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

PHM499 Research Project


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 fmolecm-2s-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 fmolecm-2s-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.


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 co
mpare 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.


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 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.