Diflubenzuron affects gamma-thioGTP stimulated Ca 2+ transport in vitro in intracellular vesicles from the integument of the newly molted american cockroach, Periplanet americana L.

Abstract

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 MgSO 4, 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 of DFB on Ca 2+ 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 Ca 2+-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and γ-S-GTP stimulated Ca 2+ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca 2+. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca 2+ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells.