Calcium Transport Essay, Research Paper
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
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):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.
247:6970-7; 1972.
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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