Membrane binding and uptake of diflubenzuron in a cell line from Manduca sexta (L.)код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 4:169-182 (1987) Membrane Binding and Uptake of Diflubenzuron in a Cell Line From Manduca sexfa (L.) Gabriele E. Klitschka, Wolfgang Witt, and Richard T. Mayer U.S. Horticultural Research Lab, Agricultural Research Service, U.S. Department of Agriculture, Orlando, Florida The binding and accumulation of the chitin synthesis inhibitor diflubenzuron (DFB) by a cell line derived from embryonic tissue of the tobacco hornworm, Manduca sexta (L.), was analyzed. A rapid and reversible binding to viable and nonviable cells suspended in the culture medium was observed at soluble concentrations of DFB for short exposure periods. Scatchard analysis gave no indication of a saturable uptake mechanism. The DFB-binding capacity of intact cells was found to be similar to that of a crude membrane preparation (70,OOOg pellet); however, plasma membrane-enriched fractions bound almost three times as much DFB as the homogenate. Repetitive shorttime incubations (up to 3 h) of suspended cells with DFB resulted in a stepwise intracellular accumulation of DFB. Treatment of growing cells with DFB at high concentrations (50 pM) of DFB for longer periods (up to 7 days) resulted in elevated intracellular accumulation of DFB, which exceeded the binding capacity of the cell membranes and the aqueous solubility of DFB. These results indicate that the intracellular crystals detected by transmission electron microscopy are precipitated DFB. N o metabolites or other chemically modified products of intracellular DFB were detected by high pressure liquid chromatography (HPLC) after a 7-day incubation. Key words: insect growth regulators, binding, plasma membrane, chitin INTRODUCTION The chitin synthesis inhibitor diflubenzuron (N-[[(4-chlorophenyl)amino]carbonyl]2,6-difluorobenzamide)interferes with the deposition of chiAcknowledgments: We thank Mr. Robert E. Droleskey for the electron microscopic material and Mr. Randy Smith for excellent technical assistance. G.E. Klitschka was supported by the Alexander von Humbolt-Foundation as a Feodor Lynen-Postdoctoral Fellow. This paper reports the results of research only. Mention of a pesticide in this paper does not constitute a recommendation by the U.S. Department of Agriculture nor does it imply registration under FIFRA. Received May 16,1986; accepted July 7,1986. Address reprint requests to Dr. R.T. Mayer, USDA, ARS, 2120 Camden Road, Orlando FL 32803. 0 1987 Alan R. Liss, Inc. 170 Klitschka, Witt, and Mayer tin in the insect cuticle [I]; however, there is disagreement about the exact biochemical mode of action [2-9]. Initially investigators suggested that DFB* directly interacted with chitin synthase . The direct interaction of DFB with chitin synthase was disproven by Mayer et al.  and Cohen and Casida [lo], who found that cell-free preparations of this enzyme were not inhibited by DFB. Recently, another site of action for DFB was proposed. Based on the finding that the thymidine incorporation into the DNA of pupae of Stornoxys cuEcitvarzs (L.) was inhibited, Deloach et al.  suggested that DFB modified membrane properties such that the permeability for certain substances, eg, nucleosides, was changed. This idea was supported by the finding that DFB inhibited the uptake of nucleosides into Harding-Passey melanoma cells [ll] and into cells from Murzducu sexta (L.) [l2]. Bishai and Stoolmiller  showed that DFB was taken up by rat glial cells and probably bound to cell membranes. They also found that DFB was not further metabolized during the 5-day incubation period. But, thus far, data on the affinity of DFB for biological membranes are unavailable. The cell line used here responds to DFB in several ways. In addition to the effect on the nucleoside transport [ll, 121, chitin synthesis is inhibited , growth rate is reduced, and morphological changes are observed (ie, normal flat cells withdrew to clumps of cells upon exposure to DFB) . In a recent communication , crystals were observed via transmission electron microscopy in cells treated with high concentrations of DFB. The results presented here indicate