Calcium transport study of SF-9 lepidopteran cells Essay Example

addition, the BSG cells were compared to the SF-9 cells for the parameters that were studied. It was found that the SF-9 cells had a calcium concentration similar to the BSG cells. Furthermore, both cell types showed the same calcium extrusion rates without Na2VO4. However, due to insufficient data, the effects of Na2VO4 could not be statistically analyzed. Based on the available data, it suggests that the Na2VO4 enhanced the rate of calcium extrusion in the BSG cells compared to the SF-9 cells.

It was observed that the calcium extruding capabilities of the SF-9 cell were impaired after two to three applications of Na2VO4, whereas it had noticeable effects on the BSG cells even after four applications. After determining these basic parameters, several questions were raised, including how SF-9 cells extrude calcium and why Na2VO4 affected the calcium efflux in SF-9 cells but not in BSG cells. SF-9 cells might have a calcium pump or exchanger that aids in calcium extrusion, and they could be highly dependent on ATP (adenosine-triphosphate) supply. This differs from BSG cells, as their calcium extrusion was unaffected by Na2VO4. Investigating the mechanism(s) of Na2VO4's action on SF-9 cells could prove useful in agriculture, such as for pest control purposes.

Materials and Methods

Chemicals and solutions, such as 4-Bromo-A23187 and Fura-2/AM, were bought from Molecular Probes in Eugene, OR. Na2VO4 was obtained from Alomone Lab in Jerusalem, Israel. Dimethyl sulfoxide (DMSO) was purchased from J. T. Baker Inc., located in Phillipsburg, NJ.

All reagents, except CaCl2, were obtained from Sigma (St. Louis, MO). The normal Ringer's solution (NRS) contains the following concentrations (mM): 125 NaCl, 5.0 KCl, 2.0 CaCl2, 1.0 MgSO4, 10.0 glucose,

and 10.0 N-[2-hydroxyethyl] piperazine-N’-[2-ethanesulfonic acid] (HEPES). The calcium-free Ringer solution (0CaNRS) is identical to the NRS, except that CaCl2 is replaced with 2.

0 mM ethylene glycol-bis(b-aminoehtyl) ether N,N,N’,N’-tetraacetic acid (EGTA). Fura-2/AM solution was prepared as follows: a stock solution of 1mM fura-2/AM in DMSO was diluted 1:500 in NRS containing 2% bovine albumin. It was then sonicated for 10 minutes. It was then kept frozen until the day of the experiment. 20 SYMBOL 109 f "Symbol" M 4-Bromo-A23187 solution was prepared by diluting a stock of 5mM 4-bromo-A23187 in DMSO 1:250 with NRS. Na2VO4 solution (VO4NRS) contained 100 SYMBOL 109 f "Symbol"M.

Na2VO4 in 0CaNRS.All experiments were performed at room temperature, 22-26 SYMBOL 176 f "Symbol"C.The above solutions were adjusted to pH 7.3 with NaOH.

Cells

BSG cells were obtained as described by Kuffler and Sejnowski (1983). BSG cells were plated and incubated at 3-10 SYMBOL 176 f "Symbol"C for up to 4 days before the experiments. The cells were plated on custom-made 3.5 cm plastic culture dishes.

A round hole, approximately half the diameter of the dishes, was cut in the center and a clear piece was inserted. The clear dishes were then treated with poly-d-lysine for one hour before plating. SF-9 cells (non-transfected) were cultured following Summers and Smith's instructions. The SF-9 cells were plated and incubated on the custom-made dishes, similar to the BSG cells, one day before the experiments. The dishes were not kept longer than two days to prevent excessive cell growth that could impact experimental measurements. Each dish contained roughly 100 SYMBOL 109 f "Symbol"l of cell suspension.

To load the cells with fura-2/AM, 100 SYMBOL 109 f "Symbol"l of fura-2/AM /BSA solution

was added for 30 minutes. Fura-2 is a fluorescence indicator of calcium used to determine free intracellular calcium concentration. In the experiments, fura-2/AM was used instead of fura-2. Fura-2/AM is an ester moiety of fura-2 that can penetrate the cell membrane (unlike fura-2), and is subsequently converted into fura-2 by esterase inside the cell. The apparatus involved a fluorescence microscope unit and a spectrofluorometer system. The fluorescence microscope unit had a 75 W Xenon arc lamp and a Zeiss inverted microscope with a Zeiss Neofluor 63X objective.

Furthermore, for perfusion, a pipette was positioned within 5mm of the sample cells. The pipette administered the solutions at a rate of 2-3 ml/min and had the capability to switch between five different solutions simultaneously. This feature facilitated quick solution switching and enhanced response speed. To measure fluorescence, the PTI Deltascan 4000 microscope system (Photon Technology International Inc., South Brunswick, NJ) was utilized. The emitted fluorescence signal was detected by the photomultiplier tube (PMT) and recorded using a NEC 286 microcomputer. The PTI Instrument Control Program, provided by Photon Technology International Inc., was employed as the software.

