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1 Molecules 2011, 16, ; doi: /molecules Article OPEN ACCESS molecules ISSN Induction of Intrcellulr C 2+ nd ph Chnges in Sf9 Insect Cells y Rhodojponin-III, A Nturl Botnic Insecticide Isolted from Rhododendron molle Xing-An Cheng, Jin-Jun Xie, Mei-Ying Hu *, Yn-Bo Zhng nd Jing-Fei Hung Lortory of Insect Toxicology, South Chin Agriculturl University, Gungzhou , Chin; E-Mils:nzi_28@163.com (X.-A.C); @qq.com (J.-J.X); fiend_wing_o@yhoo.com.cn (Y.-B.Z); hungjingfei@163.com (J.-F.H) * Author to whom correspondence should e ddressed; E-Mil: humy@scu.edu.cn; Tel.: , Fx: Received: 25 Jnury 2011; in revised form; 6 April 2011 / Accepted: 7 April 2011 / Pulished: 15 April 2011 Astrct: Mny studies on intrcellulr clcium ([C 2+ ] i ) nd intrcellulr ph (ph i ) hve een crried out due to their importnce in regultion of different cellulr functions. However, most of the previous studies re focused on humn or mmmlin cells. The purpose of the present study ws to chrcterize the effect of Rhodojponin-III (R-III) on [C 2+ ] i nd ph i nd the prolifertion of Sf9 cells. R-III strongly inhiited Sf9 cells prolifertion with time- nd dose-dependent mnner. Flow cytometry estlished tht R-III interfered with Sf9 cells division nd rrested them in G2/M. By using confocl scnning technique, effects of R-III on intrcellulr free clcium ([C 2+ ] i ) nd intrcellulr ph (ph i ) in Sf9 cells were determined. R-III induced significnt dose-dependent (1, 10, 100, 200 μg/ml) increse in [C 2+ ] i nd ph i of Sf9 cells in presence of C 2+ -contining solution (Hnks) nd n irreversile decrese in the sence of extr cellulr C 2+. We lso found tht oth extr cellulr C 2+ nd intrcellulr C 2+ stores contriuted to the increse of [C 2+ ] i, ecuse completely treting Sf9 cells with CdCl 2 (5 mm), C 2+ chnnels locker, R-III (100 μg/ml) induced trnsient elevtion of [C 2+ ] i in cse of cells either in presence of C 2+ contining or C 2+ free solution. In these conditions, ph i showed similr chnges with tht of [C 2+ ] i on the whole. Accordingly, we supposed tht there ws certin linkge for chnge of [C 2+ ] i, cell cycle rrest, prolifertion inhiition in Sf9 cells induced y R-III.

2 Molecules 2011, Key word: rhodojponin-iii; intrcellulr free clcium; intrcellulr ph; Sf9 cells rrest 1. Introduction Rhodojponin-III (R-III, Figure 1) show structure s Figure 1 is grynoid diterpene isolted from Rhododendron molle nd determined s the min insecticidl ingredient in the plnt [1]. It is n effective nturl insecticide ginst more thn 40 species of griculturl pests [2]. Previous studies indicte tht R-III shows mny nti-insect properties including potent ntifeednt, oviposition, ovicides, ntimolting, growth inhiitor, contct nd/or stomch toxicity [3], which is relted to the nervous, digestive, endocrine nd reproductive systems of insects. There hve een some studies on the mode of ction of R-III on insects, lthough the precise moleculr mechnism is not well understood. Some reserchers demonstrted tht R-III remrkly decreses the contents of cetylcholine (ACh) nd hs reversile ctivted effects on N + -K + -ATPse nd C 2+ -Mg 2+ -ATPse ctivities [4], indicting its interference with insect nervous system through locked the trnsition of nervous impulse [5], in which C 2+ s n intrcellulr second messenger plys key role. Figure 1. Structure of Rhodojponin-III. Intrcellulr free clcium ([C 2+ ] i ) is highly verstile intrcellulr second messenger nd signl trnsducer in oth excitle nd non-excitle cells. It is involved in mny functions in prolifertive cells, including gene expression, protein synthesis, cell secretion, motility, metolism, cell-cycle progression nd poptosis cell deth [6,7]. Under norml conditions, [C 2+ ] i concentrtion is mintined t nm, ut sustined C 2+ relese from intrcellulr C 2+ stores, C 2+ influx through receptor- or voltge-dependent C 2+ chnnels or lockge of re-uptke cn pertur [C 2+ ] i homeostsis [7]. A vriety of physicl, chemicl, or iologicl stimuli cn modify [C 2+ ] i which my led to cellulr physiologicl chnges such s cell rrest or cell deth [8,9]. Intrcellulr ph (ph i ) is ecoming evident to mny spects of cell physiology, nd protons my lso function s second messenger in mnner similr to tht of C 2+ [10]. Reltively smll chnges in ph i could hve profound effect on vriety of cellulr functions. For exmple, chnges in ph i tke plce in response to growth, tumor promotion, DNA synthesis [11], protein synthesis, ctivtion of the ion chnnel [12], poptosis, prolifertion nd trnsformtion [13]. High ph i cn sensiilize cellulr proteins such s enzymes, ion chnnels nd ion trnsporters [14] nd ph i shifts my hve significnt effects on clcium regultion in cells. It hs een estlished in previous reserch tht ph i nd [C 2+ ] i re closely linked. In effect, ph i hs een descried s eing le to ffect intrcellulr C 2+ homeostsis nd contriute to the length, mgnitude, nd frequency of the C 2+ signl through the

