Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Dec 12;27(50):13781-92.
doi: 10.1523/JNEUROSCI.2042-07.2007.

Cx43 hemichannels and gap junction channels in astrocytes are regulated oppositely by proinflammatory cytokines released from activated microglia

Mauricio A Retamal  1 , Nicolas Froger, Nicolas Palacios-Prado, Pascal Ezan, Pablo J Sáez, Juan C Sáez, Christian Giaume
Affiliations

Cx43 hemichannels and gap junction channels in astrocytes are regulated oppositely by proinflammatory cytokines released from activated microglia

Mauricio A Retamal et al. J Neurosci. .

Abstract

Astrocytes have a role in maintaining normal neuronal functions, some of which depend on connexins, protein subunits of gap junction channels and hemichannels. Under inflammatory conditions, microglia release cytokines, including interleukin-1beta and tumor necrosis factor-alpha, that reduce intercellular communication via gap junctions. Now, we demonstrate that either conditioned medium harvested from activated microglia or a mixture of these two cytokines enhances the cellular exchange with the extracellular milieu via Cx43 hemichannels. These changes in membrane permeability were not detected in astrocytes cultured from Cx43 knock-out mice and were abrogated by connexin hemichannel blockers, including La3+, mimetic peptides, and niflumic acid. Both the reduction in gap junctional communication and the increase in membrane permeability were mediated by a p38 mitogen-activated protein kinase-dependent pathway. However, the increase in membrane permeability, but not the gap junction inhibition, was rapidly reversed by the sulfhydryl reducing agent dithiothreitol, indicating that final regulatory mechanisms are different. Treatment with proinflammatory cytokines reduced the total and cell surface Cx43 levels, suggesting that the increase in membrane permeability was attributable to an increase in hemichannels activity. Indeed, unitary events of approximately 220 pS corresponding to Cx43 hemichannels were much more frequent in astrocytes treated with microglia conditioned medium than under control conditions. Finally, the effect of cytokines enhanced the uptake and reduced the intercellular diffusion of glucose, which might explain changes in the metabolic status of astrocytes under inflammatory conditions. Accordingly, this opposite regulation may affect glucose trafficking and certainly will modify the metabolic status of astrocytes involved in brain inflammation.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Addition of LPS-activated MG on enriched astrocyte cultures reduces GJC and increases membrane permeability in astrocytes. a1–4, Representative pictures of immunofluorescence staining in astrocyte cultures and cocultures with MG. GFAP-positive cells (red) correspond to astrocytes, and isolectine B4-positive cells (green) correspond to MG in cultures of astrocytes alone (Astrocytes, a1–2) and in astrocyte-MG cocultures (+Microglia; a3–4), both treated or not with LPS (10 ng/ml; 24 h). Scale bar, 50 μm. a5–12, Representative pictures for SL/DT with LY (a5–8) and EthBr uptake (a9–12) to measure the functional states of GJC or hemichannels, respectively. Experiments were performed with similar culture models and treatments as described above. Scale bar, 100 μm. b, c, Graphs corresponding to the quantification of LY diffusion through astrocytic gap junctions (b) and the EthBr uptake (c) either in enriched astrocyte cultures (Astrocytes) or in astrocyte-MG cocultures (+Microglia). Both culture models were treated (LPS), or not (C, control), with LPS (10 ng/ml) for 24 h. Each plotted number corresponds to the mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001; n.s., no significant difference compared with control astrocyte group; δδδp < 0.001, compared with the astrocyte-LPS group.
Figure 2.
Figure 2.
