Anti-NPPB monoclonal antibody, clone FQS4847

To maintain cellular and bodily homeostasis, cells respond to extracellular stimuli including osmotic stress by activating various ion channels, which have been implicated in many physiological and pathophysiological conditions. However, cellular osmosensory mechanisms remain elusive. Here, we report a novel voltage-dependent current in N2a cells activated by exposure to hypotonic stress. After a hypotonic challenge, N2a cells sequentially develop two distinct currents. The volume-regulated anion channel (VRAC) current emerges first and, after a delay, activation of a previously uncharacterized strongly outwardly rectifying current follows. The latter, delayed current (I-d) is insensitive to NPPB, a nonspecific blocker of Cl- channels, and intracellular Mg2+, which inhibits VRAC and swelling-activated TRPM3 and TRPM7 channels. Replacement of extracellular Na+ with NMDG(+) reduces inward tail currents, suggesting that Id is mediated by cations. Finally, Id shows voltage-dependent activation with slow activation kinetics and half-maximal activation at +76 mV. These pharmacological and biophysical characteristics of Id are distinct from those of known osmotic cell swelling-activated ion channels. In conclusion, our data identify and characterize a novel osmotically-activated, voltage-dependent ion channel in N2a cells. (C) 2017 Elsevier Inc. All rights reserved.

The effect of GABA on glutamate release from astrocytes has been studied in healthy mice and in a murine transgenic model of amyotrophic lateral sclerosis (ALS), using mouse spinal cord gliosomes labeled with [H-3]D-aspartate ([H-3]D-ASP). GABA concentration-dependently evoked the release of [H-3]D-ASP. The effect of GABA was not mimicked by GABA(A) or GABA(B) receptor agonists or counteracted by antagonists, excluding receptor involvement. However, it was prevented by the GABA transport inhibitor N-(4,4-phenyl-3-butenyl)-nipecotic acid (SKF 89976A), suggesting participation of GABA transporters type 1 (GAT1) placed on glutamate-releasing astrocyte-derived gliosomes. Accordingly, GAT1 co-expressed with glutamate-aspartate transporter (GLAST) and glutamate transporter type 1 (GLT1) in the majority of glial particles. [H-3]D-aspartate release was Ca2+-independent and not blocked by the glutamate uptake inhibitor DL-threo-bbenzyloxyaspartic acid (DL-TBOA); instead, it was abrogated by the anion channel blockers niflumic acid and 5-nitro-2-(3phenylpropylamino) benzoic acid (NPPB). The GAT1-mediated release of [H-3]D-ASP was significantly enhanced in spinal cord gliosomes from the mouse model of ALS. This excessive [H-3]D-ASP release was very precocious, largely preceding the onset of the disease symptoms. These data indicate that GAT1, GLAST and GLT1 coexist on the same gliosome in mouse spinal cord and that activation of GAT1 transporters elicits glutamate release by anion channel opening. This phenomenon might have pathological relevance, because [H-3]D-ASP release is enhanced in experimental ALS.