Abstract 2899: Ultrasound Exposed Microbubbles Cause Local Hyperpolarization of the Cell Membrane.
Background Ultrasound (US) and microbubbles have gained wide interest as a tool for local delivery of drugs and genes. However, the exact mechanisms underlying increased cellular uptake of drugs and genes are still not clear. Recently, we showed an increase in cell membrane permeability for Ca2+ in rat cardiomyoblast (H9c2) cells exposed to US and microbubbles. In this study, we hypothesized that the Ca2+ influx will have an effect on the membrane potential, and investigated whether Ca2+ dependent potassium channels (BKCa) are involved. We particularly focused on local events where the microbubble was in contact with the cell membrane.
Material and Methods H9c2 cells were cultured on US transparent membranes. US exposure consisted of bursts of 1 MHz with a peak negative pressure of 50 or 250 kPa. Pulse repetition frequency was 20 Hz, duty cycle 0.2%. Cells were exposed during 30s in the presence of Sonovue microbubbles. The membrane potential was monitored during US exposure using the fluorescent dye di-4-ANEPPS. The experiments were repeated in the presence of iberiotoxin (100 nM), a specific inhibitor of BKCa channels.
Results Surprisingly, despite the previously reported Ca2+ influx, we found patches of hyperpolarization of the cell membrane, as reflected by local increases in di-4-ANEPPS mean fluorescent intensity (MIF), shown in table I⇓ as percentage of control (%MIF/control). This hyperpolarization was caused by the activation of BKCa channels, as iberiotoxin completely prevented hyperpolarization.
Conclusion US and microbubbles elicit a Ca2+ influx, which leads to activation of BKCa channels and a subsequent, local hyperpolarization of the cell membrane. This local hyperpolarization of the cell membrane may facilitate uptake of macromolecules through endocytosis and macropinocytosis.