Calcium Transport In SF-9 and Bull Frog Ganglion Cells
Calcium transport study of SF-9 lepidopteran cells and bull
frog sympathetic ganglion cells

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 intracellular calcium levels were 58.2 and 44.7 nM
for the BSG cells and SF-9 cells respectively. The calcium
effluxes following zero calcium solution were 2.02 and 1.33
fmole·cm-2·s-1 for the BSG cells and SF-9 cells. The
calcium effluxes following sodium orthovanadate (Na2VO4) in
zero calcium solution were 6.00 and 0.80 fmole·cm-2·s-1 for
the BSG cells and the SF-9 cells. The SF-9 cells also lost
the ability to extrude intracellular calcium after 2-3
applications of Na2VO4 while the BSG cells showed no
apparent lost of calcium extruding abilities for up to 4
applications of Na2VO4.
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). However, there is not much
known about these cells' basic biophysiology. Since calcium
is involved in many cells' activities such as acting as a
secondary messenger, it is important for cells to control
their intracellular calcium level. This study was aimed
toward looking at the some of the basic properties of the
SF-9 cells such as resting calcium concentration and rate
of calcium extrusion after being calcium level being raised
by an ionophore 4-bromo-A23187. The effect of sodium
orthovanadate (an active transport inhibitor) on calcium
extrusion was also looked at. Microspectrofluorescence
techniques and the calcium-sensitive probe fura-2 were used
to measure the intracellular calcium concentration of these
cells. In addition, the BSG cells were used to compare with
the SF-9 cells for the parameters that were studied.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 Na2VO4 could not be statistically
analyzed. From the data available, it suggested that the
BSG cells' rate of calcium extrusion was enhanced by the
Na2VO4 and was greater than the SF-9 cells. It was more
important to note that the calcium extruding capabilities
of the SF-9 cell seemed to impaired after two to three
applications of Na2VO4 but it had apparent effects on the
BSG cells even up to 4 applications.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
exchanger to extrude their calcium and they may be very
sensitive to the ATP (adenosine 3'-triphosphate) supply.
This was apparently different from the BSG cells' since
their calcium extrusion were not affected by the Na2VO4..
It may be useful to find the mechanism(s) of the actions of
Na2VO4 on the SF-9 cells because it may find possible
applications in agriculture such as pest control.
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 obtained from Sigma (St. Louis, MO). 

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 CaCl2 was substituted 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 m 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 m M. Na2VO4 in 0CaNRS. All
experiments were performed at room temperature, 22-26 ° 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 ° C for up to 4 days before the experiments. The cells
were plated on custom made 3.5 cm plastic culture dishes. A
circular hole about half the diameter of the dishes were
cut out in the middle and fitted with a piece of aclar. The
aclar dishes were then treated with poly-d-lysine for one
hour before plating. SF-9 cells (non-transfected) were
cultured as described by Summers and Smith (1987). The SF-9
cells were plated and incubated (at 37 ° C) on the custom
made dishes as used for the BSG cells one day prior to the
experiments. They were not kept for more then two days to
avoid overgrowth of cells that might cause difficulties in
experimental measurement. 

Each dish contained approximately 100 m l of cell
suspension. To load the cells with fura-2/AM, 100 m 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
permeable to the cell membrane (where fura-2 is not
permeable to the cell membrane to any great extent) and is
subsequently broken down into fura-2 intracellularly by
.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
solutions at a rate of 2-3 ml/min and could switch between
the solutions from five different solutions simultaneously.
This would allow rapid switching of solutions and improved
the speed of responses 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
microcomputer. The software used was PTI Instrument Control
Program from Photon Technology International Inc. (South
Brunswick, NJ).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. 1985):[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 l =380nm obtained from calcium-free fura-2
sample and calcium-bound fura-2 sample respectively, R is
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 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 the intracellular calcium
concentration. 0CaNRS was used to decrease the calcium
concentration when the calcium level reached over 200 nM.
Once the calcium concentration was decreased and stabilized
with the 0CaNRS, 4-bromo-A23187 was added again and the
whole procedure was repeated for two to four times. Then
4-bromo-A23187 was used once again to bring the
intracellular calcium concentration up, VO4NRS now was used
instead of 0CaNRS to lower the calcium concentration. This
procedure was used for two to four cycles or until the cell
showed no response and unable to lower the calcium
concentration to the previous resting level.
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
difference between the intracellular calcium concentration
of the SF-9 and the BSG cells (P = 0.31).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 concentration. 

