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. 2009 May 15;587(Pt 10):2197-209.
doi: 10.1113/jphysiol.2008.165084. Epub 2009 Feb 2.

Bradykinin-induced astrocyte-neuron signalling: glutamate release is mediated by ROS-activated volume-sensitive outwardly rectifying anion channels

Hong-Tao Liu  1 , Tenpei Akita, Takahiro Shimizu, Ravshan Z Sabirov, Yasunobu Okada
Affiliations

Bradykinin-induced astrocyte-neuron signalling: glutamate release is mediated by ROS-activated volume-sensitive outwardly rectifying anion channels

Hong-Tao Liu et al. J Physiol. .

Abstract

Glial cells release gliotransmitters which signal to adjacent neurons and glial cells. Previous studies showed that in response to stimulation with bradykinin, glutamate is released from rat astrocytes and causes NMDA receptor-mediated elevation of intracellular Ca(2+) in adjacent neurons. Here, we investigate how bradykinin-induced glutamate release from mouse astrocytes signals to neighbouring neurons in co-cultures. Astrocyte-to-neuron signalling and bradykinin-induced glutamate release from mouse astrocytes were both inhibited by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and phloretin. Glutamate release was also sensitive to 4-(2-Butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxybutyric acid (DCPIB), a specific blocker of the volume-sensitive outwardly rectifying anion channel (VSOR). Astrocytes, but not neurons, responded to bradykinin with activation of whole-cell Cl- currents. Although astrocytes stimulated with bradykinin did not undergo cell swelling, the bradykinin-activated current exhibited properties typical of VSOR: outward rectification, inhibition by osmotic shrinkage, sensitivity to DIDS, phloretin and DCPIB, dependence on intracellular ATP, and permeability to glutamate. Bradykinin increased intracellular reactive oxygen species (ROS) in mouse astrocytes. Pretreatment of mouse astrocytes with either a ROS scavenger or an NAD(P)H oxidase inhibitor blocked bradykinin-induced activation of VSOR, glutamate release and astrocyte-to-neuron signalling. By contrast, pretreatment with BAPTA-AM or tetanus neurotoxin A failed to suppress bradykinin-induced glutamate release. Thus, VSOR activated by ROS in mouse astrocytes in response to stimulation with bradykinin, serves as the pathway for glutamate release to mediate astrocyte-to-neuron signalling. Since bradykinin is an initial mediator of inflammation, VSOR might play a role in glia-neuron communication in the brain during inflammation.

