Sphingosine 1-phosphate (S1P) induces diverse biological responses in various tissues by activating specific G proteincoupled receptors (S1P 1 -S1P 5 receptors). The biological signaling regulated by S1P 3 receptor has not been fully elucidated because of the lack of an S1P 3 receptor-specific antagonist or agonist. We developed a novel S1P 3 receptor antagonist, 1-(4-chlorophenylhydrazono)-1-(4-chlorophenylamino)-3,3-dimethyl-2-butanone (TY-52156), and show here that the S1P-induced decrease in coronary flow (CF) is mediated by the S1P 3 receptor. In functional studies, TY-52156 showed submicromolar potency and a high degree of selectivity for S1P 3 receptor. TY-52156, but not an S1P 1 receptor antagonist, inhibited the decrease in CF induced by S1P in isolated perfused rat hearts. We further investigated the effect of TY-52156 on both the S1P-induced increase in intracellular calcium ([Ca 2ϩ ] i ) and Rho activation that are responsible for the contraction of human coronary artery smooth muscle cells. TY-52156 inhibited both the S1P-induced increase in [Ca 2ϩ ] i and Rho activation. In contrast, VPC23019 and JTE013 inhibited only the increase in [Ca 2ϩ ] i and Rho activation, respectively. We further confirmed that TY-52156 inhibited FTY-720-induced S1P 3 receptor-mediated bradycardia in vivo. These results clearly show that TY-52156 is both sensitive and useful as an S1P 3 receptor-specific antagonist and reveal that S1P induces vasoconstriction by directly activating S1P 3 receptor and through a subsequent increase in [Ca 2ϩ ] i and Rho activation in vascular smooth muscle cells.Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid mediator that is mainly released from activated platelets and induces many biological responses, including angiogenesis, vascular development, and cardiovascular function (Siess,

Abstract. The effect of isoliquiritigenin (ISL), a component of licorice, on the voltagedependent, ultra-rapidly activating delayed-rectifier K + current (IKur) was examined in H9c2 cells, a cell-line derived from rat cardiac myoblasts. IKur was recorded using the whole-cell patch clamp method with a pipette solution containing 140 mM K + . Depolarizing voltage pulses of 200-ms duration were given with 10-mV steps every 10 s from −40 mV holding potential. ISL inhibited IKur in a concentration-dependent manner. The median inhibitory concentration (IC 50 ) of ISL was approximately 0.11 μM and the Hill coefficient was 0.71. Using CHO cells expressing Kv1.5 IKur channels, ISL also inhibited Kv1.5 IKur, but less potently than the IKur current in H9c2 cells. Furthermore, in H9c2 cells, the licorice extract itself inhibited IKur in a manner similar to ISL. We conclude that ISL, one component of licorice, is a potent inhibitor of K + channels, which specifically in H9c2 cells could be Kv2.1, and that this inhibition may be involved in various pharmacological effects of licorice.

Sphingosine 1-phosphate (S1P) receptors are G-protein-coupled receptors. Among the five identified subtypes S1P1-5, the S1P3 receptor expressed on vascular endothelial cells has been shown to play an important role in cell proliferation, migration, and inflammation. A pharmacophore-based database search was used to identify a potent scaffold for an S1P3 receptor antagonist by common feature-based alignment and further validated using the Güner-Henry (GH) scoring method. Assumed excluded volumes were merged into this model to evaluate the steric effect with the S1P3 receptor. Three commercially available compounds were identified as S1P3 receptor antagonists, with IC50 values <5 microM. The synthesis of further derivatives revealed that the 3,4-dialkoxybenzophenone scaffold is a potent component of an S1P3 receptor antagonist. Our results indicate that pharmacophore-based design of S1P3 receptor antagonists can be used to expand the possibility of structural modification through scaffold-hopping based on a database search.

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L-(+)-Lactate oxidase (EC 1.1.3.2) was immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure which has selective permeability to hydrogen peroxide. The lactate electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized enzyme membrane. Properties of the enzyme membrane and characteristics of the lactate electrode were clarified for the determination of L-(+)-lactic acid. The lactate electrode responded linearly to L-(+)-lactic acid over the final concentration 0-0.25 mmol/L within 30 s. When the enzyme electrode was applied to the determination of L-(+)-lactic acid in control serum, within-day precision (CV), analytical recovery, and correlation coefficient between the electrode method and the colorimetric method were 1.4% with a mean value of 4.54 mmol/L, 98.0%, and 0.986, respectively. The lactate electrode was sufficiently stable to perform 1040 assays over 13 days operation for the determination of L-(+)-lactic acid. The dried immobilized enzyme membrane retained 84% of its initial activity after storage at 4 degrees C for 12 months. Moreover, the enzyme electrode was applied to the monitoring of culture medium for human melanoma cells. L-(+)-Lactate production and D-glucose consumption were closely related to cell numbers.

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YUTAKA MAKIMOTO, Ell TAKAHASHI, IDROSHI TAKASUGI Tokyo research laboratory, Kyowa Hakko Kogyo co., Tokyo, Japan Microcarrier (M.c.) for the large-scale culture of anchorage dependent animal cells is available. We have established a micorcarrier perfusion culture (llrscale) of recombinant CHO cells to produce humanized monoclonal antibody, KM8400 and evaluated the productivity of this culture. A cell density of2.5x10 7 cells/ml was reached on day 13 and this culture maintained for 26 days. The total amount ofKM8400 produced was 3.5g.During the culture's stationary phase, specific production rate was 1.5 fold higher than in the growth phase. Surprisingly, we found that the productivity of this cell line increased with the increase of G1 phase cell population.

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