The increased EthBr permeability induced in astrocytes by conditioned medium from LPS-activated MG or TNF-α and IL-1β is sensitive to Cx43 hemichannel blocking peptides and is absent in Cx43−/− astrocytes. a, Snapshot representative pictures of fluorescent fields showing the increase of EthBr-stained nuclei observed in astrocytes cultured from Cx43 wild-type mice, but not in astrocytes from Cx43−/− mice, after 24 h application of either CM* or Mix. Scale bar, 100 μm. b, c, Graphs representing the number of EthBr-positive astrocytes after treatments with CM* (b), TNF-α, IL-1β, or Mix (c) for 30 min or 24 h applied on astrocyte cultures. During the 10 min incubation with EthBr, cells were coincubated in the presence (+) or absence (−) of the mimetic peptides gap26 or gap27 (300 μg/ml), for each proinflammatory treatment (b, c). Each plotted value represents the mean ± SEM of at least three independent experiments. **p < 0.01 and ***p < 0.001, compared with the untreated astrocyte group (C).
Figure 3.
Figure 3.
Treatment of astrocytes with conditioned medium harvested from LPS-activated MG or proinflammatory cytokines increases membrane permeability of astrocytes through Cx43 hemichannels. a, Representative time lapse of EthBr uptake in astrocytes recorded every 30 s. Each circle corresponds to one EthBr uptake determination, averaged with 20 recorded cells, measured either under untreated condition (Control; empty circles) or after 24 h incubation with either CM* (CM*; filled circles) or Mix (Mix; gray circles) in astrocyte cultures. For each group, La3+ (200 μm) was applied during the last 10 min of the EthBr uptake time lapse determination. b, Quantification of the rate of EthBr uptake in astrocytes under untreated conditions (Control; empty bar) or treated with CM* (CM*; filled bar) or Mix (Mix; gray bar). Data are expressed as the percentage of the EthBr uptake rate measured under control conditions. For each group, the dye uptake rate was measured in the presence (+) or absence (−) of La3+ (200 μm) at the end of the time lapse. Each bar represents the mean ± SEM [except for the control (−) group taken as reference] of 10 independent experiments per group. **p < 0.01, ***p < 0.001; n.s., no significant difference, compared with the indicated groups.
Figure 4.
Figure 4.
Inhibition of p38 MAP kinase or nitric oxide synthase prevents the Mix-induced membrane permeabilization through Cx43 hemichannels. a, Representative captures images showing the EthBr uptake in astrocytes treated for 24 h, either with Mix alone (a, Mix) or in presence of 1 mm l-name, 10 μm SB202190 (SB), or 10 mm DTT. In all experiments, l-name and SB202190 were coapplied for 24 h with Mix, whereas DTT was applied 10 min before dye uptake assay. Scale bar, 50 μm. b, Graph showing the inhibitory effect of l-Name, SB202190 (SB), and DTT on Mix-evoked dye uptake in astrocytes. Plotted data (mean ± SEM excepted for Mix that is taken as reference) are expressed as percentage of inhibition of dye uptake and are obtained from independent experiments. ***p < 0.001, compared with the Mix group.
Figure 5.
Figure 5.
The Mix-induced inhibition of astrocytic GJC is prevented by the inhibition of p38 MAPK but not by lowering intracellular redox state. a, Representative pictures depicting the diffusion of LY though GJC in confluent astrocyte cultures either under untreated condition (Control) or after 24 h treatment with Mix alone, Mix and 10 μm SB202190 (SB), or Mix and 10 mm DTT (DTT). Scale bar, 100 μm. b, c, Graph providing the quantification of the fluorescent area obtained with the SL/DT assay performed in astrocyte cultures treated for 24 h with Mix (b) or CM* (c). In each case, astrocytes were cotreated (or not) with 10 μm SB202190 (SB) or 10 mm DTT and compared with untreated conditions as control. Each value, expressed in AU, corresponds to the mean ± SEM obtained from seven independent measurements. n.s., No significant difference; ***p < 0.001.
Figure 6.
Figure 6.
