cAMP-Dependent Chloride Conductance Evokes Ammonia-Induced Blebbing in the Microglial Cell Line, BV-2

Abstract

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.

Chloride Channel Blockers Suppress Formation of Engulfment Pseudopodia in Microglial Cells

“…This current was present in asymmetric [21] as well as in symmetric distribution of Cl- in the extra- and intracellular solutions, which is reflected in a reversal potential close to 0 mV. NPPB (100 µM), DIOA (100 µM), FFA (200 µM), and DCPIB (5 µM) completely blocked the inward as well as the outward current.…”

“…In electrophysiological experiments Cl - -currents with biophysical and pharmacological properties similar to I Cl, swell , but induced by increased intracellular Ca 2+ or cAMP in contrast to cell swelling, are terminated by superfusion with hyperosmotic extracellular solutions [21]. In our engulfment experiments we used a similar approach; BV-2 cells were exposed to hyperosmotic conditions (400, 450 and 500 mosmol/L, respectively) for 15 min or 1 h and uptake of MBs was quantified using CLSM.…”

“…Activation of I Cl, swell is due to an osmotic imbalance, like hypotonic extracellular or hypertonic intracellular conditions [8,32]. However, a Cl - -conductance with a similar electrophysiological and pharmacological signature is also activated in normotonic conditions via an increase in intracellular Ca 2+ or cAMP and elevated PKA activity or stimulation of exchange proteins directly activated by cAMP (Epac) [15,21,33,34,35,36]. Thus, contact between a particle and a phagocyte could trigger an intracellular signalling cascade, which activates Cl - -channels in microglial cells, even at normotonic conditions.…”

P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes

“…Ca 2+ oscillations critically depend on timely entry and timely extrusion of Ca 2+ . In the absence of Ca 2+ extrusion Ca 2+ entry leads to sustained increase of cytosolic Ca 2+ activity with stimulation of apoptosis [40, 42, 48-56]. …”

Down-Regulation of Store-Operated Ca2+ Entry and Na+ Ca2+ Exchange in MCF-7 Breast Cancer Cells by Pharmacological JAK3 Inhibition

“…The Ca 2+ oscillations thus contribute to the regulation of diverse cellular functions [17,[35][36][37][38] including entering into the S and the M phase of the cell cycle [39,40] and cell survival [41,42]. Sustained increases of cytosolic Ca 2+ activity are known to trigger apoptosis [36,38,[43][44][45][46][47][48][49][50][51]. Orai and NCX dependent Ca 2+ oscillations are further instrumental in the stimulation of cell migration [52][53][54].…”