The calcium measurement methods used in the experiments were similar to Schwartz et al.'s description. In summary, the formula for determining intracellular free calcium concentration is as follows (Grynkiewicz et al. 1985): [Ca2+]i = Kd.(Fmin/Fmax). (R-Rmin)/(Rmax-R), where Kd represents the effective dissociation constant for the Ca2+-fura-2 complex. Fmin and Fmax are the fluorescence intensities at SYMBOL 108 f "Symbol"=380nm obtained from the calcium-free fura-2 sample and calcium-bound fura-2 sample, respectively. R denotes the fluorescence intensities ratio obtained with excitation at 340 and 380nm (R = F340/F380). Rmin and Rmax are the F340/F380

ratios of the calcium-free and calcium-saturated fura-2 sample, respectively. One cell of average size was randomly selected from each dish for measurement.

The cell suspension was initially washed with NRS to remove the fura-2/AM. When the intracellular calcium level was stable, it was replaced with 2-bromo-A23187 to increase the intracellular calcium concentration. To lower the calcium concentration when it exceeded 200 nM, 0CaNRS was used. After stabilizing the calcium level with 0CaNRS, 4-bromo-A23187 was reintroduced and the entire process was repeated two to four times.

Then 4-bromo-A23187 was once again utilized to elevate the intracellular calcium concentration, with VO4NRS taking the place of 0CaNRS to reduce the calcium concentration. This procedure was repeated for two to four cycles or until the cell displayed no response and was incapable of returning the calcium concentration to its previous resting level. Statistical analysis was conducted using The Student Edition of Minitab release. The results demonstrated that the intracellular calcium concentration in the SF-9 cells was 44.7  8.3 nM (mean  S.E., n = 8) in NRS. Additionally, the calcium concentration in the BSG cells measured 58.2  9.0 nM (n = 4).

According to the Student's t-test (P = 0.31), there was no significant difference in the intracellular calcium concentration between SF-9 and BSG cells. To calculate the rates of active transport of calcium out of the cells after 0CaNRS, linear regression was performed on the declining portion (from 20 to 50 seconds) following the maximum calcium concentration. The rates of calcium depletion (SYMBOL 68 f “GreekMathSymbols”C/SYMBOL 68 f “GreekMathSymbols”t) for BSG and SF-9 cells were found to be 3.92 ± 0.81 nM/s (mean ± S.E., n = 10) and 4.12 ±

...

The size of BSG cells and SF-9 cells differed, with SF-9 cells generally being smaller. When analyzing calcium flux, it is important to consider cell size. The rate at which calcium is depleted can be adjusted by taking into account the volume to surface area ratio of the cells, assuming they are spherical. The flux (J) can be calculated using the equation J = -SYMBOL 68 f "GreekMathSymbols" C/SYMBOL 68 f "GreekMathSymbols"t V/S, where J represents the flux, -SYMBOL 68 f "GreekMathSymbols" C/SYMBOL 68 f "GreekMathSymbols"t is the rate of calcium depletion, and V/S is the volume to surface area ratio of the cell (which simplifies to r/3, where r is the radius of the cell). For BSG cells, the calculated calcium efflux was found to be 2.02 ± 0.44 fmolecm-2s-1 (n = 10), while for SF-9 cells it was measured as 1.33 ± 0.26 fmolecm-2s-1 (n =7). A t-test analysis showed that there was no significant difference between these efflux values (P=0.2). Additionally, after VO4NRS applications were performed on both types of cells, their rates of calcium depletion were determined as follows: BSG cells had a rate of 9.24 ± 0.22 nM/s (n =2), while SF-9 cells had a rate of 2.46 ±0 .75 nM/s (n =3).The adjusted calcium efflux for BSG cells was found to be 6.00 ± 0.14 fmolecm^-2 s^-1 (n=2), while for SF-9 cells it was significantly lower at 0.80 ± 0.24 fmolecm^-^21 (n=3) according to table 2, after adjusting for cell size differences.

Both BSG and SF-9 cells underwent two to three cycles of VO4NRS applications. After these cycles, the SF-9 cells lost their ability to remove calcium, whereas the BSG

cells still retained this ability even after up to three to four VO4NRS applications.

Both cell types were examined for their rates of calcium depletion and calcium efflux when 0CaNRS was added. The experiment involved increasing the intracellular calcium concentration in a single sample cell using 4-bromo-A23187 and then reducing it by introducing 0CaNRS.

