Accumulating evidence indicate that the gap-junction inhibitor carbenoxolone (CBX) regulates neuronal synchronization, depresses epileptiform activity and has a neuroprotective action. These CBX ef...

In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin‐4 (AQP4), and the swelling‐activated Cl^– current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume‐regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process‐bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling‐activated Cl^– current provided evidence that in AQP4 KD astrocytes, there was a down‐regulation of chloride channel‐2 (CIC‐2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.

Background and Purpose: The ethacrynic acid derivative, 4‐(2‐butyl‐6,7‐dichlor‐2‐cyclopentylindan‐1‐on‐5‐yl) oxobutyric acid (DCPIB) is considered to be a specific and potent inhibitor of volume‐regulated anion channels (VRACs). In the CNS,DCPIBwas shown to be neuroprotective through mechanisms principally associated to its action onVRACs. We hypothesized thatDCPIBcould also regulate the activity of other astroglial channels involved in cell volume homeostasis.Experimental Approach: Experiments were performed in rat cortical astrocytes in primary culture and in hippocampal astrocytesin situ. The effect ofDCPIBwas evaluated by patch‐clamp electrophysiology and immunocytochemical techniques. Results were verified by comparative analysis with recombinant channels expressed inCOS‐7 cells.Key Results: In cultured astrocytes,DCPIBpromoted the activation of aK⁺conductance mediated by two‐pore‐domainK⁺(K_2P) channels. TheDCPIBeffect occluded that of arachidonic acid, which activatesK_2PchannelsK_2P2.1 (TREK‐1) andK_2P10.1 (TREK‐2) in cultured astrocytes. Immunocytochemical analysis suggests that cultured astrocytes expressK_2P2.1 andK_2P10.1 proteins. Moreover,DCPIBopened recombinantK_2P2.1 andK_2P10.1 expressed in heterologous system. In brain slices,DCPIBdid not augment the large backgroundK⁺conductance in hippocampal astrocytes, but caused an increment in basalK⁺current of neurons.Conclusion and Implications: Our results indicate that the neuroprotective effect ofDCPIBcould be due, at least in part, to activation ofTREKchannels.DCPIBcould be used as template to build new pharmacological tools able to increase backgroundK⁺conductance in astroglia and neuronal cells.