Western Blot analysis of ETAR and ETBR.

<p>Cardiac left ventricle tissue homogenates from control animals with untreated cardiac arrest (C group), or after 180 min of untreated return of spontaneous circulation (ROSC180 group), after 180 min of mild therapeutic hypothermia (MTH group) or 180 min after S-PBN infusion (S-PBN group) were loaded on a 12% SDS-acrylamide gel. Bands for endothelin A receptor (ETAR), endothelin B receptor (ETBR) and β-actin were detected at 25 kDa, 50 kDa and 42 kDa, respectively (A). Protein band intensities were quantified using a ChemiDoc XRS and Quantity One software and normalized to the loading control (β-actin) (B). Error bars represent standard error of the mean (SEM). Significant results (p<0.05) are marked with (*), (p<0.001) are marked with (***).</p

Western Blot analysis of nNOS and eNOS.

<p>Cardiac left ventricle tissue homogenates from control animals with untreated cardiac arrest (C group), or after 180 min of untreated return of spontaneous circulation (ROSC180 group), after 180 min of mild therapeutic hypothermia (MTH group) or 180 min after S-PBN infusion (S-PBN group) were loaded on a 4–20% SDS-acrylamide gel. Bands for neuronal NOS (nNOS) (A), endothelial NOS (eNOS) (C) and β-actin (A and C) were detected at 160 kDa, 135 kDa and 42 kDa, respectively. Protein band intensities for nNOS (B) and eNOS (D) were quantified using a ChemiDoc XRS and Quantity One software and normalized to the loading control (β-actin). Error bars represent standard error of the mean (SEM). Significant results (p<0.01) are marked with (**), (p<0.001) are marked with (***).</p

Immunohistochemistry of ETAR.

<p>Cardiac left ventricular tissue from control animals with untreated cardiac arrest (A), or after 180 min of untreated return of spontaneous circulation (B), after 180 min of mild therapeutic hypothermia (C) or 180 min after S-PBN infusion (D) were stained with anti-ETAR. The amount of ETAR-positive cardiac cells was higher in pigs treated with mild therapeutic hypothermia (C) and S-PBN (D), respectively, compared to controls (A) and untreated animals (B).</p

Effects of S-PBN on ET-1, ECE-1, ETAR and ETBR mRNA expression.

<p>Endothelin 1, ET-1 (A), endothelin converting enzyme 1, ECE-1 (B), endothelin A receptor, ETAR (C), and endothelin B receptor, ETBR (D), mRNA expression analysis was performed by real-time qPCR. The relative fold changes to control animals with untreated cardiac arrest (C group) were normalized with β-actin and calculated for animals after 180 min of untreated return of spontaneous circulation (ROSC180 group) or 180 min after S-PBN infusion (S-PBN group). Error bars represent standard error of the mean (SEM). Significant results (p<0.01) are marked with (**), (p<0.001) are marked with (***).</p

Porcine primer sequences: forward (For.) and reverse (Rev.), with transcript (RT-PCR product) length and EMBL database accession number.

<p>(ET-1) Endothelin-1; (ETAR) Endothelin A-receptor; (ETBR) Endothelin B-receptor; (ECE-1) Endothelin-Converting-Enzyme-1; (SDH) Succinate Dehydrogenase.</p>*<p>GeneBank accession number at <a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>.</p

Immunohistochemistry of nNOS.

<p>Cardiac left ventricular tissue from control animals with untreated cardiac arrest (A), or after 180 min of untreated return of spontaneous circulation (B), after 180 min of mild therapeutic hypothermia (C) or 180 min after S-PBN infusion (D) were stained with anti-nNOS. The amount of nNOS-positive cardiac cells was higher in animals treated with mild therapeutic hypothermia (C) and S-PBN (D), respectively compared to controls (A) and untreated animals (B).</p

Effects of ROSC and MTH on ECE-1 and ET-1 mRNA expression analysis.

<p>Endothelin converting enzyme 1, ECE-1 (A), and endothelin 1, ET-1 (B), mRNA expression analysis was performed by real-time qPCR. The relative fold changes to control animals with untreated cardiac arrest (C group) were normalized with β-actin and calculated for animals after 30, 60 and 180 min of untreated return of spontaneous circulation (ROSC30, ROSC60 and ROSC180 groups) or after 180 min of mild therapeutic hypothermia (MTH group). Error bars represent standard error of the mean (SEM). Significant results (p<0.05) are marked with (*), (p<0.001) are marked with (***).</p

Effects of ROSC and MTH on ETAR and ETBR mRNA expression analysis.

<p>Endothelin A receptor, ETAR (A), and endothelin B receptor, ETBR (B), mRNA expression analysis was performed by real-time qPCR. The relative fold changes to control animals with untreated cardiac arrest (C group) were normalized with β-actin and calculated for animals after 30, 60 and 180 min of untreated return of spontaneous circulation (ROSC30, ROSC60 and ROSC180 groups) or after 180 min of mild therapeutic hypothermia (MTH group). Error bars represent standard error of the mean (SEM). Significant results (p<0.001) are marked with (***).</p