Figure 1.
Schematic overview of drug interactions with the NO/sGC/cGMP pathway.
NO produced by NOS activates sGC, resulting in the production of cGMP, and subsequent relaxation of VSMCs and inhibition of platelet aggregation and leukocyte adhesion, among others. In turn, cGMP is broken down to GMP by PDE5, which can be inhibited by Sildenafil. ROS can interact at various levels with this pathway, including facilitating the oxidation of the iron in the heme-prosthetic group of sGC. The resulting heme-free sGC (apoGC) is no longer functional and a target for rapid ubiquitin-mediated proteolytic degradation. However, BAY 58-2667 can bind heme-free sGC and reactivate cGMP production independent of NO. BAY 41-2272, on the other hand, can stimulate the activity of functional (reduced-heme) sGC synergistically with NO. Nitrite, a hypoxia selective NO donor, can also be used to stimulate this pathway selectively. No cell specific effects for the compounds or enzymes are assumed, they are shown as such for simplicity only. Arrows indicate binding, interaction or induction; bold arrows indicates increased production; dashed lines indicate an inhibitory effect; bold/underlined text indicates NO/cGMP-mediated effects; ‘apoGC’ = heme-free sGC. Figure was produced using Servier Medical Art.
Figure 2.
Effect of post-treatment with BAY 58-2667, BAY 41-2272, and Sildenafil on body temperature (left) and mortality (right).
(A–B) WT mice were injected i.v. with 9.5–11 mg/kg LPS (E. coli) or 17.5 mg/kg LPS (S. abortus equi) at t = 0 and treated (+3 h or +8 h) i.v. with 100 µg/kg BAY 58-2667 or vehicle control (+3 h or +8 h). The combined results of three independently performed experiments are shown (nvehicle = 15, n+3 h = 8, n+8 h = 12). (C–D) WT mice were injected i.v. with 17.5 mg/kg LPS (S. abortus equi) at t = 0 and treated i.v. with 100 µg/kg BAY 41-2272 (+3 h or +8 h) or vehicle control (+8 h). The combined results of two independently performed experiments are shown (nvehicle = 11, n+3 h = 8, n+8 h = 8). (E–F) WT mice were injected i.v. with 17.5 mg/kg LPS (S. abortus equi) at t = 0 and treated i.v. with 1 mg/kg Sildenafil (+3 h or +8 h) or vehicle control (+8 h) (n = 5). Data are means ± SEM; temperature curves were compared to appropriate vehicle controls via repeated-measure ANOVA (see Table S1 for individual F-statistics, p- and n-values). For panel A & C, significance was calculated in relation to appropriate controls for single experiments. Survival curves were compared to controls via log-rank test for merged experiments. ****, p≤0.0001; ***, p≤0.001; **, p≤0.01; *, p≤0.05; and ns = nonsignificant.
Figure 3.
cGMP levels in kidney, heart and liver, and NOx− and IL-6 levels in plasma.
(A–C) cGMP levels in whole kidney (A), heart (B) and liver (C) homogenates, respectively, 2 h post-treatment (n = 3). (D) Circulating NOx− levels in plasma, and (E) IL-6 levels in plasma, 2 h post-treatment (n = 3). Data are means ± SEM and comparisons were made between baseline (PBS) and LPS-challenged vehicle control animals (*), as well as LPS-challenged vehicle control and treatment groups (#) via one-way ANOVA with Fisher's LSD test. ***, p≤0.001; **, p≤0.01 and *, p≤0.05.
Figure 4.
(A) The number of apoptotic cells per heart section were counted and normalized over the total surface area of the tissue section, 2 h post-treatment (n = 3). (B) Representative example of whole heart section for late (+8 h) vehicle control (top-left), and late (+8 h) BAY 58-2667 treatment (bottom-left). Representative example of data processing in BD Attovision software: calculation of total surface area (top-right) and detection of TUNEL events (bottom-right). Data are means ± SEM and comparisons were made between baseline (PBS) and LPS-challenged vehicle control animals (*), and LPS-challenged vehicle control and treatment groups (#) via one-way ANOVA with Fisher's LSD test. ***, p≤0.001; **, p≤0.01 and *, p≤0.05.
Figure 5.
Effect of BAY 58-2667 and BAY 41-2272 treatment on hemodynamic parameters.
Mean arterial pressure (MAP) and heart rate (HR) were recorded via implanted telemetry devices. (A–B) Mice were injected i.v. with 9.5–11 mg/kg LPS (E. coli), and treated i.v. with 100 µg/kg BAY 58-2667 (+3 h or +8 h) or vehicle control (+3 h or +8 h); 2 h pre- until 4 h post-treatment of data is shown (n = 4). (C–D) Mice were injected i.v. with 9.5–11 mg/kg LPS (E. coli), and treated i.v. with 100 µg/kg BAY 41-2272 (+3 h or +8 h, n = 2) or vehicle control (+3 h or +8 h, n = 1); 2 h pre- until 4 h post-treatment of data is shown. (E–F) Unchallenged mice were injected with saline (PBS), 100 µg/kg or 300 µg/kg BAY 58-2667; 4 h of data post-injection is shown (n = 4). Data are means and were compared to vehicle controls by fitting a linear mixed model (see Table S2 for fixed term statistics). ****, p≤0.0001; ***, p≤0.001; *, p≤0.05; ns = nonsignificant; trt = treatment effect and time×trt = time-treatment interaction.
Figure 6.
Effect of BAY 58-2667 and BAY 41-2272 treatment on LF (nu).
Normalized low frequency (LF (nu)) values for 40 min pretreatment were compared to 40 min post-treatment values and plotted as changing trends over time for vehicle +3 h (A), BAY 41-2272 +3 h (B), BAY 58-2667 +3 h (C), vehicle +8 h (D), BAY 41-2272 +8 h (E), and BAY 58-2667 +8 h (F).
Figure 7.
Effect of BAY 58-2667 and BAY 41-2272 treatment on scaling factors.
Scaling factors (α) for 40 min pretreatment were compared to 40 min post-treatment values and plotted as changing trends over time for vehicle +3 h (A), BAY 41-2272 +3 h (B), BAY 58-2667 +3 h (C), vehicle +8 h (D), BAY 41-2272 +8 h (E), and BAY 58-2667 +8 h (F).