Figure 1.
Connexin hemichannels open under oxidative stress independent of calcium gating mechanism.
(A). In the normal Ca++ containing extracellular medium, no significant LY uptake was observed for MC (A,E) and L2 (I,M) cells. In Ca++ free medium, both cell types show LY uptake (B,F; J,N) consistent with hemichannel behavior. In the presence of normal Ca++ medium, both cell lines allow LY uptake under oxidative stress induced by 10% CSE (C,G; K,O) and 1 mM H2O2 (D,H; L,P). Hemichannel mediated dye uptake was not observed in Cx-deficient N2A cells from Ca++ free medium (R,V) as well as under oxidative stress conditions (S,W; T,X). Scale bar: 20 µm. (B). Histogram representation of the LY uptake studies in the cell lines examined under different treatment conditions. Comparison of different treatment groups made against the control (*p<0.0001), values expressed as mean ± SE.
Figure 2.
Oxidative stress induces membrane depolarization and opens hemichannel.
(A). Plasma membrane depolarization induced by oxidative stress (H2O2/CSE) was detected by voltage sensitive dye (DiBAC4(3)). The histogram summarizes the results of change in the fluorescence intensity (indicative of membrane depolarization) as percentage of their basal fluorescence value (*p<0.0001). Error bar indicates SE of the mean. Both, Cx-expressing (MC) and Cx-deficient (N2A) cells show increase in their basal fluorescence (∼20–25%) after 3 min treatment with 1 mM H2O2 and 10% CSE with and without βGA pretreatment, while there was none-to-little increase in fluorescence (<3%) in DTT pretreated cells. (B). Quantitative estimation of the level of membrane depolarization required to open hemichannels. Membrane depolarization and hemichannel opening were assayed simultaneously using voltage sensitive dye (green) and hemichannel permeable TR. Hemichannels opened at 30 mM external K+ (E–H) and above (I–L). No dye uptake was observed at or below 25 mM K+ (A–D). Hemichannel opening at 30 mM K+ concentration corresponds to −38 mV of membrane voltage as calculated using the Nernst equation. Scale bar: 20 µm.
Figure 3.
Electrophysiological measurement of the membrane potential and dye uptake in MC.
(A). The membrane potential trace was measured using the whole-cell patch clamp technique from a cultured MC. Changing of [K+]o to various values is indicated by the bars under the trace. The pseudo-steady-state membrane potential values were −61, −42, −21 and −12 mV at [K+]o of 5, 30, 60 and 120 mM, respectively. The intracellular K+ concentration was ∼140 mM. (B). Uptake of LY dye in the extracellular solution of 30 mM KCl (C,D). The cells were incubated for 10 min before being washed with a LY dye-free buffer solution containing 5 mM KCl solution for 4–5 min. For comparison, dye uptake at 5 mM KCl is included (A,B). Scale bars: 25 µm in A,B and 10 µm in C,D.
Figure 4.
CSE cause cell death and intracellular oxidative stress.
(A). Live/dead cell assay to estimate hemichannel mediated cell death under oxidative stress. Early cell death was observed in CSE treated cells (red fluorescent cells; EtBr homodimer dye) at the end of 10 hrs (F) compared to control (E), βGA pretreated cells (G) and MFA pretreated cells (H), where more live cells (green fluorescent cells; Calcein AM dye) are seen. Cell death in F was predominantly apoptotic as evident from the morphology of the dying cells viz., cell shrinkage, fragmentation into membrane bound apoptotic bodies etc., (inset in panel F). The histogram shows the % of live cells at the end of 10 hrs in all four categories. Error bar indicates SE of the mean (*p<0.012). Scale bar: 20 µm. (B). Oxidative stress molecules enter into the cells through open hemichannels as detected by ROS sensitive dye (Carboxy-H2DCFDA). Cx-expressing cells (MC and L2), showed increase in fluorescence after 10 min treatment with CSE (B,G) and H2O2 (C, H) compared against their corresponding controls (A,F). Similarly, βGA pretreatment decreased such change with CSE (D,I) as well as with H2O2 (E,J). Cx-deficient N2A cells showed no significant change from their control (K) under same treatment conditions (L,M). Scale bar: 20 µm. Histogram summarizes the mean fluorescence intensities of different treatment conditions of all three cell lines subjected to oxidative stress. Error bar indicates SE of the mean (*p<0.0007, **p<0.0015).
Figure 5.
Hemichannel mediates cell death under oxidative stress as evidenced by gene transfection and silencing techniques.
(A). Cx43-GFP transfected N2A cells produced hemichannels and gap junctions (B, white arrow indicating typical distribution of Cx-channels in the cell-cell contact regions) and they responded to low Ca++ medium (E, LY uptake) and oxidative stress (C, EtBr uptake induced by CSE). The open hemichannels permit direct entry of ROS into the cells as detected by ROS sensitive dye (F). Live/dead assay showed significant cell death induced by CSE in Cx-transfected N2A cells (33%) (H,J) compared against their wild type N2A cells (9%) (G,I) (*p<0.0072). Scale bars (A–D): 5 µm, (E–J): 20 µm. (B). Apoptosis assay on Cx43 silenced MC after 10 hrs of induced oxidative stress (10% CSE). (A) Wild type MC in normal media (control) show live cells (blue nuclei, Hoechst 33342 stained). (B) Wild type MC in CSE show apoptotic cells (green cells, YO-PRO stained) indicated by white arrows. (C) Cx43 silenced MC show live cells (blue nuclei) and few necrotic cells (Red nuclei, Propidium Iodide) indicated by orange arrows. (D) Negative-siRNA transfected MC shows more apoptotic and necrotic cells than the wild type MC. Histogram shows the % apoptosis observed in different treatment groups (*p<0.0001). Scale bar: 5 µm
Figure 6.
Schematic of putative mechanisms involved in oxidative stress induced hemichannel opening and cell death.
Oxidative stress induced by CSE/H2O2 depolarizes the cell membrane, which opens hemichannels. Open hemichannels disturb ionic homeostasis (primarily Ca++, K+ and Na+ ions) (pathway shown in purple) [15]. Increase in free intracellular calcium would activate its dependent kinases as well as load mitochondria with Ca++ resulting in activation of intrinsic pathways of cell death. Open hemichannels permits transfer of apoptosis signaling molecules and metabolites including ATP [36], NAD [38], [52], etc., thus accelerating cell death (pathway shown in green). As shown in the present study, open hemichannels allow direct entry of ROS leading to cell death probably through apoptosis (pathway shown in maroon).