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)     

Effect of a high-protein diet on the gene expression of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide) in the pancreas

The adaptation to a high protein diet of the concentration and mRNA level of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide), which was proposed to be the mediator of the cholecystokinin release in response to protein intake, was investigated in the rat pancreas. Adult rats were placed on one of two isocaloric diets. One group was fed a 22% casein diet (control diet) and the other a 64% casein diet (high-protein diet) for 14 days. In order to quantify the monitor peptide separately from pancreatic secretory trypsin inhibitor (PSTI-II), which is highly similar in its amino acid and mRNA nucleotide sequences to the monitor peptide but has less cholecystokinin-releasing activity, we used specific assay methods: HPLC was used for determining the monitor peptide concentration in zymogen granules and a synthetic oligonucleotide probe for determining the mRNA of the monitor peptide in the pancreas. The concentrations in the zymogen granules and the mRNA levels in the pancreas of the two peptides increased in parallel during the adaptation to the high protein diet, indicating that these two peptides were under the same control during the adaptation. The concentration and mRNA level of the monitor peptide, which were measured after 0, 3, and 14 days, increased throughout the experiment period, as did the concentration of trypsin. This suggested that the monitor peptide and trypsin may respond to similar signals during the adaptation to a high protein diet and that this apparent coordination may facilitate the adaptation of the pancreas to the diet.     

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)     

Diverse effects of insulin-induced hyperpolarization on 3-O-methyl-D-glucose (3-O-MG) transport in frog skeletal muscles

It has been suggested that the insulin-induced hyperpolarization might be a mediator of the stimulatory action of insulin on glucose transport. The purpose of the present study was to investigate the relationship between the insulin-induced hyperpolarization and the stimulatory action of insulin on glucose transport in skeletal muscle. Satorius muscles dissected from bullfrogs (Rana catesbeiana) were used. Insulin induced a hyperpolarization of the membrane and an increase in the 3-O-Methyl-D-glucose (3-O-MG) uptake and extrusion. In the presence of valinomycin, insulin had no significant effect on the membrane potential. Insulin still had the stimulatory action on both the 3-O-MG uptake and extrusion even in the presence of valinomycin, under whose condition insulin had no significant effect on the membrane potential. The magnitude of the stimulatory action of insulin on the 3-O-MG uptake in the presence of valinomycin was smaller than that in the absence of valinomycin. The magnitude of the stimulatory action of insulin on the 3-O-MG extrusion was, on the contrary, larger than that in the absence of valinomycin. The abolishment of the insulin-induced hyperpolarization decreased the 3-O-MG uptake and increased the 3-O-MG extrusion. The observation in the present study concludes that insulin has two different actions on glucose transport. One of them is developed through the insulin-induced hyperpolarization, which increases the 3-O-MG uptake and decreases the 3-O-MG extrusion. The other action is irrelevant of the insulin-induced hyperpolarization and stimulates both the 3-O-MG uptake and extrusion.     

Quantitative Analysis of Endogenous Gibberellins in Normal and Dwarf Cultivars of Rice

Endogenous levels of gibberellins in shoots and ears of twodwarf rice (Oryza sativa L.) cultivars, Tan-ginbozu (dx mutant)and Waito-C (dy mutant), were analyzed and compared with thoseof normal rice cultivar, Nihonbare. The endogenous levels of13-hydroxylated gibberellins in Tan-ginbozu were much lowerthan those in Nihonbare. In Waito-C, the levels of GA₁₉ andGA₂₀ in the shoots were higher but that of GA₁ was lower thanthe levels of these gibberellins in Nihonbare. These resultssupport the hypothesis that the dy gene controls the 3ß-hydroxylationof GA₂₀ to GA₁ while the dx gene controls a much earlier stepin the gibberellin biosynthesis. Our results indicate that GA₁is the active gibberellin that regulates the vegetative growthof rice. The endogenous levels of GA₄ in the ears of the twodwarf cultivars of rice were higher than the level of GA₄ inthe ears of the normal cultivar, Nihonbare suggesting that thebiosynthesis of gibberellin is not blocked in the anthers ofthe dwarf rice. (Received April 27, 1989; Accepted July 12, 1989)     

CD16+ lymphoblastic cell lines of crab-eating monkeys (Macaca fascicularis) shared U-5 antigen and expressed natural killer activity

The CD16+ lymphoblastic cell lines of crab-eating monkeys shared the U-5 antigen recognized by a monoclonal antibody. The CD16+U-5+ cell lines expressed high natural killer activity to K562 cells, whereas the CD16-U-5- control cell line had no significant natural killer activity. A possible involvement of the U-5 antigen in natural killer function was also suggested by reduction of the natural killer activity in peripheral blood mononuclear cells of Japanese monkeys after treatment with U-5 monoclonal antibody and complement.