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. 2010 Jul 1;19(13):2567-80.
doi: 10.1093/hmg/ddq135. Epub 2010 Apr 8.

Genetic Regulation of Catecholamine Synthesis, Storage and Secretion in the Spontaneously Hypertensive Rat

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Free PMC article

Genetic Regulation of Catecholamine Synthesis, Storage and Secretion in the Spontaneously Hypertensive Rat

M L Jirout et al. Hum Mol Genet. .
Free PMC article

Abstract

Understanding catecholamine metabolism is crucial for elucidating the pathogenesis of hereditary hypertension. Here we integrated transcriptional and biochemical profiling with physiologic quantitative trait locus (eQTL and pQTL) mapping in adrenal glands of the HXB/BXH recombinant inbred (RI) strains, derived from the spontaneously hypertensive rat (SHR) and normotensive Brown Norway (BN.Lx). We found simultaneous down-regulation of five heritable transcripts in the catecholaminergic pathway in young (6 weeks) SHRs. We identified cis-acting eQTLs for Dbh, Pnmt (catecholamine biosynthesis) and Vamp1 (catecholamine secretion); enzymatic activities of Dbh and Pnmt paralleled transcripts, with pQTLs for activities mirroring eQTLs. We also detected trans-regulated expression of Vmat1 and Chga (both involved in catecholamine storage), with co-localization of these trans-eQTLs to the Pnmt locus. Pnmt re-sequencing revealed promoter polymorphisms that result in decreased response of the transfected SHR promoter to glucocorticoid, compared with BN.Lx. Of physiological pertinence, Dbh activity negatively correlated with systolic blood pressure in RI strains, whereas Pnmt activity was negatively correlated with heart rate. The finding of such cis- and trans-QTLs at an age before the onset of frank hypertension suggests that these heritable changes in biosynthetic enzyme expression represent primary genetic mechanisms for regulation of catecholamine action and blood pressure control in this widely studied model of hypertension.

Figures

Figure 1.
Figure 1.
Chromaffin cell genes involved in catecholamine biosynthesis, storage, secretion and degradation. Depicted are important chromaffin cell-expressed genes and functional relationships between them. Two different parameters are color coded: the heritability (H2) of transcript abundance as computed from the RI panel and the normalized ratio (NR) for differences between the progenitors (on the left and right side of each box, respectively). The red curved arrow represents the catecholamine biosynthetic pathway, with the intermediates also in red. The blue curved arrows represent the detected gene regulatory networks from eQTL analysis, pointing from the regulator to the regulated gene.
Figure 2.
Figure 2.
Co-localization of physiological and expression QTLs on the short arm of chromosome 3. (A) LOD plots for dopamine tissue concentration, Dbh tissue enzymatic activity and Dbh gene expression are shown. Peak LOD values are given in the inset. 95% confidence intervals (95% CIs), determined by 2-LOD drop, are shown beneath the peaks. (B) Additive/directional effect of the SHR allele on each trait at different RNO 3 loci is expressed as percent deviation from the overall trait mean. The arrow marks the location of the Dbh promoter SNP T − 551G. (C) Chromosome 3 idiogram is provided for reference. (D) Analysis of previously mapped cardiovascular QTLs overlapping with the conflated 95% CIs for the three QTLs reported here. Each bar represents a previously localized physiological QTL (data from http://rgd.mcw.edu). Bar numbers refer to Supplementary Material, Table S2 online, where details for each QTL can be found.
Figure 3.
Figure 3.
Co-localization of physiological and expression QTLs on chromosome 10. (A) LOD plots for adrenal Pnmt tissue enzymatic activity and Pnmt, Vmat1 and Chga transcript abundance. Peak LOD values are given in the inset. Bootstrap test was used to estimate the 95% confidence intervals (95% CIs) for Pnmt tissue activity and Pnmt transcript abundance—see Materials and Methods for details. Horizontal bars represent 2-LOD drop CIs; different traits are colored same as the LOD plots. SHR allele at the peak locus was associated with a trait value decrease for all four traits. The arrow points to the location of the Pnmt promoter SNP T − 529G. (B) Idiogram of rat chromosome 10 is provided for reference. (C) Analysis of previously mapped cardiovascular QTLs overlapping with the conflated 95% CI for the QTLs reported here. Each bar represents a physiological QTL (data from http://rgd.mcw.edu). Bar numbers refer to Supplementary Material, Table S3 online, where details can be found.
Figure 4.
Figure 4.
Functional studies on the single-nucleotide polymorphisms identified in the Pnmt promoter. Bioluminescent activity of luciferase was measured in rat PC12 pheochromocytoma cells transfected with ∼1 kb segment of Pnmt promoter/luciferase reporter in pGL3-Basic vector (Promega) after 16 h incubation with incremental doses of the glucocorticoid dexamethasone. Each experiment was conducted in four replicates, with luciferase results normalized to cell protein in each plate. Results are presented as -fold augmentation by dexamethasone stimulus over the signal from cells transfected with a promoterless (empty) pGL3-Basic vector. Dexamethasone stimulation elicited significant differences in promoter activity. Differences between SHR and BN.Lx promoters were dose dependent and were significant starting at concentrations of 10 nm of dexamethasone.

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