Nakagawa Y, Matsumura F
Department of Environmental Toxicology, University of California, Davis 95616.
Insect Biochem Mol Biol. 1994 Dec;24(10):1009-15. doi: 10.1016/0965-1748(94)90138-4.
To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs (Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO4, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10(-8) M). Such an inhibitory effect on DFB on Ca2+ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na+, and Li+ did not affect the calcium uptake. Among ionophores, agents disrupting H+ gradients (e.g. FCCP and NEM) totally eliminated 45Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca2+-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and gamma-S-GTP stimulated Ca2+ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca2+. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca2+ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells.
为了研究除虫脲（DFB）及其他苯甲酰基苯基脲类化合物的作用机制，我们最初推测其作用可能与胞吐作用有关：为验证这一假说，我们从新蜕皮的美洲大蠊（Periplaneta americana L.）体壁匀浆中获取细胞内囊泡制剂，匀浆置于含250 mM蔗糖（等渗）、2.5 mM MgSO4、pH 6.6的10 mM MES缓冲液中。通过研究DFB对各种离子转运活性的影响，我们发现低浓度（如10(-8) M）的DFB能显著抑制该细胞内颗粒制剂对钙的摄取。添加离子载体或膜破坏剂以及对囊泡制剂进行超声处理后，DFB对Ca2+摄取的这种抑制作用被消除。另一方面，寡霉素、蛋白质磷酸化调节剂、Na+和Li+对钙摄取没有影响。在离子载体中，破坏H+梯度的试剂（如FCCP和NEM）完全消除了囊泡对45Ca的摄取活性以及DFB的抑制作用。在钙离子调节剂中，钙调蛋白抑制剂如氯咪达唑和三氟拉嗪降低了Ca2+摄取，而膜钙通道阻滞剂维拉帕米则没有。ATP和γ-S-GTP刺激了Ca2+摄取。然而，前者仅增加了DFB不敏感部分的Ca2+摄取，而后者主要增加了DFB敏感部分的Ca2+摄取。这些数据共同支持了这样一种假说，即DFB在该制剂中的作用位点是与体壁细胞中的液泡型细胞内囊泡偶联的GTP依赖性Ca2+转运过程。
