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A. Kusuhara, M. Nakamura, A. Negi, G.M. Seigel; Nitric Oxide Rescues Retinal Neurons From Apoptosis Through a cGMP / Protein Kinase G Pathway That Activates cAMP–Responsive Element Binding Protein . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4844.
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Nitric oxide (NO) exerts an anti–apoptotic effect through a cGMP/protein kinase G (PKG) pathway in retina. cAMP–responsive element binding protein (CREB) plays an essential role in the NO/cGMP/PKG–mediated survival of rat cerebellar granule cells. We tested whether CREB is the downstream of the NO/cGMP/PKG anti–apoptotic cascade in R28 cells, a model of retinal neurons in culture.
R28 cells were induced to undergo apoptosis by 24–h serum deprivation. Varying concentrations of two kinds of NO donors, sodium nitroprusside (SNP) and nipradilol, were added to medium with or without a NO scavenger, a soluble guanylyl cyclase inhibitor, or a PKG inhibitor. The cells were stained with Hoechst 33258 and immunostained against activated caspase–3. Apoptosis was quantified by counting pyknotic or activated caspase–3 positive cells. Whole cell lysates were probed for total and phospho CREB (ser 133). Cells were transfected with a dominant–negative (DN) construct of CREB, which is defective of phosphorylation at ser133, followed by induction and detection of apoptosis as above.
SNP and nipradilol rescued R28 cells from apoptosis in a dose–dependent fashion with an optimal concentration of 1.0 and 10 µM, respectively, and showed cytotoxicity at higher concentrations. The NO scavenger and two inhibitors decreased the anti–apoptotic effect of NO donors. Both NO donors increased CREB phosphorylation, which were reversed by the two inhibitors tested. Transfection with DN–CREB interfered with the SNP’s anti–apoptotic effect.
NO exerts the anti–apoptotic effect in R28 cells via the NO/cGMP/PKG pathway, which is further mediated by CREB and eventually inactivates caspase–3.
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