that these crystals are DFB. In addition, the mechanism of the intracellular accumulation of DFB and data on the membrane affinity are presented and discussed. MATERIALS AND METHODS Cell Line and Culture Condition The insect cell line CH-MRRL was established from embryonic tissue of the tobacco hornworm, Manducu sexta (L.)  and was obtained from E.P. Marks (Fargo, ND). The cells were grown in Yunker’s variation  of Grace’s medium (Gibco, Grand Island, NY) containing 100 IUlml penicillinG-potassium, 100 pglml streptomycin sulfate, and 0.1% gentamycin in 150cm2 flasks at 27-29°C. Antibiotics were purchased from Sigma (St. Louis, MO) or Gibco. Preparation of Crude Cell Membranes Cells grown for 7 days were harvested by centrifugation at 200g. The pelleted cells were washed twice by resuspending in 20 ml Grace’s medium at room temperature and recentrifuging. Finally, pelleted cells were resus- *Abbreviations: DFB = diflubenzuron; DMSO = dimethylsulfoxide; HPLC = high pressure liquid chromatography; SDS = sodium dodecylsulfate; PAGE = polyacrylamide gel electrophoresis; TEM = transmission electron microscopy; TRIS = tris (hydroxymethyl) aminomethane. Diflubenzuron in a Manduca sexfa Cell Line 171 pended in 5 ml Grace's medium. Aliquots of the cell suspension were removed for the determination of cell populations, viability, and the DFB binding andlor content. Membranes were pre ared by homogenizing an aliquot of the cell suspension (4.8 ml, 16-22x106 F cellslml) twice for 20 s at 0°C with a Polytron PT homogenizer equipped with a 10l35 generator (Brinkman Instruments, Westbury, NY). Three milliliters of the homogenate were diluted with 5 ml of cold Grace's medium and centrifuged for 30 min at 70,OOOg. The pellet was resuspended in 8 ml Grace's medium by rehomogenizing (as above) and centrifuged at 70,OOOg for 1h. The membranes were washed once more by repeating this last step. The membrane pellet was finally resuspended in Grace's medium and the volume adjusted to 6 ml. Preparation of Plasma Membranes Cells, 7 days old, were harvested by gently pipetting them from the flasks, and were washed three times in Puck's saline A by centrifugation at 2008 for 5 min at 2°C [lq.The pellet was resuspended in 10 mM TRISIHCI (pH 7.4) at 2 x 10' cellslml, and the cells were allowed to swell for 30 min at 2°C. Cells were disrupted by sonication (Kontes, micro ultrasonic cell disrupter) for 30 s, and the unbroken cells were separated by centrifugation at 1,5008. The supernatants (homogenates) were adjusted to 1mM EDTA and layered on top of a sucrose gradient composed of 50%, 40%, 30%, and 20% (wlw) sucrose in 10 mM TRISlHCl (pH 7.4) and 1 mM EDTA. Centrifugation (50,OOOg) was performed in a Beckman L8-70 ultracentrifuge, using an SW 28 rotor, for 16 h at 4°C (50,OOOg). The fractions were collected and dialyzed 20 h against 10 mM TRISIHCI (pH 7.4) containing 1 mM EDTA. Finally, the fractions were pelleted at 50,OOOg and resuspended in a known volume of 10 mh4 TRIS/HCI (pH 7.4), 1mM EDTA. The identification and purity of the fractions were determined with marker enzymes. Acid phosphatase (EC 22.214.171.124.), a presumptive marker of lysosomes, was tested with a kit from Sigma. NADPH cytochrome c reductase (EC 126.96.36.199.) was measured according to Weirich and Adams [IS], and cytochrome c oxidase (EC 188.8.131.52) activity was determined using 100 pM cytochrome c in 50 mM potassium phosphate at pH 7.8. The ouabain-sensitive Na+lK+-ATI'ase (EC 184.108.40.206.) assay was adapted from Buff and Brundl  with 3.5 mM ATP, 1mM ouabain, and 20 mM TRISIHCI, pH 7. The cholesterol content was measured with a test kit from BoehringerlMannheim (West Germany). Long-Term Exposure of Growing Cells to DFB Cells (3x lo6) were seeded in 25-cm2 tissue culture flasks containing 3 ml culture medium. On day 1, 7.5 pl of a solution of [3H]DFB (230 mCilg; DuPhar-Solvay, B.V., The Netherlands, purified by TLC) in DMSO (50 pM, 0.06-0.18 pCi/ml) were added to cell cultures.Controls containing only DFB pure; Thompson-Hayward Chemical Co., Kansas City, KS) and culture media were run in parallel to detect any nonspecific adsorption of DFB to culture flasks or test tubes. After an incubation period