Before subjecting the segments to different experimental conditions to obtain the vasoreactive measurements described below, the aortic segments were stretched and stabilized to one of the following preloads of 10, 16, 20, 28, 30, 40, 50, or 65 mN in Krebs Ringer solution at 37°C. Aortic segments of the same mouse were stretched to different preloads, and measurements were performed in parallel. Randomization of the segments was employed to exclude non-specific effects of the changes in aortic physiology between the proximal and distal segments of the thoracic aorta.
K+ concentration-contraction curves (KCC) were measured at different preloads by gradually increasing external K+ from 5.9 to 10, 15, 20, 25, 30, 35, 40, and 50 mM by isosmotic replacement of Na+ by K+ in Krebs Ringer solution. Concentration-contraction curves for α1 adrenoceptor stimulation of the aortic segments with phenylephrine (PECC) were cumulatively determined for concentrations of 3 × 10−9 to 3 × 10−6 M. Both KCC and PECC were fitted with sigmoidal concentration-response equations with variable slope (GraphPad Prism), which revealed maximal responses (Emax) and (the logarithm of) the concentration resulting in 50% of the maximal contraction (logEC50 for PE and EC50 for K+).
Phasic contractions by PE were measured by adding 1 μM PE to the segments incubated for 3 min in Krebs Ringer solution without extracellular Ca2+ and with 1 mM EGTA. These contractions are mediated by the IP3-dependent release of contractile Ca2+ from the SR (Fransen et al., 2015 (link)). After the phasic contraction, extracellular Ca2+ (3.5 mM) was added to measure the tonic contraction induced by Ca2+ influx in the PE-sensitized segment. Next, 35 μM diltiazem was added, to block the Ca2+ influx via voltage-gated Ca2+ channels. In this way, the contribution of VGCC and NSCC to the tonic contraction can be ascertained (Fransen et al., 2015 (link)). In some experiments, segments were incubated for 5 min with levcromakalim (1 μM), a KATP channel agonist, to repolarize aortic segments to the K+ equilibrium potential.
To assess the basal release of NO, contractions by elevated K+ or PE were measured in separate segments of the same mouse in the absence and presence of 300 μM L-NAME to inhibit eNOS. We have previously shown that this is the most sensitive way to measure basal NO release (van Langen et al., 2012 (link); Leloup et al., 2015b (link)). Relaxation of 1 μM PE-precontracted segments by endogenous and exogenous NO release was measured by constructing concentration-relaxation curves for acetylcholine (ACh, 3 × 10−9 to 3 × 10−6 M) and diethylamine NONOate (DEANO, 3 × 10−10 to 3 × 10−6 M), fitting the curves with sigmoidal concentration-response equations with variable slope (GraphPad Prism) to obtain Emax- and logEC50-values.
K+ concentration-contraction curves (KCC) were measured at different preloads by gradually increasing external K+ from 5.9 to 10, 15, 20, 25, 30, 35, 40, and 50 mM by isosmotic replacement of Na+ by K+ in Krebs Ringer solution. Concentration-contraction curves for α1 adrenoceptor stimulation of the aortic segments with phenylephrine (PECC) were cumulatively determined for concentrations of 3 × 10−9 to 3 × 10−6 M. Both KCC and PECC were fitted with sigmoidal concentration-response equations with variable slope (GraphPad Prism), which revealed maximal responses (Emax) and (the logarithm of) the concentration resulting in 50% of the maximal contraction (logEC50 for PE and EC50 for K+).
Phasic contractions by PE were measured by adding 1 μM PE to the segments incubated for 3 min in Krebs Ringer solution without extracellular Ca2+ and with 1 mM EGTA. These contractions are mediated by the IP3-dependent release of contractile Ca2+ from the SR (Fransen et al., 2015 (link)). After the phasic contraction, extracellular Ca2+ (3.5 mM) was added to measure the tonic contraction induced by Ca2+ influx in the PE-sensitized segment. Next, 35 μM diltiazem was added, to block the Ca2+ influx via voltage-gated Ca2+ channels. In this way, the contribution of VGCC and NSCC to the tonic contraction can be ascertained (Fransen et al., 2015 (link)). In some experiments, segments were incubated for 5 min with levcromakalim (1 μM), a KATP channel agonist, to repolarize aortic segments to the K+ equilibrium potential.
To assess the basal release of NO, contractions by elevated K+ or PE were measured in separate segments of the same mouse in the absence and presence of 300 μM L-NAME to inhibit eNOS. We have previously shown that this is the most sensitive way to measure basal NO release (van Langen et al., 2012 (link); Leloup et al., 2015b (link)). Relaxation of 1 μM PE-precontracted segments by endogenous and exogenous NO release was measured by constructing concentration-relaxation curves for acetylcholine (ACh, 3 × 10−9 to 3 × 10−6 M) and diethylamine NONOate (DEANO, 3 × 10−10 to 3 × 10−6 M), fitting the curves with sigmoidal concentration-response equations with variable slope (GraphPad Prism) to obtain Emax- and logEC50-values.
Free full text: Click here
