Expression and activation state of signalling mediators involved in regulation of smooth muscle tone.

<p>Western blot analysis was performed for cortical and subcortical brain arterioles from 13 sheep, as described in the methods section. Differences of expression levels of signalling proteins involved in control of smooth muscle tone: (A) nNOS, P-eNOS and eNOS-protein, (B) P-CREB and CREB protein, (C) P-ERK and ERK protein was detected in relation to β-actin. As these data were not normally distributed they are presented as box plots, where boxes represent 25th and 75th percentiles, respectively. Medians are indicated by horizontal lines. Whiskers indicate 10th and 90th percentiles, respectively. 1+2, samples from two different sheep; Cx, cortex; Scx, subcortex; AU, arbitrary units; Ref. reference sample.</p

Effects of hypoxia and reoxygenation on arterial blood parameters.

<p>Values are given for baseline (min 0), during hypoxia (min 2 to min 14) and during reoxygenation (min 15 to min 23) in controls (blue) and after α1A-adrenergic blockade (red) for (A) pH, (B) partial pressure of carbon dioxide (pCO₂), (C) partial pressure of oxygen (pO₂), (D) oxygen saturation (sO₂, measured with a clinical blood gas analyzer), (E) base excess (BE), (F) lactate, (G) hematocrit (Hct) and (H) hemoglobin (Hb). Through lines separate baseline and hypoxia; dashed lines separate hypoxia and reoxygenation. Means ± SEM; * p < 0.05, ** p < 0.01 and *** p < 0.001 for trend, indicated separately for hypoxia and reoxygenation, respectively; black symbols indicate differences between treatment groups; n.s., not significant.</p

Correlation of blood flow and MABP.

<p>Cortical or subcortical CBF in the control group (blue symbols) were plotted against MABP during hypoxia (A, B) and for the reoxygenation phase (C, D), respectively. The effect of α1A-adrenergic blockade (red symbols) on the relationships of blood flow and MABP during hypoxia (E, F) or during reoxygenation (G, H) is plotted analogously. Linear regression was calculated for each data set. Best fit lines (black lines) and 95% confidence intervals (blue lines) are plotted. Correlation coefficients (<i>r</i>) and <i>p</i>-values are given in the respective panels.</p

Effects of hypoxia and reoxygenation on cortical and subcortical cerebral blood flow.

<p>Comparison of cortical and subcortical cerebral blood flow (CBF) in the control group (A) and under α1A-adrenergic blockade (B). Comparison of controls and α1A-adrenergic blockade for (C) cortex and (D) subcortex. Means ± SEM; * p < 0.05, ** p < 0.01 and *** p < 0.001 for trend, indicated separately for hypoxia and reoxygenation, respectively; black symbols indicate differences between treatment groups; n.s., not significant.</p

Cerebral blood supply in humans and sheep.

<p>Cerebral blood supply in humans and sheep.</p

Correlation of and MABP and sO₂.

<p>MABP in the control group (blue symbols) was plotted against sO₂ during hypoxia (A) and for the reoxygenation phase (B), respectively. The effect of α1A-adrenergic blockade (red symbols) on the relationships of MABP and sO₂ during hypoxia (C) or during reoxygenation (D) is plotted analogously. Linear regression was calculated for each data set. Best fit lines (black lines) and 95% confidence intervals (blue lines) are plotted. Correlation coefficients (<i>r</i>) and <i>p</i>-values are given in the respective panels.</p

Correlation of blood flow and oxygen saturation.

<p>Cortical or subcortical CBF in the control group (blue symbols) were plotted against oxygen saturation (sO₂) during hypoxia (A, B) and for the reoxygenation phase (C, D), respectively. The effect of α1A-adrenergic blockade (red symbols) on the relationships of blood flow and sO₂ during hypoxia (E, F) or during reoxygenation (G, H) is plotted analogously. Linear regression was calculated for each data set. Best fit lines (black lines) and 95% confidence intervals (blue lines) are plotted. Correlation coefficients (<i>r</i>) and <i>p</i>-values are given in the respective panels.</p

Effects of hypoxia and reoxygenation on vital parameters.

<p>(A) oxygen saturation (measured by pulse oximetry), (B) mean arterial blood pressure (MABP), (C) heart rate (HR) and (D) renal blood flow (RBF). Controls in blue and α1A-adrenergic blockade in red. Means ± SEM; * p < 0.05, ** p < 0.01 and *** p < 0.001 for trend, indicated separately for hypoxia and reoxygenation, respectively; black symbols indicate differences between treatment groups; n.s., not significant.</p

Indirubin Core Structure of Glycogen Synthase Kinase‑3 Inhibitors as Novel Chemotype for Intervention with 5‑Lipoxygenase

The enzymes 5-lipoxygenase (5-LO) and glycogen synthase kinase (GSK)-3 represent promising drug targets in inflammation. We made use of the bisindole core of indirubin, present in GSK-3 inhibitors, to innovatively target 5-LO at the ATP-binding site for the design of dual 5-LO/GSK-3 inhibitors. Evaluation of substituted indirubin derivatives led to the identification of (3<i>Z</i>)-6-bromo-3-[(3<i>E</i>)-3-hydroxyiminoindolin-2-ylidene]­indolin-2-one (<b>15</b>) as a potent, direct, and reversible 5-LO inhibitor (IC₅₀ = 1.5 μM), with comparable cellular effectiveness on 5-LO and GSK-3. Together, we present indirubins as novel chemotypes for the development of 5-LO inhibitors, the interference with the ATP-binding site as a novel strategy for 5-LO targeting, and dual 5-LO/GSK-3 inhibition as an unconventional and promising concept for anti-inflammatory intervention