of 6 days at 27-29"C, 172 Klitschka, Witt, and Mayer the cells were washed twice with 3 ml culture medium, harvested, and counted using a hemocytometer. In some cases they were allowed to continue growing in the same medium but without DFB. The harvested cells were washed four times by resuspension in Grace’s medium (4 ml) and recenhifugation at 5008 for 5 min. Concentrations of [3H]DFB in the pellets and the supernatants were determined by liquid scintillation counting. Short-Term Incubations of Cells, Crude Membranes, and Cell Fractions With DFB The insect cells were cultured, harvested, washed, and resuspended in Grace’s medium at room temperature as described above. Aliquots were removed for cell counting and protein determinations. The cells were either stored at -20°C or were used within 4 h for incubation with DFB. Cells used for this purpose were pelleted by centrifugation at 1,OOOg for 10 min and resuspended in culture medium containing [SHIDFB. Culture medium solutions of DFB were prepared by adding aliquots of [3H]DFB stock solutions in DMSO to Grace’s medium at 29°C for 10-60 min. Actual DFB concentrations were determined by liquid scintillation counting. This procedure was necessary since significant amounts of DFB adsorbed to the surface of the glass tubes at the low concentrations applied here. Concentrations and specific activities of C3H]DFB in the repetitive incubation experiments were 0.23 and 0.44 pM (56.7 and 28.3 pCi1mmol) at the beginning of the tests. In all other short-term incubation experiments [3H]DFB with a specific activity of 56.7 pcilmmol was used. The cells or the cell fractions ( 2 . 5 ~ 1 0cells ~ or the equivalent amount of protein (0.165 mg) from the membrane suspensions) were incubated at 29°C in 5 ml Grace’s medium, containing 0.2 pM f3H]DFB.After 5 min, 2 ml were applied to glass fiber filters (Gelman type AIE, 25mm, Gelman Science Inc., Ann Arbor, MI). The filters themselves and the filtrates were analyzed for radioactivity. Controls (radioisotopes and medium only) were performed to determine adventitious binding of [3H]DFJ3. Efficacy of the filtration process was controlled by determination of protein in the filtrates. Analysis of DFB by HPLC DFB-treated and control (without DFB) cells were washed twice with Puck’s saline A and homogenized in 5 ml acetonitrile with a Polytron PT homogenizer equipped with a 10135 generator. The homogenates were filtered through a sintered glass funnel, washed twice with acetonitrile (5 ml), and evaporated to dryness under vacuum. The residues were dissolved in acetonitrile: H20 (10190) applied to a Sep-Pak C18 cartridge (Waters Associates, Milford, MA). After the cartridge was washed with 5 ml acetonitrile: H20 (10/90), DFB was eluted with 5 ml acetonitrile. The solvent was evaporated and the residue dissolved in 200 pl acetonitrile for HPLC analysis  using an LDClMilton Roy HPLC (Riviera Beach, FL) equipped with a 3.9 x300-mm p-Bondapak C18 (Phenomenex, Palos Verdes Estades, CA) column. Twenty microliters of the cell extract were injected and fractions (30 s) Diflubenzuron in a Manduca sexta Cell Line 173 were collected after elution with acetonitrile: H20 (57/43)at a flow rate of 1.5 mllmin. Fractions were collected in scintillation vials, evaporated, and analyzed for radioactivity after adding scintillation cocktail. When analyzed by HPLC, standard solutions of DFB in acetonitrile gave a major peak (97%) with a retention time of 7.5 min and a minor peak (3%) at 9.1 min, which may be either an impurity or an isomer. Analytical Methods Radioactivity was measured with an LKB 1219 Rackbeta scintillation counter and was quench-corrected. [3H]DFB samples (G0.75 ml) were mixed with 10 ml scintillation fluid (PCS, Amersham Co, Arlington Heights, IL). Counting efficiency was 48%. ~) to Ways and Lipids were extracted from frozen cells ( 1 0 0 ~ 1 0according Hanahan following extraction procedure 2 . Phospholipids were determined by the procedure of Broekhuyse . SDS-polyacrylamide slab gel electrophoresis followed a modified version of the discontinuous system described by Laemmli  with 8% running gels ~241. Protein was determined by the procedure of Bradford  using bovine yglobulin