PubMed1995

Immunohistochemical study of inositol 1,4,5-trisphosphate receptor type 3 in rat central nervous system.

In the rat central nervous system (CNS), inositol 1,4,5-trisphosphate receptor (IP3R) type 3 was immunolocalized with a type 3-specific monoclonal antibody (mAb). The protein was expressed principally in prototype astrocytes, ependymal cells around the ventricle, and Bergmann glial cells in the cerebellum. These cells were stained by antibody against glial fibrillary acidic protein (GFAP), indicating the coexistence of GFAP and IP3R type 3. Immunoblot analysis using a brain homogenate detected a 240 kDa protein, verifying that the observed immunoreactivity is from the IP3R type 3 protein. IP3R type 1 and type 2 were not detected immunohistochemically in astrocytes. These results suggest that IP3-induced CA2+ release (IICR) in astroglia is directed by IP3R type 3, whereas IICR in neuronal cells is mediated by IP3R type 1.

Animals↗

PubMed1994

Monoclonal antibodies distinctively recognizing the subtypes of inositol 1,4,5-trisphosphate receptor: application to the studies on inflammatory cells.

Monoclonal antibodies were raised that specifically recognize the COOH-terminal sequences and the loop sequences between the fifth and the sixth transmembrane spanning regions of human inositol 1,4,5-trisphosphate receptor (IP3R) type 1, 2 and 3. Western blot analysis using Jurkat cells, mouse cerebellum, COS-7 expressing IP3R type 3 cDNA showed that those monoclonal antibodies reacted specifically with each of these three IP3R subtypes and that they do not cross-react. These antibodies could be used for the specific immunoprecipitation of IP3Rs. Using these monoclonal antibodies, the expression profiles of IP3R-subtype proteins were found to be different among inflammatory cells such as macrophages, polymorphonuclear cells, mast cells, eosinophils, splenocytes, thymocytes and megakaryocytic cells. Usually, more than one type of IP3R were expressed in a cell simultaneously. The observation of CMK cells under immunofluorescence confocal microscopy revealed that IP3R type 1 and type 2 are located at different subcellular fractions.

Amino Acid Sequence↗

PubMed1994

Subtypes of inositol 1,4,5-trisphosphate receptor in human hematopoietic cell lines: dynamic aspects of their cell-type specific expression.

Inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ signaling plays important roles in cellular responses to extracellular stimuli. We recently succeeded in cloning human counterparts of the three subtypes derived from separate genes. Using the cDNA sequences type-specific to these subtype receptors, we here analyzed the expression profile of IP3R subtypes in stimulated and unstimulated human hematopoietic cell lines representing T cells, B cells, neutrophils, macrophages, erythrocytes and megakaryocytes. Northern and dot blot analysis showed that each IP3R subtype is expressed differently in these cells and that the expression profile in each cell is dynamically changed upon stimuli which induce differentiation. Moreover, most of these cells were found to simultaneously express at least two different subtype receptors.

B-Lymphocytes↗

PubMed1994

Cloning and characterization of human type 2 and type 3 inositol 1,4,5-trisphosphate receptors.

We have cloned cDNAs coding for human type 2 and type 3 and part of type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs). The complete nucleotide sequences for type 2 and type 3 receptors were determined and the pharmacological properties of the latter were characterized. Human type 2 and type 3 IP3Rs are 2701 amino acids and 2671 amino acids long, respectively, and have significant sequence homologies as well as structural similarities including the six membrane-spanning regions near the C-termini when compared with the rat or mouse counterpart. COS-7 cells transfected with human type 3 IP3R showed characteristic inositol 1,4,5-trisphosphate (IP3)-binding properties with Kd values of 28.8 nM. The order of potency of competition with IP3 was Ins(1,4,5)P3 (IP3) > Ins(2,4,5)P3 > Ins(1,3,4,5)P4 > Ins(1,2,3,4,5,6)P6. Type 2 and type 3 IP3Rs were mapped to human chromosomes 12p11 and 6p21, respectively, by in situ hybridization. cDNA cloning of the human IP3Rs allowed us to identify the types of the receptor expressed in various human hematopoietic and lymphoma cell lines. The type 3 receptor was present in all of cell lines tested, while the type 1 or 2 receptor was expressed in only particular cell types. The differential expression of the IP3R types could confer the cell-specific regulation on the IP3/Ca2+ signalling.

Amino Acid Sequence↗