Fig 2: Mice with combined CathA/Scpep1 deficiency show higher increase in BP in response to systemic injection of ET-1.Sixteen week-old WT, Scpep1−/−, CathAS190A and DD male mice kept for two weeks on high-salt diet were intravenously injected with ET-1 solution in saline at a dose of 0.2 nmol/kg (A, B) and 5 nmol/kg (C, D). Changes in the systolic (ΔSBP) or diastolic (ΔDBP) blood pressure were calculated as differences between the means (±S.E) of the BP values recorded within 10 min intervals after the injections and means (±S.E) of the baseline BP values recorded within the 30 min interval before the injections. Two-way repeated measurements ANOVA was used to test differences between the mouse groups: significant differences between the mean BP values in Bonferroni post-test (* p<0.05, ** p<0.001, *** p<0.0001) are shown in the insert. N-value for each genotype is as follows: WT n = 5, DD n = 6, CathAS190A n = 6, Scpep1 −/− n = 7.

Fig 3: Mice with combined CathA/Scpep1 deficiency have reduced ET-1 degradation rate.Blood plasma, lungs and aorta were collected from WT, Scpep1 −/−, CathAS190A and DD mice 15 min post an intravenous injection of saline or ET-1 in saline (10 nmol/kg BW). The levels of ET-1 peptide were measured by ELISA in lung extracts (A), aorta extracts (B), and in blood plasma extracts (C) from mice injected with ET-1 and blood plasma extracts from mice injected with saline (D). Data are expressed as mean values (±S.E). At least 5 mice were studied for each genotype. * p<0.05 in Welch's modification of two-tailed unpaired t-test.

Fig 4: Cultured AVSMC from mice with combined CathA/Scpep1 deficiency show increased reactivity to ET-1. A. Pharmacological antagonists of ET receptors, BQ610 and BQ788, reduce MLC-2 phosphorylation in AVSMC treated with ET-1. Cultured AVSMC from WT mice were treated or not for 30 min with 2 µM BQ610 or BQ788 followed by 5 min induction with 100 nM ET-1 as indicated on the figure. Total protein extracts were analyzed by Western blotting using antibodies specific for phosphorylated (pThr18/Ser19-MLC) and total MLC-2 protein. Panel shows representative data of 3 independent experiments. Right panels show ratios (means and S.D.) of signal intensities for phosphorylated and total MLC-2 estimated with ImageQuant software. * p<0.05 in paired two-tailed t-test. B. Increased MLC-2 phosphorylation in AVSMC from double-deficient mice. Cultured AVSMC from WT, Scpep1−/−, CathAS190A and DD mice were treated for 5 min with 100 nM ET-1. Total protein extracts were analyzed by Western blotting using antibodies specific for phosphorylated and total MLC-2 protein. Panel shows representative data of 3 independent experiments. Graph below the panel shows ratios (mean values and S.D.) of signal intensities for phosphorylated and total MLC-2 protein. * p<0.05 in paired two-tailed t-test.

Fig 5: ET-1 causes significantly stronger constriction in mesenteric arteries from mice with Scpep1 deficiency.Mesenteric arteries isolated from sixteen week-old WT, Scpep1−/−, CathAS190A and DD male mice were treated with increasing concentrations of Ach (A), SNP (B) NE (C) and ET-1 (D). ET-1 caused significantly (p<0.01 two-way repeated measurements ANOVA) higher constriction of arteries from DD, CathAS190A and Scpep1−/− as compared to those from mice with other genotypes. Bonferroni post test: * p<0.05 ** p<0.01, *** p<0.001. N-value of each genotype is as follows: WT n = 8, DD n = 8, CathAS190A n = 6, Scpep−/− n = 6.