standards. Transmission electron microscopy of cells grown for 7 days with DFB was conducted as described recently . RESULTS Purification and Characterization of Plasma Membranes The centrifugation of sonicated cells on the discontinuous sucrose gradient led to three distinct bands, located at the 5040% (fraction l), 40-30% (fraction 2), and 30-20% (fraction 3) interfaces. A minor band was present at the top of the 20% sucrose layer and was numbered fraction 4. Analysis of the fractions showed ouabain-sensitive Na+/K+ -ATPase to be concentrated in fraction 3 (Table l), which also contained the highest amount of cholesterol. Cytochrome c oxidase, a marker for mitochondria, was found to be enriched in fractions 1 and 2. NADPH cytochrome c reductase, a marker for endoplasmic reticulum, was concentrated in fraction 2. Acid phosphatase was TABLE 1. Analysis of Total Homogenates and Cell Fractions of Munducu sextu L. Cells Fractiona Homogenate 1 2 3 4 NADPH Ouabain-sensitive Cytochrome Cytochrome Acid c reductase c oxidase phosphatase AWase Cholesterol (nmol/min/mg)b (pg/mg)b (pmol/min/mg)b (pmol/min/mg)b (pmol/min/mg)b 1.4 9.9 2.6 16.4 2.2 4.4 f 2.3 6.1 f 1.5 15.1 It 6.2 26.4 f 9.9 12.6 f 0.5 4.75 f 0.8 9.85 f 0.2 15.45 f 1.6 8.95 f 2.9 8.90 f 2.6 12.5 f 0.2 87.6 f 26.0 53.0 f 7.0 5.6 f 1.5 12.7 f 6.6 0.83 It- 0.14 0.52 f 0.10 1.54 f 0.11 0.79 f 0.12 1.90 f 0.88 'Fractionations were performed four times with two to five replicate enzymes or lipid analyses per fractionation. bmg = mg protein. 174 Klitschka, Witt, and Mayer equally distributed in fractions 2 and 4. This one purification step led to a sixfold enrichment in cholesterol and 11.7-fold in ouabain-sensitive Na+/K+ -ATPase, indicating the presence of plasma membranes in fraction 3. Fraction 3 was almost free of lysosomal and mitochondria1 contamination and contained some endoplasmic reticulum (Table 1). Short-Term Incubations With DFB to Determine Accumulation, Loss, and Uptake Mechanism Binding of DFB to cells and crude membrane preparations is compared in Table 2. Almost the same binding capacity was found with fresh, viable cells and frozen, nonviable cells. Nearly 75% of the DFB in fresh cells also bound to membranes prepared from these cells when incubated similarly. Table 2 also shows the binding of DFB to the cell fractions. Fractions 1and 4 had a binding rate similar to that of the homogenate, but in fractions 2 and 3, a 22.5-fold increase of binding was observed. This suggests that plasma membranes (fraction 3) have a high affinity for DFB. Considering that this was not a pure plasma membrane preparation, the increase in binding is fairly large. Fraction 2, identified as endoplasmic reticulum enriched, also shows a high level of cholesterol, which is indicative of the presence of membranes. The DFB-binding capacity of the isolated crude membranes and the cell fractions may be underestimated, since ca 18% of the membrane protein applied to the filters was detected in the filtrate. The amount of intracellular DFB for fresh cells could be increased by repeated incubations with low concentrations of DFB (Fig. 1).After the fifth incubation, the DFB content of the cells was raised about 1.5-fold the amount of DFB available at each incubation. A concomitant reduction of the DFB TABLE 2. Binding of DFB to Fresh, Frozen, and Cell Fractions of Munducu sextu (L.) Cells* pmol DFB/106cells Fresh cellsa Frozen cells Crude membranes Homogenate Fraction 1 Fraction 2 Fraction 3 Fraction 4 85.2 f 5.8 73.8 f 4.3 63.6 f 5.5 19.8 f 6.4 27.5 f 7.7b 39.3 f 7.5c 53.2 f 10.0' 22.2 f 5.9b *DFB concentration was 200 nM. Significance for the fractions was calculated by Student's t-test using the homogenate as the control value. Results reported as X f SD, n = 4. aFresh and frozen cells and crude membrane preparations were obtained using a polytron. All other preparations were obtained using a sonic cell disruptor. The two methods caused differences in the binding data. b~ < .5, not significant. 'P < .OOl, considered significantly different from control value. Diflubenzuron in a Manduca sexfa Cell Line FRESH CELLS 175 1 -0.6 -! 