It was found that the rates of calcium depletion (D C/D t)
of BSG and SF-9 cells were 3.92 ± 0.81 nM/s (mean ± S.E., n
= 10) and 4.12 ± 0.81 nM/s (n = 7) respectively. However,
the BSG cells and the SF-9 cells were generally in
different sizes in which the SF-9 cells (about 15-20 m m in
diameter) were usually smaller in sizes relative to the BSG
cells (about 25-40 m m in diameter). It is therefore
important to take into the account of the size of the cells
for the analysis of the calcium flux. 

The calcium flux (J) out of the cell can be determined by
adjusting the rates of calcium depletion with the volume to
area ratio of the cells (assuming the cells were spherical
in shape). The flux can be found by: J = -D C/D t ·V/S
where J is the flux, -D C/D 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 SF-9 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 by t-test.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-2·s-1 (n = 2) and 0.80 ± 0.24 fmole·cm-2·s-1 (n =
3) respectively (table 2)
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 three to four VO4NRS applications
(Figure. 2).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) 

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 (D C/D t) of the initial linear portion
after the maximum calcium concentration. 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) 

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 cellA time course calcium
recording of a single SF-9 cell (19 m 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 ability to extrude calcium.
Abbreviations: A, 4-bromo-A23187; N, NRS; 0, 0CaNRS; V,
VO4NRS.Figure 2. Intracellular calcium concentration of a
BSG cellIn contrast to the SF-9 cell in Figure 1, the BSG
cell (39 m 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, results are not
included in this report). However, it was later found that
the cells were not very successfully transfected. T-test
did not show any significant difference between the calcium
levels in the BSG cells and the SF-9 cells which leads to
the question of whether the transfecting process would
cause certain biophysiological changes in the cells which
led to low intracellular calcium concentrations.
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 then used to raise the calcium
concentration in the subsequent cycles. This is probably
due to the high lipidphilicity of the 4-bromo-A23187 which
enable it to partition into the cell membrane and the
internal organelles. Hence the one application of
4-bromo-A23187 would allow it to partition and remain in
the cell membrane and acted as an ionophore without the
necessity of further subsequent addition. 

The effects of the NRS at raising calcium concentration
appeared to be similar to 4-bromo-A23187's. This technique
was more economical and also reduced the effects of DMSO
(which was used to dissolve 4-bromo-A23187) on the cells. A
general discussion on of ionophores can be found in an
article by Pressman (1976). A more specific topics of
4-bromo-A23187 on use with fluorescent probes and its
action on calcium can be refered to Deber (1985) and Reed
and Lardy (1972).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 inhibitor. It
acts as a phosphate substitute for ATP and thus stops or
slows the ATP production. Without ATP, active transport
cannot be carried out. In the case of calcium, the addition
of VO4NRS would cause the cells not able to extrude the
calcium out after the application of 4-bromo-A23187. It was
indeed what was observed for the SF-9 cell (Figure 1). 

It was noted that the calcium concentration remained at a
high level and became unstable after 2 applications of
VO4NRS. It suggested that the calcium mobilization in the
SF-9 cells was closely linked to the ATP production.
Without ATP, the SF-9 cells were unable to regulate their
intracellular level in a normal manner. However, the BSG
cells showed different responses to VO4NRS (Figure 2)
compared to the SF-9 cells. After 3 applications of VO4NRS,
the BSG cell was still able to extrude calcium, despite the
abnormal high calcium concentration after the third VO4NRS

This result was not anticipated because the BSG cells had
higher calcium effluxes relative to the SF-9 cells, hence
calcium extrusion of the BSG cells were more dependent on
the ATP production. One possible explanation would be that
the BSG cells had excess organelles to store calcium
instead of extruding it. 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 understanding of the calcium transport system. Also, it
may be useful for the molecular biologists to improve the
techniques of gene expressions using the SF-9 cells.
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 BSG culture dishes
and Dr. D. R. Hampson for his kind gift of SF-9 cells.
References Deber, C. M.; Tom-Kun, J.; Mack, E.; Grinstein,
S. Bromo-A23187: a nonfluorescent calcium ionophore for use
with fluorescent probes. Anal. Biochem.
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Y. A new generation of Ca2+ indicator with greatly improved
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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 d -endotoxin from Bacillus thuringiensis:
involvement of calcium and anionic channels. Biochem.
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E. A manual of methods for baculovirus vectors and insect
cell culture procedures. Texas Agric. Exper. Sta. Bull. no
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