Effect of different peptides on the membrane potential of rat liver mitochondria measured by safranine O.

<p>Panel <b>A</b>. Shown are traces of fluorescence in the medium described in “Materials and Methods.” In all traces, 5 mM of succinate and 1 µM of rotenone were supplemented about 150 s before the addition of a peptide at t = 0 s. Control, no other additions. gA, [Glu1]gA, [Glu3]gA, [Lys1]gA, and [Lys3]gA show traces after the addition of 5 nM (i.e. about 10 nanogram/ml) of a corresponding peptide at t = 0 s. Trace “excess [Glu1]gA” was measured with 1 µg/ml peptide. Panel <b>B.</b> Dose dependence of the effect of [Glu1]gA on mitochondrial membrane potential.</p

Effect of liposomes (1 µg/ml DPhPC) on the induction of electrical current on planar lipid bilayer by gA (2 nanogram/ml, panel A) and [Glu1]gA (200 nanogram/ml, panel B).

<p>The peptides and liposomes were added at one side of the membrane. The solution was 100 mM KCl, 10 mM Tris, 10 mM MES, pH = 7.0, BLM voltage was 50 mV.</p

Effect of [Glu1]gA on mitochondrial membrane potential in renal cells.

<p>Confocal images of cultured renal cells loaded with TMRE in control (A) and after treatment with 0.01 mg/ml [Glu1]gA (B). Diagram (C) presents the mean intensity of TMRE fluorescence through 10 confocal images for each [Glu1]gA (solid bars) or gA (hatched bars) concentration.</p

Micrographs of rat kidney cell culture loaded with Lysotracker Green in the absence (A), and in the presence of 0.1 mg/ml (B) of [Glu1]gA.

<p>Micrographs of rat kidney cell culture loaded with Lysotracker Green in the absence (A), and in the presence of 0.1 mg/ml (B) of [Glu1]gA.</p

Single-channel recordings (A, D) and the corresponding current histograms (B, E) of gramicidin A (picogram/ml, A, B, and C) and [Glu1]gA (2 nanogram/ml, D, E, and F) at a voltage of 100 mV applied to the DPhPC/decane membrane.

<p>The solution was 100 mM HCl. (<b>C</b>, <b>F</b>) Open state duration histograms fitted by a single exponential with a time constant of 116 ms (<b>C</b>) and 16 ms (<b>F</b>). The records were filtered at 100 Hz (<b>A</b>, <b>B</b>, and <b>C</b>) or 1000 Hz (<b>D</b>, <b>E</b>, and <b>F</b>).</p

Dissipation of the pH gradient on membranes of pyranine-loaded liposomes by gA and [Glu1]gA (both 2 µg/ml).

<p>Inner liposomal pH was estimated from pyranine fluorescence intensity measured at 505 nm upon excitation at 455 nm. Nigericin (1 µM) was added at 400–420 s to equilibrate the pH. Control, a record without peptides.</p

Bilateral nephrectomy and gentamicin pretreatment abolish neuroprotective action of SkQR1 and RRPC.

<p>The effects of SkQR1 after bilateral nephrectomy on the brain infarct volume and brain swelling of rats exposed to MCAO are shown in (A, B) and (C) correspondingly. Nephrotoxic gentamicin pretreatment shows similar abrogation of beneficial effects of SkQR1 and RRPC on the brain infarct volume (D, E) and brain swelling (F) of rats exposed to MCAO. (A, D) Representative T2-weighted MR-images from coronal brain sections (0.5 mm thick, from rostral (top) towards caudal (bottom)) obtained 24 h after reperfusion. Hyperintensities regions refer to ischemic areas. The evaluation of the brain damage area and brain swelling were done by using MRI with analysis of T2-weighted images.</p

Hematological parameters^a.

a<p>Hematological parameters changes after treatment with 2 µmol/kg SkQR1 for 12 h: Vehicle (n = 8) and SkQR1 (n = 8); for 24 h: Vehicle (n = 6) and SkQR1, (n = 7). RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit.</p>*<p>Denotes significantly different from the vehicle group (p<0.05) (<i>t</i> tests).</p

Neurological state^a.

a<p>The neurological state scores are recorded in a blind fashion and expressed as median and interquartile ranges, the 25th to 75th percentile are shown in the parentheses. Baseline represents neurological state of intacte rats. *Denotes significant from the score at 24 h after the MCAO compared to MCAO+ VEHICLE or MCAO groups (p<0.05) (Kruskal-Wallis test with the Mann–Whitney <i>u</i>-test with Bonferroni correction post hoc or Mann–Whitney test when comparing between two groups).</p

SkQR1 and RRPC provide some features of ischemic tolerance.

<p>(<b>A</b>) Changes in erythropoietin (EPO) level in the daily urine. 1 or 2 µmol/kg SkQR1 was injected i/p 24 h before urine collection. Rats were subjected to RRPC for 24 h and gentamicin for 6 days prior to urine collection. (<b>B</b>) Detection of phosphorylated glycogen synthase kinase-3β (P-GSK-3β) in the total brain tissue. Representative Western blots with corresponding densitometry averaged over 6 blots are shown. Band densities were normalized to the density of total GSK-3β band. 1 µmol/kg SkQR1 was injected i/p 24 h before excising the brain. (<b>C1–4</b>) Detection of P-GSK-3β in cultured cortical neurons (CNs) after co-culturing with renal tubular cells (RTC) treated with 250 nM SkQR1 for 1 h before 24 h of co-culturing (C1); control CNs (C2); co-culture of CNs and RTC (C3); co-culture of CNs with RTC primed with SkQR1 (C4). (<b>D</b>) Detection of P-GSK-3β in the cultural glial cells treated with 50 or 100 nM SkQR1 for 24 h. (<b>E</b>) Detection of EPO in the entire brain tissue. Densitometry of Western EPO spots averaged over 6 blots is shown. 1 µmol/kg SkQR1 was injected i/p 3 or 24 h before excising the brain. * Denotes significantly different from the control group (p<0.05) (One-way ANOVA, followed by Tukey’s post hoc analysis or <i>t</i> tests for independent samples).</p