cAMP-dependent chloride conductance evokes ammonia-induced blebbing in the microglial cell line, BV-2.
Cell blebbing is a key feature in apoptosis. Because blebbing dynamically alters cell volume and regulatory volume changes have been linked to chloride (Cl) channels, we evaluated an association between blebbing and Cl channels activity. We used scanning electron microscopy, confocal laser microscopy, and cell sorting to quantify cell volume and blebbing and whole-cell recording to characterize Cl(-) currents. We found that blockade of Cl channel activity as well as inhibition of adenylyl cyclase or protein kinase A (PKA) activity suppressed ammonia-induced blebbing in the microglia cell line, BV-2. In further experiments, we elucidated the common mechanism of Cl channel activity and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway on cell blebbing. These experiments indicated that perfusion of cells with cAMP or the catalytic subunit of PKA activated a Cl(-) current under normotonic conditions. The pharmacological profile (sensitivity to 5-nitro-2-(3-phenylpropylamino)benzoic acid [NPPB], flufenamic acid, and [(dihydroindenyl)oxy]alkanoic acid [DIOA]), outward rectification, and kinetic of the current were identical to the swelling-activated Cl channel. Superfusion of cells with ammonia elicited an outwardly rectifying current sensitive to Cl channel blockers. We propose that ammonia induces a PKA-dependent phosphorylation of Cl channels. Localized influx of Cl(-) is followed by influx of water, required for bleb expansion.
Svoboda
Nina
N
Department of Cell Biology, University of Salzburg, Salzburg, Austria.
Pruetting
Sylvia
S
Grissmer
Stephan
S
Kerschbaum
Hubert H
HH
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000641:Ammonia / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004553:Electric Conductivity; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009579:Nitrobenzoates / Q000494:pharmacology; D010766:Phosphorylation
D018118:Chloride Channels; D009579:Nitrobenzoates; C017759:9-(tetrahydro-2-furyl)-adenine; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D000641:Ammonia; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D000225:Adenine