Carbenoxolone inhibits volume-regulated anion conductance in cultured rat cortical astroglia

Abstract

Accumulating evidence indicate that the gap-junction inhibitor carbenoxolone (CBX) regulates neuronal synchronization, depresses epileptiform activity and has a neuroprotective action. These CBX effects do not depend solely on its ability to inhibit gap junction channels formed by connexins (Cx), but the underlying mechanisms remain to be elucidated. Here we addressed the questions whether CBX modulates volume-regulated anion channels (VRAC) involved in the regulatory volume decrease and regulates the associated release of excitatory amino acids in cultured rat cortical astrocytes. We found that CBX inhibits VRAC conductance with potency comparable to that able to depress the activity of the most abundant astroglial gap junction protein connexin43 (Cx43). However, the knock down of Cx43 with small interfering RNA (siRNA) oligonucleotides and the use of various pharmacological tools revealed that VRAC inhibition was not mediated by interaction of CBX with astroglial Cx proteins. Comparative experiments in HEK293 cells stably expressing another putative target of CBX, the purinergic ionotropic receptor P2X7, indicate that the presence of this receptor was not necessary for CBX-mediated depression of VRAC. Finally, we show that in COS-7 cells, which are not endowed with pannexin-1 protein, another astroglial plasma membrane interactor of CBX, VRAC current retained its sensitivity to CBX. Complementary analyses indicate that the VRAC-mediated release of excitatory amino acid aspartate was decreased by CBX. Collectively, these findings support the notion that CBX could affect astroglial ability to modulate neuronal activity by suppressing excitatory amino acid release through VRAC, thereby providing a possible mechanistic clue for the neuroprotective effect of CBX in vivo.

Obesity Up-Regulates Intermediate Conductance Calcium-Activated Potassium Channels and Myoendothelial Gap Junctions to Maintain Endothelial Vasodilator Function

“…In this regard, in addition to gap junction effects, carbenoxolone and Gap peptides have nongap junction vascular ion channel and putative calcium store activity (Coleman et al, 2001;Tare et al, 2002;Haddock et al, 2006;Dora et al, 2008). Indeed, volume-regulated anion channel block with carbenoxolone mimics Cx43 block in astroglial cells (Benfenati et al, 2009), in a similar manner to its action in rat mesenteric artery (compare carbenoxolone data and interpretation in Tare et al, 2002;Dora et al, 2008). Thus, these nonspecific actions probably reflect some of their inhibitory effects in the present study.…”

DCPIB, the Proposed Selective Blocker of Volume-Regulated Anion Channels, Inhibits Several Glutamate Transport Pathways in Glial Cells

“…4 and 5, DCPIB has a similar IC 50 for both VRAC and connexin hemichannels composed by the major astrocytic connexin Cx43. This may not be surprising because substantial pharmacological "crossreactivity" between VRAC and Cx hemichannel blockers, including Cx inhibition by tamoxifen, has been reported in several studies (Benfenati et al, 2009;Ye et al, 2009). As previously found by Ye et al (2009), 18-aGA discriminates between VRAC and Cx permeability pathways and inhibits only connexins.…”

“…On the basis of the facts discussed above and in view of several recent reports of strong pharmacological "crosssensitivity" between VRAC and connexin hemichannels (Benfenati et al, 2009;Ye et al, 2009), we decided to test if DCPIB discriminates between VRAC and other glutamate release pathways. The effects of DCPIB on several major plasma membrane glutamate transporters and permeability pathways were explored in primary cultures of rat astrocytes and microglial cells.…”

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

“…1C) and ATP release (D) were markedly impaired in the presence of 10 M carbenoxolone, a licorice root derivative that preferentially inhibits pannexin channels over connexin hemichannels and volume-regulated anion channels (43)(44)(45). In contrast, 100 M flufenamic acid, a potent inhibitor of connexin hemichannels that displays low affinity for pannexin 1 (43,46), had no significant effect on ATP release and propidium iodide uptake in WD-HBE cells (Fig.…”