title: Low-intensity ultrasound-exposed microbubbles provoke local hyperpolarization of the cell membrane via activation of BK(Ca) channels.
abstract: Ultrasound (US) contrast agents have gained wide interest in gene therapy as many researchers reported increased membrane permeability and transfection efficiency by sonoporation in the presence of US contrast agents. We recently demonstrated an increase in cell membrane permeability for Ca2+ in rat cardiomyoblast (H9c2) cells insonified in the presence of microbubbles. In the present study, we specifically investigated whether US-exposed microbubbles have an effect on the cell membrane potential and whether Ca2+-dependent potassium (BK(Ca)) channels are involved. We particularly focused on local events where the microbubble was in contact with the cell membrane. H9c2 cells were cultured on US transparent membranes. US exposure consisted of bursts with a frequency of 1 MHz with a peak-to-peak pressure of 0.1 or 0.5 MPa. Pulse repetition frequency was set to 20 Hz, with a duty cycle of 0.2%. Cells were insonified during 30 s in the presence of Sonovue(trade mark) microbubbles. The membrane potential was monitored during US exposure using the fluorescent dye di-4-aminonaphtylethenylpyridinium (di-4-ANEPPS). The experiments were repeated in the presence of iberiotoxin (100 nM), a specific inhibitor of BK(Ca) channels. Surprisingly, despite the previously reported Ca(2+) influx, we found patches of hyperpolarization of the cell membrane, as reflected by local increases in di-4-ANEPPS mean intensity of fluorescence (MIF) to 118.6 +/- 2.5% (p < 0.001, n = 267) at 0.1 MPa and 125.7 +/- 5.9% (p < 0.001, n = 161) at 0.5 MPa at t = 74 s, respectively, compared with "no US" (100.3 +/- 3.4%, n = 52). This hyperpolarization was caused by the activation of BK(Ca) channels, as iberiotoxin completely prevented hyperpolarization. (MIF(t74) = 100.6 +/- 1.4%; p < 0.001, n = 267) and 0.5 MPa (MIF(t74) = 88.8 +/- 2.0%; p< 0.001, n = 193), compared with 0.1 and 0.5 MPa microbubbles without iberiotoxin. In conclusion, US-exposed microbubbles elicit a Ca2+ influx, which leads to activation of BK(Ca) 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. (E-mail: ljm.juffermans@vumc.nl).
authors.affiliation: Department of Physiology and Cardiology, VU University Medical Center, Amsterdam, The Netherlands. ljm.juffermans@vumc.nl
authors.forename: Lynda J M
authors.initials: LJ
authors.lastname: Juffermans
authors.forename: Otto
authors.initials: O
authors.lastname: Kamp
authors.forename: Pieter A
authors.initials: PA
authors.lastname: Dijkmans
authors.forename: Cees A
authors.initials: CA
authors.lastname: Visser
authors.forename: Rene J P
authors.initials: RJ
authors.lastname: Musters
mesh_terms: D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D045423:Microbubbles; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000000981:diagnostic imaging* / Q000187:drug effects / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects; D051381:Rats; D013459:Sulfur Hexafluoride / Q000494:pharmacology; D014465:Ultrasonics; D014463:Ultrasonography
chemical_list: D010743:Phospholipids; D024681:Potassium Channels, Calcium-Activated; C420843:contrast agent BR1; D006861:Hydrogen Peroxide; D013459:Sulfur Hexafluoride