Effects of repeated administration of pilocarpine and isoproterenol on aquaporin-5 expression in rat salivary glands.

Aquaporins are water channel proteins which enable rapid water movement across the plasma membrane. Aquaporin-5 (AQP5) is the major aquaporin and is expressed on the apical membrane of salivary gland acinar cells. We examined the effects of repeated administration of pilocarpine, a clinically useful stimulant for salivary fluid secretion, and isoproterenol (IPR), a stimulant for salivary protein secretion, on the abundance of AQP5 protein in rat salivary glands by immunofluorescence microscopy and semi-quantitative immunoblotting.

SIRPα signaling regulates podocyte structure and function.

Signal-regulatory protein-α (SIRPα) is a transmembrane protein that contains tyrosine phosphorylation sites in its cytoplasmic region; two tyrosine phosphatases, SHP-1 and SHP-2, bind to these sites in a phosphorylation-dependent manner and transduce multiple intracellular signals. Recently, SIRPα was identified as one of the major tyrosine-phosphorylated proteins in the glomeruli and found to be expressed in podocytes. In the present study, we examined the role of SIRPα expression in podocytes using knockin mice (C57BL/6 background) expressing mutant SIRPα that lacks a cytoplasmic region (SIRPα-mutant mice).

Nephrotic syndrome caused by immune-mediated acquired LCAT deficiency.

Lecithin-cholesterol acyltransferase (LCAT) is an enzyme involved in maintaining cholesterol homeostasis. In familial LCAT deficiency (FLD), abnormal lipid deposition causes renal injury and nephrotic syndrome, frequently progressing to ESRD. Here, we describe a 63-year-old Japanese woman with no family history of renal disease who presented with nephrotic syndrome.

Function of the membrane water channel aquaporin-5 in the salivary gland.

The process of saliva production in the salivary glands requires transepithelial water transfer from the interstitium to the acinar lumen. There are two transepithelial pathways: the transcellular and paracellular. In the transcellular pathway, the aquaporin water channels induce passive water diffusion across the membrane lipid bilayer.

Close association of aquaporin-2 internalization with caveolin-1.

Aquaporin 2 (AQP2) is a membrane water channel protein that traffics between the intracellular membrane compartment and the plasma membrane in a vasopressin-dependent manner in the renal collecting duct cell to control the amount of water reabsorption. We examined the relation between AQP2 internalization from the plasma membrane and caveolin-1, which is a major protein in membrane microdomain caveolae, in Mardin-Darby canine kidney cells expressing human AQP2 (MDCK-hAQP2 cells). Double-immunofluorescence microscopy showed that AQP2 is colocalized with caveolin-1 in the apical plasma membrane by stimulating the intracellular signaling cascade of vasopressin with forskolin.

Double-label immunoelectron microscopy for studying the colocalization of proteins in cultured cells.

Multiple label immunoelectron microscopy localizes and detects multiple antigens in cells and tissues. In double labeling, two kinds of primary antibodies from different animal species are used after being mixed in a single solution. To distinguish the different antigens, secondary antibodies should be labeled with colloidal gold particles of different diameter.

Pre-embedding immunoelectron microscopy of chemically fixed mammalian tissue culture cells.

Immunoelectron microscopy is one of the best methods for detecting and localizing protein molecules in cells and tissues. Gold particles of 1.4 nm in diameter (Nanogold) conjugated with Fab' fragments easily penetrate into the cell interior and are used for pre-embedding immunoelectron microscopy.

Effect of passenger position on fear of danger experienced during sudden bus stops.

The purpose of this study was to identify the effect of bus passengers' positions on their fear of danger when a bus stopped suddenly. A temporary bus running course with one bus stop was set up on the campus of the Tokyo University of Agriculture and Technology (TUAT). The bus ran the course 14 times with the bus stopping twice during the course, once at the bus stop and again just after re-starting from the bus stop.

Immunohistochemical and electron microscopic observations of stromal cells in the human oviduct mucosa.

Stromal cells in the lamina propria of the human oviduct mucosa are unique cells that can differentiate into decidual cells during ectopic pregnancy in the oviduct. The nature of stromal cells is still unknown. In the present study, we investigated human oviductal stromal cells with transmission electron microscopy and immunohistochemistry and revealed that they had ultrastructural features similar to myofibroblasts and expressed alpha-smooth muscle actin, a marker used to identify myofibroblasts.

The primary cilia of secretory cells in the human oviduct mucosa.

The human oviduct is lined with a simple columnar epithelium composed of ciliated cells and secretory cells. Primary cilia or solitary cilia usually extend from the apical surface of the secretory cells. The axoneme of the primary cilia is composed of nine peripheral microtubule doublets (9 + 0 pattern) that lack dynein arms and nexin links.

Immunolocalization of water channel aquaporins in the vomeronasal organ of the rat: expression of AQP4 in neuronal sensory cells.

The vomeronasal organ comprises a pair of narrow tubes in the mammalian nasal septum, serving as a chemosensory system for pheromones. We examined the expression and localization of water channel aquaporins (AQPs) in the rat vomeronasal organ. AQP1 was localized in blood vessels, being particularly abundant in cavernous tissues of the nonsensory mucosa.

Recent advances in fluorescent labeling techniques for fluorescence microscopy.

