Blockade of chloride channels suppresses engulfment of microspheres in the microglial cell line, BV-2.
Microglial cells, monocyte-derived cells in the brain, phagocytose apoptotic and necrotic cells during neurodegenerative processes. As uptake of particles increases cellular volume and swelling-activated chloride channels participate in volume regulation, we investigated the phagocytotic capacity of microglial cells exposed to blockers of swelling-activated chloride channels. We visualized engulfment of hydrophobic polystyrenic microspheres (4 microm in diameter) in the microglial cell line, BV-2, using scanning electron microscopy and confocal laser microscopy. Microspheres instead of apoptotic cells were used because in scanning electron micrographic images, engulfed particles were clearly discriminated from attached ones. Exposure of BV-2 cells to the chloride channel blocker, flufenamic acid (200 microM) or NPPB (200 microM), eliminated uptake of microspheres almost completely. SITS (1 mM), which blocks chloride channels and to some extend K-Cl cotransporters (KCC), had only a moderate inhibiting impact on particle uptake. DIOA, a compound that inhibits KCC as well as chloride channels, did not inhibit particle uptake in BV-2 cells. Osmotic challenge by hypoosmotic saline (60% saline) elicited a swelling-activated chloride channel sensitive to SITS (1 mM) and flufenamic acid (200 microM). Because uptake of particles requires formation of engulfment pseudopodia, we hypothesize that engulfment pseudopodia are initially nothing but local swellings and that activation of swelling-activated chloride channels participates in the formation of these swellings.
Furtner
Tanja
T
Department of Cellular Biology, University of Salzburg, Hellbrunnerstr 34, 5020, Salzburg, Austria.
Zierler
Susanna
S
Kerschbaum
Hubert H
HH
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D062365:Aminobenzoates / Q000494:pharmacology; D000818:Animals; D002461:Cell Line, Transformed; D048429:Cell Size / Q000187:drug effects; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D005439:Flufenamic Acid / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000528:radiation effects; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D018613:Microscopy, Confocal / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008863:Microspheres; D008954:Models, Biological; D009579:Nitrobenzoates; D018408:Patch-Clamp Techniques / Q000379:methods; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors
C475624:5-nitro-2-(4-phenylbutylamino)benzoic acid; D062365:Aminobenzoates; D018118:Chloride Channels; D009579:Nitrobenzoates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D005439:Flufenamic Acid