4 c z 4 c -0.42 a W a a v) f m u. -0.2 51. E Fig. 1. Repetitive incubation of fresh, viable insect cells with DFB and subsequent wastiings ~ ) incubated in 1 ml Grace's to determine accumulation and loss of DFB. Cells ( 3 ~ 1 0 were medium as described in Methods with 13H]DFB (230 nM, open symbols; 440 nM, filled symbols) for 5 min. Twenty microliters of the cell suspension were removed during this time for the determination of radioactivity. The cells were pelleted by centrifugation at 500g for 5 min. Aliquots (200 pl) of the supernatant were removed to determine the DFB concentration ( A ) by liquid scintillation counting. Subsequently, the supernatant was removed completely and the cells were resuspended in Grace's medium with the same DFB concentrations. Twenty microliters of the suspension were removed to determine the amount of [3H]DFB. After a second incubation for 5 min, the cells were centrifuged as before to measure the DFB concentration in the supernatant. These incubation/centrifugation cycles were repeated 10 times. Then the incubation continued for another five cycles at otherwise unchanged conditions except that DFB was omitted (arrow). The cell-bound DFB (0) was calculated for every incubation cycle by subtraction of the 3H content of the cell suspensions and the corresponding supernatants. concentrations in the supernatants was observed. The DFB concentration of the supernatants was constant and equal to the concentration of the added DFB solution for the next five incubations (ie, incubations 6 to 10). The cells showed no further change in the DFB content (ie, for incubations 6-10), indicating that the net accumulation had ceased. The reduced DFB content of fresh cells (Fig. 1)during the last incubation cycles probably resulted from fractionation and incomplete centrifugation of broken cells. The maximum amounts of cell-bound DFB obtained by this procedure were 0.10 nmol/106 cells at an external concentration of 230 nM and 0.21 nmol/106 cells at 440 nM. Five subsequent incubation cycles without DFB almost completely released the radioactivity (Fig. 1).Similar results were obtained for frozen cells (data not shown). The analysis of the binding of DFB to fresh or frozen cells by Scatchard plots gave no indication for a saturable uptake mechanism (Fig. 2, frozen cell plots not shown). In accordance with the finding that shortterm incubations at low concentrations resulted in a rapid, reversible bind- 176 Klitschka, Witt, and Mayer 5 - 4. 8 : 0 0 v ., A 0 32- 1- Fig. 2. Scatchard-plot of the DFB binding to insect cells to determine if a saturable uptake mechanism exists. The binding of 13H]DFB to freshly harvested cells was analyzed by the procedures described in Materials and Methods. Cells (3x 106/ml) were incubated with [3H]DFB in concentrations of 43-520 nM. ing, this result suggests that DFI3 occupies cellular binding sites by simple diffusion. Long-Term Incubations With DFB in Cell Cultures to Determine Accumulation and Loss of DFB In another series of experiments, cells were grown at standard culture conditions and were treated with DFB in concentrations that affect nucleoside uptake . After an incubation of 6 days with 50 pM DFB, 52% of the applied radioactivity was recovered in the cells (Fig. 3). This means that lo6 cells contained 14.7 nmol of DFB at these conditions. DFB was not as easily removed from the cells as were the smaller amounts incorporated during the short-term experiments. Four washings by centrifugation and resuspension of cells grown for 6 days in the presence of DFB reduced the intracellular content by 40%, but a large amount (32%of the originally applied DFB) was still retained (Fig. 3A). Nearly the same reduction of the DFB content was found when the cells were grown for an additional 7 days in fresh growth medium without DFB with five intermediary changes of medium (Fig. 3B). Diflubenzuron in a Manduca sexfa Cell Line 177 - * 60 m 50LL n n 40- a a 0 30- A 20- B 0 0 - z $ 10- 2 8 1 I s 2 3 WASHINGS 8 4 Fig. 3. Accumulation and removal of DFB in cultured insect cells after long-term incubations with DFB. Cells were grown in the presence of [3H]DFB for 6 days as described in Materials and Methods and harvested immediately (A). They were then grown for another 7 days in DFB-free medium with five