S-nitrosylation and permeation through connexin 43 hemichannels in astrocytes: induction by oxidant stress and reversal by reducing agents - PubMed (original) (raw)
S-nitrosylation and permeation through connexin 43 hemichannels in astrocytes: induction by oxidant stress and reversal by reducing agents
Mauricio A Retamal et al. Proc Natl Acad Sci U S A. 2006.
Abstract
Marked increase in cell permeability ascribed to open connexin (Cx)43 hemichannels is induced by metabolic inhibition (MI) of cortical astrocytes in culture, but the molecular mechanisms are not established. Dephosphorylation and/or oxidation of Cx43 hemichannels was proposed as a potential mechanism to increase their open probability. We now demonstrate that MI increases the number of hemichannels on the cell surface assayed by biotinylation and Western blot, and that this change is followed by increased dephosphorylation and S-nitrosylation. The increase in rate of dye uptake caused by MI is comparable to the increase in surface expression; thus, open probability and permeation per hemichannel may be unchanged. Reducing agents did not affect dephosphorylation of Cx43 hemichannels but reduced dye uptake and S-nitrosylation. Uptake was also reduced by elevated intracellular but not extracellular levels of reduced glutathione. Moreover, nitric oxide donors induced dye uptake and nitrosylation of surface Cx43 but did not affect its abundance or phosphorylation. Thus, permeability per channel is increased, presumably because of increase in open probability. We propose that increased dye uptake induced by MI is mediated by an increased number of Cx43 hemichannels in the surface and is associated with multiple molecular changes, among which nitrosylation of intracellular Cx43 cysteine residues may be a critical factor.
Conflict of interest statement
Conflict of interest statement: No conflicts declared.
Figures
Fig. 1.
MI induces dephosphorylation of Cx43 hemichannels and increases surface expression. Cultured astrocytes (≈80% confluent) were subjected to MI for different periods of time (0, 15, 30, 50, and 75 min), and relative levels of surface Cx43 were measured by Western blot analysis of biotinylated proteins from intact cells. (a) Sample blot. On the left, the phosphorylated (P3 and P2) and NP forms of total Cx43 in homogenates of control astrocytes (not biotinylated, NB) are indicated. MI induced progressive loss of phosphorylated forms as well as a progressive increase in surface expression. (b) Graph showing the densities of immunoreactive bands (P3 + P2 and NP) of surface Cx43 for astrocytes after different durations of MI (n = 4). The upper line shows the total amount of Cx43 relative to that present in the cell surface of control astrocytes (denoted by dotted line). The lower line shows the NP density. The difference between upper and lower lines is the amount of P2 + P3 (∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.001 as compared to the value of control astrocytes). (c) Immunofluorescence detection of Cx43 on the surface of control and metabolically inhibited astrocytes. In nonpermeabilized astrocyte monolayers, an anti-Cx43 E1 antibody (directed to the first extracellular loop of Cx43) labeled a few cells in control cultures (ca) but numerous cells after 75 min of MI (cb). In sister cultures, an anti-Cx43 CT antibody (directed to the C terminus, which is located intracellularly) did not react with cells either under control conditions (_ca_′) or after 75 min of MI (_cb_′). Both antibodies showed extensive Cx43 immunoreactivity in astrocytes permeabilized after 75 min of MI (cc and _cc_′). (n = 2.)
Fig. 2.
Dye uptake but not Cx43 dephosphorylation induced by MI is reduced by antioxidants. (a) Time-lapse measurements of EtdBr (10 μM) uptake during MI. Later application of 10 mM DTT reduced the rate of uptake. Mean and standard error of >20 cells in an experiment representative of seven. (b) Phase micrographs (Upper), fluorescence (Lower), from a different experiment. Astrocytes after 50 min of MI showed prominent EtdBr (100 μM) uptake during a 5-min application of the dye (MI). In sister cultures with 100 μM Trolox (MI + Trolox) added 20 min or 10 mM DTT (MI + DTT) added 10 min before the end of 50 min of MI, the dye uptake was greatly reduced (n = 3). (c) Western blot analysis of cell surface Cx43 pulled down with biotin from control astrocytes incubated for 20 min with or without Trolox (Upper Left) or 10 min with or without DTT (Lower Left) or from astrocytes subjected to 50 min of MI without or with application of 100 μM Trolox 20 min (Upper) or 10 mM DTT 10 min before the end of the period of inhibition (Lower). Representative results of three experiments are shown. The reducing agents did not prevent dephosphorylation of surface Cx43 induced by MI.
Fig. 3.
Intra- but not extracellular GSH blocks the dye uptake induced by MI. Astrocyte cultures near to confluency were subjected to a 30-min period of MI followed by a 5-min application of EtdBr (100 μM). (Upper) Phase contrast; (Lower) fluorescence. Control astrocytes did not show dye uptake (control, first column). MI induced dye uptake and changes in appearance (second column), which were prevented by cell-permeant GSH ethyl ester (10 mM, fourth column) but not by cell-impermeant GSH (10 mM, third column) applied for the last 10 min of MI. (n = 3.)
Fig. 4.
NO induces astrocyte dye uptake. (a) Confluent astrocyte cultures were photographed after incubation with 100 μM GSNO, an NO donor, for 50 min, and then exposed to 100 μM EtdBr for 5 min (Left), incubated with La3+ for the last 5 min of 50 min of GSNO and then exposed to EtdBr for 5 min (Middle), or incubated with DTT for the last 5 min of 50 min of GSNO and then EtdBr for 5 min (Right). La3+ and DTT largely prevented dye uptake, indicating rapid reduction of hemichannel permeability. n = 3. (b) Time-lapse measurement of dye uptake in 10 μM EtdBr. Dye uptake at a low basal rate was increased a few minutes after addition of GSNO. The rate of uptake was markedly reduced by DTT (10 mM) replacing GSNO at ≈48 min. Each point corresponds to mean fluorescence intensity of 21 cells in each of three independent experiments ± SE. (c) Western blot analysis of cell surface Cx43 pulled down with biotin from astrocytes under control conditions (Control), treated for 50 min with 100 μM GSNO, treated for 50 min with 100 μM GSNO, and with 10 mM DTT during the last 10 min or with 10 mM DTT for 10 min. Representative results of three experiments are shown.
Fig. 5.
An NO donor and MI increase S-nitrosylation of surface Cx43. S-nitrosylation and phosphorylation states were determined by surface biotinylation and Western blot analysis as described in Supporting Text. (a) After 50 min of treatment with 100 μM GSNO, S-nitrosylation of P2 and P3 bands of surface Cx43 (GSNO/+, right lane) was increased over basal (GSNO/−, middle lane). The left lane (Total) shows Cx43 bands of phosphorylated forms in control homogenate (5 μg of protein). (b, upper gel) Reactive bands of surface Cx43 in astrocytes under control conditions (left lane) and after 15 min (middle lane) and 50 min (right lane) of MI. (b, lower gel) Bands of S-nitrosylated surface Cx43 in astrocytes under conditions identical to those in b (upper gel). Nitrosylation was detected at 50 min of MI but not at 0 or 15 min (left and middle lanes). The lane labeled Total is as in a. (c) MI for 20 min caused nitrosylation of surface Cx43 (−DTT, middle lane), whereas DTT (10 mM) for the last 5 min of 20 min of MI prevented or reversed nitrosylation (+DTT, right lane). No basal nitrosylation was detected (left lane). The electrophoretic mobilities of the NP and two phosphorylated (P2 and P3) forms of Cx43 are indicated.
References
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