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Figures

Figure 1
Figure 1. Sensitivity of mouse astrocyte–neuron signalling induced by bradykinin to a B2 receptor antagonist and anion channel blockers
A, control experiments. a, differential interference-contrast micrograph. Squares, circles and inverted triangles designate responder astrocytes, responder neurons and non-responder neurons, respectively. b, intracellular Ca2+ changes, monitored by the percentage increase in the fluo-4 fluorescence intensity, during and after stimulation with 1 μm bradykinin (BK). The mean peak Ca2+ response in co-cultured astrocytes is not significantly different (P > 0.05) from that in astrocytes cultured alone, whereas that in co-cultured neurons is significantly different (P < 0.005) from that in neurons cultured alone. B, experiments with antagonists of bradykinin receptors (BKR). a, effects of combined application of antagonists of B1 and B2 subtypes of BKR on Ca2+ responses to 1 μm bradykinin (BK) in both astrocytes and neurons in the same co-culture. b, effects of an antagonist of the B2 receptor, 5 μm HOE140, and an antagonist of the B1 receptor, 5 μm[des-Arg10]-HOE140 (DesArgHOE140), on BK-induced Ca2+ responses in astrocytes. The mean peak Ca2+ response in the presence of DesArgHOE140 is not significantly different (P > 0.1) from that in its absence (Ab). C, experiments with blockers of VSOR (a: 200 μm DIDS, b: 100 μm phloretin). The astrocytic Ca2+ responses in the presence of DIDS (a) and phloretin (b) are not significantly different (P > 0.05) from that in the absence of drugs (Ab), whereas the neuronal Ca2+ responses are significantly different (P < 0.005) from that in Ab.
Figure 2
Figure 2. Bradykinin-induced glutamate release from mouse astrocytes and its pharmacological properties
A, the time courses of changes in the bulk extracellular concentration of glutamate released from astrocytes in the absence (Control) and presence (+BK) of 1 μm bradykinin (BK). B, pharmacological characterisation of glutamate release from astrocytes stimulated with 1 μm BK for 5 min. *P < 0.05. The drugs used are: 5 μm[des-Arg10]-HOE140 plus 5 μm HOE140 as BKR antagonists, 50 μm D-AP-5 as an antagonist of the NMDA receptor, 10 μm CNQX as an antagonist of the AMPA/kainate receptor as well as 200 μm DIDS, 100 μm phloretin, 10 μm DCPIB and 50 μm Gd3+ as anion channel blockers. The effect of extracellular hypertonicity was observed by applying hypertonic Ringer solution (350 mosmol (kg H2O)−1).
Figure 3
Figure 3. Bradykinin-induced anion currents in mouse astrocytes
A, representative whole-cell anion currents activated by 1 μm bradykinin (BK) before and during application of 100 μm phloretin. Alternating step pulses of ±40 mV (0.5 s duration, every 5 s) or step pulses from −100 to +100 mV in 20 mV increments with a pre-pulse (0.1 s duration) to −100 mV and a post-pulse (0.1 s duration) to −100 mV (at a and b) were applied from a holding potential of 0 mV. a and b, expanded traces of current responses to step pulses before (a) and after (b) application of phloretin. Arrowheads: the zero-current level. B, instantaneous current–voltage relationships measured during BK application in the absence (Control, open circles) and presence of 100 μm phloretin (filled circles) or 10 μm DCPIB (filled squares). Instantaneous currents were measured 25–30 ms after the onset of the test pulses. The current density was evaluated by dividing the current by the cell capacitance (59.2 ± 3.8 pF, n= 33). Phloretin and DCPIB significantly inhibited bradykinin-activated anion currents at all the voltages tested (except for 0 mV). C and D, sensitivity of bradykinin-induced currents recorded at +100 mV (C) and −100 mV (D) to bradykinin receptor antagonists, anion channel blockers (phloretin, DCPIB and DIDS), cytosolic ATP removal (>15 min equilibration with ATP-free pipette solution) and osmotic cell shrinkage induced by a hypertonic challenge (350 mosmol (kg H2O)−1, P < 0.001), but not to a maxi-anion channel blocker (gadolinium, P > 0.1). The percentage inhibition was calculated from the bradykinin-induced peak currents recorded at ±100 mV from astrocytes under control (isotonic, ATP-containing, and blocker-free) conditions (n= 5) and the cells under test conditions. The concentrations of drugs are the same as those in Fig. 2.
Figure 4
Figure 4. Lack of cell volume change in mouse astrocytes after stimulation with bradykinin
Effects of stimulation with 1 μm bradykinin (BK) and a hypotonic challenge (Hypotonicity, 210 mosmol (kg H2O)−1) on cell volume monitored by changes in the intracellular concentration of calcein loaded in astrocytes adhered to coverslips (A) or monitored with a Coulter-type cell size analyser in isolated astrocytes in suspension (B).
Figure 5
Figure 5. Role of ROS production by mouse astrocytes in bradykinin-induced anion currents, glutamate release and astrocyte–neuron signalling
A, effects of application (at horizontal bars) of a ROS scavenger (10 mm NAC, after 5 min pretreatment) (a), an NAD(P)H oxidase inhibitor (10 μm DPI, after 10 min pretreatment) (b), and a Ca2+ chelator (50 μm BAPTA-AM, after 30 min pretreatment) (c) on anion currents before and during application of 1 μm bradykinin (BK). Each trace is representative of 4 or 5 whole-cell experiments made as in Fig. 3A. B, effects of pretreatment with NAC (10 mm, 5 min), DPI (10 μm, 10 min) and BAPTA-AM (50 μm, 30 min) on BK-induced ROS production. The level of intracellular ROS was monitored by DCF fluorescence. NAC and DPI significantly inhibited this increase observed ≥2 min after stimulation, but BAPTA-AM never inhibited this increase observed ≤6 min after stimulation. C, effects of pretreatment with NAC (10 mm, 5 min), DPI (10 μm, 10 min), BAPTA-AM (50 μm, 30 min), apocynin (2 mm, 10 min) and tetanus toxin A (1 μg ml−1 for 24 h) on BK-induced glutamate release. *P < 0.05. D, effects of application (at horizontal bars) of 10 mm NAC after 5 min pretreatment (a) or 10 μm DPI after 10 min pretreatment (b) on BK-induced Ca2+ responses in astrocytes (open circles) and neurons (filled circles).
Figure 6
Figure 6. Schematic illustration of the mechanisms of bradykinin-induced astrocyte-to-neuron signalling
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