The Mix-induced decrease of Cx43 in surface is not prevented by SB202190 or DTT. a, Confocal representative pictures depicting Cx43 immunolabeling, made with a monoclonal Cx43 antibody, in astrocyte cultures under untreated control condition (A1) or after treatment with Mix for 24 h (a2). Moreover, astrocytes were also cotreated either with Mix and SB202190 for 24 h (a3) or with Mix for 24 h and DTT, coapplied during the last 10 min (a4). Scale bar, 50 μm. b, c, Representative Western blot analysis of total Cx43 expression (b) and cell surface biotinylated Cx43 (c) from astrocyte cultures under untreated control condition (lane c) or after 24 h exposure with Mix. SB202190 was coapplied with Mix for 24 h (lane SB), similar to DTT coapplied during the last 10 min of Mix treatment (lane DTT). In b, a total of 20 μg of protein was loaded per lane, and in c, the total amount of biotinylated protein was loaded in each lane. Western blots shown in b and c were developed for different periods of time and, thus, levels are not comparable. Similar observations were performed for both immunofluorescence and Western blot analysis in four independent experiments.
Figure 7.
Figure 7.
Conditioned medium harvested from activated microglia induces unitary current events of Cx43 hemichannels in cortical astrocytes. a, Voltage ramps from −80 to 0 mV, 3 s in duration, were applied. The ramp was initiated by a transition from 0 to −80 mV. b, c, Currents of control and CM*-treated astrocytes for 24 h, respectively. b, d, Under control conditions, no hemichannel openings were observed, and EthBr uptake was low. c, d, In astrocytes treated for 24 h with CM*, hemichannel openings were clearly observed, and this cell showed close to twice the amount of EthBr uptake compared with cells under control conditions. The boxed region in d is shown as conductance at the right bottom where two hemichannels of ∼220 pS each show transitions between closed to open states. Tilted traced along both closed, one open, and both open indicate the progressive changes in voltage during the ramp application. d, In CM*-treated astrocytes, the EthBr uptake fraction sensitive to La3+ (200 μm) was bigger than in control cells, indicating that more hemichannels were open in CM*-treated cells.
Figure 8.
Figure 8.
Proinflammatory treatment increases glucose uptake in astrocytes. a, Confluent astrocyte cultures were treated with CM* for 24 h, and then uptake of 2-NBDG, a fluorescent glucose derivative, was determined at 488 nm. a, Snapshot pictures of fields showing nuclei staining of astrocytes treated as indicated for each panel. Scale bar, 100 μm. b, Graph showing the uptake of 2-NBDG expressed as arbitrary fluorescent units in astrocytes under control conditions (Control) or treated for 24 h with MG conditioned medium (CM*). In some experiments, cells were treated with either La3+ (200 μm) or gap27 (300 μg/ml) for 10 min before dye uptake assay. Each plotted value represents the mean ± SEM of four independent determinations. n.s., No significant difference; *p < 0.05 and ***p < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Badger AM, Cook MN, Lark MW, Newman-Tarr TM, Swift BA, Nelson AH, Barone FC, Kumar S. SB 203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production, and inducible nitric oxide synthase in bovine cartilage-derived chondrocytes. J Immunol. 1998;161:467–473. - PubMed
    1. Bao L, Locovei S, Dahl G. Pannexin membrane channels are mechanosensitive conduits for ATP. FEBS Lett. 2004;572:65–68. - PubMed
    1. Battelino T, Goto M, Krzisnik C, Zeller WP. Tumor necrosis factor-alpha alters glucose metabolism in suckling rats. J Lab Clin Med. 1999;133:583–589. - PubMed
    1. Blomstrand F, Giaume C. Kinetics of endothelin-induced inhibition and glucose permeability of astrocyte gap junctions. J Neurosci Res. 2006;83:996–1003. - PubMed
    1. Boone E, Vandevoorde V, De Wilde G, Haegeman G. Activation of p42/p44 mitogen-activated protein kinases (MAPK) and p38 MAPK by tumor necrosis factor (TNF) is mediated through the death domain of the 55-kDa TNF receptor. FEBS Lett. 1998;441:275–280. - PubMed

Publication types

MeSH terms

LinkOut - more resources