3 Molecules 2011, modultion of voltge-dependent or -independent plsm memrne C 2+ chnnels nd/or through regultion of the moiliztion of C 2+ from internl stores [10]. On the other hnd, C 2+ hs een descried s inducing ph i vrition, prticulrly in neurons [15]. In severl cellulr models cytosolic lkliniztion is sufficient signl to relese clcium from intrcellulr pools [16]. Although mny studies on [C 2+ ] i nd ph i hve een crried out due to their importnce in regultion of different cellulr functions, most of the previous studies re focused on humn or mmmlin cells nd similr studies in insect cells re lcking. Studying the mode of ction of otnicl pesticide ginst insects hs een gretly simplified y the finding tht its effects cn e seen in cultured insect cells [17,18]. Therefore, the purpose of this study is to principlly chrcterize the effect of R-III on intrcellulr C 2+ nd ph i in Sf9 cells (isolted from Spodopter frugiperd pupl ovrin tissue). Otherwise, we primrily discuss the possile interctions mong chnges of [C 2+ ] i level, cell cycle nd cell prolifertion, nd the possile linkge etween chnges of intrcellulr C 2+ nd tht of ph i in Sf9 cells induced y R-III, ll of which re helpful to explore some new clues for the further study on insecticidl mechnism of R-III. 2. Results nd Discussion 2.1. Effect of R-III on the Prolifertion of Sf9 Cells To investigte the effect of R-III on the prolifertion of Sf9 cells, cell viility ws mesured y Trypn lue exclusion ssy. As shown in Figure 2, the inhiition effect of R-III on Sf9 cells ws not significnt nd survivl cell fter 24 h of tretment with 1, 10, 100 nd 200 μg/ml of R-III ws out cells/ml, similr to tht of control. After 24 h of tretments with different concentrtions of R-III, cell viility decresed in time- nd dose-dependent mnner. Figure 2. Effects of R-III on the prolifertion of Sf9 cells. The cells were grown in presence of 1, 10, 100 nd 200 μg/ml of R-III for the times shown in the figure. Survivl cell numer ws counted y mens of Trypn lue exclusion with stndrd hemocytometer. Ech result derived from the men of three repetitions. Cell numer( 10 5 /ml)

4 Molecules 2011, Survivl cell numer ws , , , cells/ml, which ws fr lower thn tht of the control ( cells/ml) fter 72 h of tretment with R-III t the concentrtions of 1, 10, 100 nd 200 μg/ml respectively. By 96 h of tretments with R-III, survivl cell numer in control ( cells/ml) hd gretly outstripped the R-III tretment. In ddition, the inhiition effect on Sf9 cells of R-III t the concentrtions of 100 μg/ml nd 200 μg/ml ws significntly higher thn tht of 1 μg/ml nd 10 μg/ml. However, the inhiition effect etween 100 μg/ml nd 200 μg/ml or etween 1 μg/ml nd 10 μg/ml showed no significnt difference. The results in this ssy indicted tht R-III strongly inhiited the prolifertion of Sf9 cells in time- nd dose-dependent mnner Effect of R-III on Cell Cycle In order to further clrify the effects of R-III on the prolifertion of Sf9 cells, we checked the effect of R-III on cell cycle y flow cytometry. As shown in Figure 3, n rrest in G2/M cell cycle phse ecme evident in Sf9 cells treted with R-III, showing time- nd dose-dependent mnner. After 24 h of tretment with 1, 10 nd 100 μg/ml of R-III, the percentges of cells in G2/M phse incresed to 29.2%,35.8%,nd 40.7% respectively. While fter 48 h of the sme tretment, the percent of cells in G2/M phse incresed to 42.2%, 39.4% nd 39.1% nd ecme 57.1%, 61.3% nd 67% respectively, fter 72 h of tretment. Compring to the treted groups, the percent of G2/M phse cells in control ws lower (28.6%, 29.9% nd 30.5% for 24, 48 nd 72 h, respectively) nd kept in stedy stte within the treted time. Results suggested tht R-III like the ntimitotic gents such s colchicine nd zdirchtin [19] interfered with Sf9 cells division nd rrests them in G2/M, showing strong inhiitory ctivity to the cell growth nd prolifertion. Figure 3. Effects of R-III on cell cycle. The cells were grown in presence of 1, 10, nd 100 μg/ml of R-III for the times shown in the figure. Cell cycle ws rrested in G2/M in Sf9 cells nd showed time- nd dose-dependent mnner. Cells tht treted with 0.1% DMSO were used s control. The error rs represent men ± SEM for dt derived from three repetitions. Tretment mens shring the sme letter were not significntly different from ech other (P < 0.05). Cell rtio of G2/M (%) h 48h 72h c 0 CK R-III (µg/ml)

