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. 2013 Jan;83(1):22-32.
doi: 10.1124/mol.112.080457. Epub 2012 Sep 25.

DCPIB, the proposed selective blocker of volume-regulated anion channels, inhibits several glutamate transport pathways in glial cells

Nicole H Bowens  1 , Preeti Dohare, Yu-Hung Kuo, Alexander A Mongin
Affiliations

DCPIB, the proposed selective blocker of volume-regulated anion channels, inhibits several glutamate transport pathways in glial cells

Nicole H Bowens et al. Mol Pharmacol. 2013 Jan.

Abstract

4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB) was identified as the selective blocker of volume-regulated anion channels (VRAC). VRAC are permeable to small inorganic and organic anions, including the excitatory neurotransmitter glutamate. In recent years DCPIB has been increasingly used for probing the physiologic and pathologic roles of VRAC and was found to potently suppress pathologic glutamate release in cerebral ischemia. Because ischemic glutamate release can be mediated by a plethora of mechanisms, in this study we explored the selectivity of DCPIB toward the majority of previously identified glutamate transporters and permeability pathways. l-[(3)H]glutamate, d-[(3)H]aspartate, and l-[(14)C]cystine were used to trace amino acid release and uptake. We found that in addition to its well-characterized effect on VRAC, DCPIB potently inhibited glutamate release via connexin hemichannels and glutamate uptake via the glutamate transporter GLT-1 in rat glial cells. In contrast, DCPIB had no direct effect on vesicular glutamate release from rat brain synaptosomes or the cystine/glutamate exchange in astrocytes. The compound did not affect the astrocytic glutamate transporter GLAST, nor did it block glutamate release via the P2X(7)/pannexin permeability pathway. The ability of DCPIB to directly block connexin hemichannels was confirmed using a gene-specific siRNA knockdown approach. Overall, our data demonstrate that DCPIB influences several glutamate transport pathways and that its effects on VRAC in vivo should be verified using additional pharmacological controls.

