Invovlement of Mitogen Activated Protein Kinases– JNK1/2 and p38MAPkinase in ET–1 Mediated Apoptosis of Rat Retinal Ganglion Cells

Abstract

Abstract: : Purpose: Previous Studies from our laboratory have shown ET–1 treatment promotes apoptosis of cultured rat retinal ganglion cells. The purpose of this study was to determine the involvement of C–Jun–N–terminal kinase (JNK1/2) and p38 mitogen activated protein kinase (p38 MAP kinase) in the ET–1 mediated apoptosis of rat retinal ganglion cells. Methods: Virally transformed rat retinal ganglion cells (RGC–5) were synchronized by thymidine treatment, which blocks the cells at G₀/G₁ phase of the cell cycle. The synchronization was confirmed by flow cytometric analysis of the DNA content. The synchronized RGC–5 cells were treated with ET–1 (100nM) a dose selected from previous dose response study, for either 5,10,15 & 30 min or long–term treatments for 12, 18 & 24 hours. The synchronized RGC–5 cells were pre–treated for 30 min with SB203580 (10µM) a specific p38 MAP Kinase inhibitor, BQ788 (1µM), a selective ET_B receptor antagonist and BQ 610 (1µM), a selective ET_A receptor antagonist and subsequently treated with ET–1 (100nM) for 5 min. Activation of JNK1/2 and p38 MAP kinase were determined by immunoblotting using anti phospho JNK1/2 and anti phospho p38 MAP kinase antibodies. In–vitro p38 MAP kinase activity assays were carried out using ATF–2 as substrate in the presence of γ³²P. The kinase assay samples were separated by SDS/PAGE (10%) and radioactivity incorporated into ATF2 was detected by autoradiography. Results: The synchronized cells had a greater number of cells in the G₀/G₁phase (95%) versus the unsynchronized cells (80%). More consistent results were obtained in the phosphorylation of p38 MAP kinase using the synchronized RGC–5 cells. An increase in the p38 phosphorylation as early as 5 min was observed following ET–1 (100nM) treatment and a subsequent increase in the phosphorylation was also observed at 24 hours. A corresponding increase in ATF–2 phosphorylation at 5 min was observed as determined by the In–vitro kinase activity assay. The activation of p38 MAP kinase by ET–1 was appreciably blocked in the cells pre–treated with SB203580. An increase in JNK1/2 phosphorylation was observed following ET–1 (100nM) treatment for 5, 10 min and a subsequent increase at 24 hrs. The activation of JNK1/2 was reduced in cells pre–treated with BQ788 a selective ET_B receptor antagonist. Conclusions: ET–1 mediated apoptosis in RGC–5 cells appears to be mediated through an ET_B receptor – activated JNK1/2 and p38 MAP kinases.

Keywords: ganglion cells • apoptosis/cell death • signal transduction