Volume 54, Pages S20-S24 (December 1998) CsA and FK506 up-regulate eNOS expression: Role of reactive oxygen species and AP- 1  Javier Navarro-Antolín, Octavio Hernández-Perera, Susana López-Ongil, Manuel Rodríguez-Puyol, Diego Rodríguez-Puyol, Santiago Lamas  Kidney International  Volume 54, Pages S20-S24 (December 1998) DOI: 10.1046/j.1523-1755.1998.06807.x Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 1 Effect of immunosuppressors cyclosporine A (CsA) and FK506 on endothelial nitric oxide synthase (eNOS) expression in cultured bovine aortic endothelial cells. Cells were incubated with 1 μm CsA or 1 μm FK506. A representative Northern blot experiment of hybridization with eNOS is shown. The blot shown is representative of three. Kidney International 1998 54, S20-S24DOI: (10.1046/j.1523-1755.1998.06807.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 2 Effects of a RNA polymerase II inhibitor on the CsA up-regulated expression of eNOS. Where indicated bovine aortic endothelial cells were preincubated 30minutes with 10 μm dichlorbenzimidazolriboside (DBR), without removing it from the incubation media during the whole experimental period with CsA or vehicle (12 hr). A representative Northern blot experiment of hybridization with eNOS is shown. The blot shown is representative of three. Kidney International 1998 54, S20-S24DOI: (10.1046/j.1523-1755.1998.06807.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Synthesis of reactive oxygen species (ROS) in cultured aortic endothelial cells exposed to CsA or FK506. Cells were incubated with 1 μm CsA or 1 μm FK506, at 37°C, and the ROS production was evaluated with a ROS-sensitive fluorescent probe, dihydrorhodamine (DHR123). Results shown are the mean ±sem of three different experiments. The symbol (*) indicates P < 0.05 versus control. Kidney International 1998 54, S20-S24DOI: (10.1046/j.1523-1755.1998.06807.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 4 Effect of catalase on the CsA or glucose oxidase up-regulation of eNOS mRNA expression in cultured bovine aortic endothelial cells. Catalase was added to the incubation media 10minutes before CsA (1 μm, 24 hr) or GO (1 mU/ml, 8 hr), without removing them from the incubation media for the whole experimental period. Lane 1, Control; lane 2, GO; lane 3, CAT + GO; lane 4, CsA; Lane 5, CAT + CsA. A representative Northern blot experiment of hybridization with eNOS and GAPDH probes is shown. The experiment shown is representative of two. Kidney International 1998 54, S20-S24DOI: (10.1046/j.1523-1755.1998.06807.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 5 Effect of CsA and glucose oxidase on the binding of AP-1 to an oligonucleotide containing the AP-1 cis-regulatory sequence of the eNOS promoter. EMSA of nuclear extracts from BAEC treated as indicated were performed as described in the Methods section. One representative experiment of 4 with nearly identical results is shown. Kidney International 1998 54, S20-S24DOI: (10.1046/j.1523-1755.1998.06807.x) Copyright © 1998 International Society of Nephrology Terms and Conditions