Udawatte C et al. (2008), Taurine suppresses the spread of cell death in ...

XB-ART-38786

Mol Vis 2008 Jan 01;14:1940-50.

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Taurine suppresses the spread of cell death in electrically coupled RPE cells.

Udawatte C , Qian H , Mangini NJ , Kennedy BG , Ripps H .

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To determine whether taurine exerts a protective effect on retinal pigment epithelium (RPE) cells exposed to a cytotoxic agent, cytochrome C (cyC), shown previously to induce apoptosis and produce cell death in electrically coupled neighboring cells. Monolayer cultures of confluent human RPE (ARPE-19) cells, which express gap-junctional proteins, were incubated in culture medium with or without taurine. After scrape loading cyC into the cells, we assayed these cells for caspase 3 activity and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to determine the spread of apoptosis. We found that cyC, too large a molecule to traverse gap junctional channels, produced apoptosis in cells injured by the scrape as well as those distant from the site of the scrape, presumably by the intercellular transmission of a toxic agent through the gap junctions that couple these cells. Incubation in taurine, or the gap-junction blocker, octanol, before application of cyC, reduced significantly the fraction of cells undergoing apoptosis. Voltage clamp recordings from electrically coupled Xenopus oocytes transfected with Cx43 showed that junctional communication was unaffected by taurine. Our results indicate that taurine can serve to suppress cell death in RPE cells independent of any effect on gap junctions. We have considered various avenues by which taurine can exert its protective effect, but the precise mechanism involved under these experimental conditions has yet to be identified.

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Species referenced: Xenopus
Genes referenced: casp3.2 gja1 rpe

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	Figure 2. Dye spread in electrically coupled cells. Gap junctions mediate the spread of Lucifer yellow (LY), indicating that junctional permeability and the system of intercellular communication are intact. Arrowheads in all panels show the location of the small injury (scrape) produced by the tip of the scalpel blade. Fluorescent images of cells incorporating both dextran rhodamine (DR) and LY show that whereas the rhodamine complex did not spread from the site of the scrape (middle panel), LY spread through the gap junctions that coupled the cells (lower panel). Scale bar (upper panel) equals 50 μm.
	Figure 3. Cell death revealed by caspase 3 activity. In the absence of taurine or the gap-junctional channel blocker octanol, an antibody to active caspase 3 shows that cell death induced by incubation with cytochome C (cyC) was widespread, extending to cells beyond the scrape (upper panels). In contrast, cell death in the presence of taurine (middle panels) or octanol (lower panels) was limited primarily to cells along the lines of the scrape. White lines indicate the position and approximate extent of the scrape. Scale bars represent 50 μm.
	Figure 4. Apoptosis visualized by TUNEL. TUNEL staining of ARPE-19 cells incubated in the presence of cytochrome C (cyC) with (middle panels) or without (upper panels) exposure to taurine also showed that significantly fewer cells were undergoing cyC-induced apoptosis in the presence of taurine. Octanol had a similar effect to taurine in blocking the spread of cell death induced by cyC (lower panels). Scale bars represent 50 μm.
	Figure 5. Quantification of cell death. Percent cell death was determined with activated caspase 3 immunocytochemistry and TUNEL assay as described in Methods. The percentage cell death was reduced by approximately 80% in the presence of taurine or the gap junctional blocker octanol, (p<0.001, comparing taurine- or octanol-treated group means to respective cyC-only control means; ANOVA with Student-Newman-Keuls post hoc testing). The reduction in percentage cell death in the presence of taurine was statistically indistinguishable from that in the presence of octanol (p>0.05). Error bars depict SEM; n=4 for caspase activity and n=3 for the TUNEL assay. “casp 3” refers to activated caspase 3 immunocytochemistry and “cyC” refers to cyctochrome C.
	Figure 6. Taurine effect on junctional conductance. Exposure to 40 mM taurine had no significant effect on the voltage sensitivity of the junctional currents recorded over the range of ±80 mV, although there was a slight current reduction at negative junctional voltages. The data (n=4 for each condition) were fit by the Boltzmann equation: Gjss = { (Gjmax – Gjmin ) / ( 1 + exp [A ( Vj –Vo ) ] } + Gjmin where Gjmax (normalized to unity) is the maximum conductance, Gjmin is the residual conductance at large values of Vj, and Vo is the Vj at which Gjss = (Gjmax–Gjmin)/2. The constant A (A=nq/kT) represents the voltage sensitivity in terms of gating charge as the equivalent number (n) of electron charges (q) moving through the membrane, k is the Boltzmann constant, and T is the absolute temperature. As shown in the inset, taurine had no significant effect on the junctional conductance between paired oocytes expressing this gap-junctional protein (p=0.5444 by Student’s t-test), and there were no significant differences in the values of A for taurine treated and control oocytes in either the positive or negative branches of the curves. These findings are a good indication that taurine does not affect Cx43 gap junctional channels.

References [+] :

Airaksinen, Taurine in normal and diseased human skeletal muscle. 1990, Pubmed