5 Molecules 2011, Effects of R-III on [C 2+ ] i nd ph i in Sf9 Cells In order to know the effects of R-III on [C 2+ ] i, Sf9 cells were exposed to Hnks, uffer solution contining C 2+. C 2+ influx took plce in Sf9 cells stimulted y R-III, which elicited significnt increse of [C 2+ ] i. As shown in Figure 4A1, compring to control, Sf9 cells showed grdul increse of [C 2+ ] i y 29.9%, 38.28%, 64.21% nd %, fter the stimultion with 1, 10, 100 nd 200 μg/ml of R-III respectively, showing dose-dependent fshion. Under these conditions, we oserved similr chnge of ph i with tht of [C 2+ ] i. As shown in Figure 4B1, Sf9 cells presented grdul increse of ph i y 22.27%, 37.13%, 69.17% nd 89.58% fter stimultion with 1, 10, 100 nd 200 μg/ml of R-III respectively, lso in dose-dependent fshion. The increse of ph i ws only 5.7% in control. In order to further investigte the effects of R-III on [C 2+ ] i in Sf9 cells, we checked the time-dependent chnges of C 2+ fluorescence signls in Sf9 cells induced y R-III. As shown in Figure 4A-, flt trce in control indicted no chnge of [C 2+ ] i in cells. A trnsient elevtion of [C 2+ ] i chrcterized y fluorescence intensity increse followed y recovery to sl level ws oserved in Sf9 cells stimulted with low concentrtion of R-III (1 μg/ml) (Figure 4A-), suggesting tht cells cn regulte [C 2+ ] i to keep intrcellulr C 2+ homeostsis in cse of slight externl stimultion. Figure 4. Effect of R-Ⅲ on [C 2+ ] i nd ph i in Sf9 cells in presence of Hnks. (A1). chnges of [C 2+ ] i in Sf9 cells stimulted y R-III t vrious concentrtions s indicted y reltive chnge of Fluo-3AM fluorescence intensity; (B1). Chnges of ph i in Sf9 cells stimulted y R-III t vrious concentrtions s indicted y reltive chnge of Snrf1M fluorescence intensity; (A d). Dynmic chnges of [C 2+ ] i indicted y dynmic trce of Fluo-3AM fluorescence intensity in cse of Sf9 cells treted with 0, 1, 100, 200 µg/ml of R-Ⅲ respectively; (B d). ph i profile in cells suject to the protocol in (A d); (A-e). Dynmic vrition of [C 2+ ] i in Sf9 cells treted with 100 µg/ml of R-III for two times; (B-e). ph i profile in cells suject to the protocol in (A-e). Arrows indicted the ddition of R-Ⅲ. Results of representtive experiment derived from three repetitions nd 4 6 cells were mesured in ech repetition. The error rs represent men ± SEM for dt derived from vlue of reltive fluorescence intensity in ech time intervl. Tretment mens shring the sme letter were not significntly different from ech other (P < 0.05). Reltive chnge of fluorescence intensity (%) A. C 2+ B. ph i c CK R-III(μg/mL) d e c CK R-III ( ug/ml) d e