intermediary changes of the growth medium and then harvested (B), or were harvested after a growth of 14 days with ten intermediary changes in DFB-free medium following the initial growth period with [3H]DFB(C). The DFB content of the freshly harvested cells and after four washings by centrifugation and resuspension in Grace’s medium, expressed as percentage of the originally applied amount of DFB, is presented. The DFB content of these cells was further reduced by subsequent washings, but less rapidly than after the 6 days‘ incubation. Even when the experiment was extended to 14 days with ten changes of the growth medium after the initial 6 days’ incubation with DFB, a large amount (16% of the originally applied 150 nmol DFB) was detectable. This amount was only slightly reduced by subsequent washings of the harvested cells (Fig. 3C). These results show that DFB incorporated during long-term incubations at high concentrations is not freely exchanged with extracellular DFB. The results also indicate that DFB is stored differently in the cells for the longterm studies than for the short-term, low-concentration experiments. In addition, cell growth (ie, increase in cell population) had no effect on the DFB content. DFB Metabolism in Cultured Cells DFB was extracted from cells grown in the presence of 50 pM DFB and analyzed by HPLC to determine whether a chemical or metabolic modification of DFB occurred under long-term incubation conditions. Figure 4 shows 178 Klitschka, Witt, and Mayer i 20000 Fig. 4. Chromatogram of HPLC analysis of a DFB extract from Manduca sexta cells. Cells were grown for 7 days with 50 pM [3H]DFB, harvested, washed, and extracted with acetonitrile. Details of the extraction and analysis procedures are given in Materials and Methods. Absorbance at 254 nm. Radioactivity = -a-. the elution pattern of the separation with a reversed phase column. The radioactivity eluted as a single peak with a retention time of 7.5 min, which corresponded to the major DFB peak in the standard. The other minor UVabsorbing peaks represent cellular substances, which were coextracted with DFB during the procedure. No major UV-absorbing peaks were detected between retention times of 3-13 min in extracts from cells grown without DEB (not shown). The same results were obtained with extracts from cells grown with DFB for 6 days and cultured subsequently for 14 days in a DFBfree medium with ten intermediary changes of the growth medium. Further Characterizationof DFB-Treated Cells Lipids were extracted from the cells and quantified to estimate the hydrophobic binding capacity of the cells. The phospholipid content was determined to be 0.32 nmol/106cells. Analysis of complete cell protein, membrane protein, and soluble protein by SDS-PAGE at reducing conditions gave no indication for changes in the overall protein pattern of cells treated with DFB in long-term incubations (not shown). The transmission electron microscopy studies of a recent communication  were continued to further elucidate the intracellular storage of DFB. It was previously assumed  that the crystals in DFB-treated cells repre- Diflubenzuron in a Manduca sexta Cell Line 179 sented precipitated DFB. This assumption was based on the evaluation of a large number of specimens. Long-term incubations of the cells with high concentrations of DFB always produced observable, intracellular crystals. In addition, crystals associated with lysosomes were also frequently observed (Fig. 5 ) . The accumulation and initial precipitation of DFB in the lysosomes may result in the formation of the large crystals reported previously . DISCUSSION A rapid and completely reversible binding of DFB was observed in shortterm incubations with low concentrations of DFB. No indications for the existence of a specific and saturable uptake mechanism were found by Scatchard analysis. These results support the view that a free diffusional exchange of the external, soluble, and the cell-bound DFB occurs at these conditions. Fig. 5. Transmission EM of cells grown for 7 days in the presence of inside lysosomes, b: Crystals in cytoplasm. x 10,336. DFB (50 pM). a: Crystals 180 Klitschka, Witt, and Mayer Since DFB is a hydrophobic