Figure 1.
Carbon monoxide liberating compound containing a central molybdenum atom.
Figure 2.
Effect of ALF186 on rotenone-induced apoptosis.
A: Representative experiment after FITC Annexin V and propidiumiodide staining and flow-cytometric analysis. 1×104 cells in each experiment were analyzed. B: ALF186-mediated effect on apoptosis analyzed by Annexin V staining (top, n = 6; mean±SD; * = p<0.001 rotenone vs. ALF186 10, 50 and 100 µmol/L+rotenone) and caspase-3 cleavage (bottom, representative Western blot of caspase-3 cleavage products). C: Flow-cytometric analysis of mitochondrial membrane potential change relative to untreated cells (ΔΨm, n = 6; mean±SD; * = p<0.05 rotenone vs. ALF186+ rotenone and ALF186+ rotenone vs. iALF186+ rotenone).
Figure 3.
The role of the heme-containing “primary” CO-target NOS in ALF186 mediated effects.
A: Representative western blot of nNOS and iNOS protein expression after ALF186 treatment. B: Quantification of nitric oxide production by colorimetric measurement of total nitrite after ALF186 incubation ±NOS-Inhibition by L-NAME (n = 6; fold change vs. untreated cells; mean±SD; * = p<0.05 ALF186 vs. L-NAME +ALF186). C: Flow-cytometric analysis of mitochondrial membrane potential change relative to untreated cells after inhibition of NOS (ΔΨm, n = 6; mean±SD; * = p<0.05 rotenone vs. ALF186+rotenone).
Figure 4.
Influence of ALF186 on ROS production and role of NADPH oxidase in ALF186 mediated effects.
A: Flow-cytometric analysis of ROS production after CM-H2DCFDA staining (n = 6; mean±SD; * = p<0.05 all vs. untreated; rotenone vs. ALF186 and vs. PMA). B: Flow-cytometric analysis of mitochondrial membrane potential change relative to untreated cells after NADPH oxidase inhibition (ΔΨm, n = 6; mean±SD; * = p<0.05 DPI vs. untreated; rotenone vs. ALF186+rotenone and ALF186+rotenone vs. DPI+ALF186+rotenone).
Figure 5.
Effect of ALF186 on cellular cGMP levels and expression of sGC.
A: ELISA analysis of cellular cGMP concentration after ALF186 treatment (n = 6; median±25th/75th percentile; * = p<0.05 untreated vs. ALF186 10, 50, 100 µmol/L). B: Analysis of cellular cGMP concentration after ALF186 treatment ± sGC-Inhibition by ODQ (n = 6; median±25th/75th percentile; * = p<0.05 untreated vs. ALF186 and ALF186 vs. ODQ +ALF186). C: Effect of ALF186 on sGC β1 subunit mRNA (top; RT-PCR analysis, n = 6; median±25th/75th percentile; * = p<0.05 untreated vs. ALF186 50 µmol/L and rotenone vs. ALF186 50, 100 µmol/L+rotenone) and protein (bottom, representative Western Blot of sGC β1 protein) expression.
Figure 6.
The role of sGC in ALF186 mediated effects.
A: Flow-cytometric analysis of mitochondrial membrane potential change relative to untreated cells after inhibition or induction of sGC (ΔΨm, n = 6; mean±SD; * = p<0.05 rotenone vs. ALF186+ rotenone, ALF186+rotenone vs. ODQ+ALF186+rotenone and rotenone vs. YC-1+rotenone). B: Flow-cytometric analysis after Annexin V staining and incubation with the cGMP-analog 8-Br-cGMP (n = 6; mean±SD; * = p<0.05 rotenone vs. ALF186+rotenone and rotenone vs. 8-Br-cGMP+rotenone). C: Flow-cytometric analysis after Annexin V staining and incubation with the PKG-inhibitor KT5823 (n = 6; mean±SD; * = p<0.05 rotenone vs. ALF186+rotenone and ALF186+rotenone vs. KT5823+ALF186+rotenone).
Figure 7.
Effect of ALF186 treatment on IRI in retinal ganglion cells in vivo.
A: Representative images from flat mounts with fluorogold-labeled retinal ganglion cells 7 days after IRI, ALF186 treatment and sGC inhibition with NS-2028. Scale bar 100 µm. B: Quantification of retinal ganglion cell density [cells/mm2] 7 days after IRI, ALF186 treatment and sGC inhibition in vivo (n = 6 per group; mean±SD; * = p<0.05 IRI vs. ALF186+IRI and ALF186+IRI vs. NS-2028+ALF186+IRI).
Figure 8.
Effect of ALF186 on retinal caspase-3, Bax, Bcl-2 and sGC-subunit gene expression analyzed by RT-PCR.
Retinal expression of caspase-3 (A; n = 6; mean±SD.; * = p<0.05 IRI vs. ALF186+IRI and ALF186+IRI vs. NS-2028+ALF186+IRI), Bax (B; n = 6; mean±SD; * = p<0.05 IRI vs. ALF186+IRI and ALF186+IRI vs. NS-2028+ALF186+IRI), Bcl-2 (C; n = 6; mean±SD), sGC α1 subunit (D; n = 6; mean±SD) and sGC β1 subunit (E; n = 6; mean±SD) mRNA expression in ischemic retinal tissue in relation to the corresponding non-ischemic retinae analyzed by RT-PCR.