Real-Time Monitoring of FM1-43-Labeled Plasma Membrane in Adrenal Chromaffin Cells Exposed to 5 ns Electric Pulses Reveals Differential Membrane Potential Changes and Ca2+-dependent Exocytosis in the Absence of Phospholipid Scrambling
AdvisorEl Zaklit, Josette
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In response to depolarizing stimuli, neurons and neuroendocrine cells secrete neurotransmitters and other bioactive molecules via Ca2+-dependent fusion of secretory vesicles and granules with the plasma membrane, a process called exocytosis. When exocytosis occurs, the surface area of the plasma membrane increases due to fusion of the secretory vesicles and granules, which can be monitored electro-physiologically in real-time by cell membrane capacitance measurements. A complementary fluorescence imaging approach for monitoring in real-time secretory vesicle and granule fusion due to exocytosis is use of the fluorescent membrane marker FM1-43. When FM1-43 is present in the bathing solution, exocytosis is observed as an increase in cell surface fluorescence. However, FM1-43 fluorescence is also known to be sensitive to other changes in the labeled membrane induced by depolarizing stimuli, such as the transmembrane potential (TMP).Our group has long been interested in examining the potential for nanosecond duration electric pulses (NEPs) to serve as a novel bioelectric stimulus of neurosecretion using adrenal chromaffin cells as our cell model. We have shown that exposing chromaffin cells to 5 ns electric pulses at an electric (E)-field intensity of 5 MV/m causes catecholamine release in a manner that relies on Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) and that occurs via Ca2+-dependent exocytosis. To further investigate this novel secretory stimulus, we recently monitored NEP-evoked exocytosis in real-time using total internal reflection fluorescence microscopy (TIRFM) in which secretory granules were labeled with the fluorescent dye acridine orange. We found that for a train of 10 pulses delivered at 1 Hz, exocytotic events occurred with a delay of several seconds as the pulse train was being delivered, contrasting with nicotinic receptor stimulation that caused exocytosis within milliseconds.The goal of the present study was to use a complementary approach to monitor NEP-evoked exocytosis in real-time using cell membrane staining with FM1-43. Initial experiments were carried out to evaluate the feasibility of using FM1-43 to monitor exocytosis using high [K+] as the depolarizing stimulus. Perfusing the cells with a balanced salt solution (BSS) containing 3 µM FM1-43 caused the dye to incorporate quickly into the plasma membrane, as detected by an increase in membrane fluorescence. After the fluorescence reached a plateau (~ 5 minutes), the perfusion of the cells with 56 mM [K+] BSS first caused a decrease in cell-surface fluorescence when the stimulus reached the cells, followed by an increase in fluorescence that was homogeneous across the surface of the cell membrane. The initial fluorescence dip was attributed to cell depolarization due to the ability of the dye to detect TMP changes, whereas the subsequent increase in fluorescence reflected exocytosis. This latter increase was Ca2+-dependent since exposing cells to high [K+] in the absence of external Ca2+ did not result in an increase in membrane fluorescence, indicating a lack of exocytosis. Application of ten 5 ns, 19.8 MV/m pulses to FM1-43 labeled cells delivered at 1 Hz also resulted in an overall increase in whole cell membrane fluorescence that was dependent on extracellular Ca2+. That is, application of the pulses in Ca2+-free medium did not cause an increase in fluorescence, indicating that the rise in membrane fluorescence was due to NEP-evoked exocytosis. However, unlike the initial homogenous decrease in cell membrane fluorescence evoked by high [K+] due to membrane depolarization, there were differential changes in FM1-43 fluorescence on the poles of the cells during the application of the electric stimulus. We observed an increase in fluorescence at the anode facing pole of the cell, while the cathode facing pole exhibited a decrease in fluorescence. These differential effects, which lasted only for the duration of the applied stimulus, were caused by NEP-induced depolarization at the cathode facing pole that induces a decrease in FM1-43 fluorescence, and hyperpolarization of the anode facing pole of the cell that induces an increase in FM1-43 fluorescence. In support of this conclusion, changing the polarity of the delivered pulses reversed the differential effects between the poles of the cells. Interestingly, the equators of the cells also exhibited a decrease in fluorescence, indicating that these regions of the cell membrane were depolarized during the stimulus. Because of these differential TMP changes that occurred during the pulse train, we could not obtain evidence of a delay in the onset of exocytosis as observed previously in the TIRFM experiments in which cells were similarly exposed to a train of ten, 5 ns pulses at 1 Hz.To assess whether the NEP stimulus train caused phosphatidylserine (PS) externalization that contributed to the increase in FM1-43 fluorescence, exposed cells were probed with the specific PS marker Lactadherin-FITC. We found that a single train of 10 NEPs did not induce PS externalization even two minutes after stimulation. However, after a second train of NEPs, PS was identified at the anode facing pole of the cell one minute after the end of the stimulus. These results suggest that PS scrambling did not contribute to the increase in fluorescence at the anode facing pole of the cell observed during a single pulse train, or at any other part of the cell two minutes post-stimulation. After the end of the pulse train, all the membrane parts of the stimulated cell, exhibited an increase in fluorescence that was observed only in the presence of extracellular Ca2+. In contrast, in the absence of Ca2+, all fluorescence curves returned to baseline. This indicates that the NEP-induced increase in membrane fluorescence is mostly driven by the Ca2+-dependent exocytosis.In summary, these results imply multiple understandings into the basic use of NEPs as a biomedical application to trigger neurosecretion. The increase in FM1-43 fluorescence occurring on all parts of the membrane of the cells between the electrodes confirms the exocytotic activity due to the influx of Ca2+ induced by the electric stimulation. The differential polarization of the stimulated cell is important to consider during the exogenous application of NEPs for biological tissues/organs. Hyperpolarization of the anode facing poles and depolarization of the cathodic poles of the cells during the application of the NEPs can vary the excitation state of the targeted cells. A novel finding in the use of 5 ns pulses reported in this study is that using a train of 10 pulses, no PS was externalized at the anode facing pole or other parts of the cells for two minutes post-stimulation. This suggested that the exposure of ten 5 ns pulses did not induce symptoms of apoptosis in the stimulated chromaffin cells. Finally, monitoring exocytosis using FM1-43 did not rule out a delay between the initiation of NEPs and the beginning of the exocytotic response. Thus, direct measurement of the NEP-induced exocytosis is needed in the future to re-investigate the nature of this delay.