6 Molecules 2011, Figure 4. Cont.

7 Molecules 2011, Figure 4. Cont. When exposed to 100 nd 200 μg/ml of R-III, Sf9 cells showed rpid rise of [C 2+ ] i, which reched to high stedy stte [Figures 4A-(c,d)]. Previous studies hve estlished tht elevtion of [C 2+ ] i my derive from extr cellulr C 2+ influx through clcium chnnels or trnsporters [20] or the C 2+ relese from intrcellulr C 2+ stores induced y intrcellulr inositol 1,4,5-trisphosphte (IP3), synthesized in response to externl stimultion [21]. Under this condition, we pplied the second stimultion of R-III, which cused [C 2+ ] i declining shrply in index fshion to stedy stte lower thn the sl level (Figure 4A-e). In this ssy, we lso found tht the chnges of ph i were in line with tht of [C 2+ ] i, s shown in the trces in Figures 4B-(,d) nd the second stimultion of R-III lso produced shrply decrese of ph i to stedy stte lower thn the sl level (Figure 4B-e). Although the mechnisms of the shrp decrese of [C 2+ ] i nd ph i re not cler yet, n interprettion from groups of Li et l. [22] who investigted the modultion effect of glutmte on [C 2+ ] i of inner hir cell of the guine pig cochle nd found similr phenomenon my enlighten us on this study: Excessive stimultion of glutmte my cuse toxicity on cells nd increse the penetrtion of plsm memrne (PM) which gives rise to [C 2+ ] i efflux. Since R-III ws otnic pesticide showing significnt toxicity to mny kinds of insect, the second stimultion of R-III possily produced toxic effect on Sf9 cells cusing [C 2+ ] i efflux, in greement with viewpoint of [22], nd [C 2+ ] i efflux exchnged for H + influx through C 2+ -ATPse in PM [23], eliciting decrese of ph i Effects of R-III on [C 2+ ] i nd ph i in Sf9 Cells in Presence of Dhnks To further clrify the effect of R-III on [C 2+ ] i nd ph i, Sf9 cells were exposed to Dhnks, C 2+ -free uffer solution, nd recoded the chnge of fluorescence intensity in Sf9 cells stimulted with R-III (100 μg/ml) t 130 s. Compring to the control with only slightly decrese of [C 2+ ] i (3.52%) (Figure 5A1, A-), [C 2+ ] i shrply decresed y 24.64% fter the stimultion of R-III (Figure 5A1), nd kept in stedy stte (Figure 5A-), indicting [C 2+ ] i efflux in Sf9 cells induced y R-III, nd no increse of [C 2+ ] i ws oserved in cse of re-ddition of CCl 2 (2 μm) to the C 2+ -free uffer solution t 500 s (Figure 5A-), suggesting tht C 2+ efflux in cells ws irreversile. Previous study finds tht lthough glucose oxidse induce rpid decrese in rt endothelil cells exposed in C 2+ free uffer, re-ddition of C 2+ to the extrcellulr uffer my ctivte store operted C 2+ entry to cuse lrge [C 2+ ] i increses [24]. However, store operted C 2+ entry in Sf9 cells ws not ctivted y the

8 Molecules 2011, re-ddition of C 2+ in this ssy. The results further proved tht it ws the C 2+ influx tht elicited the sustntil increse of [C 2+ ] i in Sf9 cells stimulted y R-III in cse of cell exposure to Hnks in the experiment ove [Figures 4A-(,e)]. Under these conditions, ph i lso showed significnt decrese (17.85%), nd sustined decrese (61.02%) ws oserved even if ddition of CCl 2 t 130 s fter stimultion (Figure 5B1). Knowing from dynmic chnge of fluorescence signls in Sf9 cells induced y R-III (Figure 5B-), R-III induced trnsient rise of ph i, followed y decline to stedy level much lower thn sl level in Sf9 cells, nd no recovery of ph i ws oserved even if ddition of CCl 2 t 130 s fter stimultion. In contrst, the control showed only slightly decrese of ph i (Figure 5B-). Interestingly, [C 2+ ] i showed no trnsient increse in the sme conditions (Figure 5A-). The results in this ssy indicted tht R-III not only induced [C 2+ ] i in Sf9 cells decline through C 2+ efflux ut lso elicited the intrcellulr cidifiction, possily through H + entry in exchnge for C 2+ extrusion y the C 2+ -ATPse in cell PM [23]. Figure 5. Effects of R-III on [C 2+ ] i nd ph i in Sf9 cells in presence of Dhnks. (A1). Chnges of [C 2+ ] i when cells were stimulted y 100 μg/ml in 130 s nd susequent ddition of 2 μm CCl 2 in 500 s, s indicted y reltive chnge of Fluo-3AM fluorescence intensity; (A-). Control; (A-). Dynmic chnges of [C 2+ ] i in the sme conditions with (A1); (B). ph i profile in cells sujected to the protocol in (A). Results of representtive experiment derived from three repetitions nd 4 6 cells were mesured in ech repetition. The error rs represent men ± SEM for dt derived from vlue of reltive fluorescence intensity (vs. control) in ech time intervl. Tretment mens shring the sme letter were not significntly different from ech other (P < 0.05). The negtive vlue ment decrese of reltive fluorescence intensity in cells. Reltive chnge of fluorescence intensity (%) A. C 2+ B. ph i Tretment 0 CK R-III CCl Tretment CK R-III CCl2 c 1

