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To obtain compounds that promote glucose uptake in muscle cells, the novel cell lines A31-IS derived from Balb/c 3T3 A31 and C2C12-IS from mouse myoblast C2C12 were established. In both cell lines, glucose consumption was induced by insulin and suppressed by the addition of Akt-activating kinase inhibitor. The A31-IS cells highly express the insulin receptor β chains, Glut4, and uncoupling protein-3, as compared to the parent Balb /c 3T3 A31 cells, and C2C12-IS cells highly express the insulin receptor β chain as compared to its parent cell line. Using A31-IS cells, we screened our library compounds and obtained three compounds, DF-4394, DF-4451, and DG-5451. These compounds dose-dependently promoted glucose consumption in A31-IS cells and facilitated [3H]-2-deoxyglucose uptake in differentiated C2C12-IS cells. The compounds that we obtained from the library screening will be good candidates for improving insulin resistance in muscle cells. Keywords:: skeletal muscle cell, insulin, glucose uptake

Novel, cell-based assays, based on bioluminescence resonance energy transfer, have been developed for FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling at receptor-proximal steps. In a stable transfectant of the HEK-293 cell line expressing human FcepsilonRIalpha, FcepsilonRIbeta, and FcRgamma-GFP2 and Syk(1-265)-Rluc fusion proteins, FcepsilonRI cross-linking markedly increased BRET2 ratios, which are the ratios of GFP2 emission to Rluc emission. These ratios reflect the FcRgamma-GFP2-Syk(1-265)-Rluc interaction in living cells. The signals are specifically inhibited by the Src-family kinase inhibitor PP2. Separately, in transient transfectants expressing GPVI, FcRgamma-GFP2, and Syk(1-265)-Rluc, the GPVI-specific ligand convulxin induced a two-fold increase in the BRET2 ratio and this increase was also inhibited by PP2. Finally, a differential assay was developed which permits the measurement of FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling in the same cell. These assays provide useful methods for monitoring FcRgamma-Syk interaction in real time in living cells and may contribute to the understanding of signal regulation through FcRgamma-containing receptors.

Corticotropin-releasing factor (CRF) increased intracellular Ca 2+ concentration in single astrocytes. The effect in increasing intracellular Ca 2+ was not observed in Ca 2+ -free solution. Furthermore, CRF at concentrations more than 10 nM stimulated 45 Ca 2+ uptake in cultured rat astrocytes. The action was blocked by α-helical CRF(9-41) in a competitive manner, but not by nifedipine and 3,4-dichlorobenzamil. On the other hand, CRF did not stimulate cAMP formation, cGMP formation and phosphoinositide hydrolysis in astrocytes. These results indicate that CRF increases Ca 2+ influx via an activation of CRF receptors in a cAMP-independent mechanism in cultured astrocytes.

Effect of endothelin-3 (ET-3) on dibutyryl cAMP (DBcAMP)-induced stellation of rat cerebral cultured astrocytes was examined. Treatment with 1 mM DBcAMP, 10 μM forskolin, 100 μM isoproterenol and 500 nM phorbol 12-myristate 13-acetate changed protoplasmic cultured astrocytes into process-bearing ones. ET-3 (1 nM) completely prevented the astrocytic stellation induced by these agents. The effect of ET-3 showed a dose-dependence, where IC 50 value and maximal effective dose were 49 pM and about 0.1 mM, respectively. ET-1 and sarafotoxin (SRTX) S6b prevented the DBcAMP-induced astrocytic stellation with potencies similar to that of ET-3. ET-3 (1 nM) did not affect the cAMP accumulation after DBcAMP treatment in cultured astrocytes. Stellate astrocytes were reversed to the protoplasmic type cells by addition of 1 mN ET-3 in the presence of DBcAMP. ET-1 and SRTX similarly reversed the astrocytic stellation. ET-3 reversed the astrocytic stellation in the absence of extracellular Ca 2+ . Pre-loading of BAPTA-AM, a permeable Ca 2+ chelator, on stellate astrocytes had no effect on the reversal by ET-3. ET-3 did not increase intracellular free Ca 2+ concentration ([Ca 2+ ] i ) of most astrocytes tested at 0.1 nM. A high concentration (100 nM) of ET-3 increased astrocytic [Ca 2+ ] i which was negated by Ca 2+ -free and BAPTA-AM loading. These results suggest that ETs modulate morphological changes in astrocytes through cAMP- and Ca 2+ -independent mechanisms.

To obtain compounds that inhibit the interaction of von Willebrand factor (vWF) and glycoprotein (GP) Ib, a novel binding assay was established. The binding of fixed platelets to vWF-R497 mutant was quantified by a solid phase assay. In this assay, fixed platelets bound to the vWF-R497 mutant, carrying the deletion of Glu497-Tyr508 and the missense mutation of Arg545 to Ala, without binding modulators such as ristocetin. The Kd value of the binding was 2.8 nM, which was consistent with the result from liquid binding assay. The binding was inhibited by aurin tricarboxylic acid (ATA) and an anti GPIb antibody, AK2. Using this binding assay, we screened our library compounds and obtained D74-3736. This compound also inhibited ristocetin-induced platelet aggregation in the human platelet-rich plasma. Keywords:: biotinylation of fixed platelets, binding assay of von Willebrand factor (vWF) to glycoprotein Ib (GPIb)