Induction of intracellular Ca2+ and pH changes in Sf9 insect cells by rhodojaponin-III, a natural botanic insecticide isolated from Rhododendron molle

Abstract

Many studies on intracellular calcium ([Ca2+](i)) and intracellular pH (pH(i)) have been carried out due to their importance in regulation of different cellular functions. However, most of the previous studies are focused on human or mammalian cells. The purpose of the present study was to characterize the effect of Rhodojaponin-III (R-III) on [Ca2+](i) and pH(i) and the proliferation of Sf9 cells. R-III strongly inhibited Sf9 cells proliferation with a time- and dose-dependent manner. Flow cytometry established that R-III interfered with Sf9 cells division and arrested them in G2/M. By using confocal scanning technique, effects of R-III on intracellular free calcium ([Ca2+](i)) and intracellular pH (pH(i)) in Sf9 cells were determined. R-III induced a significant dose-dependent (1, 10, 100, 200 μg/mL) increase in [Ca2+](i) and pH(i) of Sf9 cells in presence of Ca2+-containing solution (Hanks) and an irreversible decrease in the absence of extra cellular Ca2+. We also found that both extra cellular Ca2+ and intracellular Ca2+ stores contributed to the increase of [Ca2+](i), because completely treating Sf9 cells with CdCl(2) (5 mM), a Ca2+ channels blocker, R-III (100 μg/mL) induced a transient elevation of [Ca2+](i) in case of cells either in presence of Ca2+ containing or Ca2+ free solution. In these conditions, pH(i) showed similar changes with that of [Ca2+](i) on the whole. Accordingly, we supposed that there was a certain linkage for change of [Ca2+](i), cell cycle arrest, proliferation inhibition in Sf9 cells induced by R-III.

Figure 1

Structure of Rhodojaponin-III.

Figure 2

Effects of R-III on the proliferation of Sf9 cells. The cells were grown in presence of 1, 10, 100 and 200 μg/mL of R-III for the times shown in the figure. Survival cell number was counted by means of Trypan blue exclusion with a standard haemocytometer. Each result derived from the mean of three repetitions.

Figure 3

Effects of R-III on cell cycle.The cells were grown in presence of 1, 10, and 100 μg/mL of R-III for the times shown in the figure. Cell cycle was arrested in G2/M in Sf9 cells and showed a time- and dose-dependent manner. Cells that treated with 0.1% DMSO were used as control. The error bars represent mean ± SEM for data derived from three repetitions. Treatment means sharing the same letter were not significantly different from each other (P < 0.05).

Figure 4

Effect of R-Ⅲ on [Ca²⁺]_i and pH_i in Sf9 cells in presence of Hanks. (A1). changes of [Ca²⁺]_i in Sf9 cells stimulated by R-III at various concentrations as indicated by relative change of Fluo-3AM fluorescence intensity; (B1). Changes of pH_i in Sf9 cells stimulated by R-III at various concentrations as indicated by relative change of Snarf1M fluorescence intensity; (A a–d). Dynamic changes of [Ca²⁺]_i indicated by a dynamic trace of Fluo-3AM fluorescence intensity in case of Sf9 cells treated with 0, 1, 100, 200 µg/mL of R-Ⅲ respectively; (B a–d). pH_i profile in cells subject to the protocol in (A a–d); (A-e). Dynamic variation of [Ca²⁺]_i in Sf9 cells treated with 100 µg/mL of R-III for two times; (B-e). pH_i profile in cells subject to the protocol in (A-e). Arrows indicated the addition of R-Ⅲ.Results of representative experiment derived from three repetitions and 4–6 cells were measured in each repetition. The error bars represent mean ± SEM for data derived from value of relative fluorescence intensity in each time interval. Treatment means sharing the same letter were not significantly different from each other (P < 0.05).

Figure 5

Effects of R-III on [Ca²⁺]_i and pH_i in Sf9 cells in presence of Dhanks. (A1). Changes of [Ca²⁺]_i when cells were stimulated by 100 μg/mL in 130 s and subsequent addition of 2 μM CaCl₂ in 500 s, as indicated by relative change of Fluo-3AM fluorescence intensity; (A-a). Control; (A-b). Dynamic changes of [Ca²⁺]_i in the same conditions with (A1); (B). pH_i profile in cells subjected to the protocol in (A). Results of representative experiment derived from three repetitions and 4–6 cells were measured in each repetition. The error bars represent mean ± SEM for data derived from value of relative fluorescence intensity (vs. control) in each time interval. Treatment means sharing the same letter were not significantly different from each other (P < 0.05). The negative value meant decrease of relative fluorescence intensity in cells.

Figure 6

Effect of Ca²⁺ channels blockon [Ca²⁺]_i and pH_i in R-III-induced Sf9 cells in presence of Hanks. (A1). changes of [Ca²⁺]_i in Sf9 cells treated with 0.5 mM CdCl₂ for 200 s and10 min prior to stimulate with R-III (100 μg/mL), as indicated by relative change of Fluo-3AM fluorescence intensity; (A-a). Dynamic changes of [Ca²⁺]_i in Sf9 cells treated with 0.5 mM CdCl₂ for 200 s prior to stimulate with R-III (100 μg/mL); (A-b). Dynamic changes of [Ca²⁺]_i in Sf9 cells incubated with CdCl₂ (5 mM) for 10 min prior to stimulated with R-III (100 μg/mL); (B). pH_i profile in cells subjected to the protocol in (A). Results of representative experiment derived from three repetitions and 4–6 cells were measured in each repetition. The error bars represent mean ± SEM for data derived from value of relative fluorescence intensity in each time interval. Treatment means sharing the same letter were not significantly different from each other (P < 0.05). The negative value meant decrease of relative fluorescence intensity in cells.

Figure 7

Effect of Ca²⁺ channels block on [Ca²⁺]_i and pH_i in R-III-induced Sf9 cells in presence of Dhanks. (A1). changes of [Ca²⁺]_i in Sf9 cells treated with 0.5 mM CdCl₂ for 200 s and 10 min prior to stimulate with R-III (100 μg/mL), as indicated by relative change of Fluo-3AM fluorescence intensity; (Aa). Dynamic changes of [Ca²⁺]_i in Sf9 cells incubated with CdCl₂ (5 mM) for 200 s prior to stimulate with R-III (100 μg/mL); (A-b). Dynamic changes of [Ca²⁺]_i in Sf9 cells incubated with CdCl₂ (5 mM) for 10 min prior to stimulate with R-III (100 μg/mL) for two times; (B). pH_i profile in cells subjected to the protocol in (A). Results of representative experiment derived from three repetitions and 4–6 cells were measured in each repetition. The error bars represent mean ± SEM for data derived from value of relative fluorescence intensity in each time interval. Treatment means sharing the same letter were not significantly different from each other (P < 0.05). The negative value meant decrease of relative fluorescence intensity in cells.