9 Molecules 2011, Figure 5. Cont. C 2+ signling plys crucil role in the function of lmost ll cell types s n intrcellulr second messenger [25]. For exmple, mny reserches prove tht chnges in [C 2+ ] i homeostsis re ssocited with induction of poptotic [26] or cell deth [27]. An experimentl report coming from group of Wng et l. [28] provides convincing interprettion for the role of C 2+ in prticiption in poptotic cell deth. In their study, the uthors found tht H 2 O 2 -induced poptosis of tocco protoplsts primrily involves in the increse of [C 2+ ] i resulting from the entry of extr cellulr C 2+. In recent yers, some reports show tht clcium signl is key component of the moleculr switch mechnism in cell division cycle [29].Through the interply with severl proteins, [C 2+ ] i prticiptes in regulting key steps in the cell cycle such s reentry of quiescent cells into prolifertion nd the trnsition through the G1/S, G2/M, nd the metphse/nphse oundries [30-32]. Moreover, mitosis cn e initited y IP 3 R-induced clcium trnsients [33]. Disturnce of [C 2+ ]i homeostsis such s increse of [C 2+ ]i level in response to externl stimultion my interfere with cells division cycle, resulting in cell cycle rrest [34]. In present study, Sf9 cells showed significnt chnges of [C 2+ ]i induced y R-III [Figures 4A-(,e) nd Figure 5A-]. Otherwise, R-III lso produced cell cycle rrest in G2/M (Figure 3) nd strongly inhiited Sf9 cells prolifertion (Figure 2), lthough poptosis ws not oserved. Our results suggested tht there ws certin linkge for chnge of [C 2+ ]i, cell cycle rrest, cell prolifertion inhiition in Sf9 cells induced y R-III. Moreover, we tenttively hypothesize tht disturnce of [C 2+ ]i homeostsis in Sf9 cells induced y R-III my result in cell cycle rrest, which finlly cuses inhiition of insect cells prolifertion or even cell deth (including poptopic cell deth). This dul negtive effect would significntly decrese the solute numer of cells, nd finlly induce remrkle decrese of survivl cell numer in R-III tretment The Contriution of Intrcellulr C 2+ Stores to the Chnges of Intrcellulr C 2+ nd ph i Intrcellulr C 2+ stores such s mitochondri or endoplsmic reticulum my e the other principl source of C 2+ [35]. In present study, to exmine the contriution of intrcellulr C 2+ stores to the chnges of intrcellulr C 2+, CdCl 2, locker of C 2+ chnnels ws used to tret the cells tht were then exposed to Hnks in the following experiments. CdCl 2 (5 mm) ws pplied to tret the cells for 200 s prior to the stimultion of R-III (100 μg/ml). As shown in Figure 6A1 nd Figure 6B1, [C 2+ ] i nd ph i in Sf9 cells incuted with CdCl 2 showed slight decrese (4.52% nd 3.03% respectively).

10 Molecules 2011, After tretment with R-III, [C 2+ ] i nd ph i rose y 235% nd 40.32% respectively. As indicted in dynmic chnge of trce (Figures 6A- nd B-), [C 2+ ] i incresed immeditely, ut followed y grdul decrese when cells were stimulted y R-III, suggesting tht Cd 2+ grdully locked the C 2+ chnnels to inhiit the C 2+ influx. Figure 6. Effect of C 2+ chnnels lock on [C 2+ ] i nd ph i in R-III-induced Sf9 cells in presence of Hnks. (A1). chnges of [C 2+ ] i in Sf9 cells treted with 0.5 mm CdCl 2 for 200 s nd10 min prior to stimulte with R-III (100 μg/ml), s indicted y reltive chnge of Fluo-3AM fluorescence intensity; (A-). Dynmic chnges of [C 2+ ] i in Sf9 cells treted with 0.5 mm CdCl 2 for 200 s prior to stimulte with R-III (100 μg/ml); (A-). Dynmic chnges of [C 2+ ] i in Sf9 cells incuted with CdCl 2 (5 mm) for 10 min prior to stimulted with R-III (100 μg/ml); (B). ph i profile in cells sujected to the protocol in (A). Results of representtive experiment derived from three repetitions nd 4 6 cells were mesured in ech repetition. The error rs represent men ± SEM for dt derived from vlue of reltive fluorescence intensity in ech time intervl. Tretment mens shring the sme letter were not significntly different from ech other (P < 0.05). The negtive vlue ment decrese of reltive fluorescence intensity in cells. Reltive chnge of fluorescence intensity (%) A.C 2+ CdCl2 R-III CdCl2 R-III Tretment B. ph i CdCl2 R-III CdCl2 R-III Tretment

