Calcium Transport Term Paper

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PHM499 Research Project

Calcium transport study of SF-9 lepidopteran cells and bull frog sympathetic ganglion

cells

ABSTRACT The intracellular calcium level and the calcium efflux of the bull-frog

sympathetic ganglion cells (BSG) and the SF-9 lepidopteran ovarian cells were

investigated using a calcium-sensitive fluorescence probe fura-2. It was found that the

int

INTRODUCTION

Spodoptera frugiperda clone 9 (SF-9) cells are a cultured insect cell line derived from

the butterfly ovarian tissue. SF-9 cells are used by molecular biologists for the studies

of gene expression and protein processing (Luckow and Summers, 1988). Howe It

was found that the SF-9 cells appeared to have a calcium concentration similar to the

BSG cells. Moreover, the calcium extrusion rates of both cell types with no Na2VO4

added seemed to the same. However, due to insufficient data, the effects of Na2V

After obtaining these basic parameters, many questions raised such as how does the

SF-9 cells extrude their calcium and why the Na2VO4 affected the calcium efflux for

the SF-9 cells but not the BSG cells? The SF-9 cells may have a calcium pump or

excha MATERIALS AND METHODS

Chemicals and solutions

4-bromo-A23187 and Fura-2/AM were purchased from Molecular Probes (Eugene,

OR). Na2VO4 was purchased from Alomone Lab (Jerusalem, Israel). Dimethyl

sulfoxide (DMSO) was obtained from J. T. Baker Inc. (Phillipsburg, NJ). All other

reagents were obtaine The normal Ringer's solution (NRS) contained (mM): 125

NaCl, 5.0 KCl, 2.0 CaCl2, 1.0 MgSO4, 10.0 glucose, 10.0 N-[2-hydroxyethyl]

piperazine-N'-[2-ethanesulfonic acid] (HEPES). The calcium free Ringer solution

(0CaNRS) is the same as the NRS except CaC 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 SF-9 cells (non-transfected) were cultured as described by Summers and

Smith (1987). The SF-9 cells were plated and incubated (at 37 SYMBOL 176 \f

"Symbol"C) on the custom made dishes as used for the BSG cells one day prior to

the experiments. They w Each dish contained approximately 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.

Intracellular calcium measurements

Fura-2 is a fluorescence indicator of calcium that is used to determine the free

intracellular calcium concentration. Fura-2/AM was used in the experiments instead of

fura-2. Fura-2/AM is an ester moiety of fura-2 which has the advantages of being perm

The apparatus included a fluorescence microscope unit and a spectrofluorometer

system.

The fluorescence microscope unit consisted of a 75 W Xenon arc lamp and a Zeiss

inverted microscope with a Zeiss Neofluor 63X objective. In addition, a pipette was

placed close to the sample cells (within 5mm) for perfusion. The pipette delivered the

s The PTI Deltascan 4000 microscope system (Photon Technology International Inc.,

South Brunswick, NJ) was used to make fluorescence measurements. Emitted

fluorescence signal was detected by the photomultiplier tube (PMT) and recorded via

a NEC 286 microc The experimental methods of calcium measurements used in the

experiments were similar to the one described by Schwartz et al. (1991). In brief,

intracellular free calcium concentration can be determined through the following

formula (Grynkiewicz et al.

[Ca2+]i = Kd.(Fmin/Fmax).(R-Rmin)/(Rmax-R)

where Kd is 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 calcium-free fura-2 sample and calcium-bound fura-2 sample

respectively, R is the f One average size cell from each dish was randomly selected

for the measurement. NRS was initially perfused to wash out the fura-2/AM in the cell

suspension. When the intracellular calcium level was stabilized, it was switched to

2-bromo-A23187 to raise

Statistical Analysis

Statistical analysis was performed with using The Student Edition of Minitab release

8 (Minitab Inc., 1991).

Results

It was found that the intracellular calcium concentration in the SF-9 cells was 44.7 ±

8.3 nM (mean ± S.E., n = 8) in NRS. The calcium concentration in the BSG cells was

found to be 58.2 ± 9.0 nM (n = 4). Student's t test did not indicate a significant The

rates of active transport of calcium out of the cells following 0CaNRS were also

calculated. They were determined by performing a linear regression on the linear

portion (ranging from 20 - 50 seconds) of the decline following the maximum calcium

con However, the BSG cells and the SF-9 cells were generally in different sizes in

which the SF-9 cells (about 15-20 SYMBOL 109 \f "Symbol"m in diameter) were

usually smaller in sizes relative to the BSG cells (about 25-40 SYMBOL 109 \f

"Symbol"m in diam

J = -SYMBOL 68 \f "GreekMathSymbols"C/SYMBOL 68 \f

"GreekMathSymbols"t ·V/S

where J is 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 of the cell (V/S can be further simplified to r/3 where r is the radius of the

cell).

The calculated calcium efflux of the BSG cells and the SF9 cells were 2.02 ± 0.44

fmole·cm-2·s-1 (n = 10) and 1.33 ± 0.26 fmole·cm-2·s-1 (n = 7) respectively (table 1).

