Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian,halocynthia roretzi

The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl₂. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH₂Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH₂Cl, and tosyl-Phe-CH₂Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.     

The Effects of Ethylene on the Coordinated Synthesis of Multiple Proteins: Accumulation of an Acidic Chitinase and a Basic Glycoprotein Induced by Ethylene in Leaves of Azuki Bean, Vigna angularis

The ethylene-induced synthesis and accumulation of proteinswere studied in the primary leaves of azuki bean (Vigna angularis).Seven different proteins, designated AZ17, 23, 27, 32, 35, 36,42 according to their molecular masses, were synthesized andaccumulated in response to ethylene. AZ27 and AZ42 were purifiedto homogeneity and characterized. AZ27 was identified as anacidic chitinase and accumulated in the extracellular space.The sequence of the 40 N-terminal amino acids of AZ27 showedno similarity to that of a basic chitinase from bean and tobacco,but it was highly homologous to that of a chitinase from virus-infectedcucumber leaves. AZ42 was identified as a glycoprotein thataccumulated intracellularly. A search for proteins with sequenceshomologous to an internal sequence of 18 amino acids in AZ42was unsuccessful. Immunochemical examination revealed that auxinand abscisic acid enhanced the ethylene-induced accumulationof AZ27 but not of AZ42. In contrast, levels of AZ42 were notaffected by auxin or abscisic acid, but cytokinin suppressedthe accumulation of one of the doublet bands of AZ42. TranslatablemRNAs coding for AZ27 and AZ42 were not present in leaves thathad not been treated with ethylene, but levels of these mRNAsincreased after such treatment. (Received March 1, 1991; Accepted May 8, 1991)     

Inhibition of Auxin-Stimulated Elongation of Cells in Rice Lamina Joints by a Feruloylated Arabinoxylan Trisaccharide

A feruloylated arabinoxylan trisaccharide inhibited IAA-stimulatedelongation of cells in rice lamina joints. The de-esterifiedcompound, an arabinoxylan trisaccharide, did not inhibit suchelongation. This is the first report that feruloylated arabinoxylanfragments are involved in the regulation of plant growth. (Received September 18, 1991; Accepted January 13, 1992)     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Expression of epidermis-specific antigens during embryogenesis of the ascidian, Halocynthia roretzi

We have produced two monoclonal antibodies (Epi-1 and Epi-2) which specifically recognize epidermal cells and their derivative, the larval tunic, of developing embryos of the ascidian Halocynthia roretzi. The antigens, examined by indirect immunofluorescence staining, first appear at the early tailbud stage and are present until at least the swimming larval stage. There were distinct and separate puromycin and actinomycin D sensitivity periods for each antigen. Aphidicolin, a specific inhibitor of DNA synthesis, prevented the appearance of each antigen when embryos were exposed to the drug continuously from cleavage stages. These results suggest that the antigens are synthesized during embryogenesis by developing epidermal cells and that several rounds of DNA replication are required for the antigen expression. Early cleavage stage embryos, including fertilized but unsegmented eggs, in which cytokinesis had been blocked with cytochalasin B expressed the antigens, and blastomeres exhibiting the antigens were always of the epidermis lineage. In partial embryos produced by four separated blastomere pairs of the 8-cell embryos, the expression of antigens was seen only in those developed from the animal blastomere pairs, which are progenitors of epidermal cells. These observations indicate that differentiation of epidermal cells in ascidian embryos takes place in a typical "mosaic" fashion.     

Isolation and characterization of a mucin-type glycoprotein from the lung lavage of a patient with alveolar proteinosis

A high molecular weight glycoprotein was isolated from the lavage fluid of a patient with alveolar proteinosis by gel chromatography with Sepharose CL-4B. The glycoprotein gave a single band stainable with alcian blue and with periodate-Schiff reagent on the cellulose acetate membrane electrophoresis. The glycoprotein did not penetrate 3.3% polyacrylamide gel but moved into 1% agarose gel as a periodate-Schiff positive single band, when electrophoresed in the presence of sodium dodecyl sulfate. The chemical analysis and the results of the beta-elimination reaction showed the presence of O-linked carbohydrate chains characteristic for a mucin-type glycoprotein. These data provide the first characterization of a mucin-type glycoprotein isolated from lung in pulmonary alveolar proteinosis.