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Figures

Fig. 1.
Fig. 1.
Acute and chronic effects of DCPIB on vesicular release of glutamate from rat brain synaptosomes. Synaptosomes were preloaded with l-[3H]glutamate and then injected into basal or high K+ media containing 0 or 1.2 mM [Ca2+]o. Vesicular neurotransmitter release was determined as the Ca2+-dependent component of K+-stimulated l-[3H]glutamate release (see Materials and Methods for details). (A) acute effect of 20 µM DCPIB was measured by adding this compound into the release assay media only. n = 5 or 6 for each condition. (B) chronic effect of 20 µM DCPIB was tested by exposing synaptosomes to this agent 30 minutes prior to and during glutamate release assay. As a positive control for inhibition of vesicular release, synaptosomes were preincubated with 100 µM N-ethylmaleimide (NEM). n = 5 for control and DCPIB; n = 3 for NEM. ***P < 0.001 versus control.
Fig. 2.
Fig. 2.
DCPIB blocks the glial glutamate transporter GLT-1 but does not affect activity of GLAST. (A) uptake of l-[3H]glutamate was measured in primary astrocyte cultures to determine the effects of DCPIB on activity of the glutamate transporter GLAST. In addition to DCPIB, the GLT-1-specific inhibitor dihydrokainic acid (DHK), a broad spectrum glutamate transporter inhibitor, dl-threo-β-benzyloxyaspartic (TBOA), and Na+-free Li+-containing medium were used as positive controls. n = 18 for control, n = 9–12 for other groups. ***P < 0.001 versus control. (B) identical experiments were performed in primary microglial cells to measure the activity of GLT-1, which was determined as the DHK-sensitive component of l-[3H]glutamate uptake. n = 33 for controls, n = 9–12 for all other groups. *P < 0.05, **P < 0.01, ***P < 0.001 versus control.
Fig. 3.
Fig. 3.
DCPIB partially inhibits the cystine/glutamate antiporter (xCT). (A) xCT activity was measured in primary cultured astrocytes as uptake of l-[14C]cystine. To block activity of the Na+-dependent amino acid transporters and γ-glutamyl transpeptidase, these assays were performed in Na+-free media (Na+ substituted with Li+) and in the presence of 0.5 mM acivicin. The xCT inhibitors, dl-homocysteine (HCA) and l-serine-O-sulfate (l-SOS), were used as positive controls. n = 8 for control; n = 4-6 for other groups. *P < 0.05, **P < 0.01, ***P < 0.01 versus control. (B) a dose-response curve of DCPIB’s effects on the xCT activity. n = 3–8. **P < 0.01 versus control.
Fig. 4.
Fig. 4.
DCPIB blocks both volume-regulated anion channels (VRAC) and connexin (Cx) hemichannels. (A) comparative effects of 10 μM 18α-glycyrrhetinic acid (18-αGA) and 20 μM DCPIB on Cx-mediated glutamate (d-[3H]aspartate) release from cultured astrocytes superfused with Ca2+/Mg2+-free (CMF) medium. n = 3. ***P < 0.001, CMF versus other groups. (B) comparative effects of 10 μM 18-αGA and 20 μM DCPIB on VRAC-mediated glutamate (d-[3H]aspartate) release from cultured astrocytes superfused with hypotonic medium (40% reduction of medium osmolarity by lowering [NaCl]). n = 5. ***P < 0.001, DCPIB versus other groups. (C) a dose response curve of DCPIB’s effects on Cx (CMF)- or VRAC-mediated (Hypo) glutamate release; n = 6–21. (D) a representative trace showing the effects of 20 μM DCPIB and 10 μM 18-αGA on VRAC currents measured using the whole cell electrophysiology approach in primary astrocytes exposed to hypoosmotic medium (see Materials and Methods for details). (E) representative recording of VRAC currents in response to step voltage commands in the range of −100 to +100 mV, applied in 20-mV increments. (F) normalized VRAC current densities in the presence of 20 μM DCPIB (n = 2) and 10 μM 18-αGA (n = 4) in the experiments presented in (D). Effects of inhibitors were measured when VRAC currents were saturated under hypoosmotic conditions.
Fig. 5.
Fig. 5.
siRNA knockdown experiments confirm that DCPIB inhibits glutamate release via connexin-43 (Cx43) hemichannels. (A) qRT-PCR analysis of relative mRNA expression of Cx43 and GAPDH (negative control) in astrocytes treated with three independent siRNA constructs (Cx43-1, Cx43-5, Cx43-7). Expression levels were normalized to housekeeping gene RPL13a and compared with a scrambled negative control siRNA (NC) or untransfected cells (WT). n = 3 independent transfections. P < 0.001 versus NC siRNA-treated cells. (B) Western blot analysis of Cx43 immunoreactivity in sister cultures transfected on the same day as in (A). Representative of three independent transfections, framed box below main Western blot shows immunoreactivity on the same membranes that were stripped and probed with anti-actin antibody (loading control). (C) efflux of preloaded d-[3H]aspartate in cultured astrocytes transfected with Cx43-1 construct or negative control (NC) siRNA. One of the curves shows effect of 20 μM DCPIB on d-[3H]aspartate in cells transfected with NC. n = 7–14. ***P < 0.001 NC siRNA-treated cells versus other two groups, Wilcoxon matched pairs non-parametric test.
Fig. 6.
Fig. 6.
DCPIB potentiates glutamate release via the P2X7 receptor (P2X7)/pannexin (Pnx) permeability pathway. (A) release of glutamate (d-[3H]aspartate) was measured in astrocytes treated with the P2X7 agonist 300 μM 4-benzoylbenzyl adenosine (BzATP), the P2X7 antagonist 300 nM AZ 10606120 dihydrochloride (P2X7 AN), and the Pnx channel blocker 2 mM probenecid. n = 9–18 per group. ***P < 0.001 versus control; ###P < 0.001 versus BzATP. (B) effects of 3, 10, and 20 μM DCPIB on the BzATP-induced d-[3H]aspartate release from astrocyte cultures. n = 6-18 per group. ***P < 0.001 versus control; ###P < 0.001 versus 300 BzATP; n = 6–18.
Fig. 7.
Fig. 7.
DCPIB strongly suppresses ATP-induced glutamate release from cultured astrocytes. (A) effect of 20 μM DCPIB on glutamate (d-[3H]aspartate) release induced by stimulation with 100 μM ATP. DCPIB was present in the perfusion medium 5 minutes before and during application of ATP. n = 8 per group. ***P < 0.001, DCPIB versus control. (B) effect of the blocker of Ca2+-activated Cl− channels 100 μM niflumic acid on d-[3H]aspartate release induced by stimulation with 100 μM ATP. Niflumic acid was present in the perfusion medium 5 minutes before and during application of ATP. n = 5–7 per group. ***P < 0.001, niflumic acid versus control.
Fig. 8.
Fig. 8.
Summary of DCPIB’s effects on physiologically and pathologically relevant glutamate release pathways in neural cells. Note that for the reasons of simplicity all transporters and release mechanisms are collected in one cartoon cell. DCPIB-sensitive transport routes are depicted in red. Transporters, which are insensitive to DCPIB or potentiated by this compound, are depicted in blue. Red arrows illustrate directionality of glutamate transport under physiologic conditions. CaCC, Ca2+-activated Cl− channels, the molecular identity of which is uncertain (see Discussion).
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