Urocortin 2 lowers blood pressure and reduces plasma catecholamine levels in mice with hyperadrenergic activity.

Exaggerated adrenergic activity is associated with human hypertension. The peptide urocortin 2 (Ucn 2) inhibits catecholamine synthesis and secretion from adrenal chromaffin cells in vitro and administration to mammals lowers blood pressure (BP). The chromogranin A-null mouse (Chga-/-) manifests systemic hypertension because of excessive catecholamine secretion from the adrenal and decreased catecholamine storage. In the present study, we investigated whether systemic administration of Ucn 2 could reduce BP and adrenal and plasma levels of catecholamines in vivo. Ucn 2 peptide was administered to freely moving, conscious Chga-/- and wild-type control mice. Telemetry and HPLC measured changes in BP and catecholamine levels, respectively. In both groups of mice, Ucn 2 dose-dependently decreased BP, and this effect was mediated by corticotropin factor-receptor type 2. However, in Chga-/- mice, the maximal percentage decrease of systolic BP from basal systolic BP was 37% compared with only a 23% reduction in wild-type mice (P=0.04). In Chga-/- mice only, Ucn 2 decreased adrenal and plasma levels of catecholamines as well as adrenal levels of tyrosine hydroxylase protein and phosphorylation. In vitro mechanistic studies demonstrated that Ucn 2 reduces both catecholamine secretion and tyrosine hydroxylase promoter activity, suggesting that the exaggerated action of Ucn 2 to reduce BP in the Chga-/- mouse is mediated through inhibition of both catecholamine synthesis and secretion. The data suggest that Ucn 2 may be therapeutically useful in regulating the exaggerated sympathoadrenal function of hyperadrenergic hypertension.

Conserved regulatory motifs at phenylethanolamine N-methyltransferase (PNMT) are disrupted by common functional genetic variation: an integrated computational/experimental approach.

The adrenomedullary hormone epinephrine transduces environmental stressors into cardiovascular events (tachycardia and hypertension). Although the epinephrine biosynthetic enzyme PNMT genetic locus displays both linkage and association to such traits, genetic variation underlying these quantitative phenotypes is not established. Using an integrated suite of computational and experimental approaches, we elucidate a functional mechanism for common (minor allele frequencies > 30%) genetic variants at PNMT. Transcription factor binding motif prediction on mammalian PNMT promoter alignments identified two variant regulatory motifs, SP1 and EGR1, disrupted by G-367A (rs3764351), and SOX17 motif created by G-161A (rs876493). Electrophoretic mobility shifts of approximately 30-bp oligonucleotides containing ancestral versus variant alleles validated the computational hypothesis. Queried against chromaffin cell nuclear protein extracts, only the G-367 and -161A alleles shifted. Specific antibodies applied in electrophoretic gel shift experiments confirmed binding of SP1 and EGR1 to G-367 and SOX17 to -161A. The in vitro allele-specific binding was verified in cella through promoter reporter assays: lower activity for -367A haplotypes cotransfected by SP1 (p = 0.002) and EGR1 (p = 0.034); and enhanced inhibition of -161A haplotypes (p = 0.0003) cotransfected with SP1 + SOX17. Finally, we probed cis/trans regulation with endogenous factors by chromatin immunoprecipitation using SP1/EGR1/SOX17 antibodies. We describe the systematic application of complementary computational and experimental techniques to detect and document functional genetic variation in a trait-associated regulatory region. The results provide insight into cis and trans transcriptional mechanisms whereby common variation at PNMT can give rise to quantitative changes in human physiological and disease traits. Thus, PNMT variants in cis may interact with nuclear factors in trans to govern adrenergic activity.