11 Molecules 2011, Figure 6. Cont. Under these conditions, ph i chnged in similr fshion with [C 2+ ] i. When we used CdCl 2 (5 mm) to incute with Sf9 cells for 10 min to lock the C 2+ chnnels completely, nd then stimulted with R-III (100 g/ml), oth [C 2+ ] i nd ph i decresed shrply y rte of 33.85% nd 48.74% respectively (Figures 6A1 nd 6B1). However, in this condition, we found in dynmic chnge of trce of Figure 6A- nd Figure 6B- tht oth [C 2+ ] i nd ph i showed trnsient elevtion efore decresing shrply. Since Cd 2+ hd locked C 2+ chnnels completely nd inhiited C 2+ influx, the trnsient increse of [C 2+ ] i minly derived from C 2+ relesed from intrcellulr C 2+ stores. It is well estlished tht inositol 1,4,5-trisphosphte (IP3), synthesized in response to externl stimultion, induces the relese of C 2+ from intrcellulr stores [21]. In this ssy, stimultion of R-III my lso induce the synthesis nd increse of IP 3 to promote relese of C 2+ from intrcellulr stores through the C 2+ -ATPse. Otherwise, the C 2+ sustined relese from intrcellulr C 2+ stores my likely give rise to its depletion, which could ctivte store-operted C 2+ chnnels to promote the C 2+ influx in mmmlin non-excitle cells [36], wheres C 2+ chnnels hd een locked completely y CdCl 2, nd no C 2+ entry ut efflux chrcterized y shrp decline of [C 2+ ] i to level fr lower thn sl level occurred in this study (Figure 6A-). Under this condition, we oserved proportionl chnge of ph i with tht of [C 2+ ] i. We hypothesized tht C 2+ relesed from intrcellulr stores through the C 2+ -ATPse in exchnge for H + entry intrcellulr stores resulted in the trnsient increse of ph i, nd the C 2+ -ATPse of PM ws ctivted y the trnsient increse of [C 2+ ] i nd the sustined stimultion of R-III. [C 2+ ] i effused through C 2+ -ATPse in exchnge for H + entry intrcellulr cytosol, which cused the decrese of ph i. The results in this ssy demonstrted tht oth extr clcium influx nd C 2+ relese from intrcellulr C 2+ stores contriuted to the elevtion of [C 2+ ] i in Sf9 cells stimulted y R-III, nd ph i showed proportionl chnge with [C 2+ ] + i in response to the stimultion of R-III. To further clrify the sources of C 2+ nd the response of intrcellulr C 2+ stores in Sf9 cells stimulted y R-III, we repeted the ove experiment with the only different condition of cells eing exposed to Dhnks. As shown in Figure 7A nd Figure 7B, [C 2+ ] i nd ph i of Sf9 cells indicted only slight decline in cse of incution with CdCl 2 for 200s or 10 min. However, the susequent ddition of stimultion y R-III to Sf9 cells fter incution with CdCl 2 for 200 s gve rise to drmtic decrese of ph i y rte of 73.36% nd only slight decrese of [C 2+ ] i (11.55%) (Figure 7A1 nd Figure 7B1). Under this condition, we got the informtion from Figure 7A- tht [C 2+ ] i showed only trnsient increse followed y rpid recovery to the sl level, which explined well the only slight

12 Molecules 2011, chnge of [C 2+ ] i in this time intervl. Since cells were in presence of C 2+ free solution, nd C 2+ chnnels in PM were t lest prtilly locked, the trnsient increse of [C 2+ ] i should minly derive from intrcellulr C 2+ stores, which further prove the contriution of intrcellulr C 2+ stores to the chnges of intrcellulr C 2+. Figure 7. Effect of C 2+ chnnels lock on [C 2+ ] i nd ph i in R-III-induced Sf9 cells in presence of Dhnks. (A1). chnges of [C 2+ ] i in Sf9 cells treted with 0.5 mm CdCl 2 for 200 s nd 10 min prior to stimulte with R-III (100 μg/ml), s indicted y reltive chnge of Fluo-3AM fluorescence intensity; (A). Dynmic chnges of [C 2+ ] i in Sf9 cells incuted with CdCl 2 (5 mm) for 200 s prior to stimulte with R-III (100 μg/ml); (A-). Dynmic chnges of [C 2+ ] i in Sf9 cells incuted with CdCl 2 (5 mm) for 10 min prior to stimulte with R-III (100 μg/ml) for two times; (B). ph i profile in cells sujected to the protocol in (A). Results of representtive experiment derived from three repetitions nd 4 6 cells were mesured in ech repetition. The error rs represent men ± SEM for dt derived from vlue of reltive fluorescence intensity in ech time intervl. Tretment mens shring the sme letter were not significntly different from ech other (P < 0.05). The negtive vlue ment decrese of reltive fluorescence intensity in cells. A.C 2+ B. ph i Reltive increse of fluorescence intensity (%) CdCl2 R-III CdCl2 1-R-III 2-R-III c Tretment Tretment CdCl2 R-III CdCl2 1-R-III 2-R-III c

