Paroma Chatterjee1, P. Thomas Vernier2*, Indira Chatterjee3*, and Gale L. Craviso1* 1Department of Pharmacology, University of Nevada, Reno, Reno, NV, USA, 2MOSIS, Information Sciences Institute, University of Southern California, Marina del Rey, CA, USA and Viterbi School of Engineering University of Southern California, Los Angeles, CA, USA, 3Department of Electrical and Biomedical Engineering, University of Nevada, Reno, NV, USA *Corresponding author e-mail: INTRODUCTION
Recently we reported [1] that in electrically excitable adrenal chromaffin cells, exposure to a single 4 ns duration electric pulse at electric field intensities ranging from 2 to 8 MV/m increases intracellular calcium via entry of calcium into the cells, rather than releasing calcium from internal stores as has been reported for other cell types. We further observed that the pulse-induced influx of calcium could be blocked by a high concentration of nitrendipine, a selective blocker of L-type voltage-gated calcium channels. The results presented here provide additional insight into the mechanism underlying the nanoelectropulse-induced influx of calcium into chromaffin cells. MATERIALS AND METHODS
Fluorescence imaging was used to monitor intracellular calcium levels during nanosecond electric field exposure. For these experiments, chromaffin cells were loaded with the calcium fluorophore Calcium Green-1 (1 μM) in a balanced salt solution (BSS) containing 0.1% bovine serum albumin for 1 hour in the dark at 37 °C. Following incubation, the cells were washed and resuspended in dye-free BSS and an aliquot of the cells loaded into microelectrode chambers fabricated as described in [2] using gold instead of platinum as the electrodes. Observations of the cells during pulse exposures were made in real-time with a Nikon TE2000 epifluorescence microscope where fluorescence images obtained before, during and after nanosecond field exposure were captured and analyzed with a Photometrics CoolSNAPHQ CCD camera and SimplePCI imaging software. A fast-recovery diode-switching NanoPulser that was designed and fabricated at the University of Southern California [3] delivered 5 ns pulses directly to the microchamber electrodes on the microscope stage in ambient atmosphere at room temperature. For assessing further the involvement of L-type calcium channels in the nanoelectropulse- induced influx of calcium, cells were incubated at varying concentrations of each of two dihydropyridine L-type channel blockers, nitrendipine and nimodipine, for 30 minutes prior to pulsing. Sodium-free BSS was prepared by replacing NaCl with osmotically equivalent amounts of either choline chloride or N-methylglucamine (NMG). As previously reported, 20 µm nitrendipine blocked the pulse-induced increase in intracellular calcium. Similar results were obtained for 20 µm nimodipine. When the concentration of each dihydropyridine was reduced to 10 µm, which is still considered high for blocking cellular responses involving L-type channels in these cells, the effectiveness of each blocker to inhibit the rise in calcium was substantially decreased. Nitrendipine and nimodipine at 5 µm similarly reduced the response substantially but did not abolish it. These results showing that only a high concentration of dihydropyridines produces total inhibition of the nanoelectropulse-induced calcium response suggest more complex effects at the plasma membrane than simply activation of L-type calcium channels. Because voltage-gated calcium channels typically are activated by cell depolarization as a consequence of sodium influx via voltage-gated sodium channels, experiments were conducted in the absence of sodium to determine whether the nanoelectropulse-induced influx of calcium would be affected. Replacement of sodium with either choline or NMG produced almost total inhibition of the rise in intracellular calcium, indicating that sodium influx precedes the entry of calcium into the cell. CONCLUSIONS
The present study shows that the mechanism by which calcium enters chromaffin cells in response to a single nanoelectropulse is coupled to sodium influx. Whether this finding is an indication that the pulse causes cell depolarization awaits further investigation, and experiments to determine whether sodium influx occurs via voltage-gated sodium channels or instead via the formation of nanopores in the plasma membrane are underway. ACKNOWLEDGMENTS
This work was made possible by support from the Air Force Office of Scientific Research. P.T.V. is supported by MOSIS at the University of Southern California Information Sciences Institute. REFERENCES
[1] P. T. Vernier, Y. Sun, M.-T. Chen, M. A. Gundersen and G. L. Craviso. Nanosecond electric pulse- induced calcium entry into chromaffin cells. Bioelectrochemistry, 73:1-4, 2008. [2] P. T. Vernier, Y. Sun, L. Marcu, S. Salemi, C. M. Craft, M. A. Gundersen. Calcium bursts induced by nanosecond electric pulses. Biochem. Biophys. Res. Commun., 310:286-295, 2003. [3] A. Kuthi, P. Gabrielsson, M. Behrend, P. T. Vernier, and M. Gundersen. Nanosecond pulse generator using fast recovery diodes for cell electromanipulation. IEEE Trans. Plasma Sci., 33:1192-1197, 2005.


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