There was no significant difference between the two efflux values (P = 0.2) shown b

Similarly, the rates of calcium depletion of the BSG cells and the SF-9 cells following

VO4NRS were 9.24 ± 0.22 nM/s (n=2) and 2.46 ± 0.75 nM/s (n=3) respectively. The

adjusted calcium efflux of the BSG cells and the SF-9 cells were 6.00 ± 0.14 fmole·cm

In addition, it was observed that SF-9 cells lost the ability to extrude the calcium after

two to three cycles of VO4NRS applications (Figure 1). On the other hand, the BSG

cells did not appear to lose their abilities to extrude the calcium after up to

Table 1 Rate of Calcium depletion of BSG and SF-9 cells after the addition of

0CaNRS

BSG rate of calcium depletion (nMs-1)

BSG calcium efflux (fmole·cm-2·s-1)

SF-9 rate of calcium depletion (nMs-1)

SF-9 calcium efflux (fmole·cm-2·s-1)



2.23

1.01

4.67

1.51



0.54

0.24

4.10

1.33



4.36

1.98

3.19

1.03



8.58

3.89

7.74

2.51



5.88

2.67

5.55

1.80



1.28

5.81

2.01

0.65



5.28

2.40

1.56

0.50



7.02

4.55







2.22

1.44







2.27

1.47









Intracellular calcium concentration of a single sample cell was raised using

4-bromo-A23187 and was subsequently lowered by introducing 0CaNRS. These

data represented the rates of decline (SYMBOL 68 \f

"GreekMathSymbols"C/SYMBOL 68 \f "GreekMathSymbol

Table 2 Rate of Calcium depletion of BSG and SF-9 cells after the addition of

VO4NRS

BSG rate of calcium depletion (nMs-1)

BSG calcium efflux (fmole·cm-2·s-1)

SF-9 rate of calcium depletion (nMs-1)

SF-9 calcium efflux (fmole·cm-2·s-1)



9.02

5.85

1.05

0.34



9.47

6.14

3.59

1.16







2.74

0.89





Similar to Table 1 except VO4NRS was used instead of 0CaNRS to lower the

calcium concentration.



Figure 1. Intracellular calcium concentration of a SF-9 cell

A time course calcium recording of a single SF-9 cell (19 SYMBOL 109 \f

"GreekMathSymbols"m) with the successive applications of 4-bromo-A23187, NRS,

0CaNRS and VO4NRS. It was noted that after 2 applications of VO4NRS, the cell

was impaired in its abil Abbreviations: A, 4-bromo-A23187; N, NRS; 0, 0CaNRS; V,

VO4NRS.



Figure 2. Intracellular calcium concentration of a BSG cell

In contrast to the SF-9 cell in Figure 1, the BSG cell (39 SYMBOL 109 \f

"GreekMathSymbols"m) still maintained its ability to extrude (or decrease) calcium

after three applications of VO4NRS even at a high calcium concentration.

Abbreviations: same as in Figure 1.

DISCUSSION

In the beginning of the experiment, both the transfected and non-transfected SF-9

cells were used although only non-transfected SF-9 cells were reported here. It was

found that the transfected cells had unusual low calcium concentration (less than 20

nM Moreover, it was learned during the experiment that it was not necessary to apply

4-bromo-A23187 every cycle to raise the calcium level. It was only necessary to apply

once in the beginning of the experiment to raise the calcium concentration. NRS was

The calcium efflux after VO4NRS for the BSG cells appeared to be greater than the

SF-9 cells' (see result section). But there were insufficient data to perform a reliable

statistical test to prove such view. Vanadate is referred to an active transport Since

the SF-9 cells are commonly used for gene expressions, it is important to know the

basic biophysiology of these cells. However, there is still a lot unknown about these

cells. By studying these cells in greater details, it will improve our unders

Acknowledgments

I thank Dr. S. M. Ross for his academic and technical supports throughout this study,

and for kindly reading this manuscript. Dr. P. S. Pennefather was invaluable in

providing excellent advice during this study. I also thank B. Clark for preparing the B

References

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calcium ionophore for use with fluorescent probes. Anal. Biochem.

146(2):349-352;1985.

Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. A new generation of Ca2+ indicator 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.

Pressman, B. C. Biological applications of ionophores. Ann. Rev of Biochem.

45:501-530; 1976.

Reed, P. W.; Lardy, H. A. A23187: A divalent cation ionophore. J. Biol. Chem.

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Schwartz, J.-L.; Garneau, L.; Masson, L.; Brousseau, R. Early response of cultured

lepidopteran cells to exposure to SYMBOL 100 \f "GreekMathSymbols"-endotoxin

from Bacillus thuringiensis: involvement of calcium and anionic channels. Biochem.

Biophys.

Summers, M. D.; Smith, G. E. A manual of methods for baculovirus vectors and insect

cell culture procedures. Texas Agric. Exper. Sta. Bull. no 1555; 1987.

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