13 Molecules 2011, Figure 7. Cont. Menwhile, ph i under these conditions lso showed trnsient increse similr with tht of [C 2+ ] i, ut decline finlly to stte much lower thn the sl level (Figure 7B-), which induced high rte of ph i decrese (Figure 7A1) differing from the chnge of [C 2+ ] i. Nevertheless, cells pretreted for 10 min with CdCl 2 (5 mm) presented n mrkedly increse of [C 2+ ] i y rte of 79.43% in the first stimultion of R-III for 200 s, nd the second stimultion of R-III induced [C 2+ ] i decrese y 40.35% (Figure 7A1). Knowing from the dynmic trce in Figure 7A-, [C 2+ ] i in Sf9 cells showed trnsient elevtion followed y grdul decline to sl level in the first stimultion of R-III, which mde the pek of dynmic trce much wider thn tht of Figure 6A-, suggesting tht [C 2+ ] i ws much higher in this time intervl. In contrst, R-III induced decrese of ph i y rte of 39.85% in first stimultion nd 72.78% in the second stimultion (Figure 7B1). We found from Figure 7B- tht ph i in Sf9 cell lso showed trnsient elevtion, ut followed y rpid decline in the first stimultion of R-III. The pek of dynmic trce lso ecme wider compring to tht of Figure 6A-. Although the reson for these phenomenon ws not cler, the results in this ssy further indicted tht ph i showed proportionl chnge with [C 2+ ] i + in response to the stimultion of R-III on the whole. It is known tht the functionl reltionships nd crosstlk etween clcium nd ph receive more nd more ttention, specilly, on humn cells, ut little on insect cells. Although mny studies show tht chnges of ph i re ssocited with tht of [C 2+ ] i in numer of cell types, cler reltionships etween the stedy stte level of ph i nd [C 2+ ] i is not oserved in present ecuse interreltionships etween ph i nd [C 2+ ] i re rther complex nd depend on the cell type [37]. A few studies show tht cytosolic lkliniztion shift is ssocited with the increse of [C 2+ ] i [38] nd tht cidifiction shift is ssocited with the decrese of [C 2+ ] i [39]. More specificlly, n experimentl report on cryfish muscle fire from Kil nd Voipio [40] shows tht resting cytosolic clcium is decresed y intrcellulr lklosis. In present study, the chnges of ph i in Sf9 cells induced y R-III show distinct proportion with tht of [C 2+ ] i, which lso suggests tht cytosolic lkliniztion or cidifiction re ssocited with chnges of [C 2+ ] i, ut the specific interction mechnism of ph i nd [C 2+ ] i in these conditions remins uncler, nd need further reserches to clrify.

14 Molecules 2011, Experimentl 3.1. Regents The Rhodojponin-III (98%) ws isolted y using HPLC from the flowers of R. molle in the Lortory of Insect Toxicology, South Chin Agriculturl University. Fur-3/AM, Snrf1/AM were purchsed from Sigm nd stock solutions of ll molecules were initilly dissolved in dimethyl sulfoxide (DMSO), diluted to their finl concentrtion in phosphte uffer solution (135 mm NCl, 2.7 mm KCl, 1.5 mm KH 2 PO 4, nd 8 mm K 2 HPO 4, ph 7.2) nd stored t 20 C until used. All other chemicls were from stndrd commercil sources nd regent grde or the highest purity Cell Culture Sf9 cells were otined from Stte Key Lortory of Biocontrol, Sun Yt-sen University, nd were cultured in Grce s medium (GIBCO-BRL, Grnd Islnd, NY, USA) supplemented with 10% fetl ovine serum (het-inctivted) nd 1% penicillin/streptomycin. Cells were cultured t 27 C nd sucultured every 3 dys. Confluent cells with >95% viility (tested with Trypn lue exclusion) were used in ll experiments Cell Growth Curve Sf9 cells were seeded onto 24-well pltes ( cells per well). When cells were dherent, t concentrtions of 1, 10,100 nd 200 μg/ml ws dded to treted cells for seril times (0, 12, 24, 48, 72 nd 96 h). Cells tht cultured with 0.1% DMSO t the sme times were used s control. After hrvested, survivl cell numer for ech time point ws counted using Trypn lue exclusion test with stndrd hemocytometer. Growth curve ws mde fter nlyzing the dt Flow Cytometry Sf9 cells were seeded onto 25 cm 2 plstic tissue culture flsk ( cells per flsk). When the density ws cells/ml, cells were treted with R-III t concentrtions of 1, 10, 100 nd 200 μg/ml for seril times (24, 48 nd 72 h). After hrvested, cells were re-suspended nd wshed twice with phosphte uffer solution (PBS) (137 mm NCl, 2.7 mm KCl, 100 mm N 2 HPO 4, 2 mm KH 2 PO 4, ph 7.2). After tht, cells were fixed in cold 70% ethnol, nd stored elow 20 C over night. Prior to nlysis, ethnol ws removed nd fixed cells were wshed twice with PBS (ph 7.2). Cells were re-suspended in PI solution (50 μg/ml PI, 0.1% Triton X-100, 0.1 mm EDTA, 50 μg/ml RNse A) nd incuted for 30 min t room temperture. After tht cells were processed in FACSCliur (Becton Dickinson, USA). At lest cells were counted in ech ssy. The frction of the totl cell popultion presented in G2/M phse ws otined from DNA histogrms using Cell Quest nd Modfit Softwre (Becton Dickinson, USA). All cytometry experiments were performed on cells in log phse of growth. Cells tht cultured with 0.1% DMSO t the sme times were used s control.