The data presented in this study represent the rates of decline (SYMBOL 68 f “GreekMathSymbols”C/SYMBOL 68 f “GreekMathSymbols”t) of the initial linear portion after reaching maximum calcium concentration. The rate of calcium depletion and efflux for BSG and SF-9 cells were measured, with BSG rate of calcium depletion (nMs-1), BSG calcium efflux (fmolecm-2s-1), SF-9 rate of calcium depletion (nMs-1), and SF-9 calcium efflux (fmolecm-2s-1). Similar experiments were conducted using VO4NRS instead of 0CaNRS to lower the calcium concentration. A time course recording was performed on a single SF-9 cell, showing successive applications of 4-bromo-A23187, NRS, 0CaNRS, and VO4NRS. It was observed that after two applications of VO4NRS, the cell's ability to extrude calcium was impaired. In contrast, a BSG cell maintained its ability to extrude calcium even after three applications of VO4NRS at a high calcium concentration. These findings highlight differences between transfected and non-transfected SF-9 cells in terms of their response to different treatments.The report does not include the results, but it states that the transfected cells had a calcium concentration of less than 20 nM.

It was later discovered that the cells were not successfully transfected. T-test results indicated no significant disparity in calcium levels between the BSG cells and SF-9 cells. This implies that the transfection process potentially induced physiological alterations in the cells, resulting in decreased intracellular calcium levels. Additionally,

it was found during the experiment that there was no necessity to administer 4-bromo-A23187 with each cycle for elevating calcium levels. Administering it once at the start of the experiment proved adequate for increasing calcium concentration.

NRS was then utilized to increase the levels of calcium in subsequent cycles. This is likely because 4-bromo-A23187 has a high affinity for lipids, allowing it to distribute into the cell membrane and internal organelles. Therefore, using 4-bromo-A23187 once would permit it to distribute and remain in the cell membrane, acting as an ionophore without the need for further additions. The ability of NRS to increase calcium concentration seemed similar to that of 4-bromo-A23187. This approach was more cost-effective and reduced the impact of DMSO on the cells (which was necessary to dissolve 4-bromo-A23187).

Pressman discusses ionophores in general, while Deber (1985) and Reed and Lardy (1972) focus on the specific topic of 4-bromo-A23187 and its effects on fluorescent probes and calcium. The calcium efflux after VO4NRS treatment was higher in BSG cells compared to SF-9 cells, but there wasn't enough data for a reliable statistical test. Vanadate is known as an inhibitor of active transport.

It acts as a phosphate substitute for ATP and thus inhibits or slows down ATP synthesis. Without ATP, active transport processes cannot take place. In the case of calcium, the introduction of VO4NRS prevents cells from removing calcium after the application of 4-bromo-A23187. This was indeed observed in SF-9 cells, where it was found that the calcium concentration remained elevated and became unstable after two doses of VO4NRS. These findings indicate that ATP production is closely related to the mobilization of calcium in SF-9 cells.

The SF-9 cells were unable to regulate their intracellular level normally without ATP. However, the BSG cells exhibited different responses to VO4NRS compared to the SF-9 cells. Despite the high calcium concentration after the third VO4NRS application, the BSG cell still managed to extrude calcium. This was unexpected since the BSG cells had higher calcium effluxes compared to the SF-9 cells, indicating that ATP production played a crucial role in calcium extrusion for the BSG cells. One possible explanation is that the BSG cells had an excess of organelles for storing calcium instead of extruding it.

It is important to have knowledge of the basic physiology of SF-9 cells because they are commonly used for gene expressions. However, there is still much to learn about these cells. By studying SF-9 cells in greater detail, we can improve our understanding of the calcium transport system and potentially enhance gene expression techniques for molecular biologists.

Thanks to M. Ross for his academic and technical support throughout this study and for kindly reviewing this manuscript. Dr. P. S.

Pennefather provided invaluable advice during this study. B. Clark prepared the BSG culture dishes, and Dr. D. R. Hampson generously gifted SF-9 cells.

References

1. Deber, C. M.; Tom-Kun, J.; Mack, E.; Grinstein, S.Bromo-A23187: a nonfluorescent calcium ionophore for use with fluorescent probes. Anal. Biochem. 146(2): 352; 1985.
2. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J.

Biol. Chem. 260 :3440-3450; 1985. Kuffler, S. W.; Sejnowski, T. J.

Peptidergic and muscarinic excitation at amphibian sympathetic synapses. J.Physiol. 341: 257-278; 1983. Luckow, V. A.; Summers, M.

D. Trends in the development of baculovirus expression vectors. Biotechnology. 6:47-55; 1988.

B.

C. Biological applications of ionophores. Ann. Rev of Biochem. 45: 501-530; 1976.

W.; Lardy, H.A. A divalent cation ionophore. J. Biol. Chem. 247: 6970-7; 1972.

Schwartz, J.-L.; Garneau, L.; Masson, L.; Brousseau, R.

The text discusses the early response of cultured lepidopteran cells to SYMBOL 100 f “GreekMathSymbols”-endotoxin from Bacillus thuringiensis. It highlights the involvement of calcium and anionic channels. The source is a publication titled "Biochem. Biophys. Acta" from 1991, authored by Summers, M. D. and Smith, G. E.

A manual of methods for baculovirus vectors and insect cell culture procedures published by Texas Agric. Exper. Sta. Bull. no 1555 in 1987.