substance which has a low water solubility, the intracellular binding sites should be mostly membrane lipids and other hydrophobic membrane areas. The cell-fraction binding experiments showed that the purified plasma membrane fraction bound almost three times as much DFB as the homogenate. Also the binding to the endoplasmic reticulum-enriched fraction was significantly higher. The high level of DFB binding to plasma membranes supports the theory that DFB affects the cell membrane in such a way that chitin synthesis/ deposition is inhibited [6-81. The observation that DFB appears to have an affinity for plasma membranes becomes more significant when one realizes that chitin synthase is probably attached to the plasma membrane of insect epidermal cells. This conclusion is supported by microscopic studies of insect epidermal cells which suggest that chitin microfibrils originate at the cell surface  and by reports on yeast chitin synthase which conclusively show that the enzyme is attached to the plasma membrane [27,28]. Cells containedlbound larger amounts of DFB when incubated long-term with high concentrations of DFB. The DFB concentration used exceeded the solubility of DFB in water almost 100-fold and resulted in an almost complete precipitation of DFB in the culture flasks. Consequently, the cells were continuously exposed to a saturated DFB solution. This situation was simulated by the repetitive incubation experiments of Figure 1.The precise extent of incorporation during the long-term incubation cannot be determined from these experiments, since the cells fragmented after several consecutive incubations. The experiment does demonstrate that the continuous contact to DFB in soluble concentrations results in a cellular accumulation. We assume that during long-term incubations with high DFB concentrations the incorporation of DFB proceeds first by diffusion into cellular hydrophobic domains. However, this process cannot explain the accumulation of the large amounts of DFB after a 6-day incubation, since the cells do not have adequate hydrophobic binding sites. The amount of intracellular DFB exceeded the phospholipid content of the cells 42-fold. Klitschka et al.  suggested that the intracellular crystals represent precipitated DFB. The results presented here support this assumption. The simple fact that the hydrophobic binding capacity of the cells for a highly insoluble compound such as DFB is limited has already been mentioned above. In addition, the large amount of intracellular DFB was not as rapidly exchanged with the extracellular medium as was the small amount bound to the cells after short-term incubations with low DFB concentrations (Fig. 3). Growth of the cells for 14 days in DFB-free medium with ten intermediary changes of the growth medium reduced the DFB content of the cells by 84%. Probably, small amounts of the intracellular-precipitated DFB were dissolved and exited the cell by diffusion with each change of the medium until a new equilibrium was established. The metabolic or chemical modification of DFB to more water-soluble or reactive compounds can be excluded since DFB itself was the only radiolabeled compound detected by HPLC in extracts of cells treated for 6 days with DFB (Fig. 4). The mechanism of the intracellular crystal formation is not yet clear. Phagocytosis of extracellular crystals as the main mechanism can be excluded Diflubenzuron in a Manduca sexfa Cell Line 181 since this event was only rarely observed by TEM. We suggest another membrane-dependent process. Extracellular DFB may occupy hydrophobic binding sites in the plasma membrane and is then further transported into the cell. There the accumulated amount of DFB exceeds the solubility limit and DFB starts precipitating out of the cytoplasm. It may also be transported to lysosomes and then form crystals inside the lysosomes, a situation that was detected frequently by TEM (Fig. 5). The high affinity of DFB supports the suggestion that DFB interferes with membrane processes and that the primary site of action will be found here and not in the inhibition of the chitin synthesis itself [8,11,12]. LITERATURE CITED 1. 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