15 Molecules 2011, Fluorescence Mesurements Cell tretment: [C 2+ ] i mesurements from Sf9 cells were performed with the fluorescent C 2+ indictor fluo-3/am. Cells were cultured in 35-mm polystyrenetissue culture dishes (Nunc, Denmrk) nd wshed twice with PBS, thn loded with 1 um memrne-perment cetoxymethyl ester of the dye (fluo-3/am) for 45min t 37 C. After dye loding, cells were wshed twice with Dhnks solution ( mm NCl, 5.33 mm KCl, 4.17 mm NHCO 3, mm KH 2 PO 4, mm N 2 HPO 4, 5.56 mm D-Glucose, ph 7.2). The pre-tretment mesurement of phi ws crried out sme s [C 2+ ] i mesurements, except tht the finl concentrtion of dye solution were 10 μm (Snrf1/AM). Fluorescence mesurement: Fluo-3/AM nd Snrf1/AM is one of the most suitle C 2+ nd ph indictors for CLSM, nd widely used to monitor [C 2+ ] i nd ph i in vrious cells. It cn e excited y n rgon ion lser t 488 nm, nd its emitted fluorescence (t wvelengths 520 nm) increses with incresing [C 2+ ] i [41] or ph i. To mesure the Fluo-3/AM nd Snrf1/AM fluorescence, lser scnning confocl microscopy (Leic TCS SP2AOBS, Germny) ws used to scn the cells with good silhouette nd recorded fluorescence t intervls of 6 s for more thn 400 s, nd room temperture ws kept in C during the experiments. According to the experimentl design, drug ws dded from concentrted solutions with pipette directly into the culture dishes through smll hole on top of the cuvette lid, nd in control ssys the sme volume of DMSO ws dded. Results were nlyzed using the Leic confocl softwre, nd got time-dependent curves of clcium fluorescence signl. Although fluorescence recordings could not e clirted to count the solute vlue of [C 2+ ] i [22], [C 2+ ] i chnge could e shown y reltive chnge of fluorescence intensity Sttisticl Anlysis Dt nlysis ws crried out using SAS softwre (SAS Institute Inc.) nd Microsoft Excel softwre. Differences etween the tretments were determined y Tukey s multiple rnge tests (P < 0.05 eing considered significnt). 4. Conclusions R-III displyed strong inhiitory ctivity on the prolifertion of Sf9 cells, nd interfered with the Sf9 cell division cycle nd rrested them in G2/M. In ddition, R-III pertured [C 2+ ] i homeostsis y inducing [C 2+ ] i influx or efflux in Sf9 cells in the presence of C 2+ -contining or C 2+ -free uffer solution. In these conditions, ph i showed proportionl chnges with tht of [C 2+ ] i on the whole. According to the results nd discussion in this pper, we supposed tht there ws certin linkge for chnge of intrcellulr clcium, cell cycle rrest, cell prolifertion inhiition in Sf9 cells induced R-III nd tht cytosolic lkliniztion or cidifiction shifts re ssocited with chnges of [C 2+ ] i level in Sf9 cells induced R-III. Acknowledgements We would like to thnk Ntionl Nture Science Foundtion (Numer ) nd Reserch Fund for the Doctorl Progrm of Higher Eduction of Chin ( ) for providing funds, Xing Jing Qin (School of Life Sciences, Sun Yt-sen University, Chin) for her kind ssistnce in writing.

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