Tremendous progress in recent computer-controlled systems for fluorescence and laser-confocal microscopy has provided us with powerful tools to visualize and analyze molecular events in the cells. Various fluorescent staining and labeling techniques have also been developed to be used with these powerful instruments. Fluorescent proteins such as green fluorescent protein (GFP) allow us to directly label particular proteins of interest in living cells.

Internalization of caveolae and their relationship with endosomes in cultured human and mouse endothelial cells.

Treatment of cells with pervanadate or vanadate induces the phosphorylation of caveolin-1 and its internalization from the cell surface, but the intracellular fate of caveolae has not been fully elucidated. In the present study, we examined the fate of endocytosed caveolae in human umbilical vein endothelial cells and mouse endothelial KOP2.16 cells.

Apical localization of sodium-dependent glucose transporter SGLT1 is maintained by cholesterol and microtubules.

A GFP-labeled sodium-dependent glucose transporter SGLT1 (SGLT-GFP) was transfected into MDCK cells. SGLT-GFP was localized at the apical membrane in confluent cells. When cellular cholesterol was depleted by methyl-beta-cyclodextrin (MbetaCD) treatment, the localization of SGLT-GFP gradually switched from apical to whole plasma membrane.

Differential localization of aquaporin-2 and glucose transporter 4 in polarized MDCK cells.

Membrane water channel aquaporin-2 (AQP2) and glucose transporter 4 (GLUT4) exhibit a common feature in that they are stored in intracellular storage compartments and undergo translocation to the plasma membrane upon hormonal stimulation. We compared the intracellular localization and trafficking of AQP2 and GLUT4 in polarized Madin-Darby canine kidney cells stably transfected with human AQP2 (MDCK-hAQP2) by immunofluorescence microscopy. When expressed in MDCK-hAQP2 cells, GLUT4 and GLUT4-EGFP were predominantly localized in the perinuclear region close to and within the Golgi apparatus, similar to endogenous GLUT4 in adipocytes and myocytes.

Changes of aquaporin 5-distribution during release and reaccumulation of secretory granules in isoproterenol-treated mouse parotid gland.

Water channel aquaporin 5 (AQP5) is present in the apical membrane of the salivary gland acinar cells. We examined changes of AQP5-distribution during the fusion process of secretory granule membranes into the apical membrane and subsequent recovery process in the mouse parotid gland by administering isoproterenol (IPR) in vivo. We performed immunoperoxidase, immunofluorescence and immunoelectron microscopy.

Immunolocalization of water channel aquaporins in the nasal olfactory mucosa.

Aquaporins (AQPs), membrane water channel proteins expressed in various tissues and organs, serve in the transfer of water and small solutes across the membrane. We raised antibodies to AQPs using isoform-specific synthetic peptides and surveyed their expression in the rat nasal olfactory and respiratory mucosae. AQP1, AQP3, AQP4, and AQP5 were detected by immunohistochemical and immunoblotting analyses.

Localization of Golgi 58K protein (formiminotransferase cyclodeaminase) to the centrosome.

In vertebrate cells, the centrosome consists of a pair of centrioles and surrounding pericentriolar material. Using anti-Golgi 58K protein antibodies that recognize formiminotransferase cyclodeaminase (FTCD), we investigated its localization to the centrosome in various cultured cells and human oviductal secretory cells by immunohistochemistry. In addition to the Golgi apparatus, FTCD was localized to the centrosome, more abundantly around the mother centriole.

Differential regulation of AQP2 trafficking in endosomes by microtubules and actin filaments.

Vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct cells is critical to regulate the urine concentration. To better understand the mechanism of subcellular trafficking of AQP2, we examined MDCK cells expressing AQP2 as a model. We first performed double-immunolabeling of AQP2 with endosomal marker proteins, and showed that AQP2 is stored at a Rab11-positive subapical compartment.

Expression and immunolocalization of water-channel aquaporins in the rat and mouse mammary gland.

We examined the expression and immunolocalization of water-channel aquaporins in the mammary gland by reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry. RT-PCR and immunoblotting revealed the expression of aquaporin-1 (AQP1) and AQP3 in the lactating rat mammary gland. AQP3 was detected in the alveolar epithelium and duct system whereas AQP1 was found in the capillaries and venules.

Molecular mechanisms and drug development in aquaporin water channel diseases: water channel aquaporin-2 of kidney collecting duct cells.

Aquaporin-2 (AQP2) is one of the membrane water channel proteins expressed in principal cells of the kidney collecting ducts. In the basal state, AQP2 resides in the storage vesicles localized in the subapical cytoplasm. Upon stimulation with vasopressin, AQP2 is translocated to the apical plasma membrane by the exocytic fusion of the storage vesicles with the apical membrane.

Aquaporins in the digestive system.

Fluid transfer such as secretion and absorption is one of the major functions of the digestive system. Aquaporins are water channel proteins providing water transfer across the cellular membrane. At least six aquaporin isoforms are expressed in the digestive system.

Aquaporin-2 is retrieved to the apical storage compartment via early endosomes and phosphatidylinositol 3-kinase-dependent pathway.

Aquaporin-2 (AQP2) is one of the water-channel proteins expressed in principal cells of kidney collecting ducts, where it is stored in the intracellular compartment. Previous studies have demonstrated that AQP2 vesicles constitute a distinct intracellular compartment partially overlapping with early endosomes. In this report, we performed in vitro experiments using the renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, stably expressing AQP2 (MDCK-hAQP2).